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by the turn of the century, it was clear that the vertical transmission of hiv to children could effectively be prevented with the appropriate use of short courses of antiretroviral therapy (art). in their landmark paper on the issue, de cock et al. noted that few other aspects of hiv had, by that time, demonstrated results as dramatic as perinatal prevention. they argued that prevention of infection among children requires hiv and aids to be addressed as a disease of the family and the community and leads to consideration of other interventions, such as reproductive health care for women and support for children orphaned by the epidemic [1, p. 1181, the critical policy and programme issues needing to be addressed were highlighted as: increased hiv counselling and testing, expanded use of the most effective drug regimens and the prevention of transmission through breastfeeding. since then, all three issues have been resolved to the point of creating conditions feasible for the virtual prevention of hiv transmission from parent to child through pregnancy, delivery and infant feeding. more effective drugs are available, there is more integration among services and the drug regimens recommended are more comprehensive and better targeted. hiv counseling and testing (hct) no longer depends only on individuals stepping out of the line to be tested and risking gossip and stigmatization for doing so. in 2007, the world health organization (who) recommended provider-initiated testing and counselling (pitc) to streamline and normalize the process. pitc also helps to increase the uptake of prevention of mother-to-child transmission services (pmtct), more appropriately called prevention of vertical transmission (pvt), in order not to place blame unintentionally on women. home-based testing similarly expands services and provides for families to be tested together, as does couples testing, guidelines for which have recently been published by who. up until recently, pmtct programmes predominantly focussed on the prevention of hiv transmission to infants. however, successfully enrolling eligible pregnant women into treatment and retaining them on treatment is proving to be the most effective approach to protecting mothers, their children as well as their partners. the 2012 who pmtct technical update recommends a single universal regimen both to treat pregnant women living with hiv and to prevent transmission to her baby (so-called option b). this approach simplifies service delivery, aligns and links with art programmes enabling women to more easily transition between services and specifically targets the prevention of maternal mortality and vertical transmission both of which are disproportionately attributable to the poor health of pregnant women in need of treatment. extended prophylaxis given to infants or the continued treatment of their mothers also renders breastfeeding safer. given the dramatically increased vulnerability of young children whose mothers die or who suffer serious ill-health, treatment for women not only protects women's wellbeing but also safeguards their children. moreover, providing early treatment reduces hiv transmission between serodifferent couples. scientific and technical knowledge and programme evaluations of pvt have increased dramatically, as indicated by an overview of published literature on the topic between 1990 and 2010 (figure 1). average number of clinical, public health and programmatic papers on pvt published every five years from 1990 to 2009. despite pitc, some 10% to 20% of women refuse testing and another 20% or so either directly or indirectly avoid pvt prescriptions, procedures and follow-up. when women are not fully informed of the benefits, they may perceive pitc to be coercive. the need or perceived requirement to get their partners consent and the cost of transport and out-of-pocket expenses are some of the reasons women give for declining pvt prescriptions and procedures. a recent review highlights the loss of women between vertically provided pregnancy- and hiv-related services and stresses the need for both integration and family-focussed care. together with health service characteristics such as the availability of test kits and drugs and the negative attitudes of some healthcare providers towards women living with hiv, progress to prevent hiv infections among young children and to ensure the wellbeing of their mothers is slower than was hoped. we believe that what is needed, in addition to developments already highlighted, is to implement recommendation by de cock et al. that hiv and aids be addressed as a disease of the family and the community and that hiv interventions be integrated across the lifecycle of women, families and children. assessments of progress towards the millennium development goals show that the effects of hiv and aids are especially severe for the survival and wellbeing of mothers and young children. in addition, pvt is central to global hiv prevention efforts and remains the one area of prevention in which resounding success can be achieved. however, given current knowledge and technology to prevent almost all parent to child transmission of hiv, scale up of pvt and universal access to prevention and treatment has been slow, with especial concern for women and children's health in the worst affected countries. support for a concerted global effort to prevent hiv transmission from parent to child has been growing [2023]. at the 2011 united nations general assembly high level meeting on aids, world leaders committed to work together to achieve this goal. the aims of countdown to zero: global plan towards the elimination of new hiv infections among children by 2015 and keeping their mothers alive are to achieve a 90% reduction in the number of new hiv infections among children and a 50% reduction in aids-related deaths among pregnant women. this is the boldest plan to date to protect children from hiv and to safeguard their families, and it offers unprecedented opportunities to change the way the health sector, donors, governments and others work with affected people and communities to change the course of the epidemic. however, a modelling exercise, published before the launch of the plan, laid out how high a mountain had to be climbed to reach the targets. based on data from 25 countries with the highest numbers of pregnant women living with hiv, mahy et al. estimated that even if: (1) more effective drug regimens were implemented, (2) the current unmet need for family planning among women living with hiv was wholly met and (3) breastfeeding was limited to 12 months to curb hiv transmission, the number of new child infections averted (reckoned to be 79%) would still fall short of the target. what is critical, they argued, was for high coverage to be reached on all aspects of the pvt programme (with far fewer women lost to follow-up at each stage of what is called the pmtct cascade), safer feeding practices adopted and implementation of a comprehensive approach. such a comprehensive approach must include meeting the family planning needs of couples affected by hiv and reducing new hiv infections among women of reproductive age. that is, giving effect to all four prongs of pvt. these are (1) primary prevention of hiv among women of reproductive age; (2) reducing the unmet need for family planning among women living with hiv; (3) scaling up more efficacious arv regimens for women living with hiv and hiv-exposed infants and (4) expanding treatment and care for women, children and their families. the challenges to expanding comprehensive approaches, even in the highest burden regions, differ by country and by area within countries. ninety-one percent of pregnant women living with hiv in 2009 are in 22 countries, all but one in africa. however, huge differences exist between these 22 countries, with some classified as middle-income (such as india and south africa) and others low-income (malawi and mozambique); some stable states (for example, cameroon and ethiopia) and others with fragile political regimes (democratic republic of congo and zimbabwe). there are also differences in health service access (at least one antenatal visit: 97% in botswana, 39% in chad), hiv incidence (1.68 in south africa, 0.02 in india), unmet need for family planning (41% in uganda, 7% in namibia), coverage with any arv regimen (95% in botswana, 6% in the democratic republic of congo) and median duration of breastfeeding in the general population (34 months in rwanda, 16 in south africa). unicef has developed fact sheets on the status of national pvt responses in these 22 countries as at 2010. from the assembled data, it is clear that some countries, such as angola, must increase pvt services in antenatal care, especially in rural areas. others, such as nigeria, must expand the low reach of and access to antenatal services most of which are relatively well provisioned for pvt programmes especially amongst poor and rural women. south africa must bolster the prevention of new hiv infections among young women and improve the quality of pvt services, including the retention of women across the range of vertical prevention services. in all countries, big gaps, inefficiencies and poor quality services must be addressed. as has been recognized for some time, though, to reach the targets requires actions, not only within and by health services but also by affected women, their partners, within families and communities and in the wider society. in turn, this engagement is only possible under enabling political, legal, material and social conditions. the choices of women are affected, and their options limited, by their husbands and partners, their families and what they perceive other people think about them and how their neighbours and friends treat them. in turn, social norms are influenced by laws and policies, cultural beliefs, economic conditions and the range, quality and helpfulness of the services they have at their disposal. the same concentric circles of influence have been noted in other areas of health intervention, with the following quotation taken from cardiovascular disease: just as we have learned that it is difficult to change the behavior of individuals without changing the communities in which they live, we may be learning that it is difficult to change the behavior of whole communities without changing their broader social environment as well [27, p. 1391 scale-up emphasizes the importance of supporting community-based programmes and achieving integration with family planning, as well as other aspects of sexual and reproductive health and tuberculosis services, in order to create or enable facilitative conditions for individual and social actions (demand) to complement health service provision (supply). these programmes include the following: developing policy and legal frameworks, guidelines, tools and competencies to link services to community-based providers;defining standard packages of services to improve maternal, neonatal and child survival and health, at both health facility and at the community level;building capacity with technical and financial support to community-based organizations to deliver pvt and hiv services at facilities and in communities;promoting the active engagement of people living with hiv in advocacy and planning and delivering services;promoting male-friendly models of delivering hiv services and the participation of men in pvt and in hiv care for children; anddeveloping and implementing policies and programmes to reduce hiv-related violence, stigma and discrimination in the context of pvt and hiv care for children, including supporting women to disclose their hiv status to partners and family members [19, p. 2223]. developing policy and legal frameworks, guidelines, tools and competencies to link services to community-based providers; defining standard packages of services to improve maternal, neonatal and child survival and health, at both health facility and at the community level; building capacity with technical and financial support to community-based organizations to deliver pvt and hiv services at facilities and in communities; promoting the active engagement of people living with hiv in advocacy and planning and delivering services; promoting male-friendly models of delivering hiv services and the participation of men in pvt and in hiv care for children; and developing and implementing policies and programmes to reduce hiv-related violence, stigma and discrimination in the context of pvt and hiv care for children, including supporting women to disclose their hiv status to partners and family members [19, p. 2223]. there has been no assessment of country responses to these guidance points or progress in achieving greater women, family and community involvement in pvt. it is troubling that recommendations for community action are considerably diluted in countdown to zero. they include less specific strategies, such as developing community charters, ensuring participation of all stakeholders, maximizing community assets and identifying solutions to stigma. there is an emerging consensus that many hiv prevention programmes do not succeed specifically because they do not engage communities. instead, they are conceived by external experts and imposed on communities in top-down ways. as a result, they fail to resonate with the worldviews and perceived needs and interests of their target groupings, or to take adequate account of the complex social relations into which programmes are inserted [28, p. 1570. too often, suggest campbell and cornish, target communities are seen as passive recipients, as objects of intervention. but community engagement and participation is essential for several reasons: (1) because it is the most effective way to deliver acceptable messages and services to hard-to-reach groups; (2) because it is one of the main ways of engendering a sense of agency with which to build individual and collective health promotion; and (3) because the overall shortage of health workers means that community volunteers and workers are needed to help deliver hiv prevention, treatment and care to those who need it. the avahan experience in india, the largest prevention programme ever undertaken, demonstrates the value of involving communities, including the private sector, an oft-forgotten constituency in the public domain. at a broader societal level, laws and policies within which health services are funded and delivered emanate from social values and are subject to public opinion which, in turn influences and is influenced by all forms of media. given the importance of engaging women, families and communities to achieve an end to hiv transmission from parent to child, this special issue takes stock of current knowledge and good practice in community action to create an enabling environment, expand access and improve care of women and children in pvt programmes, as well as to reach men. the papers review and organize what is known to date, document examples of community mobilization, and the reach and effectiveness of community workers linked to health facilities. they draw attention to gender inequalities, the ways in which affected groups and their networks can expand services and the importance of their experiences and voices to unsettle complacency and compel changes in attitudes and behaviour. several things stand out from these papers: firstly, how determined community groups are to be part of the response to hiv and how energetic and innovative are their practices. secondly, how large-scale some community activities are: for example, in uganda, 750 groups of people living with hiv, working in larger coalitions, assisted 1.3 million people to access hiv-related health services (gitau-mburu, this issue); mothers2mothers reports having more than 250,000 patient encounters per month, seeing nearly 24,000 new hiv-positive women per month in 714 sites. thirdly, how comprehensive community-based services try to be as they integrate their activities to meet individual needs and family concerns (see kim et al. and patel et al. this issue), and fourth, the urgent need that community actors have for financial, policy, programme and personal support for their work (see dhlamini et al. however, what is also evident, especially from the review papers, is how little published research there is and, as a result, how limited our knowledge and use of community processes in increasing demand and supply of services and their role in the creation of an enabling environment. in addition to the papers, including the reviews and helpful conceptual frameworks offered by, amongst others, buzsa et al. firstly, the joint initiators of the global plan, dr michel sidibe, executive director or unaids, and dr eric goosby, global aids coordinator responsible for the implementation of pepfar, have provided a foreword. by doing so, they emphasize the importance of community engagement and action, and their commitment, within the global plan, to achieve it. it is a massive expression of support for community action, and it manifests the seriousness of their determination. the second unique feature is that, in addition to academic peer-reviewers, nine women living with hiv, recommended for their professional expertise and personal experiences by the international community of women with hiv/aids (icw) and the global network of people living with aids (gnp+) were invited to review terminology. their perspectives demonstrated the ways in which language can distort understanding, fuel discrimination and deeply hurt those who are directly affected by hiv. although the comments of this group of reviewers were discretionary unless independently endorsed by the journal's standard editorial process, all authors responded seriously to the issues raised. this is the first time, of which we are aware, that a scientific journal has engaged the people at the receiving end of science, policy and service in a mutual effort to find common ground and move forward together. in this sense, the special issue embraces the commitment to include community even though the common ground between science and experience is uneven, a consequence of both low use and potential misunderstanding. the special issue closes with an account of the experiences of people living with hiv, male and female recipients of our services, and an annotation by women living with hiv and their networks on language, identity and hiv. almost all the papers take as their starting point the role of community engagement in the following areas: expanded reach and supply of services, increased uptake, enhanced adherence to treatment and care regimes, improved retention in programmes and better psychosocial wellbeing of women, children and their families within an enabling environment. reviews of published research and of best practice experience endorse the need for community action in pvt. community action strategies can expand the reach of services to women and their families, increase access, boost adherence and retention, and support affected children and ensure they receive treatment. by and large, the mechanisms for community action involve the engagement, enrolment and collaboration of a diverse range of community-based volunteers, workers, counselors and social actors through independent and integrated community- and facility-based programmes. stand-out examples from the papers themselves and the work they cite are couples and home-based testing and prevention programmes [3133], mentor and peer counselors; support groups and community forums and community-based financing mechanisms that address barriers due to transport and other costs. more and more effort is being made, with success, to involve men, a strategy strongly endorsed by affected women (anderson et al. less apparent, although model studies and programmes can be found, are efforts to substantially change the social and policy environment through legal reform, mass media, community activism and demands for and implementation of mechanisms to increase accountability at the local and international levels. in a recent review of community accountability mechanisms, molyneux et al. identify three basic approaches: committees and groups, report cards and patient charters. while some impressive findings are reported for committees and groups, patient charters are less promising, offering only guidelines rather than consequences for abuses of patient rights and poor quality of health services. one of the most impressive effects of the use of report cards to improve healthcare is given by bjrkman and svensson from a randomized field experiment in fifty communities in nine districts in uganda. local organizations facilitated agreements between community members and private and government health providers on which submitted reports were based. a pre- and post-survey of 5000 households after one year suggested that the accountability mechanism was highly effective. health workers were rated as making more effort, there were large increases in utilization and health outcomes, including in child growth and a reduction of under-five mortality by 33%. it is clear that research in this area needs to be done and project and programme evaluations must be improved to pass the scrutiny of peers and be published. while there are many promising practices of the effectiveness of community action, the evidence base is still very limited. in this special issue, several different conceptual frameworks are used to review and or describe community approaches. (this issue) employ a social ecological framework that links the individual with peers and family, community and the broader social, cultural and economic environment in overlapping concentric circles. as they indicate, such conceptual frameworks are useful for illustrating the relationships between determinants that are proximal (downstream) or distal (upstream) to the person as previously described by latkin and knowlton. gulaid and kiragu (this issue) use the distinction made by rosato et al. between participation, mobilization and empowerment in an analysis of their roles in maternal, newborn and child health. see community empowerment being built up through layers of personal and collective action, starting from the individual, to small mutual groups, community organizations, partnerships and coalitions and social and political action. however, they conclude with several fundamental questions, unanswered also with respect to pvt. is community participation an essential prerequisite for better health outcomes or simply a useful but non-essential companion to the delivery of treatments and preventive health education? if essential, then is it only a transitional strategy for the poorest and most deprived populations but largely irrelevant once healthcare systems are established; or is it the critical missing component and the reason we are failing to achieve (in this case) the millennium development goals 4 and 5 regarding maternal and child mortality? community means many different things and we speak simultaneously of the international or donor community and a particular local community; we refer to community-based, community-oriented and services delivered in, for and by community (as opposed to health facility). refer to four categories of implicit constructions of community in health projects: community as setting (usually geographic), community as target (groups as compared to individuals), community as resource (frequently in terms of participation and support) and community as agent (meeting day-to-day needs). writing in 1968 of his attempts to develop a unitary approach to community intervention, jack rothman notes it was as if i had packed a large and assorted pile of conceptual clothing into a cognitive suitcase and found there was a sock or the end of a tie sticking out after i had pressed it closed [45, p. 27]. criticized for ignoring culture as a factor in determining the shape of community responses, rothman's conception remains a cornerstone for understanding community action. in short the first aims to improve service delivery through community development, bringing people together to solve local problems on a cooperative self-help basis. in this approach, participation, inclusion and consensus are critical. the second is a rationalistic approach used in policy and planning as evidenced, for example, in task shifting arguments for the role of community action. the third approach, social action, aims to aid the oppressed, promote social justice, and change society adopted, for instance, as the foundation of many aids activist groups [45, p. 27]. in practice, the three approaches may be interwoven and differentially phased, with one approach giving way to another. the treatment action campaign, gnp+ and physicians for social justice, as examples, combine assumptions, goals and processes of all three approaches, in different ways and at different times. these approaches, argues rothman, can all be applied in a way to pursue social change and human betterment in fact, they have to draw from one another because each has inherent limitations for which the others can compensate. campbell and cornish also argue for alignment of different approaches to community action but draw attention to the contexts in which such action can flourish. funding, though needed, may impose expectations and constraints that undermine the participatory ethos that is necessary for community action. similarly, mass media approaches frequently represent the views of western agencies, for example, of gender or sex work, and may miss opportunities for affected groups to challenge stigma and establish their symbolic legitimacy. lastly, community action depends also on a relational context, in which marginalized groups must have opportunities to interact with powerful local actors in their efforts to protect their health and receive services. campbell and cornish advocate what they call fourth generation social mobilization, mobilization for supportive conditions for community action at local, national and international levels. here there is a role for donors, development agencies and governments to consider how their policies and actions support or sabotage community action. in addition, they suggest, this will require that researchers also shift their attention upwards to examine, explicate and hold accountable the international community, as well as focussing downwards on the successes and failures of marginalized local communities [28, p. 1578 what is clear is that community engagement and action is necessary for the global plan to succeed. communities, holding hands with health services, are needed to contribute to creating demand for services, to help supply services and to assist in establishing an enabling environment for both to occur with maximal effectiveness.

virtual prevention of hiv transmission from parents to children is possible. this is cause for hope and renewed energy for prevention in general. the global plan is the most concerted and ambitious plan to date to protect children and to promote their care. but the inspiring and much appreciated global targets can not be achieved, nor will they be realized in spirit in addition to form, without joint action between health services, affected women, their partners, families and communities and the wider society. in turn, this engagement is only possible under enabling political, legal, material and social conditions. much has already been achieved, and community engagement can everywhere be seen in efforts to increase demand, to supply services and to create and improve enabling environments. some of these initiatives are highly organized and expansive, with demonstrated success. others are local but essential adjuncts to health services. the nature of this engagement varies because the challenges are different across countries and parts of countries. to be sustained and effective, community action must simultaneously be inclusive and supportive for those people who are affected, it must be appreciated and assigned a place within the broad systemic response, and it must promote and defend social justice.

PMC3499899

pubmed-402

stroke is a disease that causes sudden local neurological deficit symptoms due to a cerebrovascular accident1. stroke may decrease postural control and balance ability due to sensory disability, motor disturbance, muscular weakness, and asymmetric postures resulting from central nervous system damage2. reduced postural control and balance ability decreases overall physical function, making independent activities of daily living difficult for stroke patients, and increases the risk of falls. therefore, improving stroke patients balance ability is an important therapeutic objective3. with regard to this issue, a recent cross-sectional study revealed that stroke patients trunk control ability was correlated with balance, gait, and functional abilities4. exercises intended to improve stroke patients trunk control have been extensively studied in recent years. verheyden et al.2 reported that a group of stroke patients that performed trunk exercises on plinths showed better improvement in trunk control than a group that received only conventional physical therapy. pereira et al.5 conducted trunk flexion and extension, and compared trunk muscle activities measured by semg on the affected and unaffected sides of stroke patients. karthikbabu et al.6 studied the effect of exercise using swiss balls on the improvement of trunk muscles. although many exercise programs for trunk control have been implemented, there are few programs that can improve subjects balance while maintaining their interest. after the first horse riding studies were performed with patients with neurological damage, hippotherapy was used only to treat children with neurological disorders, mainly cerebral palsy, to improve their postural control and promote their normal development and functional recovery7, 8. the horse s gait is repetitive and rhythmic, and causes the center of gravity to move in anterior-posterior, lateral, and upward-downward directions. since the patterns of these movements are similar to those of the movements of the trunk and pelvis during human gait, such exercise is expected to make the subjects feel as if they were walking9. hippotherapy is effective in terms of psychological motivation because it arouses subjects interest in participating in the therapy9, 10, and helps in the recovery of many physical functions. hippotherapy increases the range of joint motion, strengthens muscles, and improves muscle tone, postural control, and balance and equilibrium ability. improvements in postural control and balance ability are achieved through stimulation of the upper motor neurons through proprioceptive sensory stimulation inputs11, 12. despite these therapeutic effects, no studies have been conducted on the changes in muscles related to stroke patients stability and balance after hippotherapy. in this study, the improvement in stroke patients trunk control and changes in abdominal muscle thicknesses after horse riding simulation training were examined. the purpose of this study was explained to patients diagnosed with stroke at d general hospital in busan, south korea. patients willing to participate in the study were recruited, and those who satisfied the selection criteria were randomly assigned to a control or experimental group (15 patients per group). subject selection criteria were as follows: patients with hemiplegia and no previous experience with hippotherapy who could follow verbal instructions, sit and walk independently (regardless of the use of walking aids), and had no restriction in the range of joint motion. then, the experimental group performed horse riding simulation training, while the control group performed trunk exercises using swiss balls. training was performed for 30 min per session, three sessions per week for a total of eight weeks10. subjects general characteristic (n=30)groupcontrol group(n=15)experimental group(n=15)age (years)56.57.555.16.1height (cm)163.38.2165.18.5weight (kg)63.58.565.49.8sex (male/female)8/77/8cause of brain damage(cerebral hemorrhage /cerebral infarction)8/76/9affected side (right/left)9/610/5data are mean sd. all subjects agreed in writing to participate in this study after receiving a detailed explanation of the experimental procedure, and possible side effects of the intervention. the experiment was conducted after receiving approval from the human subjects research ethics committee of dong-eui medical center (demcirb-2013-1001). the horse riding simulator (eu6441 core muscle trainer; panasonic, japan) used is designed to simulate the effects of horse riding exercises in an indoor environment using three-directional movements (anterior-posterior, lateral, upward-downward), and has three different programs and nine levels of exercise intensity. a therapist and an assistant monitored each subject during horse riding training. a balance ability measuring and training system (an analysis system with biofeedback, ap1153; biorescue, france) was used to evaluate subjects balance ability. static balance ability (center of pressure [cop] path length and cop travel speed) was measured while subjects stood for 60 s with their eyes open. the subjects were prevented from seeing the monitor to avoid visual feedback, and the position of the feet was maintained during repeated measurements to eliminate errors resulting from changes in foot positions. all tests were performed three times, and average values were calculated. ultrasonic imaging equipment (sonoace x4; medison, korea) subjects were instructed to bend their knees to 90 and to maintain a comfortable posture in a supine position. linear probes were positioned on both sides, midway between the bottom of the rib cage and the top of the iliac crest. the thicknesses of the external oblique, internal oblique, and transversus abdominis muscles were measured three times and their average values were calculated. the paired t-test was conducted to compare the results before and after the intervention within each group and the independent t-test was used to compare the exercise and control groups before and after the experiment. the experimental but not the control group showed statistically significant differences (p<0.01) between the cop path lengths of before and after the intervention. the differences between the two groups were not significant before the experiment, but were statistically significant (p<0.05) after the intervention (table 2table 2. changes in static balance following horse riding simulation traininggrouppre-testpost- testcenter of pressure path length (cm)control14.55.914.45.8experimental15.95.112.24.3 center of pressure travel speed (mm/s)control0.70.30.70.2experimental0.70.30.60.2 different superscripts within the same columns indicate significant differences (p<0.05)). different superscripts within the same columns indicate significant differences (p<0.05) the experimental, but not the control group, showed statistically significant differences (p<0.05) between the speeds of the cop travel of before and after the intervention. there was no significant difference between the control and the experimental group before the experiment, but the speed of cop travel decreased more in the experimental group than in the control group after the experiment, and this difference was statistically significant (p<0.05; table 2). changes in abdominal muscle thicknesses after training were observed in the experimental group. before training, tra and io did not show any significant differences between the affected and unaffected sides. in contrast, eo showed significant differences: 5.71.6 mm on the unaffected side and 4.71.4 mm on the affected side (p<0.05). after eight weeks of training, tra, io, and eo on the unaffected side did not show any changes compared to before the training. tra and io on the affected side did not show any changes after training, but the eo thickness significantly increased from 4.71.4 mm to 5.21.6 mm. total abdominal muscle thickness differed significantly before training between the affected and unaffected sides (13.52.7 mm and 14.33.0 mm, respectively). after eight weeks of training, both sides showed slight increases, with final values of 14.12.9 mm on the affected side and 14.35.8 mm on the unaffected side, but these differences were not statistically significant (p>0.05; table 3table 3. changes in abdominal muscle thickness following horse riding simulation training (experimental group; n=15)sidepre-testpost-testtraa2.50.82.70.9na2.50.62.60.6ioa6.30.156.31.6na6.00.166.11.8eoa4.71.4 5.21.6na5.71.65.71.4totala13.52.7 14.12.9na14.33.014.35.8a, affected side; na, non-affected side; abdominal muscle thicknesses unit: mm. different superscripts within the same columns indicate significant differences (p<0.05)). a, affected side; na, non-affected side; abdominal muscle thicknesses unit: mm. this study examined the effects of horse riding simulation training on the improvement of balance ability (which is an important issue for stroke patients), and the changes in abdominal muscle thicknesses of stroke patients. the distance and speed of center of gravity movements were measured using a biorescue system, and changes in abdominal muscle thicknesses were observed by ultrasonography. the cop path lengths of before and after the intervention were similar in the control group (p>0.05), but significantly decreased in the experimental group after horse riding training. the changes in the speed of cop travel showed that body sway remarkably decreased in the experimental group after training in comparison with the control group. these changes presumably occurred because the trunks of the subjects became more stable as a result of horse riding simulation training, reducing body sway and shaking. regarding abdominal muscle thicknesses, eo was thinner before training on the affected side than on the unaffected side, resulting in a difference in total abdominal muscle thickness on the affected and unaffected sides. this is consistent with the results of the study by english et al.14, which measured the abdominal muscles of stroke patients (65 years old or older) at rest. although the mean age of the subjects in our present study was somewhat lower (55.16.1 years), similar results were obtained. even though eo is mainly mobilized during trunk rotation movements, pereria et al.5 reported that eo was activated when stroke patients performed exercises that involved lifting of both legs, and this reportedly occurred to compensate for the inactivity of the rectus abdominis. similarly, in our present study the eo muscle may have activated more to compensate for the weakness of the rectus abdominal muscles on the affected side of the stroke patients. another reason for higher eo activation on the affected side could be that it is located closer to the surface layer than tra and io, which would affect thickness changes because horse riding simulation exercises induce multi-directional movements. although diverse balance training programs for stroke patients have been reported, therapeutic approaches using horse riding have rarely been studied with stroke patients as subjects. some studies have been conducted with persons with neurological disorders, in particular children with cerebral palsy7, 8. therefore, comparison of our present results with those of previous studies was difficult. future studies of horse riding simulation may lead to an improvement in stroke patients balance ability. horse riding simulation training can be fun and interesting for patients, which may increase their motivation to actively participate in exercise program and thus enhance the improvement of neurologic functions9, 10. second, ultrasonic measurement was conducted in a steady state in a supine position. third, since the damaged sites and symptoms differed among subjects, standard subjects could not be selected. therefore, in future studies, the effects of hippotherapy on stroke patients should be examined with a more efficient control and the selection of a wide range of subjects. additional information should be provided by comparison (during ultrasonic measurement) of the degrees of contraction on the affected and unaffected sides. in conclusion, horse riding simulation training may reduce the distance and speed of the center of gravity movements of stroke patients and reduce the asymmetry of the abdominal muscles. to improve stroke patients balance ability, diverse uses of horse riding simulation training should be considered.

[purpose] the purpose of this study was to assess the effects of horse riding simulation training on changes in balance ability and abdominal muscle thicknesses of stroke patients. [subjects] thirty stroke patients with hemiplegia were recruited, and they were randomly assigned to a control or experimental group. [methods] the experimental group performed horse riding simulation training, whereas the control group performed trunk exercises for 8 weeks. balance ability was measured using a biorescue system. the thicknesses of subjects external oblique, internal oblique, and transversus abdominis muscles were measured by ultrasonic imaging. [results] in the experimental group, balance ability was significantly improved after training. similarly, the thickness of the abdominal muscles on the affected side changed after training in the experimental group, whereas the control group showed no statistically significant changes. [conclusion] we suggest that horse riding simulation training is more effective than trunk exercises at reducing the center of pressure path length and travel speed and improving the asymmetry of the abdominal muscles of stroke patients.

PMC4155239

pubmed-403

surgical resections such as unilateral temporal lobectomy and amygdalo-hippo-campectomy have an established place in the management of carefully selected patients with refractory localisation-related epilepsy. adverse cognitive sequelae of epilepsy surgery have been well-recognised since the seminal report of scoville and milner in 1957 documenting the dense anterograde amnesia in patient h.m. such amnesia has been observed on occasion following unilateral surgery, reflecting preoperative damage in the unoperated, contralateral, temporal lobe, a finding which mandates careful preoperative assessment of the non-operated hemisphere, for example using sodium amytal (wada) testing and/or functional neuroimaging, to try to ensure cognitive function is preserved post-operatively. we present a patient with refractory epilepsy who, following initially apparently successful unilateral temporal lobectomy, developed recurrent seizures and profound amnesia, and in whom subsequent investigations unexpectedly suggested an autoimmune aetiology. a 36-year-old right-handed female was referred to our centre for assessment of epileptic seizures and cognitive impairment. at the age of 33, she had undergone a right (non-dominant) temporal lobectomy for refractory complex partial seizures, performed at another neuroscience centre with an established epilepsy surgery programme. the patient's seizures began at the age of 15 years; there was no history of childhood febrile convulsions. seizures were characterised by dj vu, absence, and automatisms, and were thought to arise in the right temporal lobe. mr imaging appearances were equivocal, with right temporal lobe changes thought to represent either sclerosis or a possible dysplastic lesion. because of the refractory nature of the seizures, pre-operative workup was undertaken, including fdg-pet which showed reduced uptake of tracer in the right temporal lobe. intracranial eeg (subtemporal strips) confirmed complex partial seizures arising from the lateral right temporal cortex, but there also seemed to be subclinical events arising from the left side. a sodium amytal test performed prior to surgery confirmed that the patient was left-hemisphere dominant for language and that both hemispheres supported memory function. however, 34 months post-operatively the patient deteriorated with further frequent complex partial seizures. additionally, she was noted to have symptoms suggestive of both anterograde and retrograde amnesia. by this time cognitive assessment included administration of cognitive screening instruments which showed impaired performance: on the mini-mental state examination (mmse), she scored 23/30; on the addenbrooke's cognitive examination-revised (ace-r), she scored 74/100, with 12/26 on the memory components; and on the montreal cognitive assessment, she scored 23/30 (normal 26/30). on the repeatable battery for the assessment of neuropsychological status (rbans), her delayed memory scores fell within the extremely low range. this impairment was for both verbal and visual material, with a subtle indication of slightly higher levels of delayed recall with visual information (list recall total score=0; story recall total score=1; figure recall total score=2). other domains assessed by the rbans showed the immediate memory to be in the borderline range, attention was low average, whilst language and visuospatial/constructional abilities were relatively preserved (table 1; left-hand column). in addition to the evidence of right temporal lobectomy, this also showed a high signal change in the left temporal lobe involving the hippocampus (fig. serological testing revealed a very high titre of antibodies directed against glutamic acid decarboxylase (gad). in sum, these investigations suggested a diagnosis of anti-gad limbic encephalitis (le). over the next 2 years, the patient was empirically treated with various immunomodulatory interventions including intravenous methylprednisolone, plasma exchange, and two infusions of rituximab, all without obvious clinical improvement in either seizures or cognitive function. three years after surgery, prior to embarking on a treatment trial of intravenous immunoglobulin at our centre, the patient was still receiving polytherapy for epileptic seizures (levetiracetam, pregabalin, clonazepam, and lacosamide). on cognitive testing, she now scored 17/30 on the mmse and 63/100 on the ace-r with 8/26 on the memory components. repeating the rbans, the cognitive profile was little changed, with the delayed memory score still being extremely low (table 1; right-hand column), again affecting both verbal and visual material, with the latter still at slightly higher levels (list recall total score=2; story recall total score=2; figure recall total score=8). behaviourally, she used external memory aids to record daily events since she had no recall of these after only a brief period of time. antibodies to gad have been associated with various neurological syndromes, including stiff person syndrome, cerebellar ataxia, epilepsy, paraneoplastic syndromes (encephalomyelitis, cerebellar ataxia, and le), idiopathic le, and myasthenia gravis. from the epilepsy perspective, in a cohort of 253 epilepsy patients, liimatainen et al. detected anti-gad antibodies in 15 patients (5.9 vs. 1.5% in 200 controls), most of them (90%) with temporal lobe epilepsy. in a study of patients with adult-onset (> 30 years) temporal lobe epilepsy, anti-gad antibodies were found in 5 out of 42, with evidence for pharmacoresistant epilepsy with associated memory impairment and other autoimmune diseases. temporal lobe epilepsy with anti-gad antibodies may not therefore be a rare condition, especially amongst the treatment-refractory patients referred for surgical evaluation. the precise pathogenic sequence of events in our patient remains uncertain; a number of potential explanations exist. it is possible that she had two separate problems, namely mesial temporal sclerosis followed by adult-onset anti-gad antibody le. if this is so, it might be speculated whether her temporal lobectomy surgery might have unmasked epitopes which initiated an autoimmune response that produced anti-gad antibodies and hence le. another possibility is that the entire syndrome was due to anti-gad le, albeit very long-lived. defined non-paraneoplastic anti-gad le as a chronic non-remitting disorder, with antibody titres remaining high after intravenous methylprednisolone. moreover, none of their patients became seizure free despite intense anti-epileptic drug therapy, unlike the situation in le associated with voltage-gated potassium channel (vgkc/lgi1) antibodies [10, 11]. furthermore, cognitive impairments did not improve after treatment in anti-gad le. in this context, it is of note that the neuropathological examination of our patient's temporal lobectomy specimen found evidence of chronic inflammatory change consisting almost exclusively of t cells with associated microglial activation, suggesting a chronic encephalitic process in addition to hippocampal sclerosis. to date there is little information on the efficacy of intravenous immunoglobulin in anti-gad le. response has been reported in new-onset focal epilepsy, but the chances of success must be doubtful in chronic epilepsy, as in our patient. this case may illustrate that autoimmune processes, rather than surgery, may cause bilateral hippocampal pathology resulting in profound amnesia, behaviourally akin to that seen in the classic amnesic patient h.m. [14, 15]. following bilateral anterior temporal lobectomy for intractable seizures with partial hippocampal removal, h.m. developed a profound anterograde amnesia for episodic autobiographical material but with preserved general intelligence, attention, working memory, language and perceptual skills. in contrast, the neuropsychological outcome following unilateral non-dominant hemisphere temporal lobectomy, as undergone by our patient, is usually confined to material-specific (i.e. visual rather than verbal) memory impairments. we recommend that the possibility of anti-gad le needs to be considered in all patients with refractory epilepsy of presumed temporal lobe origin, including those being considered for epilepsy surgery, especially those whose clinical course is not typical for mesial temporal sclerosis.

we describe a patient who developed significant cognitive decline with profound amnesia following non-dominant temporal lobectomy for refractory seizures, in whom the original suspicion of structural pathology was revised following the discovery of clinical and neuropathological markers of inflammation, neuropsychological evidence of bilateral involvement, and high titres of antibodies directed against glutamic acid decarboxylase (gad). this case adds to the evidence that the diagnosis of non-paraneoplastic anti-gad limbic encephalitis merits consideration in any patient with a refractory seizure disorder and cognitive decline.

PMC4249997

pubmed-404

vitamin d deficiency (vdd) is defined as serum 25-hydroxy vitamin d (25ohd) levels<20 ng/ml. vdd has been documented in more than 90% across all age groups and both sexes from india. classical manifestations of vdd is described as rickets/osteomalacia, which manifest as bony deformity/pain, decreased bone mineral density (bmd), increased risk of fracture and is associated with raised alkaline phosphatase and parathormone (pth). however, secondary hyperparathyroidism (shpt) is observed in<50% of subjects in indian and us population. subjects with same levels of serum 25ohd have varied clinical and biochemical abnormalities including some showing no abnormalities. this raises logical question do all subjects with vdd have clinical disease according to this definition? the main physiological function of vitamin d is maintenance of calcium homeostasis by its effect on calcium absorption and bone health in association with parathyroid gland. calcium is absorbed actively in the duodenum through transcellular (active transport-80%) process, which is vitamin d dependent, whereas passive absorption is a paracellular (passive diffusion-20%) process, which occurs throughout intestine independent of vitamin d and is dependent on concentration of calcium in the intestinal tract. total fraction of calcium absorbed from total intake can vary from 20 to 80%. in the event of decreased calcium availability from intake, calcium is released from bone under the effect of vitamin d-pth system to maintain its homeostasis. as per the recent institute of medicine (iom) statement the data currently suggest that fractional calcium absorption (fca) reaches a maximum between 12 and 20 ng/ml in both children and adults. in most of the studies reviewed by iom, the baseline serum 25ohd was>10 ng/ml and there was no correlation of serum 25ohd levels with calcium absorption. when we have plotted mean basal serum 25ohd levels in various studies and mean fca, there was a significant inverse correlation (r=0.75, p=0.001). there is only one study among elderly that has assessed the relation of calcium absorption and base line serum 25ohd levels ranging from 4 to 20 ng/ml. this study has clearly shown that calcium absorption decreases in the ranges from 4 to 8 ng/ml and not>8 ng/ml. similarly, in the most studies related to vitamin d supplementation, basal serum 25ohd levels were>8 ng/ml. only one study carried out in subjects with 25ohd level of 4 ng/ml showed an increase of 21% with change in 25ohd level to 24 ng/ml, whereas in those with the increase in 25ohd level from 8 to 28 ng/ml only 3% increase of calcium absorption occurred. there is no correlation of mean change in fca (increase or decrease) with either mean basal serum 25ohd levels (r=0.122, p=0.754) or increment in serum 25ohd levels. these data clearly shows that the maximum calcium absorption capacity is reached when serum 25ohd levels are>8 ng/ml. logically to maintain calcium homeostasis in the face of vdd, the first body will try to absorb maximum available calcium, rather than affecting bone. hence, calcium absorption is the first most important adaptive mechanism in patients with vdd. high fca (54-63%) has been reported from the region of china with low calcium intake (< 500 mg) compared with 25-34% in us children with high intake of calcium (> 900 mg). this suggests that the body tries to adapt to the calcium availability to bodies requirement by adjusting fca. the conventional explanation of homeostasis is by systemic adaptation, in which decreased calcium intake results in decreased calcium absorption, which leads to increase in pth levels. the pth up regulates the 1- hydroxylase enzyme, leading to increase generation of 1,25-dihydroxyvitamin d (1,25(oh) 2d) levels and increased calcium absorption and bone resorption. however, as deduced from the above discussion, the body has a tremendous reserve to increase the fca in the face of decrease in calcium intake. this suggests that calcium absorption can be kept static over a wide range of calcium intake and serum 25ohd levels by local intestinal adaptation. we hypothesize that the first adaptive mechanism in calcium homeostasis is local rather than systemic. it consists of calcium sensing receptor (casr) on intestinal brush border, which senses calcium in intestinal cells and negatively affect vitamin d system in intestinal cells to decrease active transcellular calcium transport. it also facilitates passive paracellular diffusion of calcium in the intestine, which is less efficient process. on the contrary, when there is decreased calcium intake, this feedback inhibition is removed and vitamin d dependent active calcium absorption will increase, maintaining calcium homeostasis. furthermore, there may be some genetic or epigenetic alteration in genes of 1- hydroxylase enzyme, which decreases efficiency of active vitamin d generation or vitamin d receptor (vdr) genotype affecting calcium absorption. in subjects with efficient vdr genotype for calcium absorption, local adaptation will be maintained at lower levels of serum 25ohd and vice versa will also be true. the interaction between casr and vitamin d system in intestinal cells (intestinal calcistat) will decide the level of serum 25ohd at which calcium absorption can be maintained according to the need of the body or becomes suboptimal in a given individual indicating failure of local adaptation. firstly, decrease in calcium intake<250 mg, which can not be overcome by increasing fca. this will manifest as calcium deficiency rickets on the face of normal vitamin d levels. secondly, mutation in casr, if activating, may lead to decreased calcium absorption and if inactivating, increased calcium absorption. thirdly, decreased supply of substrate below critical levels (serum 25ohd<8 ng/ml) will lead to vdd rickets. however, this level can vary according to interaction between casr and vitamin d system in an individual-intestinal calcistat. finally, genetic mutation in 1- hydroxylase [vitamin d resistant rickets-i (vdrr-i)] or vdr (vdrr-ii) will also lead to failure of local adaptive response. this will also explain the observation in vdrr-i and ii, where high intake of calcium can overcome most of the clinical manifestation of the disease. with very high intake, casrs will get saturated and will enhance passive (paracellular) calcium absorption, which will be able to fulfill the requirement of body. with increasing severity of vdd, there will be decrease in calcium absorption. the calcium levels will now be maintained by bone resorption, rather than increasing calcium absorption, which is currently believed. hence, generally held belief that increase in pth will increase calcium absorption through generation of active vitamin d metabolites, is wrong. this puts pth as a marker for systemic vdd or failure of local adaptation by the intestinal calcistat. the above hypothesis of the intestinal calcistat explains the vide variation observed in literature about relation between serum 25ohd, pth, calcium absorption and bmd. according to this hypothesis, subjects with low serum 25ohd who have normal intestinal calcistat will absorb required amount of calcium and will not mount systemic adaptive response in the form of increase in pth and 1,25(oh) 2d levels, hence will have lower 1,25(oh) 2d levels than those with failure of adaptation and bmd will not be affected. subjects with adaptive failure will have higher 1,25(oh) 2d levels and will have lower bmd. this is further supported by observation that patients with similar low serum 25ohd levels (< 10 ng/ml), bmd was lower in subjects with shpt. this will also explain the observation that about 50% of subjects with vdd do nt mount pth response because they have adequate local adaptation in intestinal calcistat. this will also explains that why there is no substantial increase in calcium absorption in with vitamin d supplementation because basal level of serum 25ohd is sufficient to supple enough substrate for generation of active vitamin d metabolites. this brings us to question that should we define vdd with a value of serum 25ohd in isolation? it is obvious from the above discussion that there are adaptive mechanisms to overcome low vitamin d levels, which can be operative over a wide range of serum 25ohd levels. failure of adaptive mechanism will lead to clinical and biochemical evidence of vdd. among them hence, the subjects with vdd defined by low vitamin d levels (< 20 ng/ml or<30 ng/ml) according to the current definition with normal pth and bmd will not have any clinical and biochemical consequence of low vitamin d levels and vice versa subjects with similar levels of serum 25ohd with raised pth or low bmd are likely to be vdd. what should we call subjects with low serum 25ohd levels without evidence of shpt or low bmd? should we call them subclinical vdd, compensated vdd, asymptomatic vdd or not call them vdd at all. further studies are required to define adverse biological consequences of vdd in this group and effects of vitamin d supplementation and comparing them with the population who already had adverse biological effects of vdd.

the main physiological function of vitamin d is maintenance of calcium homeostasis by its effect on calcium absorption, and bone health in association with parathyroid gland. vitamin d deficiency (vdd) is defined as serum 25-hydroxy vitamin d (25ohd) levels<20 ng/ml. do all subjects with vdd have clinical disease according to this definition? we hypothesize that there exist an intestinal calcistat, which controls the calcium absorption independent of pth levels. it consists of calcium sensing receptor (casr) on intestinal brush border, which senses calcium in intestinal cells and vitamin d system in intestinal cells. casr dampens the generation of active vitamin d metabolite in intestinal cells and decrease active transcellular calcium transport. it also facilitates passive paracellular diffusion of calcium in intestine. this local adaptation adjusts the fractional calcium absorption according the body requirement. failure of local adaptation due to decreased calcium intake, decreased supply of 25ohd, mutation in casr or vitamin d system decreases systemic calcium levels and systemic adaptations comes into the play. systemic adaptations consist of rise in pth and increase in active vitamin d metabolites. these adaptations lead to bone resorption and maintenance of calcium homeostasis. not all subjects with varying levels of vdd manifest with secondary hyperparathyroidism and decreased in bone mineral density. we suggest that rise in pth is first indicator of vdd along with decrease in bmd depending on duration of vdd. hence, subjects with any degree of vdd with normal pth and bmd should not be labeled as vitamin d deficient. these subjects can be called subclinical vdd, and further studies are required to assess beneficial effect of vitamin d supplementation in this subset of population.

PMC3830322

pubmed-405

collecting duct carcinoma (cdc) of the kidney is an unusual variant of renal cell carcinoma (rcc), accountings for less than 1% of all renal cancers. cdc arises precisely from the principal cells lining distal collecting ducts of epithelium and distal renal tubules that originates from mesonephros. considering that urothelial carcinoma originating from the ureter, pelvis, or calices also arises from the mesonephros, cdc might be similar to urothelial carcinoma and its radiologic and pathologic findings differ from those of other rccs. recent publications have pointed out the histological heterogeneity of this neoplasm and its extensive histological overlapping with high grade papillary tumors and urothelial carcinoma. accurate diagnosis is important for proper management. in diagnosis of cdc, it is important to distinguish between invasive papillary rcc and urothelial carcinoma. positive immunohistochemical staining for distal tubules and collecting duct markers is helpful indiscrimination of cdc from the more commonly diagnosed clear cell rcc of proximal nephron origin. cdc generally expresses broad spectrum keratins and high molecular weight (hmw) cytokeratin, which is expressed in the lower nephron and the urothelium. it also shows positive staining with e-cadherin, epithelial membrane antigen, cke12, and ck19. however, cd10, c-kit, and a-methylacylcoa racemase (amacr) show no staining. in contrast, papillary rcc showed positive results for cd10 and amacr, and it appears to be different from cdc. however, this immunohistochemistry is not specific and may be seen in medullary carcinomas and in urothelial carcinoma, including those arising in the renal pelvis. although the gross and microscopic features of the tumor are well established, diagnostic confusion can still occur. 40% of patients have already developed metastatic lesions, including lymphnodes, lungs, or adrenal glands. clinical outcome is poor, with 66% of patients dying of the disease within two years after diagnosis. various treatments have been proposed, including radiation therapy, immunotherapy, and some combinations of chemotherapy, however, results have been unsatisfactory. to date, no standard therapy for cdc has been established. the aim of this study is to conduct an investigation of the clinicopathologic findings of cdc and to determine their correlation with the disease status and prognosis. we retrospectively reviewed 35 patients diagnosed with cdc at eight korean medical centers from 1996 to 2009. data on gender, age, initial symptoms, and laboratory findings, including complete blood count profile, calcium, and urine analysis, pathological features, treatment, and patient outcome were obtained from patient medical records. diagnosis of cdc was made by examination of a nephrectomy specimen in 27 cases and by renal biopsy in eight. tumors were staged according to the 2002 american joint committee on cancer (ajcc) tnm stage classification. this study was approved by the institutional review board (irb) from each participating institution. tumor response after treatment was re-evaluated using the response evaluation criteria in solid tumors (recist ver. progression free survival (pfs) was estimated from the date treatment began to the date when disease progression was recognized, or the date of the last follow-up visit, or the date of death. overall survival (os) was estimated from the date of diagnosis to the date of death from any cause or the last follow-up visit. the cox regression model was used for multivariate analysis with factors that had been used in univariate (log rank) analysis of os and pfs. p-values less than 0.05 were considered statistically significant and all p-values correspond to two-sided significance tests. the median age of patients was 56 years (range, 29 to 82 years) and 74% of the patients were male. of 32 symptomatic patients, 16 and 11 experienced pain and gross hematuria. other presenting symptoms included weight loss, microscopic hematuria, and a palpable mass. seventeen patients had a tumor size of 7 cm or less, and 10 patients had a tumor size of 7 cm or greater. the median level of hemoglobin and calcium was 12.5 g/dl (range, 8.9 to 18.3 g/dl) and 9.30 g/dl (range, 7.9 to 11.1 g/dl), respectively. according to the immunohistochemistry finding, cdc expressed cytokeratin in nine patients (26%), hmw-cytokeratin in 14 (40%), low molecular weight-cytokeratin in three (8.6%), and cke12f in one (2.9%). it also expressed cd10 in five (14.3%) and vimentin in 11 (31.4%). at diagnosis, nine, two, four, and 19 patients had tnm stage i, ii, iii, and iv, respectively. eight patients had two or more metastatic sites of the bone (44%), lungs (39%), liver (16%), and lymph nodes (11%) as the most common sites. with a median follow-up period of 15.8 months (range, 0.6 to 88.4 months), 14 (40%) deaths were reported. during the median follow-up period of 15.8 months, 14 patients died, while nine patients (25.7%) were lost in the follow-up. twenty seven of the 35 patients underwent nephrectomy for initial treatment (curative surgery in 17, and palliative in 10), three patients received chemotherapy, and four patients did not receive any treatment (fig. palliative chemotherapy was administered for 22 persons, who were composed of eight of 14 relapsed patients, eight of 10 patients who were in stage iv and underwent palliative surgery, and four patients who did not undergo an operation (fig. median pfs and os for all patients were 5.8 months (95% confidence interval [ci], 3.5 to 9.2 months) and 54.4 months (95% ci, 0 to 109.2 months), respectively (figs. 2 and 3a). the os of the patients with stages i-iii was 69.9 months (95% ci, 54.0 to 85.8 months), while that of patients with stage iv was 8.6 months, which showed a statistical significant difference (p=0.01) (fig. 3b). in addition, among patients with stage iv, the os of patients who received a palliative treatment (immunotherapy, chemotherapy, or targeted therapy) was 18.4 months, which was higher than the os of patients without treatment of 4.5 months. the pfs of patients with stages i-iii was 6.9 months (95% ci, 1.3 to 12.4 months). recurrence occurred in 14 patients, 82% of the 17 patients who underwent a curative surgery, and their average recurrence period was 5.9 months, with a short pfs and a high relapse rate. using the log-rank method, no relationship was demonstrated between survival end points (pfs and os) and explanatory covariates, including patients ' age, gender, and initial calcium level, except for hemoglobin (p=0.005 and p=0.193, respectively) and initial tnm stage (p=0.022 and p=0.002, respectively). results of multivariate regression analysis using a cox's proportional hazards model showed that tnm stage (i-iii vs. iv; hazard ratio, 4.58; 95% ci, 1.301 to 16.135; p=0.018) was an independent prognostic factor for survival of cdc (table 3). the frequency of cdc is within 1% of the entire rcc and its radiologic and pathologic findings differ from those of other rccs. in 1976, mancilla-jimenez et al. reported on 34 cases of papillary rcc and postulated a collecting duct origin for three of these tumors based on the findings of atypical hyperplastic changes in adjacent collecting tubules. this is the first report on cdc based on medical records from eight institutions in korea. in japan, a retrospective survey was conducted in order to analyze the nature of cdc. in the study, the central pathologists confirmed cdc in 81 of 120 cases diagnosed as cdc at 66 institutions. it was a large-scale nationwide survey with an advantage of a multi-institutional central review. on the other hand, in this study, 35 patients were selected from eight different organizations nationwide in korea. although a pathological central review was not performed, there was significant detailed information on each case with the pattern of cases and treatment outcomes. thus, based on such information, the results were evaluated with regard to the types of post operational treatment and the drugs used as palliative treatment and the responses. our results are in agreement with those of previous reports showing that the median age was 56 years (range, 29 to 82 years) and that males comprised 74% of the patient population. in our study, cdc expressed cytokeratin, hmw-cytokeratin, and cke12 in many cases, however, it also expressed cd10 and vimentin, which is generally expressed in the upper nephron, and not in the lower nephron. ninety-one percent of patients had symptoms and the most common presenting symptoms were pain, hematuria, and weight loss. at diagnosis, 19 (54%) patients were tnm stage iv, and the median os period of patients with stage iv was 9.29 months (95% ci, 0.0 to 26.78 months). a summary of the clinical data on cdc gathered from published series and case reports is shown in table 4. due to the rarity of its occurrence, optimal treatment for cdc has not been established. despite past reports on striking responses to cytokines, currently, immunotherapy only has an historical role. cdc might be distinct from conventional rcc and share embryological origins and biological features with urothelial carcinoma. therefore, even if trials comparing immunotherapy with chemotherapy have not been conducted, chemotherapy currently represents the most used therapeutic approach. however, it remains unclear whether this carcinoma should be managed with a treatment similar to that for urothelial cell carcinoma or rcc. multiple chemotherapeutic and/or immunotherapeutic regimens have been tried for treatment of cdc (table 4). these data appear to suggest that chemotherapy and immunotherapy may offer only limited benefits to a selected group of patients. in our study, surgical treatment was performed as the initial treatment in 77% of patients. however, recurrence occurred in most patients who underwent surgery and a palliative treatment was administered in 75% of patients. most patients with advanced or recurrent disease were treated with immunotherapy, chemotherapy, radiation therapy, or targeted therapy. the most commonly used agents included interferon, gemcitabine, cisplatin/carboplatin, and sunitinib. it seems that patients with stages i-iii had a high relapse rate with a short pfs of 6.9 months, while seven patients (58%) with stages i-iii survived for a long time with patients in the no evidence of disease state, contributing to the increase of the os, so that there was a discrepancy between the pfs and the os. most of the long-term survivors were in stages i-iii and those who received palliative treatment after a relapse, and the treatments administered to these patients included target therapy as well as immunotherapy and chemotherapy. due to the small number of patients, the correlation between the prognosis and the treatment could not be known. however, it can be assumed that palliative treatment takes the role of extending survival. in paticular, the current standard therapy against rcc is the targeted therapy, and though it is recognized as a different disease from rcc, there were some cdc patients who were treated with sunitinib, temsirolimus, or other targeted agents, different from the past. according to an analysis of clinical aspects, treatment and prognosis in the records of seven cdc patients diagnosed with rcc in procopio's study and included in patients treated with the target therapy, five persons showed survival of four months while two patients showed long-term survival of 49 months and 19 months, respectively. in this study of the patients who were recently diagnosed and received a target therapy, one patient for whom sunitinib was used finally died, but showed a partial response during treatment. cdc is an aggressive disease with poor prognosis, however, like some patients in this study who survived for a long period of time, a study on predictive markers by which the outcomes of prognosis and therapy, especially target therapy as well as their clinical features can be predicted is needed. pfs and os were short, however, there were some long-term survivors, therefore, additional research on the predictive markers of several clinical, pathological differences and their treatments will be needed.

purposecollecting duct carcinoma (cdc) of the kidney is an aggressive disease with a poor prognosis, accountings for less than 1% of all renal cancers. to date, no standard therapy for cdc has been established. the aim of this study is an investigation of clinicopathologic findings of cdc and correlation of the disease status with a prognosis. materials and methodsfrom 1996 to 2009, 35 patients with cdc were treated at eight medical centers. the diagnosis of cdc was made based on nephrectomy in 27 cases and renal biopsy in eight cases. resultsmedian pfs and os for all patients were 5.8 months (95% ci 3.5 to 9.2) and 54.4 months (95% ci 0 to 109.2), respectively. the os of patients with stages i-iii was 69.9 months (95% ci 54.0 to 85.8), while that of patients with stage iv was 8.6 months (95% ci 0 to 23.3), which showed a statistically significant difference (p=0.01). in addition, among patients with stage iv, the os of patients who received a palliative treatment (immunotherapy, chemotherapy, or targeted therapy) was 18.4 months, which was higher than the os of patients without treatment of 4.5 months. conclusioncdc is a highly aggressive form of renal cell carcinoma. despite most of the treatments, pfs and os were short, however, there were some long-term survivors, therefore, conduct of additional research on the predictive markers of the several clinical, pathological differences and their treatments will be necessary.

PMC4022822

pubmed-406

hemolysis, elevated liver enzymes, and low platelet count (hellp) syndrome is a severe manifestation of a hypertensive disorder of pregnancy called pre-eclampsia. it affects about 10% to 20% of patients with severe pre-eclampsia (0.5% to 0.9% of all pregnancies) and causes significant mortality and morbidity, which increases in accordance with the severity of this syndrome. this syndrome is associated with increased maternal risk of developing morbidities, including cerebrovascular complications, hemorrhage, pulmonary edema, retinal detachment, hematoma/hepatic rupture, acute renal failure, liver failure, intravascular coagulopathy, placental abruption, and sepsis [35]. perinatal/infant morbidity and mortality rates are higher in pregnant women with hellp syndrome. the preterm delivery rate is about 70% in hellp syndrome patients with about 15% of cases requiring parturition before the 27 week of gestation. sufferers may refrain from further pregnancies and need psychological support, and those who attempt further pregnancies have higher risk of gestational hypertension. previously, immediate delivery was indicated for patients diagnosed with hellp syndrome, which often resulted in significant maternal and neonatal morbidity and/or mortality. later it was recognized that antepartum administration of high-dose corticosteroids can stabilize the disease indicators and prolong the gestation. although many studies have demonstrated that cort use helps raise the platelet count and reduce elevated liver enzymes, results are not consistent across all studies. moreover, evidence regarding the role of corticosteroids in improving maternal morbidity and mortality is not clear. the present study, therefore, carried out a systematic review of the relevant studies and performed a meta-analysis of all related parameters for the sake of evaluating the efficacy of cort therapy observed in studies with controlled designs. important features of the method used for the present study are summarized in table 1. several electronic databases were searched for the acquisition of required study reports by using the most relevant mesh and keywords in different logical combinations and phrases. the inclusion criterion was the studies examining the efficacy of cort therapy to treat hellp patients either in a prospective or retrospective controlled design. some studies were, however, excluded by the following exclusion criteria (table 1). important information including outcome measures and outcomes, dosage and mode of administration of cort, and obstetric and demographic characteristics were obtained from identified papers and organized on datasheets. meta-analyses of mean difference and odds ratio were carried out under the random-effects model. fifteen studies [1226] fulfilled were eligible and were included in the meta-analysis. of the included studies, 8 were randomized controlled trials and 7 were retrospective analyses. the overall population of this meta-analysis is 675 cort treated and 787 control hellp patients. age of the cort treated and control patients as mean sd (range) was 26.945.8 (23.2633.54) years and 26.355.7 (23.1630.93.3) years, respectively. gestation duration was 32.273.7 (29.13.535.12.9) weeks in cort treated and 32.423.8 (27.63.335.52.6) weeks in control hellp patients. in the cort treated group, least asymmetry was visible from the visual inspection of the funnel plots, indicative of almost no publication bias in this area of research (figure 2). the mean difference [95% confidence interval] in the change from baseline between cort treated patients and controls was 38.08 [15.71, 60.45]10/l; p=0.0009 (figure 3). on the other hand, the mean differences in the changes from baseline between cort treated and controls were 0.44 [0.76, 0.12] iu/ml; p=0.007 for ldh (figure 4) and 143.34 [278.69, 7.99] iu/l; p=0.04 for alt. however, the decrease in ast levels was not statistically significant in cort-treated patients in comparison with controls (48.50 [114.32, 17.32] iu/l; p=0.15; table 2). blood transfusion rate was significantly lower in cort-treated patients (odds ratio [95% ci]: 0.42 [0.24, 0.76]; p=0.004. hospital/icu stay was also significantly lower in cort-treated patients (mean difference: 1.79 [3.54, 0.05]; p=0.04). there was no significant difference between cort-treated and control patients in the incidence of cesarean deliveries (odds ratio [95% ci]: 1.25 [0.95, 1.63]; p=0.11), prevalence of infections (0.78 [0.19, 3.15]; p=0.73; table 2), birth weight (mean difference: 0.09 [0.11, 0.28]; p=0.38), infant respiratory distress incidence (odds ratio: 1.13 [0.50, 2.53]; p=0.78) and maternal mortality (odds ratio: 1.27 [0.45, 3.60]; p=0.65) (table 2). among the included studies, infant mortality was 23% in cort-treated patients and 8.3% in controls and 4% in cort-treated patients and 0% in controls. perinatal death was 0% in cort-treated patients and 3% in controls. despite lower frequency of morbid conditions in hellp patients treated with cort (318 vs. 418), there was no significant difference in the incidence of overall morbidity between the groups (odds ratio: 0.79 [0.58, 1.08]; p=0.14). morbid complications observed in 1 or more studies included pulmonary edema (3.6%), intraventricular hemorrhage (18%, disseminated intravascular coagulation (15%), endomyometritis (9%), ascites (13.3%), hematoma (3.3%), acute renal failure and other renal pathologies (14%, necrotizing colitis (12%, bronchopulmonary dysplasia (80%), intraventricular hematoma (20%), infant thrombocytopenia (13%), apgar score less than 7 (18%), and other hematological (36%), neurological (12%), and cardiopulmonary complications (33%). this meta-analysis of the studies with variable research designs revealed that in comparison with controls, cort therapy significantly improved the platelet count, ldh, and alt, as well as reducing ast levels non-significantly in patients with hellp syndrome. moreover, blood transfusion rate and hospital/icu stay were significantly lower in cort-treated patients. however, there was no significant difference in the maternal mortality, overall morbidity, birth weight, or infant respiratory distress between cort-treated and control patients. platelet count and serum ldh levels are reliable indicators of hellp severity, and recovery and longer recovery time is required for more severe cases. corticosteroids are thought to prevent platelet consumption and erythrocyte destruction by stabilizing the vascular endothelium and effectually reducing blood product administration requirements. the recovery of platelets is reported to start as earlier as 12 hours after cort administration. the hellp syndrome, especially in the postpartum period, is associated with high maternal morbidity. class 1 hellp syndrome patients are at higher risk of maternal mortality, and delay in the diagnosis worsens prognosis. despite improvements in biological parameters of hellp syndrome, most of the studies reported that cort treatment does not reduce maternal morbidity. the present study also found no significant difference between cort-treated and control hellp patients in the incidence of overall morbidity in a meta-analysis of 8 studies presenting 15 morbid conditions. the morbid conditions observed in the present study were also reported by many studies not included in this meta-analysis. the morbidities not reported herein include abruptio placentae, retinal detachment, adult respiratory distress syndrome, and hypoxic ischemic encephalopathy. it is believed that an imbalance between proangiogenic and antiangiogenic factors and increased proinflammatory cytokines play an important role in women with preeclampsia and hellp syndrome. higher circulating levels of anti-angiogenic proteins secreted by the placenta, such as soluble fms-like tyrosine kinase 1 (sflt1) and soluble endoglin, are found in preeclampsia patients. dexamethasone has been demonstrated to significantly decrease sflt-1, soluble endoglin, il-6, and tnf- after 24 hours of treatment in hellp patients. these soluble factors are known to stimulate angiotensin ii receptor (at1-aa) production and increase endothelin 1, which are known to play a pathophysiological role in gestational hypertension. these findings suggest that targeting immunomodulators of hellp syndrome pathology may be a novel therapeutic research strategy. management of hellp syndrome requires earlier diagnosis, mother-fetus status examination, stabilization of the indicators and symptoms, delivery at optimal time, and postpartum care in order to reduce maternal morbidity and mortality. a rather longer postpartum recovery period may be required for patients with progressively worsening hellp syndrome. corticosteroid therapy is a cost-effective medication that can be administered via different routes and reduces the length of hospitalization as compared to other treatments, such as platelet transfusion. in the present study, on average, cort therapy reduced hospital/icu stay by about 3 days in comparison with controls and this difference was statistically significant in the meta-analysis of 7 studies. thus, corticosteroids can be beneficial in carefully selected hellp patients without apparent adverse effects to mother or fetus/neonate. firstly, studies with varying designs were included because none of a particular design could make sufficient data available. secondly, clinical and methodological heterogeneity of the sample population in the form of factors such as the severity of hellp syndrome, time of cort administration, and dosage and duration of cort administration in recruited patients may have affected overall outcomes. although, the random-effects model was used to interpret the results, but multi-center randomized controlled trials will be required for clarification of these results. thirdly, the effect of some statistical procedures used to impute missing data may also have had a slight impact, as not all studies provided measures of dispersal values of the effect size of change in indicators following cort/placebo treatments. corticosteroid administration to hellp patients improves platelet count and the serum levels of ldh, besides reducing hospital/icu stay and blood transfusion rate. however, these indices are not significantly associated with maternal mortality and overall morbidity prevalence.

backgroundhemolysis, elevated liver enzymes, and low platelet count (hellp) syndrome is a severe condition of pregnancy that is associated with significant morbidity and mortality. corticoteroid (cort) therapy is common in the management of hellp syndrome. this study evaluates the efficacy of cort therapy to patients with hellp syndrome. material/methodsa literature search was carried out in multiple electronic databases. meta-analyses of means difference and odds ratio were carried under the random-effects model. resultsfifteen studies (675 cort treated and 787 control hellp patients) were included. cort treatment significantly improved platelet count (mean difference between cort treated and controls in changes from baseline, md: 38.08 [15.71, 60.45]109; p=0.0009), lactic dehydrogenase (ldh) levels (md: 440 [760, 120] iu/l; p=0.007), and alanine aminotransferase (alt) levels (md: 143.34 [278.69, 7.99] iu/l; p=0.04) but the decrease in aspartate aminotransferase (ast) levels was not statistically significant (md: 48.50 [114.32, 17.32] iu/l; p=0.15). corticosteroid treatment was also associated with significantly less blood transfusion rate (odds ratio, or: 0.42 [0.24, 0.76]; p=0.004) and hospital/icu stay (md: 1.79 [3.54, 0.05] days; p=0.04). maternal mortality (or: 1.27 [0.45, 3.60]; p=0.65), birth weight (md: 0.09 [0.11, 0.28]; p=0.38) and the prevalence of morbid conditions (or: 0.79 [0.58, 1.08]; p=0.14) did not differ significantly between both groups. conclusionscorticosteroid administration to hellp patients improves platelet count, and the serum levels of ldh and alt, and reduces hospital/icu stay and blood transfusion rate, but is not significantly associated with better maternal mortality and overall morbidity.

PMC4672720

pubmed-407

inherited breast cancers are (pleiotropic) expressions of mutations in a number of distinct genes causing other cancers as well. to some extent, the biological function of the different genes is known, and to some extent the ways breast cancers are produced when this function is lacking, have been described. the cancers associated with different genetic syndromes occur at different ages, they may differ with respect to tumour characteristics, they have different prognosis, and they respond differently to prophylactic and treatment modalities. following the clinical genetic work-up to provide health care to a woman possibly at risk for inherited breast cancer, there is a multistep approach. if she is demonstrated to be at risk, the preventive/treatment modalities she needs are a consequence of which subgroup of the inherited breast cancers she is at risk with. in most european countries, it is the understanding to discuss health care as what is to be offered to any woman who needs it. thus, the question is not what might be done or what money possibly can buy-it is about what is reasonable and affordable. it is also about tradition and culture-what is possible in the current socio-ethical context. the first detailed scientific description of inherited breast-ovarian cancer was given by paul broca in 1866. he demonstrated the transmission of the assumed underlying genetic defect, its expressions, the age-related and sex-limited penetrance, and the possibilities of modifying environmental and genetic factors. not until 10 years ago, we learned that the syndrome is produced by brca1 mutations. brca2 mutations produce breast cancers in a similar prevalence and at a similar age, but with completely different tumour characteristics and with a different set of associated cancers. a number of additional genes cause multiorgan cancer syndromes when mutated, including mutations in tp53, pten, atm and chek2. heterozygous state for atm mutation as a predisposition to breast cancer is debated, and the chek2 syndrome needs further evaluation. some assume that there have to be more dominantly inherited breast cancer genes, others disagree and conclude that they may be recessive, low penetrant or multifactorially interacting, but not dominantly inherited with high penetrance. all cancer genetic clinics are aware that " inherited breast cancer " outside demonstrated brca mutation carrying syndromes may be a fiction, nevertheless all such clinics have defined a large volume of " inherited breast cancer " cases defined by family history but lacking demonstrated dna mutations and referred them to follow-up examinations. we have demonstrated a brca mutation in but a small fraction of our at-risk families, and we have excluded brca mutations in a number of large dominantly inherited breast cancer pedigrees. none of the models for probability calculations for recurrence risk of breast cancer in breast cancer kindreds are valid after a brca mutation have been excluded-they all assume that the patient is selected from an untested population. however, many cancer genetic centres continue to estimate the probability that a given woman is a mutation carrier, after she has been tested and found not to be so. this makes sense if you-as we do-believe there are more genes. whatever we may believe, the calculations are wrong, because they can not be interpreted without correcting the probability estimates for the testing performed prior to the calculations. moreover, our activity of nesting up all the large mutation-carrying families will remove a substantial part of the mutation carriers from the population before the remaining familial clusters are referred to genetic counselling: for each passing day, the next familial cluster of breast cancer referred is less likely to harbour one of our founder brca1 mutations.. this may in principle be due to four factors: (1) selection biases, (2) improper algorithms to calculate penetrances, (3) true differences between the mutations examined and/or (4) environmental or genetic modifiers of penetrance. because the first series obviously had selection biases and because many of the families were not tested (carrier status for relatives was assumed, calculated upon and thereafter presented as results), the methodological problems may have been major. studies based upon testing rather than assuming carrier status, and studies employing sophisticated statistics to eliminate methodological problems, now agree that penetrance for breast cancer is high for all brca1 or brca2 truncating mutations. breast and ovarian cancers are competitive causes of death in a brca1/2 mutation carrier, and both are caused by the same mutation. estimating the penetrance of breast cancer (or ovarian cancer) implies the methodological problem of informed censoring: whatever you do is methodologically wrong, because you censor the data with an argument dependent on what you are examining. because there is no universally " correct " method, the solution is to formulate explicit questions specifying the assumptions to the answers. if a figure for probability for a healthy mutation carrier to contract ovarian cancer is looked for, one must censor out all mutation carriers when they contract another (breast) cancer. doing so, lifetime penetrance for ovarian cancer is high in brca1 mutation carriers, possibly as high as for breast cancer. two of the reasons for the low penetrance for ovarian cancer in some studies may be the combined effect that the families were selected for by the presence of breast cancer, and the fact that you may not contract ovarian cancer after having died of breast cancer. in addition, there may be biological differences between different mutations in the same genes. the exercise of pooling a number of small families without excluding the index cases used to ascertain the families, with different mutations, without actually testing the relatives but assuming their carrier status, without specifying the questions addressed and how the data are censored to answer that question, may give results of low practical value. a statistical flaw has made some mistakenly conclude that brca1-associated cancers have prognosis similar to other breast cancers. the flaw is that according to oncological standards for randomized trials to evaluate effects of treatment modalities, the brca1 cancers have been compared to controls selected for similar prognostic tumour characteristics (oestrogen receptor, histopathological grade, etc.). in this way, brca1 cancers have been matched with a subset of patients demonstrated to have bad prognostic signs (over-parameterization). the question of whether brca1 mutation carriers have even worse prognosis is debated, but all studies agree that brca1 cancers have worse prognosis than age-matched controls. because a germline mutation is always prior to the tumour it causes possibly, the mathematical models are pertinent, but the way the results are presented may seduce the readers to conceptional misunderstandings. brca1-associated breast cancers are, as a group, different from all other defined groups of breast cancers [6-8]. the picture is so clear that all exceptions may be sporadic cancers caused by different mechanisms in brca1 mutations carriers (age-related sporadic breast cancer may occur in brca1 mutation carriers as well). the brca1-associated breast cancers are hormone receptor negative, of histopathological high grade, and they are close to never appear as precancers (dcis) when diagnosed clinically or by mammography. attempts of early diagnosis to achieve early treatment was initially considered successful: the tumours diagnosed were small and often without spread. it turned out, however, that the prognosis was not as good as hoped for according to the stage at diagnosis: retrospective series before any attempt on early diagnosis and treatment demonstrated 5-year survival of 63% for invasive brca1 cancers. the results of the biomed2 prospective series included a 5-year survival of 63% for invasive brca1 cancers, point estimate was no effect at all. prophylactic mastectomy is an alternative, but with severe implications both on personal and professional ethical levels, and it is resource demanding. a new attempt on secondary prophylaxis has been implemented in most centres: mri obviously has the capability of demonstrating tumours invisible in mammography. we do not, however, at present know whether or not mri may diagnose the tumours before they have biologically achieved their bad prognostic propensities. we ask for time-out to retrieve this figure before we consider the alternatives. early diagnosis and treatment to improve prognosis for inherited ovarian cancer was undertaken by ultrasound and cea125 in many centres. it did not work-no report claims substantial improvement in survival. actually, there are few reports on survival, most reports mention cancer with spread at diagnosis but give no survival data. however, it became clear that brca1-associated ovarian cancer seldom occurs before the age of 40. combined with the finding that oophorectomy at that age reduced not only ovarian cancer risk by more than 90%, but also reduced breast cancer risk in brca1 mutation carriers-even in those using hormone replacement therapy -most centres advocate prophylactic oophorectomy past childbearing ages. in contrast to prophylactic mastectomy where uptake is low, the majority of postmenopausal brca1 carriers seem to choose oophorectomy. if a mean to early diagnosis and cure for ovarian cancer appears, we may soon have no brca1 carriers left to evaluate the effects, because there may not be many ovaries left in the mutation carriers aged over 40 years. moreover, the disease is so lethal and prophylactic oophorectomy past childbearing ages seems to be so well tolerated, that it would be hard to suggest a trial. in contrast to the differences in breast cancer phenotype, ovarian cancer caused by brca2 seems similar to that caused by brca1, besides that the penetrance may be lower and disease onset later in brca2 carriers. one report concluded that oophorectomy in brca1 carriers contracting cancer improved the prognosis of the breast cancer. this observation needs to be supported by an independent series, but it is in keeping with the beneficial effect of prophylactic oophorectomy to breast cancer risk in the same group. breast cancers in inherited/familial non-brca1/2 carriers have good prognosis (about 90% 5-year survival) inside early detection programmes applying annual clinical mammography. moreover, a number of cases are demonstrated as precancers (dcis), and they have reportedly 100% event-free 5-year survival. few studies have been presented on prospective survival in brca2-associated breast cancers, but these cancers seem to be comparable to sporadic breast cancers beside the young age of onset. there is no indication that the prognosis should be worse than that of non-brca1/2-associated breast cancers. because all non-brca1 carriers (including the brca2 carriers) predominantly contract hormone receptor-positive breast cancers, they should theoretically benefit from receptor blocking agents (like tamoxifen) and oestrogen production blocking agents (aromatase inhibitors). most agree that by now it would be reasonable to suggest such chemoprevention to these groups, but there is no agreement on which compound and exactly which group to address. it is advocated to give various regimens of such treatment under strict control to evaluate the effects. that is where we are today: trials and discussions, but no consensus on applying chemoprevention as standard health care. it may be expected that because as a group they have receptor-negative cancers, they should not respond either to oestrogen blockers or to oestrogen., oophorectomy prevents breast cancer, tamoxifen prevents contralateral breast cancer, and oral contraceptives induce breast cancer. it seems, however, irrational to suggest oestrogen blockers to prevent oestrogen receptor-negative tumours. in oncology, the scientific standard is a randomized trial. as is evident from the reports (beside chemoprevention) mentioned above, we have no randomized trials. you can not randomize a woman to mastectomy, and you can not deprive a mutation-carrying woman from any potential life-saving health care available. we are faced with the challenge of doing science without randomized trials, and we can not (as the mammographic screening of older women) go and get the families we want for research. this leaves us with series subjected to a number of ascertainment biases, and we should interpret the results with caution. because we are outside the framework of randomized trials, we may be better off not discussing exact figures in single reports, but rather focus on the main results, methods employed and whether or not the empirical facts are in keeping with the current paradigms for understanding. in this perspective, it may seem that we need to reconsider our paradigms for brca1-associated breast cancer. for the other groups, early diagnosis and treatment works as expected, improving early diagnosis may hopefully further improve the results obtained so far, and the principles of chemoprevention may be projected from sporadic cancers. in conclusion to the facts discussed above, most clinical genetic centres relating to inherited breast cancer today advocate annual mammography from the age of 30 onwards to women at risk for inherited non-brca1 breast cancer. it is agreed that besides brca1 carriers, oestrogen blockers/aromatase inhibitors may be beneficial-but there is no agreement on exactly how to implement such chemoprevention. we are now hoping mri to be better to avoid large numbers of prophylactic mastectomies. we are confused by the data on the effect of hormones and hormone blockers in brca1 carriers, and we all hope for chemoprevention to make the unpleasant discussion of prophylactic surgery superfluous if early detection and treatment does not work. early diagnosis does not work for ovarian cancers, but oophorectomy is beneficial and advocated at the end of childbearing ages. as is the case with most scientific reports, this contribution has focused on unsolved problems. we may remember, however, that we actually have about 90% 5-year survival of non-brca1 breast cancer with today's means, and the great majority of inherited breast cancers belong to this group. prophylactic oophorectomy at the end of childbearing ages in brca1 carriers reduces morbidity and mortality by more than 50%. the systematic attempts to prevent and cure inherited breast cancer have been undertaken for but about 10 years. the results are actually good, which is reflected in the high compliance from the affected kindreds. the attempts to prevent and cure inherited breast cancer is an example of a consumer-driven activity based on knowledge and collaboration from the patients needing our care. actually, based on knowledge two brca1 carriers with small invasive tumours without spread this year have asked me for immediate chemotherapy. trials we can not impose upon them, may soon be initiated by patients who want to know. the physicians and the researchers have the role of producing, filing, retrieving and communicating knowledge as appropriate. the choice what to do, however, should be the patient's choice, there are no scientific arguments as to whether or not to undergo prophylactic oophorectomy. as we all know, there are more arguments about what to do with your life than the doctors ' suggestions. the high compliance to our advice during the last decade most probably reflects that the families have identified their problems long ago and were waiting for our care. the high compliance to genetic testing obviously reflects the opinion that our activity may prevent and cure. to maintain the high compliance it is our obligation, however, to ensure that the advocated options are actually available to each single patient. in addition, we may produce arguments to advocate some options because they have consequences to our liking. the suggestions in table 1 may be agreed by most, and some would go further and actively advocate prophylactic mastectomy in brca1 carriers and chemoprevention for the rest. genetic counselling is to present information and options so that any given patient may be supported in exploring her values to make her choices. the challenge is to support the patients who make choices not corresponding with your own priorities. primary and secondary prevention for women at risk for inherited breast or breast-ovarian cancer

inherited breast-ovarian cancer was described in 1866. the underlying genetic defects in brca1/2 were demonstrated 128 years later. we now have 10 years of experience with genetic testing in brca kindreds. the majority of breast cancer kindreds (familial breast cancer) do not demonstrate ovarian cancer and are not associated with brca mutations. the effect of early diagnosis and treatment is monitored through international collaborations.brca1-associated breast cancer is biologically different from other breast cancers, including a worse prognosis. brca2-associated breast cancer is, beside early onset, in many ways similar to sporadic breast cancer. mammography screening of the high risk groups aiming at early diagnosis and treatment, seems promising for familial breast cancer and for brca2-associated breast cancer, but numbers included for brca2 carriers are limited. brca1-carriers have worse prognosis, and the potential benefit of mri for early diagnosis is now being explored. early diagnosis and treatment of ovarian cancer does not substantially improve survival, and prophylactic oophorectomy at the end of childbearing ages is advocated. prophylactic mastectomy is debated, and we may await the results of mri trials before recommending this option. familial breast cancer and brca2-associated breast cancers are often oestrogen receptor positive, and may be prevented by oestrogen blockers/inhibitors. oophorectomy prevents ovarian cancer, and may possibly prevent both receptor positive and receptor negative breast cancer as well, also while using hrt. oral contraceptives may reduce ovarian cancer risk and increase breast cancer risk, irrespective of initial risk and genetic subgroup.

PMC2839988

pubmed-408

stroke is still a major cause of death and long-term disability worldwide and it is associated with significant clinical and socioeconomical problems. despite the continuous efforts to develop the new pharmacological strategies, there is no effective neuroprotective therapy so far for ischemic stroke. novel approaches are needed to improve the recovery and quality of life of stroke patients. development of tissue damage after ischemic insult is dependent not only on duration and intensity of the blood flow reduction, but also on flow independent mechanisms, especially in the peri-infarct brain area. the blood flow dependent mechanisms of tissue damage develop in brain ischemic focus in the short time after onset of blood flow reduction. at that time cell death is a consequence of the acute energy failure and permanent anoxic cells depolarization is induced by loss of ionic gradients. a few hours later, the infarct expands into the adjacent penumbra, and cellular damage is mainly triggered by excitotoxicity, mitochondrial disturbances, reactive oxygen species production, and programmed cell death [1, 2]. excitotoxicity is a pathologic process based on massive activation of ampa and nmda receptors in the brain. inappropriate activation of ampa and nmda receptors is a trigger for subsequent dysregulation of calcium ions homeostasis in the neurons and finally results in neuronal loss. massive activation of those receptors is observed in many cns disorders including stroke, epilepsy, multiple sclerosis, amyotrophic lateral sclerosis, parkinson, alzheimer, and huntington diseases. the most important factor leading to ampa and nmda upregulation is glutamate. glutamate is an important neurotransmitter in physiological concentration, but in pathologically high concentration it is neurotoxic [35]. lately, it is suggested that stroke triggers immune responses leading to inflammatory cell activation and infiltration of cerebral parenchyma. in the stroke brain upregulation of a variety of cytotoxic agents like cytokines, matrix metalloproteinases (mmps), nitric oxide (no), and more ros can be detected [68]. there is also upregulation of expression of some chemokines like ccl2 in the csf [9, 10] and serum of patients with stroke. studies in experimental stroke (middle cerebral artery occlusion model (mcao)) confirmed involvement of chemokine ccl2 and its receptors ccr2 in stroke development. reported no difference in the level of ccl5 but montecucco and colleagues detected increased expression of plasma ccl5 in symptomatic as compared with asymptomatic patients. moreover, canou-poitrine confirmed that, higher systemic levels of ccl5 and cxcl10 in asymptomatic men are independent predictors of ischemic stroke. there is also a recent report from tokami et al. supporting the concept that ccl5 may be neuroprotective during stroke development. they showed upregulation of ccl5 but not ccl2, ccl3, and ccl4 on day 0 in stroke patients. this upregulation correlated with plasma concentrations of neuroprotective factors bdnf, egf, and vegf. other data from mcao model also showed upregulation of several chemokines and their receptors including ccl7, cxcl10, ccl20, and chemokine receptors cxcr4 and ccr6. et-1 induced model of stroke has been previously described by anthony et al. who induced the acute rat cerebral blood volume changes after intravenous and intracranial injections of this vasoconstrictor [21, 22]. after microinjection of et-1 into selected brain regions they observed using magnetic resonance imaging (mri) an acute reduction of local perfusion in the injected hemisphere, loss of neurons in the grey matter and a macrophage/microglia and astrocyte response. after injection of et-1 into the cortical white matter, those authors observed amyloid precursor protein-positive immunostaining (indicative of axonal disruption) and an increase in tau-1 immunostaining in oligodendrocytes. similar to the grey matter lesions, no neutrophils were present and macrophage/microglia response did not occur. additionally, no breakdown in the blood-brain barrier was detected in the white and grey matter. in this study expression of several chemokines including: ccl2, ccl3, ccl5, and cxcl2 as well as expression of markers of neuroinflammation like cd3, f4/80, and il-1 beta was studied. correlation of this expression with intensity of early neurodegeneration detected in the brain during the et-1 induced model of stroke was also analyzed. in all experiments, 8- to 12-week-old female sjl/j mice (n=five for each time point) were used. all animals were housed at the animal facility of the medical university of lodz, lodz, poland, under standard conditions. all experiments in this study have been approved by the local ethics committee for affairs experiments on animals. animal stroke model was induced by stereotactic, intracerebral injection of endothelin-1 (et-1, sigma-aldrich, poznan, poland,) (20 pmol in 1 l of pbs per mouse) into the left hemisphere of the brain. prior injection mice were anaesthetized with mixture of ketamine (1,15 mg, biowet, pulawy, poland) and ksylazine (0,1 mg, biowet, pulawy, poland) per mouse. after complete anaesthetization mice were placed in stereotactic frame (david kopf instruments, ca, usa), skin on the head was cut, and a small hole in the skull was made using surgical drill. et-1 was administered with a hamilton syringe (32 g needle) (hamilton company, bonaduz, gr, switzerland). site of injection (a-2 mm, l-1, 2 mm, d-2, 5 mm) was selected using the stereotactic atlas tissue samples were collected 24 and 72 hours after the model induction. as a controls, brains from uninjected mice and from mice injected in the same way with pbs were used. to obtain rna animals, were perfused with a saline solution. tissues were weighed and then homogenized using a mechanical homogenizer ultra turrax (ika, staufen, germany). tissues were homogenized in a volume of 1ml of trizol ls reagent (gibco brl, invitrogen, carlsbad, ca, usa). rna was isolated from the homogenates with trizol ls reagent using phenol-chloroform method described by chomczynski and sacchi (chomczynski and sacchi, 1987). after rna isolation, its concentration was estimated using the photometric method (biophotometr plus, eppendorf company, wien, austria). to obtain the proteins for the elisa assay harvested organs were weighed using a laboratory balance (radwag radom, poland) and homogenized using a mechanical homogenizer ultra turrax (ika). homogenisation was performed in a volume of 1 ml hepes buffer ph 7.4 containing: hepes 20 mm; edta 1.5 mm; benzamidyn 0.5 mm; chicken egg owoinhibitor 10 ug/ml pmsf (phenylmetylsulfonyl fluoride) 0.1 mm (sigma-aldrich, poznan, poland). supernatants were obtained after centrifugation (20 000 g, time 30 minutes at 4c mpw, warsaw, poland). analysis of the rna expression was performed using the corbett real-time pcr machine rotor gene 3000 apparatus (corbett research, sydney, australia). the key enzyme used in this reaction was taq polymerase with activity of 5 u/ml. additional reaction components were buffer for polymerase, 25 mm mgcl2, 10 mm dntps, fluorescent dye evagreen (biomibo, warsaw, poland), 10 m primers specific to the duplicated sequences, and rnase/dnase free water. for each reaction 2 l of cdna derived from the reverse transcription reaction was used and the total volume was 20 l. as a control histone h3 gene and reference rna (qpcr mouse reference total cellular rna, stratagene, la jolla, ca, usa) were used. quantitative analysis of gene expression at the protein level was performed using elisa method with commercially available immunoenzymatic quantikine kits (r&d systems, mn, usa). each set consisted of 96 well plates coated by manufacturer, standard proteins used to prepare the calibration, secondary and tertiary antibodies combined with the horseradish peroxidase enzyme, and washing buffer and color substrate for peroxidase. the assay procedure was performed according to the protocol provided by the manufacturer. after stopping the color reaction protein concentration was evaluated using a photometric reader victor2 wallac 1420 (perkinelmer, waltham, ma, usa) with for 450 nm filters, corrected at 595 nm. all samples were analyzed in duplicates. quantitative assessment of the intensity of neurodegeneration was performed using elisa method with primary antibodies directed against phosphorylated neurofilaments. the first step was the coating of 96 well maxisorb microtitre plate (nunc, roskilde, denmark) with monoclonal anti-nfh antibodies (smi35r, sternberger monoclonals, convance princeton, nj, usa) and overnight incubation at 4c. the next day tested samples and standard curve samples (neurofilament 200 kd, progen, heidelberg, germany) were added. as a secondary antibody rabbit polyclonal antineurofilament 200 antibody (sigma-aldrich, poznan, poland) was used. as a tertiary antibody swine antibodies against rabbit immunoglobulin conjugated with horseradish peroxidase (dako, glostrup, denmark) were used. the final step was the addition of color substrate for horseradish peroxidase, which was 3,35,5-tetramethylbenzidine (sigma-aldrich, poznan, poland). inhibition of the reaction was performed with 1 m hcl and finally color photometric assessment was done using victor2 reader wallac 1420 (perkinelmer, waltham, ma, usa). the analysis was performed using 450 nm filter with correction at 595 nm. all samples were analyzed in duplicates and the concentration of nfh was determined by referring to the standard curve. assessment of the localization and severity of neurodegeneration at the level of protein was performed using the fluorescent fluoro-jade c dye (chemicon, millipore, warsaw, poland). animals were perfused with 4% buffered formalin solution and tissues samples were embedded in paraffin blocks. 10 m thick sections were applied to a polished super frost slides (menzel-glaser braunschweig, germany). before final staining paraffin fluoro-jade c staining was performed according to the protocol provided by the manufacturer (chemicon). for staining of nuclei sections were counterstained using the blue fluorescent dye dapi (sigma-aldrich, poznan, poland). then the tissue was mounted and coverslipped using dpx (sigma-aldrich, poznan, poland). for the analysis and acquisition of images an inverted microscope axioobserver a1 (carl zeiss inc., the following lenses made by carl zeiss inc. were used: plan-achromat: 4x/0.10, a- plan 10x/0.25 ph1; ld a-plan 20x/0.3; ld plan-neofluar 40x/0.6, ph2 korr. the images were obtained with a digital camera, axiocam mrc5 (carl zeiss group, goettingen, germany) attached to the microscope. for image acquisition we used axio-vision rel. nonparametric kruskal-wallis and mann-whitney tests were used. for correlation analyses kendal significant upregulation of expression of t cell line marker-cd3 was observed in the et-1-injected hemisphere at 72 h after injection (p=0.03, mann-whitney test) (figure 1(a)). at that time a significant difference in expression of cd3 was detected between ipsilateral and contralateral hemispheres (p=0.019, mann-whitney test) (figure 1(a)). expression of cd3 in ipsilateral hemisphere was also increased when compared to pbs injected hemisphere (p=0.28, mann-whitney test). the expression of monocyte/macrophage lineage marker f4/80 was significantly elevated only in et-1 injected hemisphere at 72 hours after injection. at that time significant difference was observed in expression of f4/80 between et-1 injected hemisphere and untreated control group (p=0.022; mann-whitney test) (figure 1(b)). we detected also a significant difference in expression of f4/80 between ipsilateral and contralateral hemispheres of et-1 injected mice at 72 hours after injection (p=0.035, mann-whitney test) (figure 1(b)). upregulation of cytokine il-1 expression was observed in et-1 injected hemispheres only at 24 h after injection. significant difference in expression of il-1 was observed between contralateral hemispheres and normal control group at 72 hours after injection (p=0.03 and 0.019, resp.; we detected also a significant difference in expression of il-1 between ipsilateral and contralateral hemispheres of et-1 injected mice 72 hours after injection (p=0.019, p=0.019, resp.; upregulation of chemokine ccl2 expression was observed in ipsilateral hemispheres at 24 and 72 h after injection of et-1 (p=0.005, p=0.005, resp.; mann-whitney test) (figure 2(a)). at 24 h ccl2 expression in ipsilateral hemisphere was significantly higher than at 72 h (p=0.019; mann-whitney test). significant difference in ccl2 expression after et-1 injection was also observed between ipsilateral and contralateral hemispheres at 24 h and 72 h (p=0.012 and 0.036, resp.; we also showed that during early stage of et-1-injection stroke model expression of ccl3 is significantly upregulated at 24 and 72 h after model induction (figure 2(b)). there was significant upregulation of ccl3 expression in ipsilateral hemispheres of et-1 injected mice in comparison to normal controls and contralateral hemispheres at 24 h after injection (p=0.008 and 0.012, resp.; mann-whitney test). at 72 h after model induction we observed significant upregulation of ccl3 expression in et-1-injected hemispheres in comparison to normal brains and contralateral hemispheres (p=0.014 and 0.019, resp.; mann-whitney test) (figure 2(b)). ccl5 was observed in et-1-injected hemispheres in comparison to uninjected animals at 24 and 72 h (p=0.008, and 0.008 resp.; significant difference was also observed in ccl5 expression between ipsilateral and contralateral hemispheres at 24 h and 72 h after injection of et-1 (p=0.008 and 0.012, resp.; mann-whitney test) (figure 2(c)). initially increasing expression of cxcl2 in et-1 injected hemispheres was detected with the peak at 24 h and subsequent decrease at 72 h but still significantly higher than in normal controls (p=0.036, and 0.036, resp.; mann-whitney test) (figure 2(d)). at 24 h cxcl2 expression in ipsilateral hemisphere was significantly higher than at 72 h (p=0.012; mann-whitney test) and then at 24 h after et-1 injection in contralateral hemispheres (p=0.012; mann-whitney test) (figure 2(d)). the expression of cxcl12 in the et-1 and pbs-injected brains and normal controls did not show a significant difference between analysed groups (data not shown). the most severe neurodegeneration was observed in et-1-injected hemispheres at 24 and 72 h after model induction (p=0.019 and 0.029, resp.; there was also increased neurodegeneration in contralateral hemispheres of et-1 injected mice at 24 and 72 h, but it was significantly lower than in ipsilateral hemispheres (p=0.03, and 0.03, resp.; the localization of ischemic lesion was detected in et-1 injected ipsilateral hemispheres using cresyl violet staining (figure 3(b), large box). inside the ischemic focus injured neurons were abundant (detected by fluoro-jade and marked by arrows) cells nuclei counterstained with dapi are marked on the picture by arrowheads (figure 3(c)). we observed the positive correlation between expression of lymphocyte lineage marker cd3 (kendall tau=0,62; p=0.0004) (figure 4(a)) as well as monocyte/macrophage lineage marker f4/80 (kendall tau=0,56; p=0.0007) (figure 4(b)) and the severity of neurodegeneration in et-1 injected brain hemispheres. although the expression of several studied chemotactic inflammatory mediators (chemokines ccl2, ccl3, ccl5, and cxcl2) was significantly increased in the early stage of this stroke model, there was no clear correlation between this expression and intensity of neurodegeneration (data not shown). in this study we analyzed potential relationship between neuroinflammation and neurodegeneration in experimental model of ischemic stroke induced by intracerebral et-1 injection. we focused on a group of proinflammatory chemokines, especially the classical representatives of ccl subfamily. the reason for selecting these chemokines was their confirmed participation in pathogenesis of many central nervous system diseases. during the first few days of the experimental brain ischemia we observed increasing neurodegeneration in et-1 injected hemisphere. there are several studies showing that neurodegeneration occurs early during brain ischaemia [23, 24]. at that time also the relationship between those two processes is complex and still requires further studies. to measure the intensity of neuroinflammation the expression of inflammatory cells markers (cd3 for t cells and f4/80 for monocytes/macrophages) has been measured at the same time. this analysis showed increased lymphocyte migration to ischemic brain hemisphere at 72 h after model induction. similarly, infiltration of ipsilateral hemisphere by monocytes/macrophages was significantly increased at 72 h after initiation of brain ischemia. comparable observation was reported by others who showed the presence of macrophages/activated microglia at 72 h after intracerebral injection of et-1 to rat brain. in another study using mcao stroke model, the influx of mononuclear cells to the site of brain ischemia was recorded between 2 and 15 days after model induction. the presence of neuroinflammation during early brain ischemia was also confirmed in our study by elevated expression of inflammatory mediator-cytokine il-1. its presence was observed as early as 24 h after model induction. in several cell types including astrocytes, microglia, neurons, and endothelium. in another study increased production of il-1 was reported even at 36 hours after induction of brain ischemia. the peak of this expression was observed at 12 h, and it returned to baseline level after 5 days. other studies confirmed also that inflammatory mediators, such as il-1 and tnf, are important contributors to cns neural tissue damage induced by ischemia [28, 29]. in our stroke model analysis of the relationship between the infiltration of ischemic hemisphere by mononuclear inflammatory cells and this may suggest that there is close connection between neuroinflammation and neurodegeneration in ischemic stroke. migration of inflammatory cells from the blood to the ischemic brain may be at least partially induced by chemotactic cytokines-chemokines. to study this concept the highest expression of ccl2 was observed in our model at 24 h after initiation of brain ischemia. increased expression of ccl2 was still observed at 72 h but was at that time significantly lower. increased expression of ccl2 in mcao model in the ipsilateral hemisphere was observed on neurons at 12 h and on astrocytes at 24 h after cardiac arrest, suggesting that these cells are the potential source of ccl2 during ischemic stroke. minami and satoh using double in situ hybridization method pointed to microglia as the cellular source of ccl2 during mcao. it was shown in another mcao study that ccl2 leads to infiltration of the cns by monocytes and thus enhances brain damage induced by ischemia. also in human stroke patients elevated level of ccl2 was detected in cerebrospinal fluid and serum [9, 11]. the highest ccl3 expression was detected at 24 h after et-1 injection. at 72 h this expression was still increased but it was much lower than at 24 h. also at the protein level we observed significant increase in ccl3 production in the ischemic hemisphere. our results are in line with the report by gourmala et al. who observed an increase in ccl3 expression at mrna level already at 1 h after mcao in rats, with peak expression at 816 h. in addition, they observed higher expression of ccl3 during temporary mcao than in permanent mcao, suggesting the impact of reperfusion on the neuroinflammation in the damaged tissue. gourmala et al. using in situ hybridization localized the expression of ccl3 on microglial cells/macrophages during brain ischemia. in addition, another studies have concluded that ccl3 application to the brain ventricles after complete mcao enhances mcao harmful effects. we observed almost 46-fold and 30-fold increase in ccl5 expression at 24 h and 72 h, respectively, after induction of et-1 induced stroke model. there are only a few reports concerning the role of ccl5 in the development of ischemic stroke. it was suggested that ccl5 mediates blood-brain-barrier (bbb) disruption and cns tissue damage as well as inflammation after reperfusion during mcao model. these data were not confirmed by tokami and colleagues who observed neuroprotective effect of ccl5 in ischemic stroke suggesting that ccl5 is expressed during stroke mostly in neurons. in et-1 induced experimental stroke a significant increase in cxcl2 expression at 24 h after brain ischemia induction was also observed. observed increased expression of cxcl2 in the brain of rats with the permanent mcao as well as in the brain and spleen during temporary mcao in mice. in other study increased cxcl2 expression was observed at 6 h of reperfusion and decreased by almost half at 22 h after reperfusion. vikman et al. showed increased cxcl2 expression in the brain vessels in the model of subarachnoid haemorrhage and in organotypic cultures. cxcl2 involvement in the inflammatory process in the cns during mcao was also confirmed in a scid mice. mcao induced in scid mice led to development of significantly reduced area of brain damage and lower inflammatory infiltration in ipsilateral hemispheres. reduced expression of many inflammatory mediators including cxcl2 was also observed in t- and b-cell-deficient mice mcao study. unfortunately, therapy of ischemic stroke with cxcl2 receptor-cxcr2 antagonists sb225002 was not successful. in report presented by copin et al. the cxcl1/cxcl2 chemokine-binding protein evasin-3 treatment was associated with reduction in neutrophilic inflammation in mice mcao model. although in our study the expression of several studied chemokines was significantly increased at the early phase of et-1 induced stroke model, no clear correlation of this expression with neurodegeneration was observed. that they suggest that inhibition of inflammatory cell accumulation in the brain at the early stage of stroke may lead to amelioration of ischemic neurodegeneration. upregulated in the ischemic brain chemokines may be a potential target for future therapies reducing inflammatory cell migration to the brain in early stroke.

neurodegeneration is a hallmark of most of the central nervous system (cns) disorders including stroke. recently inflammation has been implicated in pathogenesis of neurodegeneration and neurodegenerative diseases. the aim of this study was analysis of expression of several inflammatory markers and its correlation with development of neurodegeneration during the early stage of experimental stroke. ischemic stroke model was induced by stereotaxic intracerebral injection of vasoconstricting agent endothelin-1 (et-1). it was observed that neurodegeneration appears very early in that model and correlates well with migration of inflammatory lymphocytes and macrophages to the brain. although the expression of several studied chemotactic cytokines (chemokines) was significantly increased at the early phase of et-1 induced stroke model, no clear correlation of this expression with neurodegeneration was observed. these data may indicate that chemokines do not induce neurodegeneration directly. upregulated in the ischemic brain chemokines may be a potential target for future therapies reducing inflammatory cell migration to the brain in early stroke. inhibition of inflammatory cell accumulation in the brain at the early stage of stroke may lead to amelioration of ischemic neurodegeneration.

PMC3844257

pubmed-409

stress urinary incontinence (sui) is defined as the complaint of involuntary leakage on effort or exertion or on sneezing or coughing. although sui is not a life-threatening condition surgical therapy is employed in patients who have severe degrees of sui or those patients in whom conservative or pharmacological treatments have failed. sling procedures for genuine sui are today the mainstay of treatment and have been used for over a century with first procedure reported by schultze in 1888. the integral theory of female urinary continence described by petros and ulmsten redefined the modern approach to anti-incontinence surgery and ushered the era of the midurethral sling. the concept was applied clinically by placing the sling to a more distal location beneath the urethra then as compared to the previous techniques. subsequently excellent results were presented using suprapubic arc system (sparc, american medical systems inc. some authors have even termed midurethral sling surgery as the new gold-standard for sui. with the available midurethral slings, the trocar has to be passed blindly either from above (suprapubically) or below (transvaginally), which increases the chances of injury to the pelvic organs and blood vessels. shlomo raz modified the technique of sling placement with opening the endopelvic fascia and passing the needle under controlled digital palpation, thereby decreasing the chances of injury to surrounding pelvic organs as well as significantly decreasing cost using tailor-made mesh. their finger-guided passing of needle was similar to the raz procedure of bladder neck suspension. we present our experience with the use of large pore polypropylene mesh/polypropylene-polyglactin mesh as a pubovaginal sling (midurethral) in the treatment of sui with the modified raz technique. a retrospective analysis was performed of consecutive 53 patients of pure sui who underwent midurethral slings procedure with the modified technique from june 2003 to december 2008 at our institute. preoperative evaluation included a history, physical examination, urine microscopic examination, and culture. severity of sui was defined by the number of pads used by the patients per day as mild (< 2), moderate (24), and severe (> 4). patients were examined in lithotomy and standing positions to demonstrate sui on cough test and valsalva maneuver. four patients of our series underwent multichannel urodynamic examination for history suggestive of mixed incontinence in accordance with nice guidelines. all patients were counseled regarding the need of postoperative clean intermittent catheterization (cic) and transient voiding dysfunction. vaginal tissue was atrophic in four patients and they were preoperatively treated with local estrogen cream. twenty patients gave previous history of hysterectomy; others had history of undergoing gynecological procedures, details of which were not available. none of the patients had previous history of surgery for incontinence or pelvic organ prolapsed. eight patients had grade 1 cystocele and two patients had grade 1 rectocele, which did not require treatment. five patients underwent simultaneous vaginal hysterectomy for gynecological indications. a polypropylene mesh (prolene) /polypropylene-polyglactin mesh (vipro) measuring 1 10 cm (ethicon, johnson and johnson, usa) was fashioned from commercially available 15 7.5 cm mesh. this strip was soaked in antibiotic saline and stitched at all four corners to 1-0 polyglactin suture. 16 fr foley catheter was placed in urinary bladder and balloon was palpated at bladder neck to estimate urethral length. two percent of xylocaine with adrenaline was infiltrated in the vaginal mucosa overlying the urethra. a 1.52 cm mid-line incision was made over the midurethra (1.5 cm proximal to external urethral meatus). vaginal mucosal flaps were dissected on either side extending into avascular plane, until endopelvic fascia was reached. blunt dissection was carried out in retropubic space by inserting a finger and the bladder was swept medially. two small punctures were made suprapubically and a double-pronged needle (cook urological inc., indiana, usa) is passed under finger control through the fascia and retropubic space. a marking catgut suture is placed in the center of the mesh along its length to ensure that mesh placement is equidistant. the polyglactin suture was pulled and tension adjusted by placing the tip of an artery forceps while positioning sling against midurethra. vaginal pack was removed after 24 hours of surgery and providine-iodine vaginal pressary was advised for 5 days. if residual urine was more than 50 ml, patient was advised cic. in sexually active patients, the patients were followed up with history and clinical examination by the operating surgeon in the opd. social dryness was defined as patient requiring 1 pad per day and acceptable leak while carrying out routine tasks. five of the patients also underwent simultaneous vaginal hysterectomy for gynecological indications with no increase in morbidity. mean duration of follow-up was 46.1 months (1278 months) [table 1]. forty-five (85%) patients were completely dry and eight (15%) were socially dry at the end of the follow-up. four patients failed voiding trial and were advised cic, which was later discontinued after 38 weeks, when their pvr fell to<50 ml. two patients complained of dull aching lower abdominal pain, which was relieved by administration of oral analgesic agents. five of our patients complained of mild dyspareunia, which was transient and did not require treatment [table 2]. none of the patients reported significant voiding dysfunction, infection, nonhealing, or erosion of the sling till their last follow-up [table 2]. over last few years, many procedures using autologous material (rectus sheath, fascia lata) or synthetic material (polypropylene, mersilene) have been reported in literature. continued refinements in materials were sought to identify an ideal compound for use in transvaginal slings that would be inert, sterile, noncarcinogenic, and mechanically durable. synthetic materials have the advantage of being readily available and do not require harvesting from another site. this decreases the operative time, discomfort, and potential donor site complications after the surgery. histological and clinical studies have shown that polypropylene is a synthetic material that is well-tolerated by the body, with little exposure of the patient to infection and vaginal or urethral erosion. cure rates using synthetic slings have been shown to be around 7395%. in a review of contemporary literature, daneshgari et al. have found complication rates ranging from 4.3% to 75.1% for retropubic midurethral slings. they have quoted postoperative obstruction ranging from 1.9% to 19.7% from various series. in these patients, resolution is commonly spontaneous; the intervening period can be managed with cic or indwelling catheter. a recent meta-analysis showed that tvt outperformed burch colposuspension both in terms of postoperative continence rates, whereas success rate efficacies were similar after tvt and pubovaginal slings. comparing tvt to the other retropubic tension-free midurethral vaginal slings, tvt was more efficacious than both intravaginal slingplasty (ivs) and sparc. some authors have even described midurethral slings as the new gold-standard for the treatment of female sui. the reasons for popularity of these procedures are effectiveness, ease, and low rate of serious complications. a recent meta-analysis of complications of these procedures have highlighted significantly high rates of bladder perforation after tvt. have questioned the authenticity of reported complication rates and have described major complications and even 10 deaths as retrieved after systemic search of food and drug administration (fda) manufacturer and user facility device experience (maude) database. rodriguez and raz have described a mid-distal urethral sling procedure in which distal urethra is defined as anything distal to the pubourethral ligament. they have explained the mechanism of action of tvt procedure by providing support as well as contributing to normal function of distal urethral complex (composed of the pubourethral ligaments, intrinsic sphincteric mechanism, extrinsic sphincter, and levator muscles located immediately distal to the pubourethral ligaments). furthermore, they have enumerated the drawbacks of current midurethral sling systems as being blind procedures with consequently higher incidences of major complications. in their modification, a sling is refashioned from commercially available mesh, which is cheap, does not require any special instrumentation, and is placed only within the retropubic space. the optimal surgical approach should minimize the risk of damage to the bladder neck, vagina, and urethra. this is achieved by developing retropubic space with blunt dissection and passing double-pronged needle under finger guidance. the procedure should augment the urethral resistance during sudden increase in the intra-abdominal pressure without preventing normal decreases in urethral pressure during voiding. placing a sling beneath the urethra increases the urethral compression and provides a plate for receiving the transmitted intra-abdominal pressure to the bladder neck and proximal urethra. the safety of the procedure has also been demonstrated in their series with no incidence of major complications. conventional guidelines recommended multichannel urodynamic studies in sui patients planned for surgery. with the advent of midurethral slings, which have shown to be effective in all types of urinary incontinence, studies have questioned the routine use of urodynamic parameters like valsalva leak point pressure in predicting outcome of sling surgery. have proposed that standard use of urodynamic investigation in the preoperative workup of midurethral slings needs to be revisited. in our series we had cure rate in all the 53 patients till their last follow-up. sling procedures that are successful at 6 months are likely to remain successful for many years. in our series, all the patients had a follow-up of more than 12 months and all these patients were doing well till their last follow-up. in a comprehensive meta-analysis of complications of midurethral slings, novara et al. have found the incidences of various complications bladder/vaginal perforation 2.919.31%; hematoma 1.453.9%, uti 3.77.5%, and cic in 77.5% of cases in various rcts and non-randomized studies. rodriguez et al. have described pelvic hematoma (not requiring treatment) in 0.33% and suprapubic pain in 0.66% of cases. three of their patients required cic for maximum of 3 months after which they were all spontaneously voiding. in our series, four (7.5%) patients required cic after failed voiding trial, which was discontinued after a period of 38 weeks. we attribute this to be the procedure-related local tissue edema/pain, which gradually subsided. other minor complications in our series were hematoma (not requiring treatment) in 1.8%, uti in 3.7%, low back pain in 3.7%, and dyspareunia in 9.4% of cases. major concerns about the erosion of the sling into the urinary tract have been diminished as a result of meticulous detail in placing the mesh through a small incision and tying the mesh loosely so as to avoid excessive compression and ischemia. we did not encounter any incidence of mesh erosion into the urethra in our series of patients. we faced none of the major injuries like vascular injury, bowel injury, necrotizing fasciitis, sepsis, or death, a fact emphasized by the shlomo raz group. one standard-sized polypropylene mesh costs around inr 1800 and polypropylene-polyglactin (vipro) mesh costs around inr 2300. this is economically friendlier as compared to custom-made mesh systems commercially available, ranging from inr 18,000 to 25,000, especially in developing country like india. the limitations of this study are lack of objective analysis and quantification of sui. outcomes were based on patients interview on opd basis by the operating surgeon and not by patient-driven questionnaires, possibly influencing the overall results. despite the limitations of the study, we believe this procedure is a cost-effective alternative to other minimally invasive procedures using commercially available kits and with comparable outcomes. polypropylene mesh as midurethral slings by modified raz technique is cost-effective, safe, and has acceptable complication rates. although our series is a not big enough to draw any formal conclusions, but we can safely infer that the results of this procedure are comparable to other techniques used in patients with pure sui.

objectives: we report our experience of pure stress urinary incontinence (sui) treated by midurethral synthetic sling placement by modified raz technique. materials and methods: fifty-three patients with pure sui operated at our institute between june 2003 and december 2008 were included in this study. midurethral sling tape, fashioned from commercially available large pore synthetic mesh, was placed using the modified raz technique. the technique consisted of placing the tape within retropubic space using double-pronged needle, which is passed under finger control through the fascia and retropubic space. outcomes were assessed on the basis of patient's interview in follow-up opd. results:mean age was 57.68 (2869) years. forty-five (85%) patients were totally dry and eight (15%) socially dry at the end of the follow-up. mean operative time was 46.5+11.3 minutes (3580 minutes). none of the patients required blood transfusion or had bladder/bowel injury. mean duration of hospital stay was 2.17 days (24 days). mean duration of follow-up was 46.1 months (1278 months). conclusions: modified raz technique is safe and cost-effective for placing midurethral sling for genuine stress incontinence.

PMC3114585

pubmed-410

this causes misalignment between the sleep and wake propensities that are controlled by hypothalamic circadian pacemaker and results into shift work sleep disorder (swsd). the reported incidence of swsd in india is about 44.8% of night-shift workers and 35.8% of rotating workers. swsd is characterized by persistent excessive sleepiness during night work and insomnia when attempting sleep in the daytime. individuals with swsd have significantly higher incidence of sleepiness-related accidents, absenteeism, depression, and missed family and social activities as compared with other night-shift workers. it is also associated with higher incidence of ulcers, cardiovascular disease, and deficit in cognition and psychomotor performance [4, 5]. the pharmacological management of swsd involves treatment with modafinil that has been shown to improve wakefulness and ability to sustain attention in these patients. however, despite the half-life of 15 hours, the wakefulness promoting effect of modafinil is found to be ill-sustained in the last one third of night shift hours. the lack of efficacy in the early morning hours and undue patient confidence in the drug can result into excessive sleepiness while commuting home. armodafinil, the chirally pure r-enantiomer of modafinil, approved by us fda in 2007 has half-life (t1/2=15 hours) three times longer than its s-enantiomer (t1/2=3 hours). despite the same half lives, comparison of the equivalent (200 mg) doses of modafinil and armodafinil, in humans has revealed that armodafinil sustains higher plasma concentrations 614 hrs postadministration than that of racemic modafinil with longer maintenance of wakefulness [810]. this was a randomized, comparative, double-blind, and multicentric study comparing the effects of modafinil 200 mg with armodafinil 150 mg in indian patients of swsd. prior approval was obtained from drug controller general of india (dcgi) and appropriate ethics committees. the study was conducted in accordance of good clinical practice guidelines (issued by central drugs standard control organization, government of india) and according to the declaration of helsinki. the trial was registered at the clinical trials registry, india (http://www.ctri.in/). after obtaining written informed consent, patients of either sex, aged between 18 and 60 years, attending outpatient clinics of the authors, and suffering from excessive sleepiness associated with swsd (assessed basis patient ' primary complaint and using the diagnostic criteria adopted from international classification for sleep disorders (table 1)) were enrolled. patients were working at least five night shifts every month for 12 hours or less, with 6 hours or more working between 10 p.m. and 8 a.m. and at least three shifts occurring consecutively. the major exclusion criteria were patients with significant liver or kidney or heart diseases, patients with clinically significant, uncontrolled psychiatric or medical condition, patients with known history of hypersensitivity to formulation, patients operating an automobile or hazardous machinery, caffeine consumption averaging more than 600 mg/day within 1 week of baseline, use of other concomitant medications which inhibit, induce, or are metabolized by cyp450, patients using sedative or cns acting drugs or medication liable to affect outcome of the study (e.g., antihistamines, selective serotonin reuptake inhibitors, tricyclic antidepressants, lithium, anti-psychotics, anticonvulsants, monoamine oxidase inhibitor, benzodiazepines, psychostimulants, and anticoagulants), pregnant and lactating mothers, females of reproductive age and expecting pregnancy or using steroidal contraception, and patients with alcohol or drug abuse. this was a multi-centric, randomized, comparative, and double-blind parallel group, clinical trial conducted over 18 sites across india. 1 ratio for test and reference products online at http://www.randomization.com/. patients received orally either armodafinil 150 mg tablet (emcure pharmaceuticals ltd., india) or modafinil 200 mg tablet (from commercial source) one hour prior to start of every night shift for 12 weeks. the test formulation was earlier found to be bioequivalent to the us fda-approved formulation of armodafinil, in 26 healthy indian volunteers. the tablets of armodafinil and modafinil were identical in shape, size, and color and were dispensed in coded, identical, and opaque packs to conceal identity and maintain blinding. patients were evaluated for sleepiness score based on stanford sleepiness scale (sss) at baseline, 4 weeks, 8 weeks, and 12 weeks. all the assessments were done in the morning hours at the end of three consecutive night shifts. the primary efficacy endpoint was proportion of patients showing at least 2 grades of improvement (responder) based on sss in both groups. the other efficacy variables included improvement in mean sss grades compared to baseline, compliance to therapy, and patients ' as well as physicians ' global assessment for efficacy. global assessment of efficacy was performed using the following grades: (i) excellent=reduction of>75% of symptoms, (ii) good=reduction of 5175% of symptoms, (iii) fair=reduction of 2650% of symptoms, and (iv) poor=no improvement or reduction in<25% of symptoms. patients ' compliance to the therapy was calculated in percentage by using following formula: (number of tablets actually taken 100)/number of tablets supposed to be taken. a general and detailed systemic examination was performed for all patients during each study visit. blood samples were collected at baseline and at the end of the study for complete hemograms, liver function tests, renal function tests, lipid profile, and fasting blood glucose levels. electrocardiograms were performed for all patients at baseline and at the end of the study. tolerability was assessed by recording patients ' global assessment about the tolerability of the drug and percent of the patients experiencing any drug-related adverse events. the global assessment of tolerability was performed using following grades: (i) excellent=no adverse drug reaction, (ii) good=mild adverse drug reaction but no interference with normal lifestyle, (iii) fair=mild adverse drug reaction which interference with normal lifestyle. however, benefits of drug therapy outweigh the inconvenience, (iv) poor=drug withdrawn. prestudy calculations showed that a sample size of 100 in each group would have 80% power to detect a difference of at least 19% in responder rate with a significance level (alpha) of.05 (two tailed). was done by comparing the proportion of patients showing excellent and good response against proportion of patients showing fair and poor response. for all statistical tests, a p value of less than or equal to 0.05 was considered as significant, after correction for any multiple comparisons. two hundred and eleven patients of swsd were recruited with 105 subjects in armodafinil group and 106 subjects in the modafinil group (figure 1). both modafinil and armodafinil significantly improved sleepiness grades as compared to baseline (p<.0001) (figure 2). responder rates with armodafinil (72.12%) and modafinil (74.29%) were comparable (p=.76). at the end of therapy, compliance in both modafinil group (99.31% 3.06%) and armodafinil group (99.13% 2.35%) was found to be good and comparable (p=.63) indicating adequate patient adherence to therapy. both physicians ' and patients ' assessment of efficacy was found to be comparable between armodafinil and modafinil group (figure 3). the intention-to-treat analysis showed that the adverse event incidences in modafinil (40.57%) and armodafinil (42.87%) groups were similar (p=.78). the adverse effect profile of both drugs was found to be similar with headache, nausea, and dry mouth being the common adverse effects (table 3). no incidences of accidents or absenteeism from work were noted during the study period as assessed from patient history. physicians ' and patients ' assessment of tolerability was found to be comparable between armodafinil and modafinil group (figure 4). the baseline and after-therapy biochemical values were within normal range and similar between two groups, except that there was slight increase in mean sgpt in both armodafinil and modafinil groups as compared to baseline (p=.008 and .0007) without inter-group significance and mean blood urea value in armodafinil group increased (p=.002) compared to baseline. however, the increased values were within normal limits. in both groups, electrocardiograms were within normal at baseline and after completion of therapy in all patients. the adverse events that led to discontinuation were palpitation, anxiety, hypertension, depression, nervousness, and depressed mood in a patient receiving armodafinil and vomiting along with dizziness in another patient receiving modafinil. the present study confirms the efficacy of armodafinil 150 mg in patients of swsd. the efficacy of armodafinil was found to be comparable to 200 mg of modafinil in maintaining wakefulness. both modafinil and armodafinil caused a slight increase in liver enzymes, and armodafinil caused a slight increase in blood urea nitrogen. this was not of clinical significance as the increased values were within normal laboratory limits. armodafinil 150 mg was comparable to modafinil 200 mg, which indicates that armodafinil is 1.33 time more potent than racemic modafinil. the use of r-enantiomer of modafinil avoids unnecessary use of s-isomer and exerts less metabolic load on the body. in previous studies, 200 mg of armodafinil was shown to provide more sustained plasma concentrations late in the day as compared to 200 mg of modafinil and monophasic plasma elimination kinetics as compared biphasic for modafinil. we chose the 150 mg dose of armodafinil, as this was the approved dosage for the present indication. our study demonstrated no difference in the efficacy of 150 mg of armodafinil over 200 mg of modafinil. the comparative efficacy of 200 mg of armodafinil with modafinil in swsd has not yet been assessed. a limitation of the present study is that the assessment of sleep latency and polysomnography throughout the nightshift could not be done due to unavailability of patients and investigators. the study did not demonstrate any difference in efficacy and safety between armodafinil 150 mg and modafinil 200 mg, and both drugs were comparable.

aim. to compare the efficacy and safety of armodafinil, the r-enantiomer of modafinil, with modafinil in patients of shift work sleep disorder (swsd). material and methods. this was a 12-week, randomized, comparative, double-blind, multicentric, parallel-group study in 211 patients of swsd, receiving armodafinil (150 mg) or modafinil (200 mg) one hour prior to the night shift. outcome measures. efficacy was assessed by change in stanford sleepiness score (sss) by at least 2 grades (responder) and global assessment for efficacy. safety was assessed by incidence of adverse events, change in laboratory parameters, ecg, and global assessment of tolerability. results. both modafinil and armodafinil significantly improved sleepiness mean grades as compared to baseline (p<.0001). responder rates with armodafinil (72.12%) and modafinil (74.29%) were comparable (p=.76). adverse event incidences were comparable. conclusion. armodafinil was found to be safe and effective in the treatment of swsd in indian patients. the study did not demonstrate any difference in efficacy and safety of armodafinil 150 mg and modafinil 200 mg .

PMC3135062

pubmed-411

lignocaine (n-diethylaminoacetyl-2,6-xylidide) was first synthesized by lofgen a swedish chemist in 1943 and was first introduced into clinical use in 1948. lignocaine hydrochloride (c14h22n2o.hcl) is most soluble in water and so this is most commonly used injectable solution as local anesthetic agents. the efficacy profile of lidocaine as a local anesthetic is characterized by a rapid onset of action and intermediate duration of efficacy. lidocaine or lignocaine along with adrenaline has the advantage of a rapid onset of action. epinephrine (adrenaline) vasoconstricts arteries, reducing bleeding and also delays the resorption of lidocaine, almost doubling the duration of anesthesia. for surface anesthesia, several available formulations can be used, e.g. for endoscopies, before intubations, etc. topical lidocaine has been shown in some patients to relieve the pain of postherpetic neuralgia (a complication of shingles), though not enough study evidence exists to recommend it as a first-line treatment. although not completely curing the disorder, it has been shown to reduce the effects by around two-thirds. lidocaine is also the most important class-1b antiarrhythmic drug; it is used intravenously for the treatment of ventricular arrhythmias (for acute myocardial infarction, digoxin poisoning, cardioversion, or cardiac catheterization) if amiodarone is not available or contraindicated. lidocaine should be given for this indication after defibrillation, cardiopulmonary resuscitation, and vasopressors have been initiated. a routine prophylactic administration is no longer recommended for acute cardiac infarction; the overall benefit of this measure is not convincing. the onset of action of lidocaine is about 4590 s and its duration is 1020 min. it is about 95% metabolized (dealkylated) in the liver mainly by cyp3a4 to the pharmacologically active metabolites monoethylglycinexylidide (megx) and then subsequently to the inactive glycine xylidide. megx has a longer half-life than lidocaine but also is a less potent sodium channel blocker. the elimination half-life of lidocaine is biphasic and around 90120 min in most patients. this may be prolonged in patients with hepatic impairment (average 343 min) or congestive heart failure (average 136 min). lidocaine is excreted in the urine (90% as metabolites and 10% as unchanged drug). bupivacaine (1-butyl-2, 6- pipecoloxylidide) was first synthesized in 1957 by ekenstam, a scandinavian chemist, and was first introduced into clinical use in 1963. it is the longer side chain with four methylene groups on the piperidine ring that is responsible for the different properties of bupivacaine when compared to lignocaine. it has a molecular weight of 288.4 and an empirical formula of c18h28n2o and exists as white crystalline base with a melting point of 251258c. the base is not soluble in water, but the acid salt, bupivacaine hydrochloride (c18h28n2o.hcl) is slightly soluble. the rate of systemic absorption of bupivacaine and other local anesthetics is dependent upon the dose and concentration of drug administered, the route of administration, the vascularity of the administration site, and the presence or absence of epinephrine in the preparation. duration of action (route and dose-dependent): 29 h. half-life in neonates is 8.1 h and in adults is 2.7 h. time to peak plasma concentration (for peripheral, epidural or caudal block) is 3045 min. the rate of systemic absorption of local anesthetics is dependent upon the total dose and concentration of drug administered, the route of administration, the vascularity of the administration site, and the presence or absence of epinephrine in the anesthetic solution. a dilute concentration of epinephrine (1:200,000 or 5 mcg/ml) usually reduces the rate of absorption and peak plasma concentration of bupivacaine, permitting the use of moderately larger total doses and sometimes prolonging the duration of action. the onset of action with bupivacaine is rapid and anesthesia is long lasting. the duration of anesthesia is significantly longer with bupivacaine than with any other commonly used local anesthetic. it has also been noted that there is a period of analgesia that persists after the return of sensation, during which time the need for strong analgesics is reduced. local anesthetics are bound to plasma proteins in varying degrees. in general, the lower the plasma concentration of drug the higher the percentage of drug bound to plasma proteins. the rate and degree of diffusion are governed by (1) the degree of plasma protein binding, (2) the degree of ionization, and (3) the degree of lipid solubility. fetal/maternal ratios of local anesthetics appear to be inversely related to the degree of plasma protein binding because only the free, unbound drug is available for placental transfer. bupivacaine with a high protein binding capacity (95%) has a low fetal/maternal ratio (0.20.4). depending upon the route of administration, local anesthetics are distributed to some extent to all body tissues, with high concentrations found in highly perfused organs such as the liver, lungs, heart, and brain. pharmacokinetic studies on the plasma profile of bupivacaine after direct intravenous injection suggest a three-compartment open model. the second compartment represents the equilibration of the drug throughout the highly perfused organs such as the brain, myocardium, lungs, kidneys, and liver. the third compartment represents an equilibration of the drug with poorly perfused tissues, such as muscle and fat. the elimination of drug from tissue distribution depends largely upon the ability of binding sites in the circulation to carry it to the liver where it is metabolized. after injection of bupivacaine hydrochloride for caudal, epidural, or peripheral nerve block in man, peak levels of bupivacaine in the blood are reached in 3045 min, followed by a decline to insignificant levels during the next 36 h. various pharmacokinetic parameters of the local anesthetics can be significantly altered by the presence of hepatic or renal disease, addition of epinephrine, factors affecting urinary ph, renal blood flow, the route of drug administration, and the age of the patient. the half-life of bupivacaine in adults is 2.7 h and in neonates 8.1 h. in clinical studies, elderly patients reached the maximal spread of analgesia and maximal motor blockade more rapidly than younger patients. amide-type local anesthetics such as bupivacaine are metabolized primarily in the liver via conjugation with glucuronic acid. patients with hepatic disease, especially those with severe hepatic disease, may be more susceptible to the potential toxicities of the amide-type local anesthetics. when administered in recommended doses and concentrations, bupivacaine hydrochloride does not ordinarily produce irritation or tissue damage and does not cause methemoglobinemia. local anesthetics block the generation and the conduction of nerve impulses, presumably by increasing the threshold for electrical excitation in the nerve, by slowing the propagation of the nerve impulse, and by reducing the rate of rise of the action potential. in general, the progression of anesthesia is related to the diameter, myelination, and conduction velocity of affected nerve fibers. clinically, the order of loss of nerve function is as follows: (1) pain, (2) temperature, (3) touch, (4) proprioception, and (5) skeletal muscle tone. systemic absorption of local anesthetics produces effects on the cardiovascular and central nervous systems (cns). at blood concentrations achieved with normal therapeutic doses, changes in cardiac conduction, however, toxic blood concentrations depress cardiac conduction and excitability, which may lead to atrioventricular block, ventricular arrhythmias, and cardiac arrest, sometimes resulting in fatalities. in addition, myocardial contractility is depressed, and peripheral vasodilation occurs, leading to decreased cardiac output and arterial blood pressure. recent clinical reports and animal research suggest that these cardiovascular changes are more likely to occur after unintended intravascular injection of bupivacaine apparent central stimulation is manifested as restlessness, tremors and shivering progressing to convulsions, followed by depression and coma progressing ultimately to respiratory arrest. however, the local anesthetics have a primary depressant effect on the medulla and on higher centers in most of the studies 0.5% bupivacaine with adrenaline 1:200,000 and 2% lignocaine with adrenaline 1:200,000 were used as local anesthetic agents for third molar surgery as they are roughly equipotent. studies have proved that bupivacaine is superior to lidocaine plus diflunisal in controlling postoperative pain after lower third molar surgery. surprisingly there have been records of patients with no or mild pain at 8 h after the bupivacaine procedure was almost twice as many as after the lidocaine procedure, but is explained by the reason for preference being shorter duration of the anesthetic agent. in an australian study, most of the patients preferred the long-acting anesthetic agent bupivacaine. many authors suggest that it seems reasonable that the combination of a long-acting local anesthetic with a weak analgesic is more efficient in relieving pain than the combination of a rather short-acting local anesthetic and a weak analgesic. bupivacaine is considered to have a therapeutic ratio of 2:0 while lignocaine in combination with adrenaline has a therapeutic ratio of 2:3. however, it has shown that the injection route alters the relative toxicity of local anesthetics. in a study by de jong and bonin in 1980 they found out that the bupivacaine has a greater therapeutic ratio than lignocaine when used for surgical removal of impacted third molars. studies have proved that long-acting bupivacaine can be safely administered for surgical removal of lower third molar and it does have a long period of nalgesia postoperatively compared to lidocaine, but the cardio depressant property of bupivacaine should be kept in mind and should be administered judicially. even studies have also shown that bupivacaine combined with methylprednisolone reduced the postoperative pain and swelling compared with the use of lidocaine and placebo, lidocaine and methylprednisolone, or bupivacaine and placebo. right now clinical trials are going on bupivacaine versus lidocaine anesthesia under university of british columbia 2014 with the idea that the longer duration of anesthesia offered by bupivacaine when administered preoperatively in elective outpatient hand surgeries, will offer more effective postoperative pain control compared to using lidocaine only. it has been found that both bupivacaine and lignocaine have their merits and demerits but beyond any doubt it has been proven by the clinical trials that bupivacaine provides better and prolonged analgesia and anesthesia post operatively during minor surgical procedures done at chair side along with surgical removal of impacted third molars. hence, bupivacaine can be regularly used as the anaesthetic solution along with adrenaline 1:200,000 for surgical removal of impacted third molars provided care being taken regarding the dosage and the cardiodepressant property of bupivacaine. right now, further studies are going on.

one of the most important goal in minor surgical procedures is to achieve proper and sufficient anesthesia and analgesia preoperatively, intraoperatively and in the immediate postoperative period. several local anesthetic agents have been cited in the literature and studied. bupivacaine is one of the most common long-acting anesthetic agents being used for surgical removal of impacted third molars. lignocaine is one of the commonest short-acting anesthetic agents being used for the same procedure. in this review article, the analgesic and anesthetic abilities of the bupivacaine versus lignocaine have been reviewed while surgical removal of impacted third molars.

PMC4439680

pubmed-412

bacterial infections as well as the emergence and spread of antibiotic resistance in human pathogens are serious public health problems in hospital and community settings across the globe. new strategies to prevent and treat bacterial infections are needed, including methods to overcome antibacterial resistance that results from the outer membrane permeability barrier in gram-negative organisms and targeting approaches that afford species- or pathogen-specific therapeutics. almost all bacterial species have a metabolic iron requirement and therefore employ various strategies to acquire this metal ion when colonizing. siderophores are high-affinity fe(iii) chelators that are produced by bacteria under conditions of iron limitation, such as those encountered in the vertebrate host, to scavenge this metal ion from the environment. siderophore producers also express dedicated ferric siderophore import machinery and employ various mechanisms to release siderophore-bound iron following cellular uptake (e.g., reductive and/or hydrolytic release mediated by reductases and/or esterases, respectively). numerous studies support the importance of siderophore-based iron acquisition during bacterial infections. thus, the potential of using siderophores, or targeting siderophore biosynthetic and transport machineries, in therapeutic development continues to attract significant interest. of particular relevance to the advances described herein are prior investigations pertaining to the development of siderophore this strategy has received particular attention for the delivery of antibiotics into gram-negative bacteria because these organisms are inherently less sensitive to many antibiotics used in the clinic as a result of the outer membrane permeability barrier. both native siderophores and synthetic siderophore mimics have been evaluated as platforms for therapeutic development. in the clinic, the native siderophore desferrioxamine b is used for iron-chelation therapy in patients with iron overload. several antibiotic small molecules found in nature called sideromycins provide inspiration for synthetic siderophore the sideromycins are secondary metabolites comprised of a siderophore moiety and a toxic cargo; the siderophore portion targets sideromycins to bacterial strains expressing the appropriate siderophore receptor. antibiotic conjugate produced by a clinical isolate of klebsiella pneumoniae, is an 84-residue antibacterial peptide with a glucosylated enterobactin (ent, figure 1) derivative attached to its c-terminus that exhibits enhanced antibacterial activity against strains expressing the enterobactin receptor fepa. from the standpoints of antibacterial activity and therapeutic potential, studies of synthetic siderophore antibiotic conjugates have provided the community with mixed results, causing some skepticism about the potential of siderophore-based approaches despite the successful utilization of such molecules by nature. structures of enterobactin (1, ent) and a generalized enterobactin cargo conjugate. many of the failures encountered with early and recent studies of siderophore-based antibiotic delivery may be attributed, at least in part, to (i) use of non-native siderophores with relatively low fe(iii) affinities and/or compromised receptor recognition; (ii) modification of antibiotics such that the antibacterial activity is attenuated or lost completely; (iii) bridging the siderophores and antibiotics with problematic linkers, including linkers designed for drug release that are either too stable or too labile, the latter of which promotes premature release; and (iv) antibiotic resistance. nevertheless, the lessons of many unsuccessful studies highlight the complexity of siderophore-based therapeutic development and provide a foundation for inventing improved next-generation approaches. many of the issues described above may be overcome by careful molecular design and biological evaluation. in particular, the selection of appropriate native siderophore platforms and modification of these platforms in ways that do not compromise iron binding or receptor recognition, installation of an antibacterial cargo in such a manner that antibacterial activity is retained, and the development and application of assays that afford insight into the fate of siderophore antibiotic conjugates are critical to the overall success of this approach. along such lines, a recent and insightful study by pfizer addressed complications associated with using relatively low-molecular-weight siderophore mimics in vivo. their results indicate that competition between the siderophore-conjugated monobactam mb-1 and native siderophores resulted in poor in vivo efficacy against pseudomonas aeruginosa and provide support for designing and evaluating siderophore one recent and successful example based on a native siderophore platform is a mycobactin artemisinin conjugate that exhibits enhanced antibacterial activity against mycobacterium tuberculosis compared to unmodified artemisinin. enterobactin (ent, figure 1) is a triscatecholate siderophore biosynthesized by enteric bacteria and used for iron acquisition in the vertebrate host. motivated by the importance of ent in the host/microbe interaction as well as the decades of investigations pertaining to its (bio)synthesis, coordination chemistry, and biology, in prior work we reported a synthetic route to monofunctionalized ent platforms. moreover, we established that the native ent platform, when monofunctionalized at the c5 position of one catecholate ring (figure 1), affords delivery of nontoxic small-molecule cargo across the outer membrane of gram-negative organisms that express ent uptake machinery (e.g., fepabcdg of escherichia coli). as described herein, this proof-of-concept study motivated us to demonstrate that ent effectively delivers antibacterial cargo to organisms that utilize ent for iron acquisition, thereby providing antibiotic targeting to specific sub-populations and a means to address antibiotic resistance that results from the gram-negative outer membrane permeability barrier. in this work, we present the syntheses and characterization of siderophore antibiotic conjugates based on the native ent platform that harbor the clinically relevant -lactam antibiotics ampicillin (amp) and amoxicillin (amx). these antibiotics block cell wall biosynthesis by inhibiting transpeptidases, also named penicillin binding proteins (pbps), located in the periplasm of e. coli. we report that the ent--lactam conjugates exhibit significantly enhanced antibacterial activity (up to 1000-fold) against pathogenic e. coli and provide more rapid cell-killing than the parent -lactams as a result of ent-mediated delivery to the periplasm. moreover, in proof-of-concept studies for species-specific killing, these conjugates selectively kill e. coli in the presence of staphylococcus aureus, a gram-positive organism that is more susceptible to the parent -lactams. these studies support the notion that native siderophore platforms provide an effective means to target molecular cargo to siderophore-utilizing organisms and to hijack siderophore uptake machinery to deliver cargos, including antibiotics, across the outer membrane permeability barrier of gram-negative microbes. dimethylformamide (dmf) and dichloromethane (ch2cl2) were obtained from a vac solvent purification system (vacuum atmospheres). anhydrous dimethyl sulfoxide (dmso) was purchased from sigma-aldrich and used as received. l-ent 1, the d-enantiomer of benzyl-protected ent-co2h 2, and the benzyl-protected ent-peg3-n33 were synthesized according to previously reported procedures. 11-azido-3,6,9-trioxaundecan-1-amine was purchased from fluka. all other chemicals and solvents were purchased from sigma-aldrich or alfa aesar in the highest available purity and used as received. benzyl-protected ent-azide 3 (80 mg, 55 mol) and pentamethylbenzene (pmb, 147 mg, 990 mol) were dissolved in 5 ml of anhydrous ch2cl2 to give a light yellow solution. this solution was cooled to 78 c in an acetone/dry ice bath under n2, and bcl3 (660 l of 1 m solution in ch2cl2, 660 mol) was added slowly along the flask wall. after the solution was stirred for 1.5 h, dipea (300 l, 1.73 mmol) was added to the flask, followed by meoh (2 ml) to quench the reaction. the reaction was then warmed to room temperature, and the solvents were removed under reduced pressure. the resulting white solid was dissolved in 5:3 meoh/1,4-dioxane and purified by preparative hplc (33% b for 5 min and 3360% b over 20 min, 10 ml/min). the product eluted at 17 min and was lyophilized to yield compound 4 as white solid (13.9 mg, 28%). h nmr (dmso-d6, 500 mhz): 3.353.57 (16h, m), 4.384.41 (3h, m), 4.634.69 (3h, m), 4.894.96 (3h, m), 6.74 (2h, dd, j=7.5, 8.0 hz), 6.97 (2h, d, j=7.5 hz), 7.35 (2h, d, j= 8.0 hz), 7.46 (1h, s), 7.94 (1h, s), 8.338.35 (1h, m), 9.12 (2h, d, j=6.0 hz), 9.29 (1h, d, j=6.0 hz), 9.44 (2h, bs), 9.76 (1h, bs), 11.6 (2h, bs), 11.9 (1h, bs). c nmr (cdcl3, 125 mhz): 50.1, 51.5, 63.6, 69.1, 69.4, 69.8, 69.8, 69.9, 69.9, 115.3, 115.4, 115.4, 117.7, 118.5, 118.7, 119.4, 125.2, 145.9, 146.3, 148.7, 148.7, 150.8, 166.0, 168.4, 169.1, 169.6, 169.7. ir (kbr disk, cm): 3389, 2954, 2928, 2868, 2111, 1754, 1645, 1589, 1535, 1460, 1384, 1329, 1266, 1176, 1132, 1074, 992, 846. 11-azido-3,6,9-trioxaundecan-1-amine (36 l, 181 mol) and d-bn6ent-cooh (2, 177 mg, 142 mol) were dissolved in 5 ml of dry ch2cl2. pyaop (147 mg, 283 mol) and dipea (98.5 l, 568 mol) were added to give a light yellow solution. the reaction was stirred for 4 h at room temperature and concentrated, and the crude product was purified by preparative tlc (50% etoac/ch2cl2) to afford 5 as white foam (159 mg, 77%). h nmr (dmso-d6, 500 mhz): 3.33 (2h, j=5.2 hz), 3.623.69 (14h, m), 4.024.06 (3h, m), 4.154.18 (3h, m), 4.914.94 (3h, m), 5.045.21 (12h, m), 6.96 (1h, s), 7.117.45 (36h, m), 7.657.67 (2h, m), 7.857.85 (1h, m), 7.977.97 (1h, m), 8.508.54 (3h, m). c nmr (cdcl3, 125 mhz): 25.6, 29.5, 38.8, 40.0, 45.3, 51.3, 51.4, 63.9, 64.1, 69.8, 39.8, 70.0, 70.3, 70.4, 70.4, 71.2, 71.2, 76.3, 76.3, 116.8, 117.5, 120.4, 123.0, 124.3, 125.4, 126.1, 126.2, 127.6, 127.6, 127.9, 128.2, 128.3, 128.4, 128.4, 128.5, 128.5, 128.6, 128.6, 128.8, 128.8, 128.9, 129.0, 130.1, 135.4, 135.7, 135.9, 136.0, 136.1, 146.8, 146.9, 149.1, 151.6, 151.8, 164.3, 164.9, 164.9, 165.8, 168.9, 169.0, 169.1, 176.2. ir (kbr disk, cm): 3357, 3062, 3032, 2958, 2923, 2859, 2104, 1751, 1551, 1576, 1515, 1455, 1375, 1345, 1299, 1264, 1204, 1126, 1082, 1040, 1018, 957, 915, 854, 811. hrms (esi): compound 6 was synthesized from 5 (153 mg, 105 mol) following the same procedure as for compound 4. the crude reaction was purified by preparative hplc (33% b for 5 min and 3360% b over 20 min, 10 ml/min). the product eluted at 17.0 min and was lyophilized to yield compound 6 as white solid (31 mg, 33%). h nmr (cdcl3, 500 mhz): 3.343.56 (16h, m), 4.394.41 (3h, m), 4.614.66 (3h, m), 4.884.94 (3h, m), 6.74 (2h, dd, j=7.8, 7.8 hz), 6.96 (2h, d, j=7.8 hz), 7.33 (2h, d, j= 7.8 hz), 7.44 (1h, s), 7.91 (1h, s), 8.318.33 (1h, m), 9.129.13 (2h, m), 9.279.28 (1h, m), 9.50 (2h, bs), 9.84 (1h, bs), 11.6 (2h, bs), 11.9 (1h, bs). c nmr (cdcl3, 125 mhz): 50.1, 51.5, 63.6, 69.1, 69.4, 69.8, 69.8, 69.9, 69.9, 115.3, 115.4, 115.4, 117.7, 118.5, 118.7, 119.4, 125.2, 145.9, 146.3, 148.7, 148.7, 150.8, 166.0, 168.4, 169.1, 169.6, 169.7. ir (kbr disk, cm): 3390, 2958, 2925, 2863, 2110, 1754, 1645, 1589, 1535, 1460, 1384, 1342, 1262, 1176, 1117, 1074, 841, 800. hrms (esi): [m+na]m/z calcd 936.2506, found 936.2512. 5-hexynoic acid (113 l, 1.00 mmol) and thionyl chloride (1.00 ml, 13.8 mmol) were combined and refluxed for 1 h. the reaction was cooled to room temperature and concentrated under reduced pressure, and the resulting crude acyl chloride was dissolved in acetone (0.5 ml) and carried on to the next step without purification. ampicillin sodium salt (186 mg, 0.500 mmol) was dissolved in a solution of nahco3 (210 mg, 2.5 mmol) in 4:1 water/acetone (2.5 ml) and cooled on ice, to which the acyl chloride was added slowly with stirring. the reaction was subsequently warmed to room temperature and stirred for 1 h. water (3 ml) was added to the reaction, and the aqueous phase was washed with etoac (2 10 ml), acidified to ph 2 by addition of hcl, and extracted with etoac (20 ml). the resulting organic phase was washed with cold water (2 5 ml), dried over na2so4, and concentrated under reduced pressure. the crude reaction was triturated with hexanes, which afforded a yellow solid (180 mg, 77%). h nmr (dmso-d6, 500 mhz): 1.41 (3h, s), 1.55 (3h, s), 1.641.69 (2h, m), 2.132.16 (2h, m), 2.292.32 (2h, m), 2.772.78 (1h, m), 4.20 (1h, s), 5.39 (1h, d, j=4.0 hz), 5.52 (1h, dd, j=4.0, 8.0 hz), 5.70 (1h, d, j=8.0 hz), 7.257.43 (5h, m), 8.57 (1h, d, j=8.0 hz), 9.11 (1h, d, j=8.0 hz). c nmr (dmso-d6, 125 mhz): 17.4, 24.4, 26.6, 30.4, 33.8, 55.5, 58.1, 63.7, 67.3, 70.3, 71.5, 84.2, 127.2, 127.6, 128.2, 138.2, 169.0, 170.2, 171.5, 173.5. ir (kbr disk, cm): 3297, 3058, 3023, 2970, 2937, 2863, 2626, 2526, 2120, 1780, 1688, 1518,1455, 1437, 1390, 1373, 1324, 1295, 1208, 1139, 1027, 1001, 843. compound 8 was synthesized as described for compound 7 except that amoxicillin (760 mg, 2.1 mmol) was used instead of ampicillin sodium salt. compound 8 was obtained as light yellow solid (533 mg, 56%) after trituration and employed without further purification. h nmr (dmso-d6, 500 mhz): 1.42 (3h, s), 1.56 (3h, s), 1.631.68 (2h, m), 2.122.15 (2h, m), 2.252.29 (2h, m), 2.762.78 (1h, m), 4.19 (1h, s), 5.39 (1h, d, j=4.0 hz), 5.515.54 (2h, m), 6.69 (2h, d, j=8.5 hz), 7.19 (2h, d, j=8.5 hz), 8.41 (1h, d, j=8.0 hz), 8.93 (1h, d, j=8.0 hz), 9.40 (1h, bs). c nmr (dmso-d6, 125 mhz): 17.6, 24.5, 26.8, 30.4, 34.0, 55.4, 58.2, 63.9, 71.6, 71.7, 84.4, 115.1, 128.4, 128.7, 128.8, 157.0, 169.2, 170.9, 171.7, 173.8. ir (kbr disk, cm): 3356, 3294, 3045, 2970, 2928, 1770, 1738, 1650, 1615, 1515, 1457, 1373, 1208, 1009, 945, 839, 815. hrms (esi): [m+na]m/z calcd 460.1537, found 460.1534. compound 7 (60 mg, 0.13 mmol) was dissolved in 1:1 h2o/mecn (5 ml), and tfa was added to a final concentration of 1%. the solution was incubated at 37 c for 24 h and purified by preparative hplc (2050% b over 25 min, 10 ml/min), which afforded a white powder (12 mg, 25%). the white powder is a diastereomeric mixture of products, and no further separation was performed. h nmr (dmso-d6, 500 mhz): (mixture of two diastereomers) 1.221.23 (3h, pair of s), 1.541.58 (3h, pair of s), 1.621.68 (2h, m), 2.122.14 (2h, m), 2.282.30 (2h, m), 2.77 (1h, s), 3.183.24 (0.5h, m), 3.303.36 (0.5h, m), 3.443.49 (0.5h, m), 3.553.60 (0.5h, m), 3.92 (0.5h, s), 4.01 (0.5h, s), 4.67 (0.5h, dd, j=6.7, 6.5 hz), 4.78 (0.5h, dd, j=5.2, 5.2 hz), 5.455.48 (1h, m), 7.277.40 (5h, m), 8.508.61 (2h, m). c nmr (dmso-d6, 125 mhz): (mixture of two diastereomers) 18.1, 25.0, 27.6, 28.1, 28.4, 29.5, 34.4, 42.2, 56.8, 56.8, 72.1, 72.2, 84.8, 127.9, 128.2, 128.9, 139.2, 139.4, 171.2, 171.4, 172.0. ir (kbr disk, cm): 3297, 3071, 3041, 2967, 2938, 2535, 2124, 1734, 1653, 1527, 1456, 1427, 1375, 1299, 1199, 1137, 1070, 1027, 836. hrms (esi): compound 10 was synthesized as described for compound 9 except that compound 8 was used instead of 7 (60 mg, 0.13 mmol). the product was purified by preparative hplc (2050% b over 25 min, 10 ml/min), and obtained as white powder (14.5 mg, 24%). h nmr (dmso-d6, 500 mhz): (mixture of two diastereomers) 1.26 (3h, s), 1.551.59 (3h, pair of s), 1.611.67 (2h, m), 2.122.14 (2h, m), 2.242.27 (2h, m), 2.77 (1h, s), 3.203.26 (0.5h, m), 3.343.39 (0.5h, m), 3.423.47 (0.5h, m), 3.563.60 (0.5h, m), 4.03 (0.5h, s), 4.12 (0.5h, s), 4.68 (0.5h, dd, j=6.5, 6.5 hz), 4.79 (0.5h, dd, j=5.5, 5.5 hz), 5.285.31 (1h, m), 6.69 (2h, d, j=8.5 hz), 7.17 (2h, d, j=8.5 hz), 8.368.50 (3h, m). c nmr (dmso-d6, 125 mhz): (mixture of two diastereomers) 17.5, 24.3, 26.9, 27.5, 27.7, 28.8, 33.8, 41.4, 55.7, 55.8, 71.4, 71.6, 84.2, 114.8, 115.0, 128.5, 128.7, 128.8, 156.9, 158.3, 158.6, 171.2, 171.3. ir (kbr disk, cm): 3301, 3071, 3028, 2973, 2928, 2548, 2111, 1737, 1662, 1606, 1593, 1515, 1435, 1377, 1197, 1139, 837. hrms (esi): ampicillin-alkyne 7 (120 l of an 80 mm solution in dmso, 9.6 mol) and ent-peg3-n34 (250 l of a 13 mm solution in 1,4-dioxane, 3.3 mol) were combined, and 400 l of dmso was added. cuso4 (100 l of a 90 mm solution in water, 9.0 mol) and tris[(1-benzyl-1h-1,2,3-triazol-4-yl)methyl]amine (tbta, 200 l of a 50 mm solution in dmso, 10 mol) were combined, and 100 l of dmso was added to give a blue-green solution, to which naasc (400 l of a 90 mm solution in water, 36.0 mol) was added. this solution became light yellow and was immediately added to the alkyne/azide solution. the reaction was shaken on a benchtop rotator for 2 h at room temperature, diluted by 3- to 4-fold with 1:1 mecn/water, centrifuged (13,000 rpm 10 min, 4 c), and purified by semi-preparative hplc (20% b for 5 min and 20%50% b over 11 min, 4 ml/min; 0.005% tfa was used in the solvent system to prevent decomposition of the -lactam). the hplc fractions containing 11 were collected manually and flash frozen in liquid n2 immediately after collection to prevent -lactam decomposition. h nmr (dmso-d6, 500 mhz): 1.40 (3h, s), 1.54 (3h, s), 1.781.81 (2h, m), 2.27 (2h, t, j=6.8 hz), 2.58 (2h, t, j=6.5 hz), 3.48 (12h, m), 3.76 (2h, s), 4.19 (1h, s), 4.384.44 (5h, m), 4.644.66 (3h, m), 4.914.92 (3h, m), 5.39 (1h, d, j=3.5 hz), 5.515.52 (1h, m), 5.72 (1h, d, j=7.5 hz), 6.74 (2h, dd, j=7.8, 7.8 hz), 6.96 (2h, d, j=7.5 hz), 7.267.35 (5h, m), 7.427.45 (3h, m), 7.81 (1h, s), 7.92 (1h, s), 8.33 (1h, s), 8.55 (1h, d, j=7.5 hz), 9.12 (3h, d, j=7.0 hz), 9.29 (1h, d, j=6.5 hz), 9.42 (2h, bs), 9.74 (1h, s), 11.6 (2h, s), 11.9 (1h, bs), 13.35 (1h, bs). hrms (esi): [m+na]m/z calcd 1379.4021, found 1379.4046. compound 12 was synthesized as described for 11 except that compound 8 was used instead of compound 7. h nmr (dmso-d6, 500 mhz): 1.40 (3h, s), 1.54 (3h, s), 1.781.80 (2h, m), 2.23 (2h, t, j=6.5 hz), 2.57 (2h, t, j=6.5 hz), 3.47 (12h, m), 3.76 (2h, bs), 4.18 (1h, s), 4.394.43 (5h, m), 4.634.65 (3h, m), 4.90 (3h, bs), 5.38 (1h, s), 5.525.56 (2h, m), 6.68 (2h, d, j=8.5 hz), 6.73 (2h, dd, j=7.8, 7.8 hz), 6.96 (2h, d, j=7.5), 7.19 (2h, d, j=8.5 hz), 7.33 (2h, d, j= 7.5 hz), 7.44 (1h, s), 7.80 (1h, s), 7.92 (1h, s), 8.338.39 (2h, m), 8.94 (1h, d, j=8.0 hz), 9.119.12 (2h, m), 9.29 (1h, bs), 9.389.43 (3h, m), 9.75 (1h, s), 11.6 (2h, bs), 11.9 (1h, bs). compound 13 was synthesized as described for 11 except that compound 6 was used instead of compound 4. hrms (esi): [m+na]m/z calcd 1379.4021, found 1379.4022. compound 14 was synthesized as described for 12 except that compound 6 was used instead of compound 4. hrms (esi): [m+na]m/z calcd 1395.3970, found 1395.3995. compound 15 was synthesized as described for 11 except that compound 9 was used instead of compound 7. hrms (esi): [m+h]m/z calcd 1331.4409, found 1331.4389. compound 16 was synthesized as described for 12 except that compound 10 was used instead of compound 8. hrms (esi): [m+na]m/z calcd 1369.4177, found 1369.4191. general microbiology materials and methods, including details of ent-amp/amx stock solution preparation and storage, are provided as supporting information. overnight cultures of the bacterial strains (table s1) were prepared in 15-ml polypropylene tubes by inoculating 5 ml of lb media with the appropriate freezer stock. the overnight cultures were incubated at 37 c for 1618 h in a tabletop incubator shaker set at 150 rpm and housing a beaker of water. each overnight culture was diluted 1:100 into 5 ml of fresh lb media containing 200 m 2,2-dipyridyl (dp) and incubated at 37 c with shaking at 150 rpm until od600 reached 0.6. g/l) with or without 200 m dp to achieve an od600 value of 0.001. a 90-l aliquot of the diluted culture was combined with a 10-l aliquot of a 10 solution of the antibiotic or ent-antibiotic conjugate in a 96-well plate, and the covered plate was wrapped in parafilm and incubated at 30 c with shaking at 150 rpm for 19 h in a tabletop incubator housing a beaker of water. bacterial growth was determined by measuring od600 (end point analysis) using a biotek synergy ht plate reader. each well condition was prepared in duplicate and at least three independent replicates using two different synthetic batches of each conjugate were conducted on different days. the resulting mean od600 values are reported, and the error bars are the standard error of the mean (sem) obtained from the independent replicates. these assays were performed with e. coli atcc 35218 and k. pneumoniae atcc 13883 following the general procedure except that sulbactam (sb) or potassium clavulanate (pc) were mixed with ampicillin or amoxicillin and the ent-amp or ent-amx conjugates, respectively. the molar ratios of the inhibitor/-lactam mixtures were sulbactam/amp or ent-amp, 1.5:1, and potassium clavulanate/amx or ent-amx, 0.9:1. these assays were performed with e. coli k-12 and cft073 following the general procedure except that varying concentrations (1100 m) of synthetic l-ent were mixed with ent-amp/amx. this assay is based on a published protocol and was conducted with e. coli cft073. each overnight culture was serially diluted into m9 minimal medium to provide 1010 cfu/ml. lipocalin 2 was diluted in pbs to a concentration of 10 m upon arrival, aliquoted, and stored at 20 c until use. a 90-l aliquot of the diluted culture was added to each well of a 96-well plate that contained varying concentrations of lipocalin 2, ent-amp, and ent, and the final volume was adjusted to 100 l with sterile pbs. the 96-well plate was incubated at 37 c for 24 h in a tabletop incubator set at 150 rpm, and bacterial growth was determined by measuring od600 using a plate reader. each well condition was repeated at least three times independently on different days and with different batches of lipocalin 2. the resulting mean od600 is reported, and the error bars are the sem. a 5-ml overnight culture of e. coli k-12 or cft073 was grown in lb (vide supra) and diluted 1:100 into 5 ml of fresh lb media containing 200 m dp, and this culture was incubated at 37 c with shaking at 150 rpm in a tabletop incubator housing a beaker of water until od600 reached 0.3. the culture was centrifuged (3000 rpm 10 min, rt), and the resulting pellet was washed twice by resuspension in 50% mhb and centrifugation (3000 rpm 10 min, rt). the resulting pellet was resuspended in 50% mhb with or without dp, and the od600 was adjusted to 0.3. a 90-l aliquot of the resulting culture was mixed with a 10-l aliquot of a 10 solution of amp/amx or the ent-amp/amx in a 96-well plate, which was covered, wrapped in parafilm, and incubated at 37 c with shaking at 150 rpm. the od600 values were recorded at t=0, 1, 2, and 3 h by using a plate reader. in a parallel experiment, a 10-l aliquot of the culture was taken at t=0, 1, 2, and 3 h, serially diluted by using sterile phosphate-buffered saline (pbs), and plated on lb-agar plates for colony counting (cfu/ml). the resulting mean od600 or cfu/ml is reported, and the error bars are the sem. a 5 ml overnight culture of each bacterial strain was grown in lb, diluted 1:100 into 5 ml of fresh lb media containing 200 m dp, and incubated at 37 c with shaking at 150 rpm in a tabletop incubator housing a beaker of water until od600 reached 0.6. each mid-log-phase culture was diluted to 10 cfu/ml in 50% mhb with or without 200 m dp. for experiments requiring a mixture of two species, a 1:1 mixture was prepared (10 cfu/ml for each strain) in 50% mhb with or without 200 m dp from the mid-log-phase cultures. to confirm cfu/ml of each culture, the single- and double-species cultures were serially diluted by using sterile pbs, and aliquots were plated on a chrom-uti plate (starter-culture plate). for each cell-killing experiment, a 90-l aliquot of each culture was combined with a 10-l aliquot of a 10 m solution of the antibiotic or ent-antibiotic conjugate in a 96-well plate, which was covered, wrapped in parafilm, and incubated at 30 c with shaking at 150 rpm for 19 h. bacterial growth was assayed both by measuring od600 using the plate reader and by plating on chrom-uti plates after serial dilution (assay plate). each well condition was repeated at least three times independently on different days. the resulting mean od600 is reported, and the error bars are the sem. we aimed to harness our enterobactin-mediated cargo delivery strategy to enable the transport of toxic cargo across the outer membrane of e. coli. to address this goal, we linked the -lactam antibiotics ampicillin (amp) and amoxicillin (amx) to a monofunctionalized ent scaffold where ent is derivatized at the c5 position of one catechol ring via a flexible and stable peg3 linker. we selected amp and amx as antibacterial cargo for several reasons: these molecules are commercially available and amenable to synthetic modification, retain antibacterial activity when appropriately modified, possess periplasmic targets in gram-negative bacteria and must cross the outer membrane to be active against these species, and have relatively low molecular weights. we selected low-molecular-weight antibiotics because our prior studies of ent-mediated cargo transport indicated that the ent transport machinery of e. coli k-12 imports ent-cargo conjugates harboring relatively small cargos (e.g., cyclohexane, naphthalene, phenylmethylbenzene) to the cytosol readily, whereas large cargos (e.g., vancomycin) are not transported to the cytosol. moreover, in prior studies, various -lactams including amp and amx have been linked to simple catechols and more complex catechol-containing siderophores or mimics thereof, which provides the opportunity to compare the outcomes obtained for different siderophore-inspired design strategies. in scheme 1, we present the syntheses of ent-amp 11 and ent-amx 12, which feature installation of alkyne-modified -lactam warheads onto ent-azide 4 via copper-catalyzed azide-alkyne cycloaddition (hereafter click reaction) in the final step. the catechol moieties of benzyl-protected ent-azide 3 were deprotected by using bcl3 at 78 c to achieve ent-azide 4 as a white powder in 28% yield following purification by reverse-phase preparative hplc. catalytic hydrogenation using hydrogen gas and a pd/c catalyst is typically employed to deprotect ent catechols; however, we observed that amp/amx decompose under these conditions and poison the pd/c catalyst. moreover, deprotection of the enterobactin catechols prior to installing the -lactams requires preservation of the azide moiety, and we therefore employed bcl3 for this reaction. initial attempts at assembling ent-amp/amx using standard conditions for the copper-catalyzed click reactions with ent-azide 4 and the alkyne-modified -lactams 7/8, prepared by thionyl chloride coupling of 5-hexynoic acid to the amino group of amp/amx, failed because of copper-mediated -lactam decomposition. this problem was overcome by including the metal-ion chelator tris[(1-benzyl-1h-1,2,3-triazol-4-yl)methyl]amine (tbta) in the click reactions, and ent-amp and ent-amx were obtained as white powders in high purity and yields of 66% and 76%, respectively, following semi-preparative hplc purification. it was necessary to perform hplc purification with eluents containing only 0.005% tfa to prevent decomposition of the acid-sensitive -lactam moieties. this synthetic route was likewise employed to prepare the d-enantiomers of ent-amp/amx 13 and 14 (scheme 1). deprotected ent-azide 4 enables alkyne-functionalized molecules to be covalently linked to ent via a click reaction, including molecules that are incompatible with reaction conditions required to deprotect the ent catechols. moreover, the deprotected ent-azide may be employed to append ent to surfaces, other materials, or biomolecules harboring alkyne groups. indeed, very few examples employing the copper(i)-catalyzed click reaction with fused -lactams are reported in the literature, and it is likely that this paucity stems from the fact that -lactams are incompatible with standard conditions for copper-catalyzed azide-alkyne cycloaddition. the conditions defined in this work employing tbta allow for copper-catalyzed triazole formation and preserve the -lactam warhead. in the absence of fe(iii), ent-amp/amx exhibit an absorption band centered at 316 nm resulting from catecholate absorption and the solutions are colorless (meoh, rt). addition of 1.0 equiv of fe(iii) to methanolic solutions of ent-amp and ent-amx causes the solution to immediately change from colorless to purple-red, and a broad absorption feature in the 400700 nm range appears (figure s1), confirming that both ent-amp/amx readily chelate fe(iii). to ascertain whether ent-amp/amx provide antibacterial activity against e. coli, including pathogenic strains, we preformed antimicrobial activity assays using six strains (table s1, figures 2, s2s7). e. coli atcc 25922 is a laboratory susceptibility test strain originally obtained as a clinical isolate. coli uti89 and cft073 are both pathogens of the human urinary tract (upec).e. coli atcc 35401 (serotype o78:h11) is an enterotoxigenic (etec) strain that was isolated from human feces. e. coli atcc 43895 (serotype o157:h7) is an enterohemorrhagic (ehec) strain that was isolated from raw hamburger meat implicated in a hemorrhagic colitis outbreak. both etec and ehec strains produce virulence factors and toxins and cause diarrhea in humans. all e. coli strains biosynthesize ent for iron acquisition and express the ent receptor fepa. some e. coli strains employed in this work also have the capacity to produce and utilize salmochelins, c-glucoyslated ent derivatives. these molecules are produced by salmonella spp. and pathogenic e. coli strains for iron acquisition. the iroa gene cluster (irobcden) encodes proteins required for the biosynthesis and transport of salmochelins, and iron is the outer membrane receptor for salmochelins encoded by the iroa cluster. studies with salmonella indicate that iron has the ability to transport ent as well as its glucosylated forms. of the strains considered in this work, e. coli cft073 and uti89 harbor the iroa gene cluster. e. coli h9049 does not produce salmochelins, and a blast search using available e. coli genomes reveals that the e. coli 43985 genome does not contain the iroa cluster. the genome for e. coli atcc 25922 is unpublished; however, this strain is reported to be sensitive to lipocalin-2 (vide infra), which suggests that its genome does not encode the iroa cluster. lastly, it should be noted that e. coli cft073 is celebrated for having redundant iron import machineries, and this strain also harbors the iha gene, which encodes the outer membrane ent receptor iha that is distinct from fepa. antibacterial activity of ent-amp/amx against various e. coli strains that include human pathogens. all assays were performed in 50% mhb medium supplemented with 200 m dp to provide iron-limiting conditions (mean sem, n 3). the data for assays performed in the absence of dp are presented in figures s2s7. we performed antibacterial activity assays using a 10-fold dilution series to compare the abilities of ent-amp/amx and unmodified amp/amx to kill e. coli (figures 2, s2s7). these assays were conducted in 50% mhb and in the absence or presence of 200 m dp. the latter growth conditions provide iron limitation and result in expression of the ent uptake machinery fepabcdg. amp/amx exhibit minimum inhibitory concentration (mic) values of 10 m against these e. coli strains regardless of the presence of dp in the growth medium (figures 2, s2s7). all six e. coli strains are more susceptible to ent-amp/amx than amp/amx under conditions of iron limitation (figure 2). based on the 10-fold dilution series, ent-amp/amx are 100-fold more potent against e. coli 25922, uti89, and h9049 under iron-limiting conditions. although the mic values for 35401 and 43895 are only ca. 10-fold higher than for amp/amx in this assay, a significant reduction in growth is observed at 100 nm ent-amp/amx, whereas this concentration of amp/amx affords no growth inhibition. the enhanced sensitivity of e. coli cft073 to ent-amp/amx is remarkable. this strain exhibits the greatest sensitivity to ent-amp/amx, providing a 1000-fold decreased mic value (10 nm), and growth inhibition in the presence of 1 nm of the conjugate. moreover, in the absence of dp, e. coli cft073 exhibits the greatest susceptibility to ent-amp/amx (figures s2s7). a noteworthy characteristic of cft073 is its multiple mechanisms for iron acquisition, and we hypothesize that the presence of multiple receptors that recognize and transport ent (fepa, iron, iha) contributes to this enhanced sensitivity. cft073 and uti89 both express iron, which may indicate that iha is responsible for the enhanced susceptibility of cft073; however, we can not rule out the possibility that the conserved receptors function differently or exhibit different expression levels depending on the strain. moreover, other unappreciated mechanisms may contribute to the potent bactericidal action exhibited by ent-amp/amx against cft073 and other strains. to gain further insight into the mechanism of ent-amp/amx antibacterial action, we performed a series of experiments with the standard laboratory strain e. coli k-12. we employed the same 10-fold dilution series to compare the activities of ent-amp/amx and amp/amx against k-12. in the absence of dp, ent-amp/amx and amp/amx exhibit comparable mic values, with complete killing observed at 10 m. at lower concentrations, ent-amp/amx exhibit slightly greater antibacterial activity than unmodified amp/amx (figure 3a). this phenomenon is most evident at a conjugate/drug concentration of 1 m, where ent-amp/amx inhibit e. coli k-12 growth to varying degrees and amp/amx do not affect bacterial growth. under conditions of iron limitation, a 100-fold reduction in mic value (10 m to 100 nm) for ent-amp/amx is observed, and ca. 50% growth inhibition occurs at 10 nm of each conjugate (figure 3b). these trends are comparable to those observed for e. coli atcc 25922, h9049, and uti89 (figures 2, s2, s3, and s5). when the antibacterial activity assay was performed using a 1:1 ratio of native l-ent and amp/amx, no reduction of amp/amx mic values was observed (figure 3c), which suggests the conjugation between ent and the -lactams is required for the enhanced bactericidal action. moreover, treatment of e. coli k-12 with the iron-bound forms of ent-amp/amx, obtained by pre-incubating each conjugate with 1 equiv of ferric chloride, afforded the same mic values as observed for apo ent-amp/amx. these results indicate that the enhanced antibacterial activity does not result from iron chelation in the growth media (figure 3d). in total, the data obtained for e. coli k-12 as well as the six other e. coli strains demonstrate that the antibacterial activity of ent-amp/amx against e. coli k-12 is enhanced under conditions of iron limitation and support a model of ent-mediated delivery of antibacterial cargo to the e. coli periplasm. antibacterial activity of ent-amp/amx against wild-type and mutant e. coli k-12. (a, b) growth inhibition of e. coli k-12 by amp/amx and ent-amp/amx in the absence (a) and presence (b) of dp. (c) growth inhibition of e. coli k-12 treated with a 1:1 molar ratio of ent/amp and ent/amx. (d) growth inhibition of e. coli k-12 treated with ferric ent-amp/amx. (e g) growth inhibition of fepa- (e), fepc- (f), and fes- (g) by amp/amx and ent-amp/amx. (h, i) growth of e. coli k-12 in the presence of 1 m ent-amp/amx (conjugate) and mixtures of ent-amp/amx (1 m) and 1, 5, or 20 equiv of exogenous ent in the absence (h) and presence (i) of dp. the** indicates od600<0.01. all assays were performed in 50% mhb medium with or without 200 m dp (see panels) (mean sem, the data for additional assays performed in the absence of dp are presented in figure s8. to evaluate transport of ent-amp/amx into e. coli, we investigated the antimicrobial activity of ent-amp/amx and amp/amx against three single-gene knockout e. coli k-12 strains obtained from the keio collection, fepa-, fepc-, and fes- (figures 3e we selected these mutants to ascertain how components of the enterobactin transport and processing machinery contribute to ent-amp/amx antibacterial activity. e. colifepa- lacks the outer membrane ent receptor fepa that allows periplasmic delivery, fepc- lacks the atpase component of the inner membrane ent permease that transports ent into the cytosol, and e. colifes- lacks the cytoplasmic esterase fes responsible for hydrolysis of the ent macrolactone for iron release. on the basis of our studies with wild-type k-12 and other e. coli strains, we hypothesized that the activity of ent-amp/amx would be attenuated for the fepa- mutant. moreover, we questioned whether loss of fepc or fes would modulate the antimicrobial activity. overnight cultures of fepa- and fepc- reached od600 values (0.15) similar to that observed for wild-type k-12 when grown in 50% mhb supplemented with 200 m dp. in contrast, the fes- strain exhibited a severe growth defect under these conditions (od600 0.04). treatment of fepa- with ent-amp/amx afforded the same mic values as for amp/amx (figure 3e) and hence a 100-fold reduction in activity as compared to wild-type k-12. iron deprivation is deleterious to e. coli, and we contend that the growth inhibition observed at 10 m ent-amp/amx results from iron starvation rather than an antibacterial activity of the amp/amx cargo. analysis of 50% mhb by inductively coupled plasma optical emission spectroscopy (icp-oes) revealed a total iron concentration of ca. 4 m (table s2), which can be compared to 10 m of a high-affinity extracellular iron chelator. indeed, we previously observed similar growth inhibition of fepa- with 10 m of an ent-vancomycin conjugate (extracellular iron chelation) and also 10 m d-ent (iron chelator that can not be used for iron acquisition) under these growth conditions. these data confirm that fepa is essential for the potent antibacterial activity of ent-amp/amx against e. coli k-12. in contrast to fepa-, the growth of fepc- and fes- was completely inhibited with 100 nm ent-amp/amx under iron-limiting conditions (figure 3f, g), comparable to what was observed for the wild-type strain. the targets of -lactam antibiotics are penicillin binding proteins (pbps), which are located in the periplasm of gram-negative bacteria. after crossing the outer membrane through fepa, ent-amp/amx enter the periplasm where covalent capture by the pbps presumably occurs. thus, it is reasonable that the downstream ent transport and processing steps involving fepcdg and fes do not affect the antimicrobial activity of the conjugates if they are trapped in the periplasm as a result of pbp binding. although it is possible that the ent uptake machinery (e.g., periplasmic binding protein fepb) competes with the pbps for ent-amp/amx, no improved antibacterial activities were observed for the fepc- and fes- mutants compared to wild-type k-12. this observation indicates that ent-amp/amx bind to pbps and are trapped in the periplasm. it should be noted that the fepb- mutant, which lacks the periplasmic binding protein, was also considered in this work; however, this strain exhibited a severe growth defect and afforded inconsistent results. to probe interaction between fepa and ent-amp/amx, we performed growth inhibition assays employing mixtures of ent-amp/amx and varying concentrations of unmodified ent (figure 3h when these assays were performed under conditions of iron limitation, the presence of exogenous ent attenuated the antibacterial activity of ent-amp/amx. a 1:1 molar ratio of ent-amx/ent afforded an od600 value comparable to that of the untreated control, whereas higher equivalents of ent were required to block the antibacterial action of ent-amp. the origins of this difference are unclear and may indicate that the hydroxyl group of amx has a negative effect on the transport efficiency of the conjugate. in total, these ent addition assays suggest that competition for ent-amp/amx and ent occurs at the receptor(s) and that the conjugates are delivered into the bacteria via the same uptake machinery as ent. l-serine is a biosynthetic building block for ent, and a role for chiral recognition in ent transport has been probed in prior studies. in one series of investigations, e. coli fepa was found to bind ferric l-ent and ferric d-ent with similar affinities (kd=21 and 17 nm, respectively; ascertained by measuring the binding of fe-loaded siderophores to e. coli bn1071 cells). a lack of transport of ferric d-ent into e. coli bn1071 was also reported in this work. a later study probed ent uptake in bacillus subtilis, and transport of both l- and d-ent analogues was observed to occur with similar efficiency. 2457 t to hydrolyze l-ent and d-ent was evaluated, and fes did not accept d-ent as a substrate. as a result of the transport studies in b. subtilis and enzymatic activity assays with s. flexneri fes, a model in which both ent enantiomers are transported and chiral taken together, these studies suggest that the ability to transport d-ent may vary between species and even between strains of a given species, and more studies are required to address such possibilities. based on the observation that e. coli fepa binds d-ent and that b. subtilis transports d-ent analogues, we synthesized the d-enantiomers of the ent--lactam conjugates (13 and 14, scheme 1) and evaluated the antibacterial activity of these conjugates against e. coli k-12, 25922, cft073, 35401, and 43895 (figures s9s13). d-ent-amp/amx exhibited reduced antibacterial activity relative to ent-amp/amx for k-12, 25922, cft073, and 43895. under conditions of iron limitation, complete growth inhibition was observed with 1 m d-ent-amp/amx for e. coli k-12, 25922, and 43895, compared to 100 nm for ent-amp/amx. likewise, a 10-fold reduction in antibacterial activity was observed for e. coli cft073, where 100 nm d-ent was required to inhibit growth completely. in contrast, a negligible difference in antibacterial activity of the l- and d-isomers was observed for e. coli 35401. regardless of enantiomer, all four ent-amp/amx conjugates provide enhanced antibacterial activity against these e. coli strains relative to amp/amx. nevertheless, these data suggest that d-ent-amp/amx are less readily transported into various e. coli strains than the l-isomers. although this modification provides no appreciable benefit for this -lactam delivery system with periplasmic targets, it is possible that ent-antibiotic conjugates based on d-ent may be desirable for delivering cargos to the cytosol, precluding concomitant delivery of nutrient fe(iii). with support for ent-mediated delivery of ent-amp/amx to the e. coli periplasm, we sought to confirm the essentiality of the -lactam warheads in antibacterial action. we therefore designed and prepared hydrolyzed ent-amp/amx analogues 15 and 16 (scheme 2) where the -lactam structure is destroyed. hydrolysis of the amp/amx-alkynes 7 and 8 was achieved in the presence of 1% tfa with heating at 37 c, and the decomposition products 9 and 10 were obtained as diastereomeric mixtures (scheme 2). the formation of these species followed the reported degradation pathways for ampicillin, where hydrolysis and subsequent decarboxylation occur. the diastereomeric mixtures were employed to prepare the hydrolyzed conjugates ent-hydro-amp 15 and ent-hydro-amx 16 via a copper-catalyzed click reaction. (a, b) antibacterial activity assays against e. coli k-12 (a) and cft073 (b) using ent-amp/amx and ent-hydro-amp/amx. (c) antibacterial activity assays against e. coli atcc 35218, which expresses a class a serine -lactamase, using ent-amp/amx in the absence and presence of the -lactamase inhibitors potassium clavulanate (pc) and sulbactam (sb). all assays were performed in 50% mhb supplemented with 200 m dp (mean sem, n 3). we employed ent-hydro-amp/amx in antibacterial activity assays against e. coli k-12 and observed negligible growth inhibition (figure 4a). when e. coli cft073 was treated with ent-hydro-amp/amx in the presence of dp this result indicates that ent-hydro-amp/amx are transported into the cytoplasm of e. coli cft073, where nutrient iron is released. we also performed a series of antibacterial activity assays with e. coli atcc 35218, a strain that expresses a class a serine -lactamase. similar to unmodified amp/amx, ent-amp/amx were inactive against e. coli atcc 35218 (mic>10 m) in the absence and presence of dp (figures 4c and s14). slight growth inhibition was observed at 10 m under conditions of iron limitation, which may be attributed to iron chelation. the addition of -lactamase inhibitors restored the activities of ent-amp/amx and amp/amx, and the conjugates exhibited greater antibacterial activity than the parent antibiotics (figures 4c and s14). in total, the assays with ent-hydro-amp/amx and strains expressing -lactamase demonstrate that an intact -lactam is required for the antibacterial activity of ent-amp/amx. moreover, these studies indicate that the -lactams retain their original function and inhibit pbps when conjugated to ent. the remarkable sensitivity of e. coli cft073 to ent-amp/amx (figure 2c) motivated us to investigate the relative cell-killing kinetics of ent-amp/amx and amp/amx to determine whether these conjugates kill e. coli cft073 more rapidly than the unmodified drugs. for comparison between e. coli strains, ent-amp/amx provide more rapid cell death than unmodified amp/amx (figure 5), and this behavior is most apparent for e. coli cft073, where the od600 value was almost reduced to the baseline value after 1 h incubation with 5 m ent-amp/amx, corresponding to a 2-fold log reduction in cfu/ml. in contrast, the change in od600 and cfu/ml for e. coli cft073 treated with 50 m unmodified amp/amx is negligible over this time period. the time-kill kinetics for e. coli k-12, conducted with 50 m of both unmodified and modified -lactams, indicate a slight increase in kill kinetics for ent-amp/amx relative to amp/amx, and that the kinetics of cell-killing are slower for k-12 than cft073 (figure 5). these results support a model whereby ent modification facilitates uptake of amp/amx relative to the unmodified drugs. this effect is more dramatic for e. coli cft073 than k-12, which is in accordance with the enhanced antibacterial activity observed for cft073 relative to the other e. coli strains considered in this work. time-kill kinetic assays for treatment of e. coli k-12 (top panel) and cft073 (bottom panel) with amp/amx and ent-amp/amx. e. coli k-12 (10 cfu/ml) was treated with 50 m of amp/amx or 50 m ent-amp/amx. e. coli cft073 (10 cfu/ml) was treated with 50 m of amp/amx or 5 m ent-amp/amx. the assays were conducted in 50% mhb medium containing 200 m dp at 37 c (mean sem, n=3). to determine whether ent-amp/amx exhibit broad-spectrum or species-selective activity, we performed antibacterial activity assays with two additional gram-negative and two gram-positive species in both the absence and presence of dp. these species include klebsiella pneumoniae atcc 13883, pseudomonas aeruginosa pao1, s. aureus atcc 25923, and bacillus cereus atcc 14579. k. pneumoniae is a gram-negative species that biosynthesizes and utilizes ent for iron acquisition. p. aeruginosa is a gram-negative bacterium that captures ent as a xenosiderophore and expresses two ent receptors pfea and pira.s. aureus and b. cereus are both gram-positive bacterial species, and the ability to utilize ferric ent as an iron source is reported for both species. in contrast to gram-negative bacteria, where the pbps are located in the periplasm, the targets of -lactam antibiotics are in the extracellular peptidoglycan of gram-positive organisms. k. pneumoniae atcc 13883 has a chromosomally encoded class a -lactamase (shv-1) and lacks sensitivity to amp/amx. we observed no effect of 100 m amp/amx on k. pneumoniae growth under our assay conditions (figure s15). only 50% growth inhibition was observed when k. pneumoniae was treated with high concentrations (10 m) of ent-amp/amx in the absence of dp, and the ent-amp conjugate provided the greatest activity under conditions of iron limitation with 90% growth inhibition at 10 m. when -lactamase inhibitors were included in the assays, k. pneumoniae exhibited greater sensitivity to amp/amx (mic=100 m) and ent-amp/amx (mic=10 m); however, we observed some growth inhibitory activity of the -lactamase inhibitor sulbactam alone under these assay conditions (100 m sb, figure s15). thus, the possibility of a synergistic effect from the inhibitors and conjugates can not be ruled out completely. the lack of activity of ent-amp/amx against k. pneumoniae atcc 13883 is reminiscent of results obtained during investigations of amp/amx-functionalized tripodal triscatecholate ligands. these compounds were inactive against k. pneumoniae, and the behavior was attributed to either an inability of the k. pneumoniae iron transport machinery to import the conjugates or the development of resistance over the course of the assay. an alternative explanation is that -lactamase expression by k. pneumoniae resulted in inactivation of the -lactams. the p. aeruginosa pao1 strain employed in this work exhibited little sensitivity to both amp/amx and the conjugates under the antibacterial assay conditions (figure s16). amp/amx exhibited no activity up to 100 m, whereas ent-amp/amx provided growth inhibition at 10 m in both the absence and presence of dp. whether these results indicate that ent-amp/amx will be ineffective against multiple p. aeruginosa strains is unclear. p. aeruginosa strains exhibit different phenotypes, and highly variable and strain-dependent mic values have been reported for triscatecholate-lactam conjugates against p. aeruginosa. we previously reported that p. aeruginosa pao1 imports ent-cargo conjugates, and we speculate that the lack of activity observed for this strain stems from its inherent insensitivity to amp/amx. b. cereus atcc 14579 was also insensitive to amp/amx, which only afforded growth inhibition at 100 m. some growth inhibition was observed for b. cereus treated with 10 m ent-amp/amx, which may result from iron sequestration (figure s17). s. aureus atcc 25923 is susceptible to amp/amx, with complete growth inhibition observed at 1 m. in this case, a 10-fold reduction in antibacterial activity was observed for ent-amp/amx relative to unmodified amp/amx (figure 6). although the origins of this attenuation are unclear, we speculate that ent-amp/amx may have trouble penetrating the thick peptidoglycan of s. aureus. an alternative possibility is that recognition of ent-amp/amx by the s. aureus ent receptor diverts the -lactams from the pbps. in total, the results from these assays indicate that ent-amp/amx exhibit antibacterial activity enhancements that are species-selective, providing increased potency against e. coli strains and not for the other strains evaluated in this work. we therefore reasoned that ent-amp/amx, at low concentrations, should selectively kill e. coli in the presence of other less sensitive species. we treated co-cultures of e. coli cft073 and s. aureus with ent-amp/amx or amp/amx and analyzed the species composition following a 19-h incubation using harty-uti plates. these agar plates are employed in medical microbiology laboratories for the diagnosis of urinary tract infections and provide species identification by the colony color. when grown on harty-uti plates, s. aureus are off-white and e. coli are purple-pink. in figure 6, we present representative images of the colonies that resulted from treating co-cultures of e. coli and s. aureus with amp/amx or ent-amp/amx. in the absence of antibiotic, the cultures provide a mixture of off-white and purple-pink colonies, indicating that both e. coli and s. aureus grow when cultured together. when the co-cultures are treated with 1 m amp/amx, only purple-pink colonies are present, which reveals that only e. coli survives. in contrast, treatment of the co-cultures with 1 m ent-amp/amx results in only off-white colonies from s. aureus. these comparisons demonstrate that ent-amp/amx selectively kill e. coli in the presence of s. aureus and that the siderophore modification reverses the inherent species selectivity of the parent antibiotics. achieving such species-selective and single-pathogen antibiotic targeting is an important goal and unmet need for pharmaceutical development that will allow for treating disease with minimal perturbation to the commensal microbiota. ent-amp/amx selectively kill e. coli cft073 in the presence of s. aureus atcc 25923. (a, b) antimicrobial activity assays against s. aureus atcc 25923 in the absence (a) and presence (b) of 200 m dp. (c, d) bacterial growth monitored by od600 for cultures of e. coli only, s. aureus only, and 1:1 e. coli/s. aureus mixtures treated with amp/amx or ent-amp/amx in the absence (c) and presence (d) of 200 m dp. (e) representative photographs of colonies from mixed cultures of e. coli cft073 and s. aureus atcc 29523 treated with ent-amp/amx (1 m) or amp/amx (1 m) in the presence of 200 m dp. all assays were conducted in 50% mhb medium (t=19 h, 30 c) (mean sem, n 3 for a d). we evaluated the cytotoxicity of ent-amp against the human t84 colon epithelial cell line. cell survival was evaluated by mtt assay after a 24 h treatment with apo or iron-bound ent-amp, amp, or ent. the iron-bound forms were assayed to determine whether iron chelation in the growth medium is a factor. no cytoxicity was observed for amp or ent-amp, whereas apo ent itself decreased the survival of t84 cells by approximately 30% at the highest concentration evaluated. when pre-loaded with fe(iii), percent cell survival quantified by mtt assay after a 24 h treatment with apo or iron-bound ent, ent-amp, and the parent antibiotic amp in the absence and presence of 1 equiv of fe(iii) (mean sem, n=3). lipocalin-2 (lcn2, also known as siderocalin or ngal) is a 22-kda protein produced and released by neutrophils and epithelial cells. it has a hydrophobic binding pocket and coordinates ferric ent with sub-nanomolar affinity. by sequestering ferric ent, this host-defense protein contributes to the metal-withholding response and prevents bacterial acquisition of this essential nutrient. to determine whether lcn2 also binds ent-amp/amx and thereby blocks antibacterial activity, we performed antibacterial activity assays with e. coli cft073 in m9 minimal medium supplemented with lcn2 or bovine serum albumin (bsa). under these conditions, up to 1 m of lcn2 had no effect on the growth of e. coli cft073. addition of 1 m lcn2 to the medium rescued the growth of e. coli cft073 treated with 100 nm of ent-amp (figure 8) whereas addition of 1 m bsa had no effect on ent-amp cell killing. these results suggest that lcn2 binds ent-amp and blocks its recognition and uptake. to ascertain whether lcn2 binds ent-amp in the presence of exogenous ent, we performed a series of experiments where e. coli cft073 were treated with fixed concentrations of ent-amp (100 nm) and lcn2 (1 m) and the concentration of ent was varied (0, 0.5, and 1 m). moreover, when ent-amp was combined with a 1:1 molar ratio of ent and lcn2 at 10-fold excess over the conjugate, no e. coli growth was observed, which suggests that lcn2 preferentially binds ent. prior work demonstrated that lcn2 can not bind glucosylated ent, which was attributed to a steric clash between the glucose moieties and the ent binding site of the protein, and decreased hydrophobicity of the siderophore may also be a factor. thus, our data suggest that the nature of linker attachment at c5 and the peg3 moiety of ent-amp/amx do no abrogate lcn2 binding as effectively as the glucose moieties exhibited by the salmochelins. antibacterial activity of ent-amp against e. coli cft073 in the presence of lcn2 or bsa. (a) e. coli cft073 treated with 100 nm ent-amp and varying concentrations of lcn2 or bsa control. (b) e. coli cft073 treated with ent-amp, varying concentration of ent, and varying concentrations of lcn2 or bsa control. the assays were performed in m9 minimal medium (24 h, 37 c) (mean sem, n 3). ent-amp/amx are two siderophore-lactam conjugates based on the native enterobactin scaffold. these molecules hijack siderophore-based iron uptake pathways and provide potent antibacterial activity against various e. coli strains, including human pathogens. our investigations of ent-amp/amx establish the following: (i) ent-amp/amx provide up to 1000-fold enhanced antibacterial activity against e. coli strains; (ii) ent-amp/amx are transported into e. coli by fepa and potentially other catecholate siderophore receptors (iron, iha) employed by pathogenic ctf073 and uti98; (iii) ent-amp/amx are captured by pbps in the periplasm, which results in pbp inhibition and cell death; (iv) selective killing of e. coli in the presence of less susceptible organisms such as s. aureus is achieved because of the enterobactin scaffold; (v) ent-amp/amx exhibit negligible cytotoxicity to human t84 intestinal epithelial cells; and (vi) although lcn2 has the ability to bind ent-amp/amx, this siderophore-scavenging protein prefers to capture native ent. in total, these studies demonstrate that modification of antibiotic cargo with the native enterobactin platform provides many desirable features for antibiotic delivery and efficacy. the large molecular weight of the conjugates resulting from the native ent scaffold (as opposed to a smaller mimic) enhances rather than diminishes uptake for gram-negative e. coli. moreover, we observed no evidence for the development of resistance to ent-amp/amx over the course of the antibacterial activity assays performed during these investigations. our studies confirm that the enhanced antibacterial activity observed for ent-amp/amx requires both enterobactin recognition by outer membrane receptors and an intact -lactam moiety. the results are reminiscent of the dramatic antibacterial activity enhancements observed for albomycin, a secondary metabolite produced by actinomyces subtropicus. albomycin is comprised of the siderophore ferrichrome and a trna synthetase inhibitor, and it exhibits antimicrobial activities that are 30,000-fold greater than those of the unmodified trna synthetase inhibitor against e. coli and s. aureus. nonetheless, the ent-amp/amx cell-killing mechanism may be more complex than only more efficient -lactam delivery across the gram-negative outer membrane. binding of ent-amp/amx to the pbps presumably results in accumulation of ferric enterobactin in the e. coli periplasm for some period of time, which may have deleterious consequences. a recent study of an e. colitolc- mutant revealed that enterobactin accumulation in the periplasm affords growth defects and abnormal cellular morphologies. a fascinating observation that stems from our current work is the variable susceptibilities and responses of different e. coli strains to ent-amp/amx, which contrast the effects of unmodified amp/amx. such differences are manifest in the mic values to some degree and time-kill kinetics; however, the results presented in figure 2 indicate that mic values alone do not provide a full description of how ent-amp/amx susceptibility differs between e. coli strains. these results suggest underlying complexity in microbial physiology related to iron-uptake pathways that can not be fully explained by the presence or absence of a gene for a particular receptor (i.e., fepa, iron). the heightened sensitivity of uropathogenic e. coli cft073 is particularly noteworthy, and it will be interesting to decipher the physiological origins of this effect as well as the differential behavior of various e. coli pathogens toward ent-antibiotic conjugates. there is a clear and unmet need for new antibacterial agents to treat bacterial infections in humans, including antibiotics that target specific bacterial sub-populations. preventing undesirable consequences of antibiotic treatment on the commensal microbiota, which contributes to human health in beneficial ways, is a challenge that needs to be addressed. such targeted therapeutics will be valuable not only for treating bacterial infections when the causative agent is known (e.g., urinary tract infection and e. coli, cystic fibrosis lung infection and p. aeruginosa) but also for other pathologies that involve microbial dysbiosis, such as irritable bowel disease. our studies of ent-amp/amx provide one step toward addressing species-specific antibiotic targeting as well as overcoming gram-negative outer membrane permeability. from the standpoint of the host environment, commensal e. coli employ ent for acquiring iron in the host, and thus further elaboration of this strategy to specifically target pathogenicity and evade host responses (e.g., lcn2) is desirable.

the design, synthesis, and characterization of enterobactin antibiotic conjugates, hereafter ent-amp/amx, where the -lactam antibiotics ampicillin (amp) and amoxicillin (amx) are linked to a monofunctionalized enterobactin scaffold via a stable poly(ethylene glycol) linker are reported. under conditions of iron limitation, these siderophore-modified antibiotics provide enhanced antibacterial activity against escherichia coli strains, including uropathogenic e. coli cft073 and uti89, enterohemorrhagic e. coli o157:h7, and enterotoxigenic e. coli o78:h11, compared to the parent -lactams. studies with e. coli k-12 derivatives defective in ferric enterobactin transport reveal that the enhanced antibacterial activity observed for this strain requires the outer membrane ferric enterobactin transporter fepa. a remarkable 1000-fold decrease in minimum inhibitory concentration (mic) value is observed for uropathogenic e. coli cft073 relative to amp/amx, and time-kill kinetic studies demonstrate that ent-amp/amx kill this strain more rapidly at 10-fold lower concentrations than the parent antibiotics. moreover, ent-amp and ent-amx selectively kill e. coli cft073 co-cultured with other bacterial species such as staphylococcus aureus, and ent-amp exhibits low cytotoxicity against human t84 intestinal cells in both the apo and iron-bound forms. these studies demonstrate that the native enterobactin platform provides a means to effectively deliver antibacterial cargo across the outer membrane permeability barrier of gram-negative pathogens utilizing enterobactin for iron acquisition.

PMC4353011

pubmed-413

colorectal cancer (crc) is one of most common cancers and leading causes of mortality in the usa, accounting for approximately 136,000 new cases and 50,000 deaths per year.1 for metastatic or unresectable crc, standard first- and second-line treatments typically involve a combination of cytotoxic chemotherapies (eg, folfiri [5- fluorouracil+oxaliplatin+irinotecan])2,3 and molecular targeted agents (eg, bevacizumab, cetuximab, panitumumab),46 which can help to improve progression-free survival and overall survival. however, due to the nature of the disease, many patients will progress through guideline-recommended standard regimens while maintaining a good performance status. regorafenib, an oral multikinase inhibitor, which targets angiogenic, stromal, and oncogenic receptor tyrosine kinases, was approved by the us food and drug administration in 2012 for the treatment of patients with metastatic crc who have been previously treated with fluoropyrimidine-, oxaliplatin-, and irinotecan-based chemotherapy, an anti vascular endothelial growth factor (vegf) therapy and, if kras wild-type, an anti epidermal growth factor receptor (egfr) therapy.7 this approval was based on the correct study, which demonstrated a median overall survival of 6.4 months with regorafenib vs 5.0 months with placebo (hazard ratio, 0.77; 95% confidence interval, 0.640.94; p=0.0052).8 among the most common adverse effects were fatigue, hand a case of an elderly patient on regorafenib who achieved stable disease for over 11 months with strategic dose modifications was presented. no ethics approval was needed for the care of this patient as all treatments were in accordance with institutional best practices. verbal consent to appear in this case study was given by the patient to dr seery. a 73-year-old indian female with a past medical history of hypertension and hyper cholesterolemia the patient had no history of smoking, alcohol consumption, or any family history of cancer. the patient initially had symptoms of hemoptysis for approximately 2 weeks and then subsequently developed hematochezia for 3 days. due to these symptoms, the patient underwent a colonoscopy, which revealed left-sided colon cancer. a left hemicolectomy was performed; pathology revealed a stage iiib well-differentiated, kras wild-type adenocarcinoma, with one out of 15 lymph nodes testing positive. the patient moved back to india and did not receive adjuvant chemotherapy due to patient preference. in march 2011, the patient returned to the usa with complaints of mild abdominal distention, and a subsequent positron emission tomography/computed tomography (pet/ct) scan showed evidence of recurrence, with ovarian and peritoneal metastases. the patient began treatment with capecitabine, oxaliplatin, and bevacizumab, and developed an anaphylactic reaction in the sixth cycle. she continued with single-agent capecitabine for an additional two cycles and completed therapy in june 2011 as there was no further evidence of disease on the pet/ct scans. the patient did not receive any additional therapy until early 2012 when she was restaged and was again noted to have diffuse metastatic disease in the peritoneum, with imaging consistent with moderate to extensive carcinomatosis. the patient received cetuximab and folfiri from february 2012 through august 2012 and achieved an excellent response, with the pet/ct scan showing no evidence of measurable disease, with some minimal hypodensities in the liver that were not pet avid. the patient did well until april 2013 when she experienced increased abdominal distention, bloating, and difficulty with defecation. the patient received a repeat ct scan that showed progression of the disease, with a large pleural-based mass in the right lower lobe with bilateral pleural effusions, ascites, and multiple peritoneal implants, as well as omental infiltration along with a possible l3 lytic lesion. also, the patient s carcinoembryonic antigen (cea) values had increased to 23. as a result, the patient was restarted on cetuximab and folfiri in may 2013; however, a pet/ct scan 2 months later revealed a mixed response, with persistent fluorodeoxyglucose-avid right pleural disease, but also some areas of decreased activity with stable nodal disease in the left axilla, retroperitoneum, and pelvis, and increased activity in the left inguinal and right retrocrural nodes. the patient continued with cetuximab and folfiri until august 2013, and a repeat pet/ct scan in september 2013 revealed progressive disease in the right pleural mass along with increase in size of the retrocrural and left inguinal lymph nodes and liver (figure 1a). the patient was started on regorafenib 160 mg daily on september 12, 2013, but required a treatment interruption due to hypertension with consistent systolic blood pressure readings in the range of 170 mm hg and diastolic blood pressure in the range of 100 mm hg, as well as grade 3 fatigue during cycle 1. the patient was started on antihypertensive medications, and regorafenib was held for 2 weeks to resolve these adverse events. the dose was then reduced again due to grade 3 fatigue, and the patient s dose was stabilized at 80 mg daily. a repeat pet/ct scan in february 2014 showed a mixed response, with increased activity in non-target lesions noted in multiple omental, peritoneal, and pelvic lesions, but decreased activity in target lesions, specifically the thoracic and liver lesions, with new activity in the right tenth rib (figure 1b). specifically, a right pleural-based mass had decreased from 27.6 mm to 26.7 mm, and standardized uptake value decreased from 14.4 to 3.4 in conjunction with a decrease in disease measuring from 58.0 mm to 43.6 mm in the central pelvic mesentery superior extending to the uterine fundus. the patient s cea decreased to 3.7, while eastern cooperative oncology group (ecog) performance status improved from 2 to 1, and treatment was therefore continued at 80 mg daily. the patient s most recent pet/ct scan in august 2014 showed frank progression of disease, with an increase in number and activity of multiple metastatic pulmonary and hepatic lesions along with increased activity in omental and mesenteric implants. as of august 2015, the patient has been on regorafenib for 11 months with progression; however, her ecog performance status remains at 1, and she would like to continue taking the medication as she does not have any side effects and feels her quality of life has improved. verbal consent was obtained from the patient prior to her departing our facility for hospice care. the patient s identification has been protected and any information which could potentially be used to reveal the identity of the patient has been removed. we reported on a case of an elderly woman with kras wild-type, refractory metastatic colon cancer who has received multiple chemotherapy regimens, most recently regorafenib for 11 months with minimal side effects and palliation of symptoms. the report shows that regorafenib can provide a decrease in tumor burden and improve quality of life with prolonged survival if an appropriate dose is used in terms of balancing side effects with benefits of symptom palliation. moreover, the report shows that continuing therapy beyond disease progression is no longer an absolute contraindication. this case report additionally demonstrates that close and frequent follow-ups by the primary oncologist is needed in order to monitor side effects and to find the effective and tolerable dose. in the correct study, the frequency of adverse events was shown to peak during the first cycle and gradually taper off with subsequent cycles.8 this report suggests that regorafenib can be a viable option for patients who still have a good performance status and have progressed through other regimens. importantly, patient and financial support services are in place through the reach program to help patients manage the cost of this therapy.

regorafenib, an oral multikinase inhibitor, was approved in september 2012 by the us food and drug administration for the treatment of patients with metastatic colorectal cancer. since this time, however, few case reports outlining real-world usage have been published in the literature. here, we detail the clinical history of an elderly woman with kras wild-type colon cancer who received regorafenib after prior treatment with other agents. we show that by employing dose modification strategies to address adverse events, this patient was able to remain on therapy for 11 months and achieve stable disease.

PMC4657800

pubmed-414

the treatment and surgical background of gynaecomastia has history of evolution over and above 60 years. in 1928, dufourmental described a semi circular intra-areolar incision which was later documented by webster in 1946, who recognised the fact that the conspicuous scarring left by older techniques is more embarrassing than the original condition. the categories were classified by simon in 1973 and later on, liposuction was introduced in 1983. ultrasound-assisted liposuction remains the accepted standard that gained much popularity for addressing dense adipose tissue for the management of relatively glandular or fibrous gynaecomastia. it is significant to mention the wide range of alternatives and advancements with minimally invasive approaches developed to eliminate the condition of gynaecomastia in a more refined way. it has been observed that liposuction technique alone has its limitation with removal of adipose tissue only and unable to address the problem of tough glandular component; therefore, combining liposuction with different kinds of direct incisions was introduced to deliver the fibrous architecture of the breast by many authors. a minimally invasive approach was advocated by some authors that consist of minimal access incisions in either peri-areolar, circum-areolar or trans-areolar region, while some proposed arthroscopic shavers to break down the fibrous capsule of the glandular tissues and deliver it with liposuction. but they have few drawbacks such as less access to the fibro-glandular tissue due to the minimal incision, visible unattractive scarring at nipple areola complex left after the peri-areolar incision as shown in figure 1, dermal necrosis and subsequent scarring due to arthroscopic shavers and poor results. scarring left after peri-areolar incision the gynaecomastia surgery is basically sought for aesthetic purpose and such operation should not leave any tell-tale signs of the procedure. there had to be a solution for the smooth delivery of fibro glandular tissues without added scarring. this article presents an innovative approach that consists of a criss-cross trans-nipple incision to retrieve the fibro-glandular tissues following liposuction method for excellent outcomes. no additional scarring is visible, even on the operation table and even in a close up view right after the procedure with this trans-nipple approach. in all the cases, the trans-nipple incision is proved to give no signs of surgery over the nipple areola complex after a recovery period of three to six months following the surgical procedure. between the duration of january 2012 to october 2013, 28 candidates suffering from various degrees of gynaecomastia (except grade-4, which definitively required skin excision) were surgically treated by this method. after proper consultation and examination, it was concluded that a surgical approach would be needed and all of them underwent the process of routine medical investigations for the anaesthesia fitness. right before the procedure, markings were made to limit the boundaries of treatment [figure 2]. the procedure started with liposuction to retrieve adipose tissues through a tiny hole (approx 6-7 mm) at both sides of the chest using number 4 liposuction canullae. after performing thorough liposuction of the marked area, a small criss-cross incision was made right on the top of the nipple with the help of no-11 blade [figure 3]. pre-operative marking of the treatment boundaries for liposuction the criss-cross incision at the centre of the nipple through a fine curved hemostat, the tough fibro-glandular tissues were delivered while creating slight pressure with thumb and forefinger (broad pinching) over the base of nipple areola area [figure 4a and 4b]. only tough tethering bands, which obstructed the path, were cut with a fine curved scissors [figure 4c]. care was taken to keep the point of scissors away from the dermis of areola. an amount of 3-5 mm tissue beneath the areolar skin was preserved to prevent any vascular complications [video 1]. removal fibro-glandular tissues through the incision cutting the tethering band through scissors keeping tip away from areolar skin to preserve at least 3-5 mm tissue beneath areolar skin the incision was then closed with 4-0 monocryl in a single purse-string suture manner [figure 5]. the liposuction incisions were left open to drain any collection of fluid without any freshening of margins or stitches. the compression garment was applied on the operation table right after the procedure and was replaced with new compression garment on seventh day of the surgery when the patient was called for first follow up. all the patients were advised to do proper massage of the chest area after 7 days of procedure with any moisturiser of their choice and to wear the compression garment for the next three months following the surgery. serial medical photographs-front, lateral and semi-lateral views were taken prior to the procedure, after three, six and twelve months from the surgery to assess the final outcomes. it was observed in a close view that there was no visible scar on the nipple areola complex even on the operation table after the surgery [figure 6] and on long-term follow-up of six months and one year [figures 7 and 8]. none of the 28 patients reported any alteration of sensation in the nipple areola region after 3 to 4 months. trans-nipple removal of fibro-glandular tissue is an easy, innovative approach in gynaecomastia surgery that removes all the tough fibro-glandular tissue without leaving any visible scars over the nipple areola complex. it produces the most rewarding results and maximum satisfaction to the patient right from their procedure. close-up view right after the surgery showing practically no disturbance of nipple-areola complex architecture example cases showing follow-up between six months to one year after surgery. see the close up view of practically scar less nipple-areola complex after recovery gynaecosmastia usually requires no attention, unless patient feels uncomfortable or embarrassed due to aesthetic reasons. chances of having this condition can be same in a slender patient as well as in a healthy or over-weight patient. the difference is only the degree or severity that can be noticed differently in obese patients or who are relatively fat can develop significant visibility or enlargement. though the root cause of this problem is unknown in most of the cases and commonly idiopathic, heredity, consumption of certain drugs, obesity or hormonal changes can be one of the various other reasons. many techniques and refined approaches have been advocated through the years to address the issue that leave visible scars. through the recent years, the surgical techniques that have been supported by many authors advocate liposuction or ultrasound-assisted liposuction alone or in combination with direct incision of the breast tissue through a peri-areolar incision or a pull-through technique, and various other methods have been advocated to eliminate remnant tough fibro-glandular tissue of gynaecomastia and achieve more aesthetic results. it is observed that gynaecomastia causes a great mental discomfort and impairs the self-confidence of the patient. they tend to over react and sometimes become extra-conscious about their condition and start to notice even a mild elevation which is left in most cases of liposuction alone methods. this slight elevation can be acceptable for normal people but becomes great matter of mental stress for the patients suffering from this problem. it has been noticed that slight bulges are left as remnant after doing liposuction and it is evident on operation table during the surgery that significant elevation is left after doing liposuction only [figure 9a]. this elevation is formed by the leftover fibro-glandular tissues that causes visible bulges and can not be retrieved through only liposuction. that is why liposuction alone can not serve the purpose as it has its limitations and is only ideal for removal of adipose tissue. in most of the cases where patients are more conscious even about a mild elevation, there remains a need of removal of this fibro-glandular tissue to achieve flat chest appearance [figure 9b]. the invasive approaches along with liposuction are able to address this issue but they leave some conspicuous scarring (peri-areolar or trans-areolar incisions) or loss of sensation of the nipple areola complex, compromised vascualrity of overlying skin and subsequent complication (arthroscopic shavers or modified liposuction shaver canullae), leading to inadequate outcomes. per-operative view showing visible bulge below nipple-areola complex just after completing liposuction due to remaining tough fibro-glandular tissue per-operative view of same case showing flat chest just after removal of tough fibro-glandular tissues through trans-nipple approach we consider a trans-areolar incision to remove these tough glandular tissues to achieve flat chest and eliminate any elevation caused by fibro glandular component over the nipple areola complex [figure 9c]. our technique is relatively simple and retrieves any volume of the tough fibro-glandular breast tissue without leaving any visible scars on the nipple areola complex, unlike peri-areolar incision or other invasive techniques. the tough fibro-glandular tissues are removed through a criss-cross incision over the centre of nipple right after performing liposuction of the marked area. the key to prevent any complication is to cut only the tethering band coming in way of delivery of fibro-glandular tissue and preservation of 3-5 mm tissue beneath the areolar skin. the technique offers the perfect control over nipple position as there are absolutely no chances to re-positioning or traction on the nipple due to subsequent scar that sometimes causes nipple malpositioning or inversion in peri-areolar incision technique. on table final result right after the procedure in our study, the presented patients had different categories of gynaecomastia and patient-a [figure 10a] delivered little amount of tough fibro-glandular component (approx 50 g) and while patient-b [figure 10b] delivered huge amount (approx 250 g). both patients were operated with the same modality of treatment, i.e., liposuction for adiposal component and trans-nipple approach for removal of tough fibro-glandular tissue to treat their condition and discharged on the same day of their operation. both were advised to do regular massage and wear compression garment for three to four months. they both enjoyed flat chest, very faint and thin scar of liposuction incision site and practically invisible scars over the nipple areola complex after 6 months of follow up. example patients-a (before and after) with fewer amounts of fibro-glandular tissues example patient-b (before and after) with large amount of fibro-glandular tissues trans-nipple approach is an easy, innovative and practically excellent technique to remove any amount of the residual glandular fibrous tissue after liposuction without leaving any additional visible scars on the nipple-areola complex in gynaecomastia surgery and results in best aesthetic outcomes. all the 28 patients who were operated by this approach reported very satisfactory aesthetic outcomes after their recovery. the procedure offers a promising approach for all the patients of gynaecomastia, especially for the patients who want to keep secret of having any surgical procedure done to their chest.

context: the established techniques that have been used to treat gynaecomastia are said to have relatively less patient satisfaction rate as they leave some visible scars or mild elevation over the nipple areola complex, resulting in aesthetically unsatisfactory results. even the slightest elevation or smallest scar over nipple areola complex leave patients extremely self conscious and in a dilemma of having a second intervention to get rid of that blemish.aims:the aim of the study is to achieve-a flat chest without adding a scar and with no chances of re-occurrence of the condition. this article suggests an innovative approach to address the problem. materials and methods: the author presents trans-nipple incision approach for the delivery of fibro-glandular tissue component following liposuction for maximum patient satisfaction. this method consists of a unique small criss-cross incision right on the nipple itself for retrieving any volume of tough fibro-glandular tissues. between the duration of january 2012 to october 2013, 28 male patients of different ages were operated with this technique. results:the surgery resulted in well-shaped, symmetric chest contour without any visible elevation or additional scars on nipple areola complex. no complications were noticed in any of the patients. conclusions:the presented technique is proved to have a high patient satisfaction rate and to be promising method to achieve good aesthetic results in gynaecomastia surgery.

PMC4075217

pubmed-415

many problems occur while we are trying to improve the quality of life and material wealth by the development of modern society. among them, air pollution is a serious problem in western and developing countries. long-term exposure to air pollution has a high correlation with the incidence of cardiovascular disease, respiratory diseases, and diabetes1,2,3, threatening health. in particular, air pollution is a major cause of respiratory diseases such as asthma and allergic diseases4, 5 and it has a negative effect on the healthy growth and development of growing children and adolescents. another problem caused by the development of modern society is the reduction in physical activity across all generations throughout the world. the time spent in physical activity by children has decreased significantly compared to before6, 7. the time spent using visual media and watching tv has increased8, 9, and physical inactivity due to excessive academic pressure and the influence of parents10, has increased obesity, incorrect posture, and muscle weakness. in addition, energy consumption has increased due to environmental, metabolic, and genetic factors11. thus, children living in modern society are showing gradually decreasing physical fitness. the change in the environment due to the development of modern society is a threat to the health of growing children. according to the korea education development institute, respiratory system abnormalities have been much higher than those other systems over the last three years. reduced basic physical fitness and reduced pulmonary functions are seriously threating the physical development of korean elementary, middle, and high school students. although the physical fitness and lung health of growing children and adolescents is threatened, some studies have reported a relationship between air pollution and pulmonary function12, 13. however, there has been insufficient research on the relationship between exercise and lung function. most studies on exercise have only suggested lung function is improved by regular exercise14, 15. the present study is to our knowledge the first to investigate the relationship between basic physical fitness and lung function. the purpose of the present study was to determine the relationship between basic physical fitness and pulmonary function in healthy korean school students to enable us to present an alternative method for improving their pulmonary function. a total 240 healthy children and adolescents aged 617 years who lived in busan in korea were recruited for the present study. the participants had a body mass index (bmi)< 25 kg/m, and no respiratory system abnormalities. the sample comprised 20 healthy students (10 boys and 10 girls) of each age from 6 to 17. the participants who did not meet the bmi criteria or who showed significantly low pulmonary test values were excluded, and replacements were recruited in order to conduct the analysis with the same numbers of subjects. the participants were divided into the early period of elementary school (68 y), the late period of elementary school (911 y), the period of middle school (1214 y), and the period of high school (1517 y) in order to investigate the rapid changes in body composition and basic physical fitness with growth. all participants gave their informed consent and the experimental protocol was approved by the ethical committee of dong-a university. body composition including height, weight, bmi, and body fat (%) was measured using a body composition analyzer venus 5.5 (jawon medical, korea) while subjects were fully relaxed. muscle strength, muscle power, flexibility, and balance were measured to evaluate basic physical fitness. all participants were given a full explanation about the correct posture and procedure of each measurement. hand-grip strength was evaluated using a grip-d grip strength dynamometer (takei, japan) with 0.1 kg accuracy of both the right and left hands. flexibility was evaluated by measuring sit and reach using a helmas iii trunk forward flexion instrument (o2run, korea) with 0.1 cm accuracy. muscle power was evaluated by measuring the sargent jump height using a helmas iii sargent jump instrument (o2run, korea) with 0.1 cm accuracy. balance was evaluated by measuring the eyes-closed single-leg standing time using a helmas iii blind single-leg stand instrument (o2run, korea) with 0.1 sec accuracy, and the participants performed the test using their preferred leg. pulmonary function tests were conducted under standard laboratory conditions (temperature: 2225 c, relative humidity: 5560%). all spirometric tests were conducted by the same technician to reduce inter-observer variability and to prevent the failure of the measurement due to the young age of the subjects. the participants were given sufficient explanation about the method and instrument use, and the tests were performed in a sitting position while wearing a nose clip. the forced vital capacity (fvc) and the forced expiratory volume in one second (fev1) were measured using a quark pft (cosmed, italy). all of the pulmonary tests were conducted following the standards presented by the american thoracic society/european respiratory society16. the data were analyzed using the statistical package for the social sciences (spss version 22.00) and the results are presented as the mean standard deviation. the significance of differences between boys and girls were examined using the independent t-test. the relationships between body composition and pulmonary function, and basic physical fitness and pulmonary function were analyzed using simple linear regression analysis. table 1table 1.the differences in body composition between groups (gender and age) and within group (n=240)groupsage (yrs)height (cm)weight (kg)bmi (kg/m)body fat (%) malefemalemalefemalemalefemalemalefemaleperiod of early elementary school6123.7 4.43121.6 4.9624.8 4.5622.9 3.4816.1 2.0415.4 1.748.9 3.0113.0 5.067127.7 4.66126.2 3.9728.8 4.3726.2 5.0717.6 2.2016.4 2.619.6 4.4216.5 3.618133.0 4.57133.9 5.4631.7 6.6730.8 5.4117.8 2.9117.2 2.4310.5 6.1717.9 3.461,2<3**1<2<3***1<3*1,2<3**period of late elementary school9137.3 4.62139.6 5.7135.9 7.7634.8 6.7718.9 3.1817.6 2.2313.1 5.9617.8 4.5110143.9 5.80147.1 9.4245.2 11.9236.8 10.1121.6 4.1216.7 2.8818.2 7.8017.0 4.7611155.5 6.65154.5 2.8648.2 11.5946.8 6.1419.9 3.9919.6 2.2014.7 3.8221.4 5.171<2<3***1<2<3***1<3*1,2<3**period of middle school12164.9 6.43158.3 4.9149.3 10.8347.4 6.9519.5 2.9718.9 2.5913.1 8.4720.4 4.0813165.9 3.41159.1 3.3055.1 11.1250.8 6.4520.0 3.9320.3 2.1612.8 7.6021.0 5.8114169.6 5.17158.0 3.0864.9 10.9153.2 5.4421.7 1.7921.3 2.0716.8 3.9721.1 6.401,2<3*period of high school15176.5 7.30164.1 3.8160.3 7.9258.6 2.2419.3 1.4921.8 1.5311.5 4.8126.1 2.9416174.3 5.63162.5 5.8057.6 3.5453.1 5.8720.6 0.5720.0 1.4514.7 3.0024.2 2.3617172.8 4.01163.1 5.0162.4 8.1857.0 5.3220.9 2.3821.4 1.5715.5 5.8326.1 2.06values represent means sd. bmi: body mass index. *: significant difference within group (* p<0.05,** p<0.01,***p<0.001) shows the differences in body composition between groups (gender and age) and within groups. height was significantly different in the early period of elementary school (boys: p<0.01, girls: p<0.001) and the late period of elementary school (p<0.001 for both boys and girls). weight was significantly different in the periods of early and late elementary and middle school for boys (p<0.05), and in the periods of early and late elementary school for girls (p<0.01). *: significant difference within group (* p<0.05,** p<0.01,***p<0.001) table 2table 2.the differences in basic physical fitness between groups (gender and age) and within group (n=240)groupsage (yrs)basic physical fitnesspulmonary functionright hand grip (kg)left hand grip (kg)sit and reach (cm)sargent jump (cm) single-leg stance (sec)fvc (l)fev1 (l)boysperiod of early elementary school68.42.508.81.954.75.2823.23.808.46.071.360.321.300.20710.62.3610.52.678.05.8326.64.5615.911.371.730.451.540.4089.32.6010.92.594.44.8226.02.5910.97.181.840.351.720.44period of late elementary school912.51.4714.13.325.66.3528.13.6925.819.002.000.381.850.411017.82.7517.02.765.66.7230.15.6544.224.672.550.442.220.321119.14.1018.73.897.99.1738.56.5933.118.672.590.532.390.531<3***1<2,3*1,2<3**1<2,3*1<3*period of middle school1220.97.5221.89.112.29.6238.55.5226.317.333.430.703.190.621326.64.3029.94.385.27.6141.26.5626.222.533.540.563.530.291436.69.3937.97.137.89.1145.35.7530.813.514.270.493.840.511<2,3**1,2<3**1,2<3*period of high school1531.96.1232.67.117.05.6343.64.4131.425.804.180.654.010.571636.30.3538.50.8512.12.2543.50.5052.56.504.600.724.390.661736.83.0438.32.8611.59.5147.69.8236.422.784.120.703.850.65girlsperiod of early elementary school67.71.878.52.059.43.3122.14.1411.17.901.460.261.230.2178.81.589.21.9510.83.7825.96.0916.315.781.430.271.310.2789.31.829.33.456.13.7525.03.134.32.091.660.281.570.283<1*period of late elementary school910.23.0310.93.226.46.2327.94.4223.917.791.830.411.580.311012.72.6813.84.009.88.5430.04.8329.621.722.040.591.930.531116.83.5317.43.9910.87.4030.48.6735.422.812.540.392.330.341<3***1,2<3**1<3*1<3*period of middle school1218.92.5520.01.9313.48.5333.65.8512.65.732.630.442.490.371321.74.8322.64.8310.38.7329.82.5727.820.372.610.322.580.241420.12.7021.73.6311.78.3428.44.0337.422.332.510.572.300.50period of high school1523.62.4225.53.9910.911.3129.87.8023.319.162.780.372.680.371622.23.9324.14.0415.310.6629.64.1330.215.853.010.472.850.371720.23.2323.03.4110.311.7028.95.0524.76.582.920.212.810.16values represent means sd. fvc: forced vital capacity, fev1: forced expiratory volume in 1 second. *: significant difference within group (* p<0.05,** p<0.01, ***p<0.001) shows the differences in basic physical fitness and pulmonary function between and within groups. for boys, right and left hand-grip strength were significantly different in the late period of elementary school (right: p<0.001, left:<0.05) and middle school (p<0.01). the sargent jump height was only significantly different in the late period of elementary school (p<0.01) for boys. in the case of girls, right and left hand-grip strength significantly different in the late period of elementary school (p<0.01), and the sit and reach distance was significantly different in the early period of elementary school (p<0.01). for boys, fvc was significantly different in the late period of elementary school and middle school (p<0.05), and fev1 was significantly different only in the late period of elementary school (p<0.05), and for girls, fvc and fev1 were significantly different in the late period of elementary school (p<0.05). fvc: forced vital capacity, fev1: forced expiratory volume in 1 second. *: significant difference within group (* p<0.05,** p<0.01, ***p<0.001) table 3table 3.the results of simple linear regression analysis between pulmonary function and body compositionfvcfev1rrboysheight0.827***0.758***weight0.677***0.658***bmi0.168***0.144***body fat (%) 0.048*0.037girlsheight0.756***0.764***weight0.728***0.733***bmi0.455***0.462***body fat (%) 0.392***0.400**** p<0.05,** p<0.01,***p<0.001 shows the results of the simple linear regression analysis that was performed for fvc and fev1 against height, weight, bmi, and body fat (%). for boys, fvc significantly correlated with height (r=0.827, p<0.001), weight (r=0.677, p<0.001), bmi (r=0.168, p<0.001), and percent body fat (r=0.048, p<0.05) in descending order. fev1 significantly correlated with height (r=0.758, p<0.001), weight (r=0.658, p<0.001), and bmi (r=0.144, p<0.001); however, there was no significant correlation with percent body fat (r=0.037, p=0.077). in the case of girls, fvc significantly correlated with height (r=0.756, p<0.001), weight (r=0.728, p<0.001), bmi (r=0.455, p<0.001), and percent body fat (r=0.392, p<0.001) in descending order. fev1 significantly correlated with height (r=0.764, p<0.001), weight (r=0.733, p<0.001), bmi (r=0.462, p<0.001), and percent body fat (r=0.400, p<0.001) in descending order.*p<0.05,** p<0.01,***p<0.001 the results of the comparison between boys and girls revealed that height and weight were similarly correlated. however, bmi (fvc: 0.455 vs. 0.168, fev1: 0.462 vs. 0.144) and percent body fat (fvc: 0.392 vs. 0.048, fev1: 0.400 vs. 0.037) showed higher correlations with pulmonary function in girls than in boys. table 4table 4.the results of simple linear regression analysis between pulmonary function and basic physical fitnessvariablefvcfev1rrboysright hand grip strength 0.774***0.794***left hand grip strength 0.747***0.762***sit and reach distance 0.0420.039sargent jump height0.573***0.584***single-leg standing time 0.058*0.048*girlsright hand grip strength 0.619***0.652***left hand grip strength 0.607***0.641***sit and reach distance 0.0200.015sargent jump height0.129**0.121**single-leg standing time 0.0180.020*p<0.05,** p<0.01,***p<0.001 shows the results of the simple linear regression analysis that was performed for fvc and fev1 against right hand-grip strength, left hand-grip strength, sit and reach distance, sargent jump height, and single-leg standing time. for boys, both fvc and fev1 showed high correlations with right hand-grip strength (r=0.774, r=0.794, p<0.001), left hand-grip strength (r=0.747, r=0.762, p<0.001), sargent jump height (r=0.573, r=0.584, p<0.001), and single-leg standing time (r=0.058, r=0.048, p<0.05) in descending order. in the case of girls, both fvc and fev1 showed high correlations with right hand-grip strength (r=0.619, r=0.652, p<0.001), left hand-grip strength (r=0.607, r=0.641, p<0.001), and sargent jump height (r=0.129, r=0.121, p<0.01) in descending order, whereas the sit and reach distance and single-leg standing time showed no significant correlations.*p<0.05,** p<0.01,***p<0.001 the results of boys and girls were similarly in descending order. however, basic physical fitness parameters were more highly correlated with pulmonary function for boys than for girls, especially the sargent jump height (fvc: 0.573 vs. 0.129, fev1: 0.584 vs. 0.121). the purpose of this study was to determine the relationship between basic physical fitness and pulmonary function in healthy korean school students, in order to present an alternative method for improving their pulmonary function. an investigation of the development of korean students was conducted by the ministry of education in 2014. the results show that boys grow 56 cm per year from an average height of 121.5 cm in the first grade of elementary school. the biggest growth was found among 6th grade elementary school students and 1st grade middle school students. girls grow around 6 cm per year from an average height of 120.3 cm in the first grade of elementary school. their degree of growth decreases rapidly after 6th grade elementary school compared to boys. body weight of boys showed the biggest differences between the first and second grades of middle school (5.9 kg), and between the 5th and 6th grades of elementary school among girls (5.5 kg). the results of the present study show that the height of both boys and girls rapidly increased in the elementary school period. however, weight dramatically increased in the period of elementary school and middle school among boys, and in the period of elementary school among girls. the growth of children and adolescents who participated in this study seems to be representative of korean children and adolescents since the results appear to be similar to the findings of the ministry of education. however, bmi was somewhat different since we selected healthy students, excluding obese children as subjects. basic physical fitness is closely related to health. especially, it has a negative correlation with the prevalence of obesity17, hypertension18, and cardiovascular disease19. in this respect, the korean government has implemented the physical activity promotion system (paps) in order to systematically measure the physique and fitness, as well as to prescribe physical activity for individuals, highlighting the need of regular exercise. however, accurate measurement, evaluation, and prescription of exercise seem insufficient. in the present study, basic physical fitness was measured using the correct postures and methods by a fully trained technician. muscle strength increased sharply in the late period of elementary and middle school in boys, and in the late period of elementary school in girls. in addition, muscle power of boys increased sharply only in the late period of elementary school. pulmonary function was reported as being correlated with age20, 21, in addition, height, weight, area of body surface, percent body fat, smoking status, and residential environment also have effects on spirometry22,23,24,25. height is a factor positively influencing lung function at all ages26, whereas age and body fat mass sometimes have an inverse correlation in accordance with the age of subjects. the present study revealed that pulmonary function increased with age for subjects during the growth period. in addition, pulmonary function was influenced by height, weight, bmi, and percent body fat, in descending order. height, area of body surface, and weight correlate with pulmonary function in descending order27, a result which is consistent with the results of our present study. girls showed higher correlation than boys between percent body fat and pulmonary function in the present study. however, the percent body fat showed slight or no correlation with pulmonary function for boys, a result which differs from those of previous studies. rossi et al.28 found that body fat and lung function showed an inverse correlation in obese female adults. gonzalez-barcala et al.29 conducted a study of children aged 6 to 18 years whose bmi was under 30 kg/m. they suggested that lung function can be difference depending on individual differences. in the present study, subjects had normal weight and bmi<25 kg/m. park et al.30 reported that it was difficult to explain the effects of bmi, percent body fat, and muscle mass on pulmonary function. therefore, different results of pulmonary function could be occurred depending on the body composition of the individual subjects. thus, aerobic exercise and resistance exercise are effective ways of improving lung function. besides the present study revealed that pulmonary function is highly correlated with right hand-grip strength, left hand-grip strength, and sargent jump height in descending order. therefore, exercises for muscle strength and power could be effective at improving school students pulmonary function. in summary, in order to improve the pulmonary function of growing children and adolescents, aerobic exercise and exercise programs to increase muscle strength and power are needed, and they would be especially effective if they were to begin in the late period of elementary school when the muscle strength and power of students are rapidly increasing.

[ purpose] the purpose of the present study was to determine whether there was a correlation between basic physical fitness and pulmonary function in korean school students, to present an alternative method for improving their pulmonary function. [subjects and methods] two hundred forty healthy students aged 617 years performed physical fitness tests of hand-grip strength, sit and reach, sargent jump, single leg stance, and pulmonary function tests of forced vital capacity (fvc) and forced expiratory volume in one second (fev1) using a quark pft. [results] muscle strength and power of boys improved in the late period of elementary school and middle school. muscle strength of girls improved in the late period of elementary school. analysis of factors affecting pulmonary function revealed that height, weight, bmi, and body fat significantly correlated with spirometric parameters. right hand-grip strength, left hand-grip strength, and sargent jump also significantly correlated with fvc and fev1. [conclusion] in order to improve the pulmonary function of children and adolescents, aerobic exercise and an exercise program to increase muscle strength and power is needed, and it should start in the late period of elementary school when muscle strength and power are rapidly increasing.

PMC4616070

pubmed-416

noise-induced hidden hearing loss (nihhl) refers to any functional impairment seen in subjects with noise exposing history but no permanent threshold shift (pts). this is different from the conventional definition of noise-induced hearing loss (nihl), which is based on changes in auditory sensitivity or threshold shift. therefore, noise exposure recommendations are based on the likelihood that a particular dose of exposure will result in a pts. physiologically, variations in auditory sensitivity following exposure to noise are largely due to the functional status of outer hair cells (ohcs) in the cochlea, which provide mechanical amplification of soft sounds [2, 3]. noise exposures that result in only a temporary threshold shift (tts) have a reversible impact on ohc function, which is manifested by the recovery of otoacoustic emissions (oae) [46] and cochlear microphonics (cm) [711]. the functional changes in these measures parallel the recovery of hearing thresholds, as well as the repair of structures such as stereocilia and the tectorial membrane [7, 12]. by contrast, noise exposure at higher levels and/or for longer durations can cause permanent damage to, or even the death of, ohcs and, hence, lead to pts. therefore, the ohcs and the structures surrounding them, including the tectorial membrane and the supporting cells, are considered to be the major loci of cochlear damage that result in noise-induced threshold shifts [13, 14]. although some early reports claimed that reversible noise-induced ihc pathologies were responsible for tts [15, 16], ihcs are relatively insensitive to noise-induced cell death. however, it has long been recognized that the synapse between ihcs and primary spiral ganglion neurons (sgns) can be damaged by noise [1719]. these early studies showed that this manifests mainly as damage to the postsynaptic terminals; however, there is clear evidence from more recent studies that noise induces damage to both pre- and postsynaptic structures. more importantly, disruption of the synapses can be permanent, resulting in degenerative death of sgns. the finding that damage to ribbon synapses can occur without pts is significant because of the potential impact of such damage on hearing function. because the physiological damage is not accompanied by a permanent shift in hearing threshold, it would likely be missed by a standard (i.e., threshold-based) hearing assessment and has thus been referred to as nihhl. nihhl first manifests as reduced output of the auditory nerve at high sound levels, without affecting the hearing threshold. this reduction has been found in both animals [6, 2023] and human subjects with a history of noise exposure but with normal audiograms. since the thresholds of the auditory nerve remain unchanged, the function relating compound action potentials (cap) amplitude with sound levels in nihhl animal is different from that in animals with threshold changes. schematic curves of cap input/output functions are presented in figure 1 for a comparison across normal control and those with different pathologies. theoretically, if the damage is restricted to ohcs, the major change in cap input/output (i/o) curve is restricted around threshold and the amplitude reaches the control value at high sound levels. in the case of nihhl, cap reduction is mainly at high sound level, with no difference at low sound level, suggesting a suprathreshold deficit. when the damage occurs at both ohcs and the ihc-sgn synapses, the reduction of cap amplitude is seen across all sound levels. as nihhl is initiated at the synapse between the ihcs and sgns, which silences the auditory nerve fibers (anfs) that extend from them, the corresponding disorder is categorized as a cochlear neuropathy (i.e., cochlear synaptopathy) [25, 26]. presumably, the reduction in the amplitude of the auditory nerve response without threshold elevation is due to selective loss of anfs that have high thresholds, which is supported by single-unit recording studies [20, 27]. given the important features of those low-spontaneous-rate anfs in auditory coding, the neuropathy or synaptopathy in hidden hearing loss is not simply a reduction in the number of functional anfs. furthermore, the synaptopathy in nihhl is likely to be related to the synaptic repair after initial damage by noise, rather than a simple initial loss. in addition, the functional deficits seen in nihhl may also involve the contribution from central auditory plasticity [26, 2832]. in this review, we summarize the available data for noise-induced damage and repair around ihc sgn synapses and discuss the evidence for the contributions of cochlear malfunction and central plasticity to nihhl. accumulated evidence has shown that the synapses between ihcs and type-i sgns are sensitive to noise and the damage to this synapse is likely to be the bases for nihhl. the synapse is characterized by presynaptic dense bodies termed ribbons [3335], which are spherical or ellipsoidal in shape, 100200 nm in diameter, and surrounded by synaptic vesicles. the ribbons are built up from ribeye protein subunits [37, 38] and anchored to the active zone of the presynaptic membrane via bassoon proteins [3941]. the functional role of ribbons has been recognized as tethering and conveying synaptic vesicles to the active zones [42, 43], where the release of neurotransmitters at these synapses is modulated by a specific l-type calcium ion channel (i.e., cav1.3) [44, 45]. noise exposure causes damage to both the presynaptic ribbons and postsynaptic nerve terminals of the ribbon synapses [6, 22, 23, 4648]. the damaged synapses exhibit various degrees of swelling of the terminals, resulting in disruption of the synaptic connections between ihcs and sgns [20, 46, 48]. immunohistological staining has revealed similar losses for ribbons and terminals [6, 22, 23, 49]. the mechanism for the damage to the postsynaptic terminal is glutamate-mediated excitotoxicity (reviewed in). one possible mechanism of ribbon loss is the loss of cell-cell contact that is required for the maintenance of the pre- and postsynaptic complexes [5053]. our electron microscopy evaluations did not reveal any residual presynaptic complexes without ribbon and postsynaptic terminals. therefore, it is likely that the entire presynaptic structure breaks down when the postsynaptic terminal is damaged. a brick assembly model, in which a ribbon is built up from multiple ribeye subunits, has been proposed for ribbon construction in retina photoreceptor cells. moreover, the ribbons in retina sensorial cells can be partially broken down by light, but they rapidly reassemble in the dark, probably serving as a mechanism of adaption to bright light [5458]. in the retina, the ribbon size appears to be a determining factor for the quantity of neurotransmitter released. however, the dynamic disassembling/reassembling process has not been identified in the cochlea, and changes in the ribbon size and the relationship with the release of neurotransmitters have not been investigated in the cochlea. additionally, disassembly and reassembly, as well as ribbon size, are modulated by ca signaling involving cav-channels, presynaptic ca levels and storage, and guanylate cyclase-activating protein-2 (gcap2; see the review by schmitz). interestingly, optical stimulation of photoreceptors causes hyperpolarization of the presynaptic membrane and a decrease in [ca]i, as opposed to depolarization and the large increase in [ca]i in ihcs in response to sound. the decrease in [ca]i in photoreceptor cells is followed by a conformational change of gcap2, which results in the disassembly of the ribbons. in the cochlea, it is not known whether there is a gcap-mediated pathway that controls ribbon size. as the membrane potential of ihcs is depolarized with increasing sound levels, resulting in an influx of ca, the role of ca in ribbon assembly is unlikely to be the same as it is in the retina. the first quantitative study of noise-induced ribbon synapse damage in cba mice reported that the number of ribbon synapses was reduced to 40% compared with the control 1 day after brief noise exposure that did not lead to pts. the synapse count recovered to 50% within 1 week, but no further recovery was observed, and this 50% loss of synapses was considered permanent. sgn death observed 2 years after the noise was found to match the 50% permanent loss of synapses. however, a study on guinea pigs carried out by the same research group found a similar loss of ribbon synapses 2 weeks after exposure to noise that did not cause pts, but this study found a much smaller final loss of sgns. this suggests that some sgns, which had originally lost their synapses with ihcs, survived and reestablished synapses with ihcs. our studies on guinea pigs have revealed a clear recovery in the synapse count following a massive initial loss induced by noise exposure that did not lead to pts [22, 23]. although this recovery was not complete, approximately 50% of the initial loss of paired ribbon and postsynaptic density (psd) puncta in the basal half of the cochleae was seen 1 day after noise, and the loss was recovered to<20% within 1 month. comparing the aforementioned data from mice and guinea pigs, it appears that there may be some species difference in the ability to regenerate synapses following noise-induced hearing damage. however, a recent study of c57 mice reported that the loss of ribbon synapses induced by non-pts-inducing noise was largely reversible. this discrepancy in synapse regeneration following noise exposure requires further investigation. in a recent review, it was argued that the recovery of ctbp2/psd counts in guinea pig cochleae following noise exposure reported in our studies may be attributable to up/downregulation of the synaptic protein rather than regeneration of synaptic connections. however, there are several lines of evidence for the possibility of synapse repair following noise-induced damage. first, it has been reported that plastic changes occur in the presynaptic component, including the existence of multiple presynaptic ribbons around an active zone and the changes in the size and location of ribbons following noise exposure. second, the change in the amplitude of the compound action potential (cap) corresponded to the changes in ribbon/psd counts: a large initial reduction in cap amplitude and synapse counts were followed by a significant recovery after the noise exposure. third, changes in many single-anf coding activities were not seen at the time that the synapses were damaged but rather manifested later (see section 3) with the recovery in both the cap and synapse number, suggesting that those changes occurred in the anfs that connect ihcs via repaired/reestablished synapses. further work is required to determine the mechanisms and factors that influence the repair of both pre- and postsynaptic components. ribbon synapses exhibit spatial differences around ihcs; that is, the synapses at the modiolar side of an ihc have relatively small ribbons but larger postsynaptic terminals, whereas those at the pillar side have relatively large ribbons but smaller terminals. liberman et al. reported that anfs are functionally categorized by their spontaneous rate (sr), which is inversely related to the fiber's threshold and dynamic range [6365]. it is widely accepted that low-sr anfs exhibit synapses with ihcs on their modiolar side, whereas high-sr units exhibit synapses on the pillar side (this is based on data obtained using intracellular tracer injections). the low-sr units are considered critical for hearing in noisy environments due to their larger dynamic range, higher thresholds, and the ability to follow the quick change of the amplitude of acoustic signals. by contrast, high-sr units are responsible for the sensitivity to quiet sounds and are saturated by high-level background noise [26, 63, 64, 67, 68]. in nihhl, low-sr anfs appear to be more vulnerable to noise than high-sr units. selective loss of low-sr anfs has been found following exposure to noise that did not lead to pts. presumably, this selective loss of low-sr units should produce coding deficits, which can be predicted based on the unique features of those units. however, no coding deficits were examined and reported in this study. on the other hand, we reported a time delay in the development of coding deficits by single anfs in guinea pigs following a similar noise exposure that did not cause pts; these deficits were attributed to intensity coding and temporal coding as summarized in sections 3.1 and 3.2. intensity coding in the cochlea is defined as the ability of anfs to encode the sound intensity or the change of sound intensity. this ability is determined primarily by the spike rate (or the change of spike rate) of individual anf in response to sound intensity change and the number of functional anfs. therefore, the intensity coding deficits can be evaluated in both evoked field potential and single-unit recordings. deficits in intensity coding were first suggested by a reduction in wave i of the auditory brainstem response (abr) [6, 49], as well as a reduction in the amplitude of the cap, as this is likely due to the loss of functional anfs following synapse disruption. the fact that the reduction is more significant at higher sound levels has been considered evidence for selective damage to low-sr fibers, which have higher thresholds [25, 26, 69]. the deterioration in intensity coding following no-pts noise exposure was manifested as a reduction in the driven spike rates (peak, sustained, and total rates) of anf units that were tested only at one sound level. such changes are significant only in low-sr anf units and are seen at a later time rather than immediately following exposure. this time delay in the development of coding deficits suggests that (1) the reduction in driven spike rates occurs in the anfs to which the synaptic connections to the ihcs are reestablished following the initial disruption and (2) the repaired synapses are functionally abnormal, with less efficient neurotransmitter release. temporal processing ability in the cochlea as well as in the whole auditory pathway is defined as the ability to follow the quick change of acoustic signals. in human subjects, the process involves both bottom-up and top-down mechanisms; but in animal models, only bottom-up mechanisms are tested (see reviewed by). many different tests have been used to detect the bottom-up mechanisms of temporal coding, some of them based on the peristimulatory changes of firing rate showing latency and adaptation. as reviewed above, the major function of presynaptic ribbons in ihcs is to facilitate the synaptic transmission. indeed, such deficits were manifested as an increase in response latency of anfs in animals with nihhl. this was first demonstrated as a significant delay in cap peak latency and then further supported by the delayed latency of peak in psth (or peak latency) of anfs in our single-unit study. in another very recent report, such delay was reported in abr as the marker of cochlear synaptopathy. we also found a reduction in the ratio of peak to sustained rates in animals that were exposed to noise. this ratio is considered an index of the ability of a neuron to encode dynamic signal changes (see review by). using a paired-click paradigm, we found that the anf response of noise-exposed animals to the second click recovered more slowly from the masking effect of the first click. these results reveal poorer coding to the transient features of acoustic signals by anfs, which were examined in previous studies to show the deterioration in of temporal coding in animals with bassoon mutation [39, 41]. whereas an increase in peak latency was seen shortly after exposure to noise, changes in the peak rate and the peak/sustained spike ratio, as well as a slower recovery of the spike rate to the second click, were not seen until later, suggesting an association between the deficits and the synapse repair. a temporal deficit in phase-locking responses has been proposed based on selective loss of low-sr units and the functional features of this group of anfs [25, 26, 69], but it has not been tested at the single-unit level. so far, there appear to be two models for the development of coding deficits in nihhl. one model suggests that the coding deficit or synaptopathy is simply due to the loss of low-sr anfs. since those units have unique functions in signal coding, the loss of those functions is predicted as the consequences. that is, the coding deficits are developed as the result of unhealthy synaptic repair after initial disruption. we found that the noise-induced synaptic damage in guinea pigs under nihhl is largely repairable, leaving only a small amount of synapses not being reestablished. therefore, the coding deficits or synaptopathy can not be simply attributed to the loss of sr units. since the coding deficits are seen at the time when the synapse counts are largely recovered, we believe that the coding deficits likely occur in the repaired synapses (most of them innervating low-sr anfs). studies are needed to verify which model is more likely the case in human subjects. it has long been recognized that subjects with normal audiograms may have perceptual difficulties, and this is especially true in the elderly. age-related hearing loss with threshold elevation is termed peripheral presbycusis, whereas the perceptual difficulties seen in the elderly without threshold shift are usually termed central presbycusis. for example, temporal processing deficits and difficulties of hearing in noisy environments are two major problems experienced by the elderly. these problems were recognized long before the discovery of cochlear damage associated with nihhl and were considered to be the result of central auditory processing disorders [7175]. it was generally accepted that any perceptual deficits observed without changes to hearing thresholds and cognitive functioning can be attributed to central dysfunctions. based on recent progress in functional deficits in cochlear coding, such separation between peripheral and central presbycusis is likely to be incorrect. the so-called central presbycusis may, at least in part, result from disorders in the auditory periphery. the coding deficits related to the loss of low-sr anfs had been described as a type of auditory neuropathy and/or synaptopathy even before any of the predicted deficits were identified. data on changes in the sr distributions of anfs suggest the reestablishment of synapses following an initial disruption that was selective to low-sr units. although our data revealed abnormalities in some aspects of coding in the auditory nerve, further work is required to investigate coding deficits in nihhl. such studies can not be replaced by speculation based on the selective loss of low-sr fibers; for example, one can not be certain how the auditory nerve changes its response to amplitude modulation until it is measured at the single-unit level. two possibilities must be considered: (1) the surviving anfs may change their function and (2) the initially lost low-sr fibers may be repaired but with changed function. it should be noted that there is now a tendency in the literature to consider nihhl to be a purely peripheral issue, a result of the overcorrection of the central presbycusis. however, despite the strong evidence for a peripheral contribution, the central contribution to the problems seen in nihhl should not be neglected. in other words, it may be more constructive to assume that there are both peripheral and central contributions to nihhl. it is well known that hearing loss (with elevated threshold) can induce central changes, which can result in deteriorations in signal processing. studies aiming to distinguish the role of central plasticity from that of ribbon synapse damage are rare. one such report found that an increase in central gain was responsible for tinnitus in human subjects with typical damage seen in nihhl (i.e., reduced auditory nerve input to the brain (measured as a smaller abr wave i)) but normal hearing threshold. in an earlier study in rats, tinnitus was found 6 months after exposure to noise that caused minimal loss of hair cells and pts but significant loss of anfs. one of the central impacts of hearing loss due to damage to peripheral auditory organ is imbalance between excitation and inhibition, resulting in hyperactivity and/or hyperresponsiveness in the central auditory system (see reviews in [7780]). the types of hearing loss producing such central enhancement include cochlear ablation, drug- and noise-induced damage. while direct effect of drugs and noise on central neurons needs to be differentiated, a similarity across those hearing loss models is the reduction of cochlea output to the auditory brain, which may be the main initial factor causing the imbalance between excitation and inhibition. in this sense, while most of studies in central plasticity using nihl model correlated the central enhancement with the amount of threshold shifts [29, 8185], at least one study has reported central enhancement in mice exposed briefly to noise at a moderate level that did not cause pts, presumably producing only nihhl. unfortunately, the reduction in auditory input from the cochlea was not quantified in this study. taken together, available data suggest that cochlear damage, with or without threshold elevation, can lead to central plasticity by reducing input from the auditory nerve. further work is required to establish the central contribution to coding/perception difficulties in nihhl, and previous studies on central processing disorders in subjects with nihl should be reevaluated to differentiate the central contributions from the peripheral ones. in a brief summary, we use figure 2 to summarize the available data for the mechanisms of perception difficulty experienced by subjects with history of noise exposure but normal or near normal thresholds. in this schematic diagram, we include the two potential models of noise-induced synaptopathy in cochleae. in model 1, the coding deficits are speculated based on the role of low-sr anfs in signal coding. both models result in a reduction in the cochlear output to the auditory brain, which in turn will result in plastic reorganization of the brain. auditory signal processing disorders experienced by subjects with long-term nihhl should include what are inherited from the coding deficits developed in the auditory peripheral and those associated with the plastic changes of auditory brain. although more studies on the impact of noise on human hearing showing no changes in auditory sensitivity are required, evidence suggesting the occurrence of nihhl in human subjects is being accumulated. this is supported by thorough research on the signal perception deficits experienced by subjects with a history of noise exposure but normal thresholds. since the deficits are demonstrated at suprathreshold levels, it is clear that normal hearing thresholds do not guarantee normal hearing functions, especially in subjects with history of noise exposure [24, 86]. the second line of evidence is the reduction in the output of the auditory nerve in subjects with a history of exposure to noise. interestingly, the combination of a reduction in wave i and an increase in wave v/i ratio may be considered evidence of increased central gain and is likely responsible for the generation of tinnitus in hidden hearing loss [32, 87, 88]. the third line of evidence comes from the age-related sgn degeneration seen in the examination of human temporal bones. unfortunately, there is, as yet, no clear human evidence that degeneration of sgns is expedited by exposure to noise that does not cause threshold elevation. the clinical implications of nihhl are manifested by the fact that noise exposure causing nihhl occurs frequently in daily life and impacts much more general population. such noise exposure has been generally considered to be safe according to current safety standards for exposure to noise. the evidence from the studies reviewed here indicates that the resulting damage to the ribbon synapses from noise that did not induce pts can be repaired even though the repair is incomplete. more importantly, the signal coding deficits are developed in association with the synapse repair. since the damage and repair occur repeatedly, the damage on signal coding can be accumulated during aging and likely contributes to the perceptual difficulties experienced by the elderly. this impact of noise exposure on signal coding is obviously different from the contribution made by the hearing loss defined by threshold shifts. in future, the coding deficits and related synaptic repair in nihhl should be further investigated in a laboratory setting. since the ribbon synapse is the first gating point for temporal processing in auditory pathway, the observed coding deficits suggest a clear peripheral origin for the decline in temporal processing and perceptual difficulties during aging. whether and how the synaptic damage will impact the central auditory processing need to be investigated in a manner that is clearly differentiated from the impact of hearing threshold shift. moreover, the coding function of anfs should be observed over a long period of time following exposure to noise to determine whether the coding deficits are temporary or persistent. we are currently collecting data using electron microscopy, as well as conducting an analysis on the potential changes of the molecular structures of ribbons and psds, in an attempt to elucidate the morphological/molecular mechanisms responsible for functional changes of repaired ribbon synapses. it would also be interesting to understand the reasons for the extreme sensitivity of low-sr synapses to noise, as well as elucidate possible methods to prevent damage. laboratory studies should also aim to explore the mechanisms of synaptic repair in the cochlea, as well as reveal the factors that influence repair in order to promote it. to translate the knowledge to clinic, investigation is needed to establish good measures for detecting nihhl in human subjects. although abr wave i is useful for evaluating synaptopathy caused by noise that does not induce pts, its reliability and sensitivity are questionable in human subjects where the abr amplitude is small, and other methods should be explored. a very recent report suggests the use of abr latency as the marker of nihhl. the study tested human subjects with normal hearing thresholds and reported a big variation in the threshold of envelope interaural timing difference, which was negatively correlated with the shift of abr wave v latency by background noise: the higher the threshold (poorer sensitivity), the smaller the shift. the observation of the latency shift with masking is supported by the fact that the low-sr anfs have longer latency than high-sr fibers and are resistant to background noise [91, 92]. it is not clear why the study did not report the change in wave v amplitude by masking. theoretically, the masking should produce greater reduction in wave v amplitude in subjects with selective loss of low-sr units. moreover, no information about the history of noise exposure was reported and it is not clear whether the poorer performance in temporal cue detection was due to noise-induced synaptopathy or other reasons. to date, the most promising methods for diagnosing cochlear synaptopathy are related to selective loss of low-sr anfs, the subcortical steady state responses (sssr) [93, 94]. based on the animal studies, this test should be carried out using amplitude-modulated signals at relatively high intensity and a shallow modulation depth. the input intensity of the driving signal should fall within the saturation range of the high-sr fibers. high frequency carrier waves with a high intensity and with shallow amplitude modulation are especially useful for evaluating the function of low-sr fibers. this is supported by modeling the loss of low-sr fibers. to differentiate the sssr contribution from the auditory nerve from that of central neurons a recent mouse study found that the modulation frequency close to 1 khz was optimum with a high frequency carrier without concern of modulation depth. however, a recent human study reported a successful detection of the low-sr unit loss using off-frequency maskers and a shallow modulation depth.

recent studies on animal models have shown that noise exposure that does not lead to permanent threshold shift (pts) can cause considerable damage around the synapses between inner hair cells (ihcs) and type-i afferent auditory nerve fibers (anfs). disruption of these synapses not only disables the innervated anfs but also results in the slow degeneration of spiral ganglion neurons if the synapses are not reestablished. such a loss of anfs should result in signal coding deficits, which are exacerbated by the bias of the damage toward synapses connecting low-spontaneous-rate (sr) anfs, which are known to be vital for signal coding in noisy background. as there is no pts, these functional deficits can not be detected using routine audiological evaluations and may be unknown to subjects who have them. such functional deficits in hearing without changes in sensitivity are generally called noise-induced hidden hearing loss (nihhl). here, we provide a brief review to address several critical issues related to nihhl: (1) the mechanism of noise induced synaptic damage, (2) reversibility of the synaptic damage, (3) the functional deficits as the nature of nihhl in animal studies, (4) evidence of nihhl in human subjects, and (5) peripheral and central contribution of nihhl.

PMC5050381

pubmed-417

they have been suggested as the main cause of time off from work, reduced school performance, and low quality of life1,2,3. furthermore, they have led to personal, familial and societal burdens, and significant healthcare problems globally4, 5. the prevalence of headaches is estimated at 13% of the united states population6, 20% of the australians1, and migraines are estimated at 11%, with tension-type headaches at 78% of the population world-wide5, 7. according to the international headache society, headaches can generally be divided into two categories which are primary and secondary headaches on the basis of the underlying pathology8. primary headaches are not associated with pre-existing medical conditions and there are three types: migraines, tension-headaches and cluster-headaches6. the remaining headache-sufferers discontinue medications due to adverse side-effects or excessive use of abortive medications. these can lead to a refractory condition of medication overuse headache, which means a consequent worsening of the headaches10. as a result of these shortcomings, complementary and alternative medicine has recently become common practice in current headache management6, 10,11,12,13. yoga exercises are considered to be complementary and alternative medicine and are practiced by approximately 5% of the adult population in the united states and 12% of australians for alleviating headaches14. yoga has been reported as a safe and cost-effective intervention for managing pain1, 14. evidence for the efficacy of yoga exercise for a number of conditions is emerging. a growing body of evidence also supports the belief that yoga benefits physical and psychosocial health through the mechanisms of down-regulation of the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system15,16,17. as a result, yoga plays an important role in reducing sympathetic activity, increasing parasympathetic activity, improving quality of life, and decreasing pain levels18, 19. as stated, there is evidence of the benefit of yoga in reducing pain20, 21. however, rigorous methodology and quality of the evidence needs to be examined to establish whether or not we can assert yoga can be used as a complementary and alternative therapy for sufferers of headaches22, 23. therefore, the aim of this review was to assess the evidence for the effectiveness of yoga exercises in the management of primary headaches. the review was planned and conducted in accordance with the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines24, and the consolidated standards of reporting trials (consort) guidelines for reporting parallel group randomized trials25. the cochrane library, cinahl, embase, psycinfo, pubmed, and koreamed electronic databases were searched to identify rcts published between 1966 and january 2015. all potentially eligible studies were retrieved and the full texts of the articles were reviewed to determine whether they met the following selection criteria. to be eligible, studies had to meet the following conditions. 1) population: participants in the trials had to meet diagnostic criteria according to the international classification of headache disorders, 3rd edition (beta version) published by the international headache society 20138; primary headaches. 2) intervention: randomized controlled trials were included that used yoga as an intervention to review or reduce symptoms associated with headaches or migraines compared with no yoga. 3) outcomes: primary outcomes were headache intensity, frequency, and duration; secondary outcomes were anxiety and depression scores, and symptomatic medication use. quality assessment of the articles was conducted using the critical appraisal, cochrane risk of bias tool for rcts, which was recommended by the cochrane handbook for systematic reviews of interventions26. the cochrane risk of bias tool is a six-item list designed to assess sequence generation, allocation concealment, blinding, incomplete outcome data, selective outcome reporting, and other potential sources of bias. each item is rated as yes, no, or unclear. according to the cochrane handbook, the quality of clinical trials can be divided into three levels27. when the study design fully meets the preceding six criteria, it is considered a level, which means a low risk of bias. b level is assigned when one or more criteria are partly met, and when one or more criteria are not met, the study is defined as c level, implying high risk of bias. a total of 179 titles related to the search terms were screened. among these, there were 32 potential trials identified from chinal, 52 from koreamed, 43 from psycinfo and 52 from the pubmed databases. after the titles had been retrieved a total of 121 studies were excluded either because they were duplicates or they were case studies, commentaries, review articles, or had no target concepts, which means no headaches or migraines. thirty-four potential trials were identified in the search conducted in january 2015. thirty-four potentially relevant papers were retrieved for evaluation of the full text. after evaluation of the 34 full texts, 33 studies were excluded, because 30 studies had no randomized trials and 3 studies had no full text of rct.. the characteristics of the included study are also presented (table 1table 1.characteristics of included randomized controlled trialsauthor, year, location participantsinterventionsoutcome measuresmain resultsadverse events limitationsexperimental groupcontrol grouppopulation sample sizen (n; eg/cg)n mean age (years; eg/cg)n drop out n (%) interventions delivery method duration interventionistjohn et al.,2013, india/ rajasthangeneral person72 (36/36)34.2 (34.3/34.2)7 (9.7)yoga postures, pranayama, kriya 60 min per day, 5 days per week 3 months yoga therapistself-care education once a month 3 months handouts headache intensityheadache frequencyheadache durationanxiety-depression scoressymptomatic medication use(p<0.001)(p<0.001)(p<0.001)(p<0.001)noneabsence of a placebo groups.no blinding.all subjective outcome measures.no long-term follow-up dataeg: experimental group; cg: control group; n: number). flowchart of included studies through the literature searches eg: experimental group; cg: control group; participants were recruited from a headache clinic of the nmp medical research institute by advertising in local newspapers. the participants mean age was 34.2 years, and they had primary headaches with migraines. the yoga program comprised yoga postures, breathing and pranayama, and kriya etc. yoga postures included physical exercises such the stretching of the neck, shoulder and back muscles, followed by relaxation, toning, strengthening, and flexibility. breathing and pranayama means conscious breathing, kriya was practiced as a jalaneti (nasal water cleansing) and kapalbhanti (forced exhalations). program length, frequency, and duration of one trial was 60 minutes a day, 5 days per week for 3 months. one trial was identified that compared a control group with a yoga intervention group and evaluated the effect on headaches. headache intensity (p<0.001), headache frequency (p<0.001), anxiety and depression scores (p<0.001), and symptomatic medication use (p<0.001) were significantly lower in the intervention group then in the control group (table 1). neither included trial reported data on adverse effects of treatment (table 1). assessments of each methodological quality item of the one included trial are described (table 2table 2. methodological quality summary of included trialsstudy, yearrandom allocationallocation concealmentblindingincomplete outcomeselective reportingother biasquality leveljohn et al. the quality of the one trial was level b. +: criteria met; -: criteria not met;?: unclear whether criteria were met the purpose of this review was to assess the evidence for the effectiveness of yoga interventions for primary headaches when compared to no yoga. a meta-analysis combining results from all the trials was not possible because only one study was identified. its interventions included yoga poses, pranayama, and kriya to manage headaches or migraines. one trial reported a significant decrease in headache intensity, headache frequency, anxiety and depression scores, and symptomatic medication use in the trained group. if required, participants were allowed to take acute medication prescribed by neurologists during the trial. the effects of the medication could have diminished the efficacy of yoga exercises for alleviating headaches. in spite of both groups having received medication, reduction in the outcome of the yoga group was significantly higher than that of the control group. as stated in previous studies, these results support yoga practice as a means of evidence-based positive management of headaches or migraines20, 22, 28, 29. the quality rating of the trial included in this review had a moderate methodological quality, and the trial did not mention blinding. however, no strong conclusion can be made due to the number of small trials and other methodological considerations. major strengths of this group of studies include the study, the use of randomization, and the quality of measurement tools. second, the trial did not mention blinding, lack of which may have threatened the internal validity of the trial. third, all the outcome measurements were questioner-based and subjective; objective parameters were lacking. therefore, evidence-based research employing objective outcome parameters is needed to identify the efficacy of integrated yoga therapy for headaches. fourth, the trial had no long-term follow-up data concerning the durability of the treatment effect. finally, the generalizability of the findings was limited due to the number of small trials and their partially limited quality. however, this one trial does provide a strong basis for future studies and suggests that yoga exercises could provide a safe, cost-effective therapy for the growing public health issue of headaches. furthermore, this review contributes to the development of knowledge in physical therapy about how sufferers with primary headaches can manage themselves. in conclusion, although this review retrieved only a limited number of small trials, of partially limited quality, its findings suggest that yoga practice can effectively alleviate symptoms associated with primary headaches. however further rigorous methodological and high-quality rcts are needed to confirm and further comprehend the effects of standardized yoga programs aiming to control pain intensity and frequency, symptoms, and medication use etc.

[ purpose] to assess the evidence for the effectiveness of yoga exercises in the management of headaches. [subjects and methods] a search was conducted of six electronic databases to identify randomized controlled trials (rcts) reporting the effects of yogic intervention on headaches published in any language before january 2015. quality assessment was conducted using the cochrane risk of bias tool. [results] one potential trial was identified and included in this review. the quality critical appraisal indicated a moderate risk of bias. the available data could only be included as a narrative description. headache intensity and frequency, anxiety and depression scores, and symptomatic medication use were significantly lower in the yoga group compared to the control group. [conclusion] there is evidence from one rct that yoga exercises may be beneficial for headaches. however, the findings should be interpreted with caution due to the small number of rcts. therefore, further rigorous methodological and high quality rcts are required to investigate the hypothesis that yoga exercises alleviate headaches, and to confirm and further comprehend the effects of standardized yoga programs on headaches.

PMC4540885

pubmed-418

staphylococcus (s.) intermedius and s. pseudintermedius are both staphylococcus intermedius group (sig) members. these microorganisms are opportunistic pathogens that cause skin and nosocomial infections in dogs and cats. s. pseudintermedius is the predominant sig species in korea and until recently was misclassified as s. intermedius. increased identification of methicillin-resistant sig (mrsig) isolates has emphasized the importance of public health precautions for veterinary staff and pet owners. many current studies have evaluated methicillin-resistant characteristics and the staphylococcal cassette chromosome mec (sccmec) of sig isolates, particularly s. pseudintermedius. although type i to type viii sccmec types have been identified, there is no significant studies have been conducted to examine sccmec types isolated from veterinary hospitals located nationwide in korea. sccmec types may produce staphylococcal exotoxins including staphylococcal enterotoxins (ses), exfoliative toxins (ets) including s. intermedius exfoliative toxin (siet), and toxic shock syndrome toxin 1 (tsst 1); these toxins are associated with atopic dermatitis, mastitis, food poisoning, canine pyoderma, and chronic otits. siet (encoded by the siet gene), first described by terauchi et al., plays a potential role in the pathogenesis of canine pyoderma and chronic otitis. there may also be a greater chance of sig isolates to colonization and/or expand in veterinary staff, companion animals, and hospital environments. therefore, this study is focused on the genetic identification of staphylococcal exotoxins, sccmec types, and genetic relatedness by pulsed-field gel electrophoresis (pfge) of the sig isolates in korea. samples of s. pseudintermedius (n=167) and s. intermedius (n=11) were isolated; their identity was confirmed by gram-staining and biochemical testing such as coagulase, dnase production and hydrolysis. polymerase chain reaction (pcr) was carried out with primers targeting s. intermedius nuclease and 16s rrna genes. to differentiate s. pseudintermedius from s. intermedius and s. delphini, pcr-restriction fragment length polymorphism analysis was performed using pta gene-specific primers (pta_f1:5'-aaa gac aaa ctt tca ggt aa-3 ', and pta_r1: 5'-gca taa aca agc att gta ccg-3 '), and the restriction enzyme mboi (new england biolabs, usa). samples were collected from eight veterinary hospitals from four different regions in korea between november 2006 and january 2010 as previously described. pcr analysis to detect the methicillin-resistance gene (meca) pcr was also performed to amplify genes encoding sea (sea), seb (seb), sec (sec), sed (sed), see (see), seg (seg), seh (seh), sei (sei), tsst 1(tsst-1) and ets (eta, etb, etd, and siet) using primers and pcr conditions used in other previous studies. fri913, fri 361, fri 472, fri 569, mnhoch, and rn4220 strains were used as either positive or negative controls for superantigen genes. a superantigen gene obtained in this study was further analyzed by dna sequencing using the vector nti align x program (invitrogen, usa). all mrsig isolates including 49 methicillin-resistant s. pseudintermedius (mrsp) and three methicillin-resistant s. intermedius (mrsi) were subsequently classified as sccmec type i to viii by either single or multiplex pcr assays using protocols in other previous studies including primers and pcr conditions. genomic dna plug kits (bio-rad, usa) with smai (new england biolabs, usa) digestion were used for the pfge analysis of 39 s. pseudintermedius isolates from a private referral veterinary hospital collected at different times (eight isolates from november 2006, 24 isolates from april 2008, four isolates from june 2009, and three isolates from october 2009) and six s. intermedius isolates (all collected during april 2008). pfge was performed according to protocol used in previous study and the manufacture's instruction (bio-rad, usa) except for an initial pulse time of 5 sec and final time of 40 sec for 21 h. analysis of the sig isolates (n=178) for ses and tsst 1 genes showed that only a single s. pseudintermedius isolate from a veterinary staff member (h1-23) harbored the enterotoxin c (sec) gene (fig. 2) revealed that the amplified sec gene the canine type c se gene (seccaine). a total of 166 out of 178 sig isolates (155 s. pseudintermedius and 11 s. intermedius) isolated from veterinary staff members (n=38, 95%), companion animals (n=107, 93%), and veterinary hospitals (n=21, 91.3%) harbored siet originally detected in s. intermedius. however, other et genes such as eta, etb, and etd, known to originate from s. aureus, were not identified. sccmec typing of the 49 mrsp isolates identified 38 type v isolates, one type iv isolate, and 10 non-identifiable isolates. pfge analysis showed that sig isolates from a private referral animal hospital (collected between 2006 and 2009) recovered from veterinary staff, companion animals, and the environment had the same band patterns (fig. 3). six s. pseudintermedius isolates (two from companion animals collected in september 2006, three from veterinary staff members, and one from companion animals collected in april 2008) and two s. pseudintermedius isolates (one from a veterinary hospital environment isolated in april 2008, and one from a companion animal isolated in june 2009) showed the same band patterns (fig. since sig is closely related to s. aureus, studies have been performed to determine whether sig has adapted the eta, etb and etd toxins from s. aureus by pcr test with specific primers. however, 166 out of 178 (93.3%) sig isolates harbored the siet toxin originating from s. intermedius. this result and those of other studies imply that the majority of sig isolates harbor the siet gene. although the siet gene was present in 93.3% of the sig isolates (from 108 dogs and two cats) in this study, only 14 dogs had a history of various skin disease including allergy and prolonged inflammation lesion in skin (n=13) or otitis (n=1) (data not shown). therefore, other factors such as the general health of an animal and existence of other sig virulence factors may play an important role in outbreaks of various kinds of skin disease or otitis. the sec gene was detected in a single isolate in the present study, which was identified as seccanine by dna sequencing. however, this isolate was isolated from a veterinary staff member, and no additional seccanine isolates were identified in the veterinary hospital where this individual worked. this suggested that the isolate might be transmitted from an in- or outgoing companion animal with which the veterinary staff was in contact. the low incidence of toxins in this study could be secondary to the small number of isolates collected from companion animals that a history of skin disease or otitis. only 11.3% of s. pseudintermedius in a previous study had exotoxins although all samples were taken from patients diagnosed with pyoderma or chronic otitis and referred to a veterinary teaching hospital. although the majority of sccmec types were type v (78.9%), one isolate was type iv. in a previous study, 23 isolates (85.2%), three isolates (11.1%) and one isolate (3.7%) from veterinarians, staff, students, companion animals and environment in the veterinary hospitals were determined to be an mrsp hybrid sccmec type i~ii, type v, or non-identifiable, respectively. a previous european and north american study identified 75 hybrid sccmec type ii~iii isolates (72.8%), two type iii isolates (1.9%), six type iv isolates (5.8%), 14 type v isolates (13.6%), four type vii isolates (3.9%), and two non-identifiable isolates (1.9%) from diseased and healthy dogs in veterinary diagnostic laboratories of different countries. this demonstrated that the majority of mrsig isolates in korea harbor the sccmec type v whereas the hybrid type ii~iii is the main sccmec type found in veterinary hospitals in japan, europe, and north america. in the present study, pfge analysis of the 39 s. pseudintermedius and six s. intermedius isolates from a private referral veterinary hospital (collected during november 2006, april 2008, june 2009, and october 2009) showed that 20 isolates (lines 1~6, lines 7~9, lines 10~15, lines 16~18, and lines 19~20) had the same band patterns. moreover, some isolates obtained on different sampling dates showed the same band patterns (six isolates from lines 12~17, and two isolates from lines 21~22). these results suggest potential contamination or expansion of s. pseudintermedius and s. intermedus isolates among veterinary staff, companion animals, and veterinary hospital environments, and colonization by these specific strains for more than 13 or 18 months in the same hospital. in conclusion, eta, etb, etd genes were not detected but siet toxin was found in 166 isolates in the current study. pfge analysis results of isolated from h animal hospital showed that s. pseudintermedius isolates collected in over a period of 13 and 18 months from veterinary staff, companion animals, and the hospital environment had the same band patterns. s. pseudintermedius infections in humans, spread of mrsp populations, and association of sig with canine pyoderma and chronic otitis has been previously reported. therefore, sig, especially mrsig, may have significant clinical implications for companion animals with skin infections or chronic otitis that is of concern for veterinary staff, companion animal owners, and healthy companion animals.

the staphylococcus (s.) intermedius group (sig) has been a main research subject in recent years. s. pseudintermedius causes pyoderma and otitis in companion animals as well as foodborne diseases. to prevent sig-associated infection and disease outbreaks, identification of both staphylococcal exotoxins and staphylococcal cassette chromosome mec (sccmec) types among sig isolates may be helpful. in this study, it was found that a single isolate (one out of 178 sig isolates examined) harbored the canine enterotoxin sec gene. however, the s. intermedius exfoliative toxin gene was found in 166 sig isolates although the s. aureus-derived exfoliative toxin genes, such as eta, etb and etd, were not detected. sccmec typing resulted in classifying one isolate as sccmec type iv, 41 isolates as type v (including three s. intermedius isolates), and 10 isolates as non-classifiable. genetic relatedness of all s. pseudintermedius isolates recovered from veterinary staff, companion animals, and hospital environments was determined by pulsed-field gel electrophoresis. strains having the same band patterns were detected in s. pseudintermedius isolates collected at 13 and 18 months, suggesting possible colonization and/or expansion of a specific s. pseudintermedius strain in a veterinary hospital.

PMC3165150

pubmed-419

benign neoplasms of the salivary glands are frequently encountered in dental practice. these account for 3% of the tumors involving the head and neck. the majority of them occur in the parotid gland, and 80% of them are benign. of these benign neoplasms, 50-80% are pleomorphic adenomas and 5-20% are warthin's tumors (wt). however, warthin's tumor is the most frequent monomorphic adenoma of the major salivary glands. this is a curious benign neoplasm with its intimidating histological name, papillary cyst adenoma lymphomatosum. however, the eponym wt has been extensively used ever since aldred warthin reported two cases of this tumor in 1929. earlier in the literature this was also referred to as adeno-lymphoma, papillary cyst adenoma, cystadeno-lymphoma, and epitheliolymphoid cyst. wt is generally a disease of elderly men, with the highest incidence in the sixth and seventh decades and the male: female ratio is 4.6:1. the typical features on cytology of wt include oncocytic cells in cohesive, monolayered sheets; background lymphocytes; and amorphous, cystic debris. histopathologically, it has a cystic appearance with a double layer of oncocytes surrounding a lymphoid stroma. the following case presentation deals with wt of the left parotid gland and highlights its clinicopathologic concepts along with its therapeutic management. a 65-year-old male patient visited the department of oral medicine, with the chief complaint of swelling below the left ear lobe since six years. patient was a known smoker since the past 25 years and there was no history of alcohol consumption. on examination, the lesion extended from the left ear lobule to the lower border of the ramus of the mandible superoinferiorly and also extended behind the left ear [figure 1]. it was approximately 5 cm in greatest dimensions; smooth contoured, was firm in consistency and had well-defined borders. stimulation of the parotid glands yielded normal salivary flow with normal consistency, quantity and color. based on the history and clinical examination, a provisional diagnosis of warthin's tumor was given. a differential diagnosis of pleomorphic adenoma, a low-grade parotid malignancy, lipoma and neurofibroma arising in the salivary gland were included. the investigatory workup included complete hemogram, extra-oral radiograph, ultrasonography, computed tomography and excisional biopsy of the lesion. ultrasonographic finding showed a well-defined hypoechoic mass in the lower pole of the left parotid gland. the rest of the parotid gland parenchyma was normal and there was no evidence of ductal dilatation. computed tomography examination revealed a rounded and well-defined cystic lesion involving the superficial lobe of the left parotid gland [figure 3]. ultrasonograph showing well-defined hypoechoic mass computed tomography examination showing the lesion later, excisional biopsy of the lesion was planned using partial parotidectomy as the technique of choice [figure 4]. the tissue obtained was fixed in 10% of neutral buffered formalin, and processed routinely. the sections stained with hematoxylin and eosin revealed cystic spaces lined by a papillary epithelial proliferation which was bilayered. the cells of the epithelial lining appeared intensely eosinophilic. at the core of papillary projections a variable amount of lymphoid tissue with mature lymphocytes was observed [figure 5]. tumor after superficial parotidectomy microscopic picture (10) the patient did not present with any post-surgical complications. the most accepted hypothesis about the origin of wt is that it develops from salivary duct inclusions in the lymph nodes, after the embryonic development of the parotid gland. this hypothesis is further supported by the frequent detection of salivary gland tissue in the peri- and intraparotidal lymph nodes. in the parotid region, lymph nodes the tumors presenting epithelial differentiations similar to those observed in wt develop outside lymph nodes and have no lymphoid stromal component. benign tumors have only rarely been associated with cigarette smoking, which focuses attention on the nature of the underlying neoplastic process and how it may differ from other benign tumors. although generally believed to be an adenoma, wt, as suggested by allegra, may be a delayed hypersensitivity reaction. an interesting fact that caught the attention of the pathologists is that a decline in the incidence in men and a concurrent increased incidence in women has been observed in recent years. the change is probably due to decline in the smoking habit in men and a reverse trend in women. the increased frequency of adenolymphoma has been ascribed to the association of adenolymphoma with smoking and the proportional increase in female smokers. studies conducted among atomic bomb survivors suggest that radiation may also be implicated in the tumorigenesis barr virus (ebv), because of the ebv dna found in tumor cells in some studies has not been substantiated. clinically, wt occurs almost exclusively in the parotid glands, in its superficial lobe and rarely in the deeper lobe (10%). the other preferred locations include the buccal mucosa, submaxillary gland, lip and palate. the patients can be asymptomatic or can have facial pain, rarely, facial nerve palsy may be seen in tumors associated with inflammation and fibrosis, which can be mistaken for malignant tumor. ipsilateral earache, tinnitus and deafness are uncommon ear symptoms that might be seen in some patients. the size is variable, from a few millimeters to centimeters, averaging 2 to 4 cm in diameter, with a preferred location in the lower pole of the gland (in the jaw angle). it has been reported predominantly in whites, less frequently in orientals, and rarely in blacks. the incidence rate is higher than that of salivary gland cancer but is lower than that of benign mixed tumors (pleomorphic adenoma). macroscopically wt presents as a spherical or ovoid mass, with a dense fibrous capsule and displaying multiple cystic compartments filled with a viscous yellow or dull brown material. however, eveson and cawson found 77% cases with an incomplete capsule, a full capsule in 8% and 16% tumors in which there was no evidence of capsule. the cytological smears in our case showed variable amounts of cellularity, ranging from barely optimum cellularity to occasional hypercellularity. there was an admixture of epithelial fragments, occasional single epithelial cells, and abundant lymphocytes noted in a granular cystic background. the epithelial cells were oncocytic in appearance with large nuclei, prominent nucleoli, and moderately abundant granular cytoplasm. since wt can be multifocal, a preoperative diagnosis by means of fine needle aspiration biopsy is mandatory and complete bilateral screening of the gland by mri is needed to program surgery.. an experienced cytopathologist can reliably distinguish malignant salivary pathologies from benign, but a histological classification based on only aspiration is an unrealistic goal. computerized tomography and magnetic resonance imaging enable accurate assessment of tumor extension, compression or infiltration of adjacent structures, presence of nodal metastases and better planning of the therapeutic approach. dynamic dual-phase scinti-scanning with technetium-99, a recognized method of identifying adenolymphoma, could be used more frequently in these selected patient groups. lesion vascularity on initial power doppler examination is often relatively sparse, but wt that did contain areas of vascularity on initial examination showed a reduction in this vascularity as the tumor size reduced. with regard to luminal cells of the tumor lining the lymphoid stroma the cells reveal a similar aspect to the striated ducts of the normal salivary glands and have numerous mitochondria. these cells, called oxifile or oncocytic cells are swollen epithelial cells, with abundant eosinophilic granular cytoplasm, rich in mitochondria and enzymes. an increased number of oncocytic cells are also observed in the normal salivary glands once the person gets older. the diffuse proliferation of the oncocytes without other changes has no pathologic significance and is called oncocytosis or oncocytic metaplasia. the epithelial cells, the oncocytes, are disposed on two layers, a luminal layer of oncocytic columnar cells, supported by a discontinuous layer of oncocytic basal cells. the nuclei of the luminal cells appear uniform and display palisading towards the free surface. the basal cells possess round to oval nuclei, centrally located, small, with conspicuous nucleoli. the lumen of the cysts contains thick proteinaceous secretions, cellular debris, cholesterol crystals, and sometimes, laminated bodies that resemble corpora amylacea. seifert recognizes four subtypes: subtype 1 (classic wt) is 50% epithelial (77% of all wt); subtype 2 (stroma-poor) is 70-80% epithelial (14% cases); subtype 3 (stroma-rich) is only 20-30% epithelial (2%); and subtype 4 is characterized by extensive squamous metaplasia. however, presence of cellular atypia and a pseudoinfiltrative appearance of the metaplastic squamous epithelium in the residual tumor often can be mistaken for squamous cell or mucoepidermoid carcinoma. squamous metaplasia of wt usually lacks keratinization, which is seen in most squamous cell carcinoma. in contrast to low-grade mucoepidermoid carcinoma, there is no definite infiltrative growth and the tumor cells appear more frankly squamous. a differential diagnosis must be made also with a variant of papillary thyroid carcinoma recently reported as warthin-like. the microscopic characteristic is a prominent lymphoid stroma and oncocytic metaplasia of the epithelium, but the nuclei have chromatin clearing, inclusion and groove-formation and the epithelial cells show immunohistochemical expression of thyroglobulin. the differential diagnosis of this malignancy should be performed preferably with pleomorphic adenoma and cystoadenoma. the anatomico-pathological diagnosis is generally easy, but it also should be distinguished from canalicular adenoma, sialadenoma as well as from branchial cyst when involving the parotid gland. sunardhi-widyaputra and van darmne in 1993 immunohistochemically studied the presence of tenacin, a molecule in the mesenchyme of salivary glands believed to play a role in the embryogenesis and development of tumors, in papillary cystadenoma lymphomatosum and in oncocytoma. they found the protein to be abundant in papillary cystadenoma lymphomatosum, prominent in the proximity of the basement membrane, beneath the oncocytic epithelial components. tenacin staining in oncocytoma was focal although oncocytes are the actively proliferating cells in this tumor. the presence of oncocytic myoepithelial cells both in papillary cystadenoma lymphomatosum and in oncocytoma surrounded by tenacin suggested that both tumors may arise from stem cells that are capable of differentiating into aberrant epithelial cells (oncocytes), myoepithelial cells in variable proportion or both. recent molecular studies have shown that the epithelial component is polyclonal and does not exhibit clonal allelic losses, suggesting that this tumor is not a true neoplasm. recent studies have also reported the presence of b-cells (cd20), nk (cd56) and t (cd3), including helper subtypes (cd4) and suppressor (cd8) in the tumor's stroma, something similar to that of normal or reactive lymph nodes. also, it was found that cd20-positive b-lymphocytes were located in the germ centers and peripheral b-area while cd3-positive t-lymphocytes are located interfollicularly. surgeons are traditionalists, and the early experience of our peers has colored current surgical opinion and slowed the introduction of conservative surgery for the benign parotid lump. this situation is now changing, and centers with experience of treating parotid tumors increasingly recognize that benign tumors can be removed safely by techniques much less invasive than a formal parotidectomy. this surgical modality is based on meticulous dissection immediately outside the tumor capsule with preservation of the facial nerves. in view of the possible association of wt with extra-salivary neoplasms, extensive workup of the patients harboring multiple wt is, therefore, indicated and long-term follow-up is mandatory, due to the possible occurrence of metachronous salivary and extra-salivary tumors even after prolonged time intervals. rarely, either the epithelial or lymphoid component of wt can undergo malignant transformation with an estimated incidence of less than 0.1%. in order of frequency, the commonest carcinomas are squamous cell carcinoma, oncocytic carcinoma, adenocarcinoma, undifferentiated carcinoma, mucoepidermoid carcinoma and merkel cell carcinoma. complications must be unusual and of low frequency for the surgical resection of a wt, including some complications considered of minor importance, such as paresis of the ear lobe resulting from manipulation and/or section of the auricularis magnus branch of the superficial cervical plexus. the auricularis magnus nerve, in its path toward the ear lobe, may pass through the tumor, hampering the dissection. another complication of lesser importance is the change of facial contour due to resection of a large portion of the parotid gland.

warthin's tumor undoubtedly is the most frequent monomorphic adenoma of the major salivary glands. clinically, it appears as a slow-growing tumor often fluctuant on palpation due to its cystic nature. the treatment of choice is complete excision with wide tumor-free margins. this article highlights a case of warthin's tumor of the parotid gland in an elderly male patient along with a review of the literature on the aforementioned pathology.

PMC3783811

pubmed-420

broomrapes (orobanche spp.) are fully parasitic flowering plants that lack chlorophyll; hence they penetrate roots of susceptible hosts, removing water, minerals and sugars. (egyptian broomrape) attacks dicotyledonous crops cultivated around the mediterranean, causing massive yield losses. broomrape attached to the host by means of tubercle, a swollen organ which may be simple or composite. with the exception of the case of transgenic target-site, herbicide-resistant host plants, meant to be a temporary measure until other effective control means are found. orobanche generally maintains a close relationship with the host and so it is unreasonable to attack it using herbicides because the latter may adversely affect the nontarget host. despite research on orobanche spp. for over three decades, yield losses still abound because there is no sustainable method for controlling the parasite. fusarium compactum (wollenw.) the inundative biocontrol approach, with repeated applications of the biocontrol agent, generates a state of equilibrium with a very low level of weed density as a result of the artificial inoculation of the biocontrol agent but the fungi are not sufficiently virulent for field release, regardless of the amount used. a series of experiments were conducted using f. compactum, a biological control agent that infects orobanche without affecting the roots of tomato. pectins are complex polysaccharides and are one of the major components of the plant cell wall of dicotyledonous plants, where they control the ionic status, cell expansion, and separation. some cellulase producing fungi includes acremonium sp, aspergillus spp and fusarium sp,, trichoderma spp. [6, 7], zymomonas, and mutant penicillium. in this paper, a hydrolytic enzyme plays an important role in the pathogenicity of plants by facilitating fungal penetration through the host cell wall [10, 11]. experiments using mycoherbicidal organisms plus pectinase (ec 3.2.1.15) or cellulase (ec 3.2.1.4) indicate that enzyme enhances the weed control of pathogenic fungi. here, pectinolytic and cellulolytic enzymes have been used to enhance the virulence of f. compactum on tomato plants infested with broomrape. a semiaxenic polyethylene bag system was used that allowed easy visual observation of the fungal infection of the tubercles. this study reports that the addition of pectinase and cellulase alone or in mixtures enhanced the virulence of f. compactum on broomrape. f. compactum was cultured on potato glucose agar (pda, pronadisa) in petri dishes incubated at 25c. subcultures were grown in 100 ml potato glucose broth (pdb, pronadisa) in 250 ml erlenmeyer flasks. the cultures were left on a rotary shaker (brunswick scientific) at 150 rpm for 48 h. f. compactum mycelia were harvested on miracloth (calbiochem, la jolla, ca), rinsed with distilled water to remove remaining spores and excess medium, and harvested by vacuum filtration. the washed hyphae were chopped at 6,000 rpm for 2 min with a homogenizer (ika t18 basic ultra-turrax usa), resuspended in sterile water, and the propagule concentrations of chopped mycelia were estimated after serial dilution and plating. surface sterilizing seeds ensure that the fungal infection on the seeds is from deliberate infection. thus, about 13 mg seeds in small bags formed of miracloth were wetted and surface sterilized in 80% ethanol for 1 min and in a mixture of 1% sodium hypochlorite in 0.01% aqueous tween 20 for 10 min. tomato transplant plugs at the two to three leaf stages in speedling insert trays were purchased from hishtil inc, ashkelon, israel. the pathogenicity of f. compactum was tested in the semiaxenic polyethylene bag system; briefly about 13 mg of dry surface-disinfected seeds (up to 1,500) were sprinkled on wet whatman gf/a glass-fiber sheets (whatman int. the broomrape seeds were conditioned for a 7 d period on the wet glass-fiber sheets. a tomato seedling with three or four expanded leaves and washed roots was fixed inside each polyethylene bag containing conditioned broomrape seeds. the plant roots in each bag were moistened by capillary action with forty ml of modified hoagland's solution in the base of each bag. modified hoagland's solution fourteen-hour photoperiods were provided by a photosynthetically active light intensity of 65 e/m/s (li-cor, inc., photometer, model li-188b) produced by six 40 w cool white fluorescent tubes suspended 35 cm above the benches. two ml of 5-g ml gr-24 (synthetic germination stimulant) were added to each bag with a pipette to augment the tomato root exudates. the broomrape seeds germinated, attached to tomato roots, and formed small tubercles during the following 2 weeks. allocation of treatment to orobanche-infested tomato plants were in such a way that the tubercle numbers and sizes were almost the same. the virulence of the fungus was determined with and without various concentrations of either pectinase (ex fungal origin, 1.1 u mg, sigma) and/or cellulase (cellulysin, ex trichoderma viride, 10 u mg calbiochem-behring corp., la jolla, ca 92037). the effect of cellulase concentration (10 to 20 u ml) on tubercle death was similarly determined at a constant inoculum level. thereafter, the virulence of the fungus with the two enzymes was determined in combination at varying ratios. control plants were mock-inoculated with either sterile distilled water containing 0.01% tween 20 or 4 to 20 u ml of single or combined enzyme preparations but without fungal mycelia. tubercles on the tomato plants infested with broomrape were counted and the diameters were measured with a ruler, with the assumption that the tubercles are perfectly spherical. the treatments consisted of f. compactum or f. compactum plus cellulase (4 to 20 u ml). one ml of aqueous fungal cellulase samples (10 units mg), freshly made for each experiment as a solution containing 10 u ml, and was checked for pectinase activity. pectinase activity was measured in a reaction mixture consisting of 533 l of 1% polygalacturonic acid (pectin), 400 l of 50 mm sodium acetate buffer at ph 5.0 and 67 l of the cellulase. the mixture was incubated at 37c for 10 min as outlined by tonukari et al.. a 100-l aliquot of the reaction mixture was mixed with 1.5 ml of 1% 4-hydroxybenzhydrazide (fluka, fluorescence grade) in 0.5 m naoh. the mixture was heated at 100c for 10 min, and cooled on ice water. absorbance was measured at 410 nm against the zero time blank as outlined by lever, using a spectrophotometer with a versamax tunable microplate reader. pectinase activity of the cellulase was calculated from a standard curve prepared with a d-galacturonic acid (sigma). one unit of enzyme forms 1 mol of galacturonic acid from polygalacturonic acid in 1 min under the conditions of the assay. broomrape tubercle deaths were recorded at 24 h intervals after fungal inoculation, for 8 to 11 d. tubercles were visually scored as healthy (translucent, dense, and intact), infected (diseased), or dead (black and soft). the levels of infection caused by f. compactum plus pectinase in both experiments were always better than those achieved by the fungus without pectinase (figures 1(a) and 1(b)). the numbers of broomrape tubercles infected continuously increased over time throughout the period of observation. this was not only due to inoculum buildup in the tomato root system but also to the enzyme action. this study wanted to ascertain whether the added pectinase would have an effect on suboptimal f. compactum inoculum. therefore, subthreshold levels of inoculum (10 propagules ml) were investigated in the presence of pectinase. f. compactum alone killed 30% of broomrape tubercles at the suboptimal inoculum levels (figure 1(a)). at the lowest inoculation density, f. compactum (3.84 10 propagules ml) combined with 11 u ml pectinase killed more broomrape tubercles than the f. compactum alone (figures 1(a) and 1(b)). the killing of broomrape tubercles indicated that pectinase enhanced the virulence of f. compactum (figure 2). since both enzymes separately enhanced the severity of tubercle infection, it was ascertained whether the pathogenicity of f. compactum (1.4 10 propagules ml) on broomrape tubercles could be synergistic through the joint action of the enzymes (10 u ml of each enzyme) (figure 3). the broomrape tubercles on the tomato roots were large and healthy in the absence of fungal treatment but, at times, a few naturally brown tubercles could be seen (figure 3(a)). f. compactum alone infected all the inoculated tubercles but did not kill any significant number (figure 3(b)). infested tomato roots that were inoculated with mycelia plus pectinase (20 u ml) (figure 3(c)) had over 50% tubercles dead one week after treatment. those inoculated with mycelia plus cellulase (20 u ml) (figure 3(d)) had above 60% mortality. a mixture of both enzymes with f. compactum increased fungal infection of broomrape by f. compactum (figure 3(e)). the response of the infected tubercles varied from 100% kill to mild infection, depending on tubercle size. mycelia plus cellulase (20 u ml) mix provoked about the same level of infection as mycelia in solution with cellulase and pectinase (10 u ml of each enzyme) (figure 3(f)). it is logical to think collective mixing of pectinase and cellulase to f. compactum will effectively enhance the fungal biocontrol potential: thus, it was tested. at various enzyme compositions, the f. compactum treatment (1.05 10 propagules ml) that caused only a hypersensitive reaction (9% death) on the tubercles caused about 35 to 85% tubercle death when the tubercles were treated with mixtures that contained mycelia, pectinase, and cellulase in various ratios (figure 4). higher tubercle infection by f. compactum was observed with a high ratio of cellulase to pectinase. beginning from day 4 after f. compactum inoculation on broomrape infested tomato plants; statistically significant differences among treatments were observed (figure 4). broomrape infested roots coinoculated with chopped mycelia plus pectinase and cellulase had substantial tubercle infection and subsequent large numbers of dead tubercles (3585%) (figure 4). this system ensures easy examination of seedling attachment to the tomato roots and easy observation of tubercle infection in the course of the experiment. the activity catalyzed by an enzyme is a measure of the amount of enzyme present. values pooled from three sets of experiment showed that in a 1 mg (10 u mg) cellulase preparation, 0.055 mg pectinase (1.1 u mg) is present. this means the 10 u cellulase has a 0.06 u of detectable pectinase. the basis for this study stems from previous studies which examines single enzyme-assisted effect on fungi pathogenicity. the orobanche were at three main phases of their life cycle (seed, germination, and parasitic phases) at fungal inoculation. tubercles are usually present at the germination phase and later become parasitic. in this study, in apparently healthy tubercles, it may be possible to find diseased tubercles (as depicted in figure 2); such tubercles deaths were induced by accidental infestation. in total, these results demonstrate that exogenous pectinase as well as cellulase can contribute to pathogenicity. the mycelia plus enzyme mix killed some broomrape tubercles, thus they could not form new seeds to replenish the orobanche seed bank. the rationale of approach is also applicable to soil grown tubercles where the presence of enzyme may be exogenous. this is well taken care of in nature because the tubercles are borne on the roots and roots are mostly below the soil surface. the temperature in the growth chamber was kept at 25c to induce infection by f. compactum. as demonstrated in this study, descriptions of actions of cell wall degrading enzymes are given by wanjiru et al.. f. compactum can infect the tubercle by degrading the cell wall components and invading the tissues and cells. pectinolytic and cellulolytic activities of the enzymes (in this case of fungal origin) result in a loss of structural integrity in the tubercle and characteristic damage. the enzyme substrate range and mechanism of action could partly explain the interaction between f. compactum and the enzymes on broomrape tubercles. the pathogenicity of f. compactum plus the enzyme mix observed could rarely be observed without pectinase and cellulase at the inoculum level used. besides, neither pectinase nor cellulase was ordinarily able to injure and kill broomrape at the concentration used (max. 20 u ml), demonstrating that the broomrape tubercle infection and death observed are due to the addition of a mycoherbicidal organism. the role of pectinase and cellulase in the degradation of cell wall material as observed in this study corresponds with the reports of many authors [10, 11, 18, 19]. in the previous paper, the results reported by sasaki and nagayama showed that the fungi pathogenicity was not always proportional to the enzyme activity. it is known that the level of -glucosidase in an enzyme preparation may affect the result of cellulase assays, for example, for the estimation of activities of extracellular cellulase enzymes produced by trichoderma. as pointed out by kumpoun and motomura, the pectinase used in their study is mainly pectinase, but some glycosidases were also present. the presence of -glucosidase or other enzymes, such as cellobiose phosphorylase, which are required for cellobiose metabolism and to enhance cellulose hydrolysis but which are not, strictly speaking, cellulases or rhamnosidase and -glucosidase activities in pectinase could further complicates research findings. care must be taken in interpreting the results of pathogenicity tests as the enzyme preparation may contain a battery of enzymes. this result may suggest the need to study the presence of other enzymes in an enzyme of interest even if the enzyme of interest is from a commercial source. contrary to some of the findings associated with enhanced f. compactum infection on broomrape, f. oxysporum pathogenicity on broomrape was not enhanced by pectinase and/or cellulase. thus, although f. oxysporum and f. compactum belong to the same genus, they may have different mycoherbicidal mechanisms. mycelia of f. compactum did not penetrate nor show apparent damage to the tomato roots as also observed in this study. in conclusion, pectinolytic and cellulolytic activities are widely exhibited by bacteria and fungi. the enzymatic activities can predispose broomrape tubercles to infection by fungi, in this case, f. compactum. the latter fact adds to the commercial value of f. compactum as a potential mycoherbicide when sufficiently virulent.

the use of enzyme could facilitate pathogen penetration into plant host. here the combination of cellulase and pectinase was ascertained on the pathogenicity of f. compactum (1.4 106 propagules ml1) on broomrape tubercles. f. compactum alone infected all the inoculated tubercles but did not kill any significant number. infested tomato roots that were inoculated with mycelia plus pectinase (20 u ml1) had over 50% tubercles dead one week after treatment. those inoculated with mycelia plus cellulase (20 u ml1) had above 60% mortality. mixtures of mycelial plus the two enzymes (10 u ml1 of each enzyme) showed synergy. the activity catalyzed by an enzyme is a measure of the amount of enzyme present. it was shown that, in a 1 mg (10 u mg1) cellulase used, 0.055 mg pectinase (1.1 u mg1) is present. this explains why mycelial plus cellulase mix contends with mycelial plus the two enzymes.

PMC3026981

pubmed-421

isotopic signatures of environmental plutonium are generally used to assess the origin of the material. while decay counting is restricted to pu, pu, and pu, all mass spectrometric methods can in principle detect the isotopes pu (t1/2=24.1 ka), pu (6.65 ka), pu (14.4 a), pu (373 ka), and pu (80.8 ma). however, there are several publications available concerning isotopic concentrations of the minor plutonium isotopes pu, pu, and pu in environmental samples. has assessed the global distribution of pu and pu, while uses pu to distinguish chernobyl plutonium from global fallout. in we have shown how the isotopic vector of pu (pu, pu, pu, pu) can be measured by combining the respective best suited method for each isotope. due to the strong interference of u in mass spectrometers, pu can generally be measured with alpha-spectrometry only. also, while the combined activity of pu and pu can be determined efficiently by decay counting, their similar -energies are not readily separated, thus a mass spectrometer is usually required to determine the isotopic ratio pu/pu. the short half-life of pu renders -decay counting by lsc (liquid scintillation counting) the most efficient method. for the long lived isotopes pu and pu only mass spectrometric methods are suitable. among the mass spectrometric methods, ams provides the most sensitive measurements, probably since in most cases the limit is imposed by background from u rather than by detection efficiency. the destruction of molecules by stripping and the combination of several spectrometers, permits ams to suppress background better than other methods. if only ams is used, pu can not be measured, and a lower efficiency is achieved for pu. however, since no additional sample processing is required, the measurement of pu can be done at very little additional effort if the concentration of pu in the sample is high enough. during the last few years, we have carried out several studies on environmental pu [57]. in most cases, the measurement of the pu/pu ratio was the main interest. however, if we encountered samples with sufficiently high count rates for these isotopes, we additionally performed measurements of the minor pu isotopes pu, pu, and in some cases even pu. dating with plutonium can be done very precisely by using the ratio am/pu (e.g.). the parent nuclide pu (t1/2=14.4 yr) and the daughter am are different chemical elements and thus this ratio yields the date of either the irradiation or that of the last chemical separation. while this is a useful method for applications in the scope of nuclear safeguards, chemical fractionation occurring while the sample resides in the environment compromises its use for environmental studies. without the presence of the daughter isotope am in the sample, the age since the irradiation can be assessed only if the initial isotopic abundance of pu is known. this abundance, however, depends strongly on the production process, and thus is generally ambiguous. we propose to use the isotopic ratios of the other plutonium isotopes to estimate the initial pu content. to check the accuracy of this estimate, we have applied the method to measured literature values for a thermonuclear weapons test, and for the chernobyl accident where we adopt the numbers obtained by given by. additionally, we have simulated thermal neutron irradiation of u. to demonstrate the practical applicability, we have measured environmental samples of different origin. the short lived nuclide pu (t1/2=5 h) decays in a reactor before pu is produced, which results in cm being the isotope of mass 244 produced in a reactor. in a nuclear explosion, however, in which all nuclear reactions happen on a time scale of microseconds, pu can be produced directly via neutron captures on plutonium isotopes, and also by captures on uranium and subsequent -decays after the explosion. results have been published for the thermonuclear weapons test ivy mike (pu/pu=(11.8 0.7) 10) and for the low-yield test (pu/pu=(2.3 0.4) 10). local weapons test fallout in bikini atoll sediments and soils has been measured with ams recently by, yielding pu/pu ratios between 2.8 10 and 5.7 10. in the outer layer of a deep-sea manganese nodule a pu/pu ratio of approximately 1 10 has been observed (taken from fig. 3 of), which the authors attribute to a global fallout. samples were obtained from three different projects. in depth profiles of pu and pu (among others) were obtained from the region of nassfeld (salzburg, austria). while cs and sr in this region stem from the reactor accident in chernobyl, pu/pu and am/pu activity ratios as well as the pu/pu isotope ratio determined by ams identified global fallout as the source of plutonium. the samples used for this work (t2f and t2 g in) were collected on the mountain pasture nafeld-alm at an altitude of 2530 m asl, at 47.018 n, 13.012 e on august 25th, 1999. the chemical separation of the samples (separating am from pu) was carried out in summer 2006, the ams measurement in august 2006. the discharge history of pu from sellafield is well documented. though releases extended from 19521992, the peak discharge occurred between 1970 and 1980 (fig. 1). a marine sediment core was collected in 1993 at 54.416 n, 3.563 w by the research vessel gauss, federal maritime and hydrographic agency, germany. this core was investigated for a number of isotopes in and recently for pu isotopes and u with ams at vera. generally, the chronology of the 48 cm long core is unclear, since mixing of the sediment is suggested by the oceanographic situation of the sampling site and hence expects an average integral ratio of the discharges in the core; despite this, clearly discriminated peaks in the pu, pu, and pu are observed in. for the present work, the ams sputter targets prepared for were re-measured for pu, pu, pu, pu and pu. the chemical sample preparation was performed in november 2009, while the ams re-measurement was done in december 2011. for some of the samples, pu/pu and pu/pu data have already been given in, however the data in the present manuscript stems from the new measurement. the garigliano nuclear power plant was situated at sessa aurunca near the garigliano river in campania, italy. it consisted of a boiling water reactor with a thermal power of 506 mw, and was in operation from 1964 to 1978, and is presently in the decommissioning phase. though mainly operated with 2.3% enriched uo2, mixed oxide fuel, consisting of uo2 and puo2, was also used. during operation and decommissioning, a core from the sediment inside the (now dry) drain channel (41.257 n; 13.832 e) and another one from the entry point of the channel into the river was collected in may 2006; pu was separated in autumn 2006, and the ams measurement was performed in august 2007. based on the pu depth profiles, in studies on the yield of heavy elements in thermonuclear explosions a general exponential trend with increasing mass was observed. this trend can be understood under the simplifying assumption of an identical cross section for all involved (n,) reactions; the chance to produce a mass n masses above the initial nucleus is then the probability of absorbing exactly n neutrons, which is described by a poison distribution. as long as the neutron absorption probability is small, furthermore, the higher binding energy of even-even nuclei leads to a higher abundance of isotopes with even masses; a factor of 1.58 was observed for the ivy mike device. thus the initial ratio between mass 241 and 239 will be the same as that between mass 242 and 240:(1)241pu239pu0=242pu240puand thus we obtain the time t since irradiation(2)t=t1/2ln2ln(241pu239pu)(242pu240pu)with t1/2=14.4 yr the half-life of pu. despite the present-day availability of improved nuclear data for a more detailed simulation, calculation of the actual isotope yields is complicated by the fact that a constant neutron flux throughout the uranium or plutonium used in the devices can not be assumed. the resultant distribution will generally exhibit a higher yield of the heavier masses (more frequently produced in the high flux spots) than indicated by the lighter masses. simulations are possible for simplified cases, especially for thermal neutrons, which corresponds roughly to the case of reactor production. additionally, we assume that the duration of the irradiation is much shorter than the half life of pu. both neutron capture and fission are taken into account, the corresponding cross sections were taken from the endf/b-vii.0 database. the system of differential equations was solved numerically with mathematica (wolfram research, champaign, il, usa) for different assumed burn-up levels. the methods to extract the plutonium from the environmental samples varied between the different projects; the (dry) sample mass was 1 g for sellafield, 212 g for garigliano, and 20 g for salzburg samples, respectively. generally, the samples were leached, and plutonium was extracted with ion exchange. after co-precipitation with 1 to 3 mg iron, the sample was calcined to produce plutonium oxide embedded in an iron oxide matrix. the ams facility vera is especially equipped for the measurement of heavy ions. uranium pilot beams are used for tuning the spectrometer, which is then scaled to the various pu isotopes for the measurement. the switching times between the different isotopes is typically 15 s, therefore fast variations of the ion source output are not completely averaged out and contribute to the uncertainty of the measured isotopic ratios. the high-energy beam analysers are designed to allow the transport of 5+actinide ions at a terminal voltage of 3 mv. oxygen is used as a stripper gas which results in a stripping yield of 5% for this charge state. for most previously published data a time-of-flight (tof) detector with 25% efficiency was used to further suppress potential background from u; but we have never observed such background, so measurements can also be carried out without tof, resulting in a theoretical detection efficiency of 4 10. however, under typical sputter source conditions, for 10 pu atoms in the sputter target we obtain a count rate of 30 s (for puo2 only 1/10 of this count rate is observed). since the sputter targets last for many hours, the available accelerator beam time usually limits the detection efficiency. we hope that we can improve on this by reducing the amount of iron matrix used, which requires new handling procedures. counting statistics were no limitation for the sellafield marine core samples, since their concentration of radionuclides is exceptionally high; as already reported in, to avoid saturation of the detectors by the very high count rates we had to reduce the source output strongly, by running the cesium reservoir essentially at room temperature instead of the usual 150 c. therefore, these sputter targets appeared almost untouched after the measurement. all data was normalized to a pulser running at a fixed frequency to correct for any detector dead time that arose as a result of high counting rates. since all pu isotopes are measured in the same ionization chamber, we assume the same ion optical transmission. the ams device can in principle introduce large fractionation, since each isotope is essentially measured with a different tuning of the (relatively complex) mass spectrometer, and the tunings may differ in quality. since we obtain our plutonium tunings by scaling from the u pilot beam the heavier plutonium masses are more likely to suffer from ion optical losses. the resultant uncertainty is difficult to assess, since no plutonium standard with isotopic ratios suitable for ams is available so far, which would allow to monitor and correct machine fractionation for every measurement. however, reference materials measured in the past suggest a typical precision better than 10%. to monitor whether the beam tuning degrades as a result of drift in the components, the u/u ratio of our in-house uranium standard vienna-kku is measured using three sputter targets per turn of the sample wheel. it should be noted that systematic trends in the ion optical transmission of the different pu isotopes will partly cancel out in the double-ratio used in eq. we expect little fractionation during chemical sample preparation, and blank material allows any laboratory contamination to be traced. the beam analysers will not distinguish between the in-growing am and the remaining pu. however, the time span between the separation of am and the ams measurement is still relevant. am (most likely) has a different negative ion yield to pu, which introduces an uncertainty into the mass 241 determination. we are not aware of any published ionization yield for am, but has investigated other monoxide ions of actinides: the relative negative-ion formation probability of tho, uo, npo, and puo is approximately 1:3:5:7. as an example, we assume that am does form negative monoxide ions even two times better than pu, and that the measurement is carried out one year after chemical separation. since 5% of pu will have decayed into am, the observed count rate for mass 241 will appear 5% higher, which corresponds to a 1 yr lower age. as another extreme example, if we assume that am does not form any negative monoxide ions at all (which is definitively not the case, see), the sample will appear one year older-as it actually is. since no information is available on the actual ion yield of am, which is moreover expected to vary with the matrix of the sputter target and with ion source conditions, we assume an additional uncertainty of 1 year for the age determination per year between separation and measurement. fig. 2 and table 1 shows the results measured in this work, in comparison to the numbers given for the ivy mike 43 counts of pu were successfully measured on salzburg soil within 1.5 h. the resultant isotopic ratio for pu/pu is (5.7 1.0) 10. a contribution of reactor material in our samples, in particular from the chernobyl accident, could cause a lower ratio. however, a significant contribution from chernobyl was excluded in. additionally, while a pu/pu ratio of 2.7 10 is expected for chernobyl debris in 2011, our measured ratio of (1.43 0.06) 10 is more than one order of magnitude lower, and lies perfectly on the eye guide for global fallout (through masses 239 and 242 of salzburg soil). we think that our value better represents the northern hemisphere global fallout ratio than that taken from. a possible explanation for the higher ratio observed in the deep sea crust (va132, 918 n, 14603 w, 4830 m depth) would be influence from the various nuclear test sites in the south pacific. we think this assumption is supported by the pu/pu of 0.25 observed in the nodule sample (also taken from fig. 3 in), while the accepted northern hemisphere global fallout average is 0.185; this value is also observed in our salzburg soil samples. (2) for the measurements from literature and the simulations for thermal neutrons is shown in table 2 and fig. since all ratios were calculated for the time of production, or corrected for pu decay, an age of 0 is expected in all cases. while the mike test appears 6 years older than expected, the simulated thermal neutron results appear too young, and the deviation grows with the assumed burn-up. the chernobyl data appears 5 years too young; a more accurate estimate is obtained if we assume production by thermal neutrons. in this case (2), but fit the simulation to the measured pu/pu ratio, which results in a total thermal neutron fluence of 0.0025 b, and in an estimated age deviating by less than 2 years from the known calendar date of the chernobyl explosion. this agreement is surprising, considering that the processes in the actual reactor deviated significantly from a simple thermal neutron irradiation. for the salzburg soil, an age of 52.5 1.5 yr is obtained, corresponding to the year 1954. this age is 8 yr before the maximum of the nuclear weapons tests in 1963, and the deviation is similar to that for the ivy mike test. if this trend holds for all material produced in nuclear explosions, a correction could be applied reducing the deviation. the garigliano results suffer from a larger statistical uncertainty, due to lower pu concentration in the samples. the age is 42 8 yr, which corresponds to the year 1965, in agreement with expectations. our new pu/pu data for the sellafield core agrees with previous measurements on the same sputter targets presented in, while the three pu/pu ratios given there have significantly lower precision. however, one sample (st60 k) shows a deviation of about 25% from the previously published data, which is much more than allowed by counting statistics (see table 1). we attribute this outlier to variations in the transmission of pu through the spectrometer, limiting the reproducibility of the present method. since a deviation of 25%, corresponding to a dating error of about 5 years, is relevant for the present work, this emphasizes the need for an ams standard which would allow to trace and correct for such variations. our sellafield dataset is well suited to illustrate the advantage of using the pu information for dating. (2) compared to an estimate of the initial pu/pu which is based only on pu/pu:(3)241pu239pu0=240pu239pu2 while the age shift due to the neglected even odd offset of this simpler method could probably be handled, the pu information establishes a monotonous age-depth curve for the sediment core, however the data covers only a short time span of seven years. the time range 1977 to 1984 falls well into the later part of the sellafield discharges; however, if the age is calibrated against a simulated thermal neutron irradiation, a fluence of 0.0015 b fits best, and leads on an estimated age correction of 5 years. the conventional approach towards source assessment of an unknown material is to measure elemental concentrations and isotopic ratios, and to compare them with a database of all viable sources. this works also for environmental plutonium isotopes, and probably would lead to the same source assignments as the method proposed in this work. however, we think that the calculation of predictive values like burn-up, irradiation date, or production mode (i.e. reactor vs. explosion) makes the assessment more straightforward. the measurement of pu, pu, pu, pu and pu allows for more precise assessment of the history and origin of environmental plutonium. additionally we have shown that the use of pu and pu allows dating of the time of irradiation accurate to within 6 years, while our data on the irish sea sediment core suggest that relative dating of material from the same source is possible with a precision of less than 2 years. the information contained in the other plutonium isotopes may allow further improvement in the precision. when a reactor origin can be assumed, possibly based on the absence of pu, and by calibrating the data against simulated thermal neutron data with the same pu/pu, an accuracy of 2 years is suggested by our data.

vera, the vienna environmental research accelerator, is especially equipped for the measurement of actinides, and performs a growing number of measurements on environmental samples. while ams is not the optimum method for each particular plutonium isotope, the possibility to measure 239pu, 240pu, 241pu, 242pu and 244pu on the same ams sputter target is a great simplification. we have obtained a first result on the global fallout value of 244pu/239pu=(5.7 1.0) 105 based on soil samples from salzburg prefecture, austria. furthermore, we suggest using the 242pu/240pu ratio as an estimate of the initial 241pu/239pu ratio, which allows dating of the time of irradiation based solely on pu isotopes. we have checked the validity of this estimate using literature data, simulations, and environmental samples from soil from the salzburg prefecture (austria), from the shut down garigliano nuclear power plant (sessa aurunca, italy) and from the irish sea near the sellafield nuclear facility. the maximum deviation of the estimated dates from the expected ages is 6 years, while relative dating of material from the same source seems to be possible with a precision of less than 2 years. additional information carried by the minor plutonium isotopes may allow further improvements of the precision of the method.

PMC3617651

pubmed-422

penile squamous cell carcinoma (pscc), the predominant histological type (> 95%) of penile cancer, is a relatively rare malignant tumor in western countries and japan. pscc is a rare disease, making it difficult to establish a standard of care in any of the clinical stages, particularly in advanced disease. multimodal treatments, including surgery, chemotherapy, and radiation therapy, should be considered for patients with advanced pscc. however, the optimal chemotherapeutic regimen is unknown, although cisplatin-containing chemotherapy is the mainstay of combination chemotherapy. recent studies have suggested that taxanes in combination with cisplatin and fluorouracil (5-fu) have a significant effect on unresectable and recurrent penile cancer. malignant wound, results in a decline in the quality of life because of the presence of bleeding, exudates, and/or strong odor. mohs chemosurgery is a technique of chemical fixation of a cutaneous tumor and subsequent excision. several studies have shown the efficacy of mohs paste for maintaining malignant wounds in advanced squamous cell carcinoma of the breast, skin, head, and neck [4, 5]. we report a case of a pscc with advanced lymph node metastasis treated with combination therapy consisting of taxane-based chemotherapy, irradiation, and mohs paste. an 80-year-old male presented to a community hospital with pain and redness in his left inguinal region. he had undergone penectomy and bilateral inguinal lymphadenectomy 1 year ago because of primary pscc (pt1pn0). a large solid mass with bleeding and a smelly exudate was observed in his left inguinal region. macroscopically, the tumor mass of the left inguinal region was 7.0 cm in diameter with an ulcer, redness, and exudate (fig. a ct scan revealed a huge mass (5.9 5.8 cm) in the left inguinal region along with his left femoral vessels (fig. he was administered combination therapy consisting of a taxane-based chemotherapy, irradiation, and mohs chemosurgery. the chemotherapy regimen consisted of 60 mg/m docetaxel administered over 3 h on day 1; 750 mg/m 5-fu on days 15; and 70 mg/m cisplatin on day 4. the patient also received 50-gy external-beam radiation therapy to the left inguinal region, initiated on the same day of chemotherapy. mohs paste was applied every morning with a surrounding gauze to avoid attaching with normal skin (as previously reported). mohs paste included 50 ml of zinc chloride-saturated aqueous solution, 10 g of zinc powder, and 15 ml of glycerin. radiation therapy was temporarily deferred for 12 days beginning on day 9 because of neutropenia and general fatigue. the necrotic tissue fell off and the wound flattened on day 20 after starting chemotherapy. 2b), and a ct scan showed that the tumor had decreased by 70% in diameter at 1 month after the first course of chemotherapy (fig. 3b). because of socio-economic reasons, the patient declined additional treatment. there was no progression or metastasis on the chest and abdominal ct for 8 months. however, the wound grew and became erosive again 10 months after discharge, and the patient died because of the progression of local recurrence 1 year after chemotherapy treatment. in our patient with inguinal recurrence of pscc, multimodal treatment, consisting of combination chemotherapy, irradiation, and mohs chemosurgery, was responsible for the tumor shrinkage and the successful local control of a malignant wound. although this field is limited by a paucity of clinical trials or prospective data, the available single institutional retrospective reviews indicate that multiagent cisplatin-based chemotherapy regimens fight significantly against pscc. in 1991, dexeus et al. reported a triple-drug chemotherapy regimen, which soon became a standard regimen for pscc with tolerable adverse effects. the regimen consisted of 20 mg/m cisplatin on days 26, 200 mg/m methotrexate on days 1 and 15, and 10 mg/m bleomycin on days 26. they reported a response in 10 of 14 (71%) patients (with moderate side effects). in their follow-up studies, a lower rate of complete responses with severe toxicities was reported [8, 9]. with respect to scc of the head and neck, in which pscc is historically similar, a randomized trial revealed that the addition of docetaxel significantly improved progression-free, and overall survival in patients with unresectable scc compared to the standard regimen of cisplatin and 5-fu; therefore, docetaxel, cisplatin and 5-fu have become the current standard induction regimen for advanced scc of the head and neck. in pscc, pizzocaro et al. they reported a high activity of this regimen, with 5 of 6 treated patients showing a response. based on these findings, we chose docetaxel for taxane-based combination therapy for the patient, although there are few reports demonstrating its efficacy against pscc. further studies are needed to determine which taxane has a better response and less toxicity in patients with pscc. radiation therapy has been used for many years in the treatment of pscc for the primary tumor, inguinal metastases and distal metastases. it may also play a role in the treatment of locally advanced penile cancer, particularly when inguinal adenopathy is initially unresectable. there are no randomized trials that have evaluated the impact of radiation on the prognosis and the local control of patients with inguinal metastasis originating from pscc. however, ravi et al. revealed that palliative radiation therapy showed an amelioration of symptoms in 56% of patients with inguinal metastasis in growing pscc. in addition, the radiosensitizing effect of cisplatin, 5-fu, and docetaxel has been demonstrated in several types of cancers [13, 14, 15]. therefore, it is plausible that radiation in combination with cisplatin, 5-fu, and the docetaxel regimen is a promising option for multimodal treatment of pscc. malignant wounds from primary or metastatic carcinoma are generally incurable, and palliative methods to manage the wounds are needed. frederic e. mohs developed and published a technique for the chemical fixation of a cutaneous tumor in 1941. mohs paste is effective for the hemostasis of bleeding, odors, and exudates, thereby contributing to the patient's quality of life. in japan, kakimoto et al. reported on 5 patients with breast cancer who were successfully treated with mohs paste. showed the efficacy of radical surgery followed by systematic therapy and mohs paste in patients with breast cancer. in our case, the tumor started to decrease in size after treatment and the necrotic tumor fell off by day 20, without any resection. to the best of our knowledge, this is the first report demonstrating the advantage of mohs paste for inguinal recurrence of pscc. our result shows that mohs paste may be an effective and reliable option for multimodal treatment of inguinal recurrence. it is difficult to define which treatment modality was the most effective for the patient, because we used a combination of 3 different modalities. although our result suggests that each treatment was synergistically effective, it is also important to delineate which modality is the most effective and then choose a suitable modality for each patient with pscc. secondly, the socio-economic background of the patient did not allow him to continue systemic treatment and/or maintenance therapy. the study showed that paclitaxel was well tolerated, with a moderate activity against pscc. therefore, the patient may have been able to avoid cancer progression for a much longer time if he had received additional maintenance chemotherapy. in conclusion, we report a case of successful local control of a recurrent inguinal mass of pscc treated with multimodal therapy. to the best of our knowledge, combination treatment with taxane-based chemotherapy, external beam radiation therapy, and mohs paste is a reliable option for the induction therapy for recurrent pscc. further studies are needed to investigate maintenance treatment after effective induction therapy for recurrent pscc.

abstractpenile squamous cell carcinoma (pscc) is a rare disease, making it difficult to establish a standard of care, particularly in the advanced stage. we report a case of pscc with advanced lymph node metastasis treated with multimodal therapy consisting of combination chemotherapy, irradiation, and chemosurgery using mohs zinc chloride-containing paste. an 80-year-old male with a past history of local treatment for penile cancer presented with a large painful inguinal mass with an ulcer and exudates. the patient underwent multimodal treatment with combination chemotherapy, irradiation, and mohs paste. the combination chemotherapy consisted of cisplatin, 5-fluorouracil, and docetaxel. the patient received 50-gy external-beam radiation therapy to the left inguinal region along with daily local treatment with mohs paste. after the initiation of treatment, the pain and bleeding in the inguinal region considerably ameliorated. the wound became dry and flattened 20 days after the initiation of chemotherapy. a ct scan showed that the tumor had decreased 70% in diameter 1 month after the initiation of chemotherapy. after the first course of chemotherapy, the patient and his family decided not to continue treatment because of socio-economic reasons. the patient underwent no additional treatments; nevertheless, he had no local progression of the inguinal tumors for 8 months. we report a case of successful local control of recurrent inguinal pscc treated with multimodal therapy. combination treatment with taxane-based chemotherapy, external-beam radiation therapy, and mohs paste is an option for the management of recurrent pscc.

PMC4164061

pubmed-423

the majority of women living with hiv are in their reproductive years (ages 1549) [1, 2]. the dramatic decrease in the risk of mother-to-child hiv transmission (mtct) is leading to normality in the lives of couples affected by hiv, who want own children. in europe, the reduction in mtct to less than 1% is mainly due to highly active antiretroviral therapy (haart). effective haart is resulting in suppressed viral load (vl); thus, a vaginal birth can be as safe as a planned caesarean section [3, 4]. avoidance of breastfeeding and postnatal neonatal postexposure prophylaxis (pep) further supports the effective reduction in mtct [35]. the literature suggests that there is no increased rate of fetal malformations due to the hiv infection or haart [6, 7]. a pregnant woman with hiv infection large studies of noninvasive prenatal screening have already indicated that it will lead to a decrease of invasive prenatal screening procedures such as amniocentesis (ac) or chorionic villi biopsy (cvs). if invasive prenatal testing is necessary, it can be done, but in these circumstances, haart should be started prior to the procedure to suppress the vl below the limit of detection. in these cases current, evidence suggests that mtct is very unlikely; however, studies reporting on the risk of mtct in invasive prenatal testing are limited due to small study size. haart is given during pregnancy for two reasons, first to women with an own indication for haart (they require treatment for their own health) and secondly to pregnant women starting therapy purely as a prophylactic treatment to reduce mtct. the aim of our study was to investigate if pregnant hiv-positive women get referred for special prenatal ultrasound screening services in our tertiary referral center, but also if and at what point the prenatal ultrasonography is performed. pregnant hiv-positive women usually have a combined antenatal care in a tertiary referral center and with their own gynaecologists. as well as the prevalence of prenatal ultrasound screening, prenatal, and postnatal finding was recorded. we hypothesized that the fetal anomaly rate in women with hiv-infection is as low as in all other pregnancies (35%) [12, 13]. hiv-positive pregnant women who presented in our tertiary referral center between january 1, 2002 and december 31, 2012 were included in this retrospective cohort study. three categories were used: very preterm delivery (24+0 to 33+6 weeks of gestation), preterm delivery (34+0 to 36+6 weeks of gestation), and term delivery (37 weeks of gestation). all data regarding early prenatal screening (as, e.g., nuchal translucency measurements) and fetal anomaly scan at 20 weeks of gestation or at first presentation in our center were recorded. only scans which were performed in our center were included, reflecting the fact that hiv-positive pregnant women are high risk pregnancies, and high-risk pregnancies are referred to a tertiary center or an equivalent specialized center for prenatal screening [1417]. an early anomaly scan was defined as a first trimester scan; in the study period, the fetal nuchal thickness was assessed; a formal nuchal translucency measurement was included if measured by appropriately qualified sonographers. a fetal anomaly scan was defined as a detailed scan in the second trimester (usually between 20 to 22 weeks of gestation). all the scans performed at a later gestation in our department prior to birth are recorded separately as late scans in the third trimester. malformations were any fetal/neonatal disease, which required either surgery or special pediatric care including chromosomal anomalies. all cases with an ac, mtct, and any intrauterine or postnatal death were evaluated. maternal information included age, ethnicity, gestational age at delivery, gravidity and parity, haart already before the pregnancy, vl (copies/ml), cd4 count (cells/l) prior to birth, and other risk factors such as coinfection with hcv. the last recorded vl prior to the delivery was used and classified in three risk groups. in the study, a vl below 50 is considered as negative/undetectable. the last cd4 count prior to birth was noted, and again three categories were used. the mode of delivery was classified as (1) planned caesarean section; (2) in cases of rupture of membranes and/or contraction it was recorded as elective caesarean section in labour; (3) emergency caesarean section; (4) caesarean section after planned vaginal birth; (5) vaginal birth; (6) unplanned vaginal birth and (7) instrumental vaginal delivery. in the unit the first planned vaginal birth was recorded in 2009. before that time, women were offered elective caesarean section at around 37+0 weeks of gestation [3, 8, 19]. with evidence for the safety of the vaginal birth with undetectable vl, the policy in the unit shifted towards planned vaginal birth, and if caesarean section was offered in these cases, the delivery was delayed until>37 weeks of gestation according to the german-austrian guidelines [3, 4, 8]. the following neonatal data were included: apgar score, arterial cord ph (aph), cord base excess (be) and neonatal weight (stratified according to 10th, 1090th and>90th percentile). a weight below the 10th percentile was considered to be intrauterine growth retardation (iugr). information about scan findings was obtained from the record of the ultrasound department, and further information was collected from maternal case notes, pediatric notes, and discharge letters. ethics approval for the retrospective study was obtained from the ethics committee at the j. w. goethe university, frankfurt (number 30/13). for categorical variables and nominal variables, frequency tables were used for descriptive statistical analysis. for ordinal and quantitative data, mean and these data were further analyzed using the wilcoxon-mann-whitney test, kruskal-wallis test, spearman-correlation, chi-test, and fisher's exact test as appropriate. in addition, multivariate logistic regression analysis was performed to identify factors associated with a woman having an early anomaly scan. overall 330 pregnancies were recorded, with 322 singleton pregnancies (97.6%) and in eight twin gestations (2.4%). one twin pregnancy was conceived due to ivf with first diagnosis of the hiv-infection in the early second trimester. there were 122 preterm deliveries (36.5%) and 90 (26.9%) of these were between 34 and 36+6 weeks of gestation. maternal and neonatal characteristics are presented in table 1, stratified by pregnancy duration in table 2. two thirds of women (66.4%) were of african ethnicity. in one quarter of women, more than three quarters 257 (77.4%) of the births were elective caesarean section. in 29 cases (8.7%), women delivered vaginally. the cd4 count (cells/l) prior to birth was in the majority of 175 (62.5%) 350, in 76 (27%) between 200 and 349, and in 30 (10.7%)<200. the vl (copies/ml) in most women 168 (55.8%) was suppressed below 50 copies in 88 (29.2%) 50399 and in 45 (15%) 400. one hundred and eight women (37.4%) were on no haart treatment at the beginning of the pregnancy. in 25 (8.9%), a positive anti-hcv test was recorded. thirty newborns (9%) were classified as below the 10th percentile. in 100 of the 330 pregnancies (30.5%) the nuchal translucency was measured in 67 (20.3%) of the 330 cases (nt median 1.22 mm (range 0.63 mm)). a multivariate analysis for factors influencing a woman having an early anomaly scan (table 3) showed that african ethnicity and first diagnosis of hiv during the ongoing pregnancy were factors which significantly could be related to not having early prenatal ultrasound screening (figure 1). invasive testing (ac) was done in three (0.9%) of 330 cases. only one case was done at 25 weeks in our department, and we started haart and performed the ac after vl was fully suppressed. two cases were done for advanced maternal age without control of vl and without specific precautions for example, haart, and both revealed a normal karyotype. in all of three cases, no mtct occurred. in the second trimester in 252 (74.5%) of 330, a detailed anomaly scan at 2022 weeks was done. in 18 (5.5%) patients, the scan was performed in the third trimester due to late presentation in our unit. in table 4, fetal and neonatal malformations as well as chromosomal anomalies are presented. in seven cases of 330 cases (2.1%), we diagnosed a fetal malformation. postnatally, all of the seven cases were confirmed, and eight further malformations and two cases with trisomy 21 were detected. the chromosomal anomalies were not suspected. both women, 33 and 39 years of age, had no early scan or biochemical screening but a scan in our unit (late in the second trimester with no anomalies seen). there were three cases with a skin tag, one nevus sebaceous of the occiput, and one case with a socalled sucking blister on the hand, all considered to be minor. each of these cases had at least one scan in our department prior to the birth. however, the sucking blister and the nevus were leading to an upgrade in neonatal pep due to breaking down of protective skin barrier, and one newborn presented with a small omphalocele which was not seen prior to birth.. the overall fetal malformation rate (including the minor anomalies) was 4.5%. in table 5, the fetal and postnatal mortalities are recorded. in our cohort, we had six cases of intrauterine or postnatal loss and all were born by caesarean section. all of the three newborns were delivered by caesarean section, and all were preterm (33+6, 36+3 and 36+4 weeks of gestation). in all cases, the vl was detectable, all women were on haart, and one woman was coinfected with hcv. one woman had already a vertically infected child, and she had a poor compliance. there are conflicting results regarding the risks for hiv-positive mothers for possible adverse effects in their pregnancies [5, 6]. in our study, we confirm the low fetal malformation rate of 4.5% in women living with hiv. there are different national registers collecting data on haart and pregnancy outcome (e.g., apr: antiviral pregnancy registry; nshpc: national study of hiv in pregnancy and childhood (uk); ecs: european collaborative cohort; epf french perinatal cohort) [4, 2124]. these registers confirm the same malformation rate in women taking haart as in the general population (35%) [12, 13]. the postnatal anomalies were minor ones (skin tag, sucking blister) or missed due to minimal extend (omphalocele). the two cases with postnatal trisomy 21 were missed prenatally but were not seen in typical screening periods. there were no anomalies in the unscreened population. a change in treatment policies is evident over the 11 years of the study, reflected in the changes in delivery mode over time and the gestational age at delivery. a high preterm delivery rate is confirmed by other groups. in our population, 26.9% are late preterm deliveries (3436+6 weeks of gestation) and are mostly iatrogenic due to early caesarean section as in other studies and in the past. the updated national german-austrian guidelines now delay caesarean section to term in women with suppressed vl. the numbers of women with fully suppressed vl (vl<50 copies/ml) (p<0.001) and cd4 cells 350 (p>0.20) prior to birth increased over the last years. the first is early screening which should take place between 11+0 and 14+0 weeks of gestation. this early screening was introduced by nicolaidis in 1992 as a combined method of screening (including ultrasound screening and two maternal biochemical markers: free human chorionic gonadotropin (free hcg) and pregnancy-associated plasma protein a (papp-a) [2628]. in germany this test is not covered by the national health system and there for is paid by the woman herself. usually at that time an early anomaly ultrasound scan can be performed, which is covered by the health system. the second screening interval is the anomaly scan at 2022 weeks of gestation [17, 29]. the prevalence of first trimester screening of 97.5% in a low risk general population has been demonstrated. we demonstrate that prenatal screening is offered and available, but that the early screening interval is missed, as only 30.5% women get referred for early anomaly scan. even so some women may have chosen not to undergo testing for ethical and cultural reasons. as a limitation of our data it could be that the screening which is done at the community-based care is missed, but as indicated usually, it warrants a referral to a highly qualified and specially trained team [16, 29]. in our study, population the majority of 188 (66.4%) women were of african origin, and in 79 (24.2%), the diagnosis hiv-infection occurred in the pregnancy, both factors were significantly related to having no early prenatal screening. tariq et al. are reporting on late booking for antenatal care in non-caucasian women compared with caucasian women regardless of time of diagnosis of hiv-infection. three cases of mtct are low (0.9%) and confirmed by other groups (reporting mtct rates of 0.1%1.3%) [3, 4]. however, looking back in our data, the viral control has improved dramatically over the last 11 years; in 55.8% of all pregnancies, the vl is<50. national health systems vary, and a complete first trimester screening (with inclusion of biochemical serum markers) has not been established on a national basis for high risk pregnancies in some european countries. in our cohort, two cases of trisomy 21 occurred, and the question remains open if these two cases could have been traced in a complete first trimester screening. in one pregnancy, an ac was performed due to suspected chromosomal anomaly, which revealed a normal karyotype. due to the time required to initiate haart and to have a suppressed vl in hiv-positive pregnancies, invasive testing will be very likely to happen in the second trimester which will then raise the difficult ethical questions about late termination of pregnancy when an abnormal result is obtained [32, 33]. first trimester screening (including maternal markers as free hcg and papp-a) has been investigated in pregnant women living with hiv. some groups feel that maternal markers could be less reliable than those in hiv-negative women [34, 35]. in our study data from 2002 to 2012, in the first years, the nuchal translucency was assessed but not formally measured. this could be due to the delay in having certified specially trained sonographers involved. in the future, the new methods of chromosome-selective sequencing of maternal plasma cell-free fetal dna (cfdna) in noninvasive prenatal testing (nipt) are valuable especially for our study group due to no risk of mtct. at present this interesting method is not widely available, and more data of this new method are needed.

objective. to assess the prevalence of prenatal screening and of adverse outcome in high-risk pregnancies due to maternal hiv infection. study design. the prevalence of prenatal screening in 330 pregnancies of hiv-positive women attending the department for prenatal screening and/or during labour between january 1, 2002 and december 31, 2012, was recorded. screening results were compared with the postnatal outcome and maternal morbidity, and mother-to-child transmission (mtct) was evaluated. results. one hundred of 330 women (30.5%) had an early anomaly scan, 252 (74.5%) had a detailed scan at 2022 weeks, 18 (5.5%) had a detailed scan prior to birth, and three (0.9%) had an amniocentesis. in seven cases (2.12%), a fetal anomaly was detected prenatally and confirmed postnatally, while in eight (2.42%) an anomaly was only detected postnatally, even though a prenatal scan was performed. there were no anomalies in the unscreened group. mtct occurred in three cases (0.9%) and seven fetal and neonatal deaths (2.1%) were reported. conclusion. the overall prevalence of prenatal ultrasound screening in our cohort is 74.5%, but often the opportunity for prenatal ultrasonography in the first trimester is missed. in general, the aim should be to offer prenatal ultrasonography in the first trimester in all pregnancies. this allows early reassurance or if fetal disease is suspected, further steps can be taken.

PMC3803124

pubmed-424

diabetes mellitus is a global health problem and an important cause of mortality and morbidity in many countries. the trend of increasing diabetes prevalence seems to prevail among developing countries. in brazil, diabetes affected 11.3 million people in 2011, and this number is likely to triple by 2030. estimates suggest that the diabetes rate in less developed countries will increase by 69% between 2010 and 2030. diabetes imposes a burden for society such as high socioeconomic costs that have an impact on productivity as well as life and health quality. this situation seems to be worse in developing countries, where the healthcare system often fails to meet demand. studies have concluded that a western dietary pattern, sedentary lifestyle, and genetic factors play a central role in diabetes development. the brazilian ministry of health has followed the world health organization's recommendations and has taken some actions to monitor diabetes such as an annual telephone-based survey. socioeconomic disparities might contribute to some degree of heterogeneity in measures of prevalence between regions. a study demonstrated that diabetes prevalence across the brazilian states ranged from 11% to 25%, with an overall rate of 16% in 2001. brasilia, the capital of brazil, is located in the central-west region of the country. the city has the highest human development index in brazil, but it has one of the highest levels of social inequality compared with other brazilian regions [11, 12]. these characteristics of brasilia warrant further investigation in many aspects, including the health status of its population. thus, the goal of this study was to estimate the prevalence of diabetes and its associated risk factors in adults of brasilia, brazil. the present cross-sectional population-based study was conducted in brasilia, brazil, from february to may 2012. the sample size was calculated based on an estimation of 16% of self-reported diabetes cases. considering a 95% confidence interval (ci), precision of 2.25%, and a design effect of 1.8, we added 10% of the sample size to compensate for any eventual attrition, which resulted in a final sample of 2,019 individuals. participants were selected by a two-stage probability sampling process by cluster and were stratified by sex and age. a total of 220 census tracts were randomly selected from 3,886 urban tracts with more than 200 inhabitants. up to 10 households were selected from each census tract. in total, one adult per household was selected following the predefined quotas of sex and age to answer the interview. trained professionals surveyed all of the participants in their homes using a semistructured questionnaire. to ensure reliability, 20% of the interviews were audited by telephone. to test the understanding and acceptability of the questionnaire, 150 pilot interviews were held prior to data collection. the dependent variable was self-reported diabetes. independent variables included demographic characteristics (age group, sex, marital status, living arrangements, and household location), socioeconomic characteristics (level of education, occupation, and social class), chronic health conditions (self-reported hypertension, depression, respiratory diseases, cardiovascular diseases, and other chronic diseases), access to healthcare (health insurance, medical consultation, and hospitalization), and perceived health status (mobility, self-care, usual activities, pain/discomfort, and anxiety/depression). the stratification was based on the brazilian criterion of economic classification, which defines five classes, with a being the wealthiest group and e being the poorest. in all of the analyses, self-reported diabetes prevalence in the population was then calculated at a 95% ci. to identify factors related to diabetes prevalence, we calculated prevalence ratios (pr) using bivariate analysis and calculated the adjusted pr by a poisson regression model with robust variance. in this model, we preferred to use this more conservative model that included all of the variables to allow for better confounding adjustment. other models that included only the most significant variables were tested and did not change the significance of the variables. associations were considered to be statistically significant when p<0.05. the stata software version 10.1 was used for all of the calculations. approximately 60% of the participants were women, and 57% were aged between 35 and 60 years. most of the participants belonged to economic class c, had completed high school, were married or cohabitating, lived with at least one more person in the household, and dwelled in a satellite town. diabetes was self-reported by 10.1% (95% ci: 8.5%11.6%) of the adult population in brasilia. table 1 depicts diabetes prevalence and prevalence ratios (pr) before and after adjustment by poisson regression. the age group of 3565 years, hypertension, respiratory disease, cardiovascular disease, and pain/discomfort were significantly associated with diabetes. sex, marital status, living arrangements, social class, education level, employability, living location, health insurance, medical consultation, hospitalization, physical mobility, self-care, usual activities, and anxiety/depression revealed no significant association. figure 2 illustrates differences in diabetes prevalence between all persons and the population with comorbidities. diabetes prevalence in the age range 3065 years is higher among individuals with cardiovascular disease, followed by those with hypertension and those with respiratory diseases. this result suggests that the likelihood of diabetes increases with age and is greater in persons with comorbidities. an age of 35 years and over, presence of pain or discomfort, cardiovascular disease, hypertension, and respiratory disease were positively associated with diabetes in the adult population of brasilia. the main limitations of our study were the self-reported assessments of the primary outcome and independent variables. self-reported diabetes might be a source of bias because individuals need to be aware of the diagnosis prior to answering, which could result in disease underestimation. however, performing a clinical test for diagnosing diabetes is not always possible in population-based studies. thus, self-reported answers regarding diabetes have been a common practice according to the literature [17, 18]. another shortcoming was the cross-sectional design of the study, which hampers a causal relationship between diabetes and the significantly associated factors identified herein. a previous population-based study developed in brazil in 2008 used telephone interviews to investigate self-reported diabetes prevalence and found low prevalence rates in brasilia. another study found that brasilia was the region with the highest diabetes prevalence compared with other brazilian regions from 2002 to 2007. research identified a significant increase in self-reported diabetes in the brazilian population because it ascended from 3.3% in 1998 to 5.3% in 2008. in south and central america, the estimated diabetes prevalence in 2013 was 8.0%; brazil demonstrated the highest prevalence, followed by colombia and argentina. the variability of diabetes prevalence may be due to a poorer diet and a lack of physical activity, or it could be related to better access to diagnostic testing. as expected, our results demonstrated that the likelihood of having diabetes increases with age. from a healthcare policy perspective, diabetes prevention and management programs should target young people and not only the elderly population. diabetes prevalence was higher among individuals with cardiovascular disease, hypertension, and respiratory disease compared with the general population. there is convincing evidence of the association between diabetes and hypertension, which increases the risk of a cardiovascular event. a 2003 study conducted in so luis, a city located in one of the poorest areas of brazil, observed a positive association between diabetes and hypertension. a cross-sectional study conducted between 2004 and 2005 in so jose do rio preto, a city in the brazilian southeast region, revealed that the diabetes prevalence was almost threefold higher in a population of hypertensive individuals compared with the general population. a cohort study performed in women between 1988 and 1996 throughout 11 states in the united states found that chronic obstructive pulmonary disease was a diabetes risk factor. a retrospective cohort study conducted in northern california reported that individuals with diabetes are at a greater risk of developing asthma, chronic obstructive pulmonary disease, fibrosis, and pneumonia. in contrast, a systematic review of 10 studies suggests that growing up in a socioeconomically disadvantaged environment may contribute to diabetes in later life. an australian study also described a positive association between socioeconomic variables and diabetes in adults aged 45 years and over. the perceived health dimensions physical mobility, self-care, usual activities, and anxiety/depression were not associated with diabetes in our sample. in 2012, a literature review found that diabetes was considered a potential risk factor for the poor performance of daily life activities among individuals aged 50 years and over. a study conducted with older adult other than depression, this finding might depict an association between diabetes and activities of daily living, which may be developed at older ages. diabetes is a common health condition in adults living in brasilia and is positively associated with older age, cardiovascular disease, hypertension, respiratory disease, and presence of pain or discomfort.

aim. the aim of this study was to estimate the prevalence of diabetes and its associated risk factors in adults from brasilia, brazil. methods. the present cross-sectional population-based study consisted of interviews with individuals aged 1865 years. participants were selected through two-stage probability sampling by clusters and stratified by sex and age. demographic and clinical data were collected directly with participants from february to may 2012. self-reported diabetes prevalence was calculated at a 95% confidence interval (ci). prevalence ratios (pr) were adjusted by poisson regression with robust variance. results. in all, 1,820 individuals were interviewed. diabetes prevalence in the adult population of brasilia was 10.1% (95% ci, 8.5%11.6%). variables associated with diabetes were an age between 35 and 49 years (pr=1.83; 95% ci, 1.192.82) or 50 and 65 years (pr=1.95; 95% ci, 1.173.23), hypertension (pr=4.04; 95% ci, 2.666.13), respiratory disease (pr=1.67; 95% ci, 1.112.50), cardiovascular disease (pr=1.74; 95% ci, 1.152.63), and pain/discomfort (pr=1.71; 95% ci, 1.212.41). conclusion. diabetes is a prevalent condition in adults living in brasilia, and disease risk increases with age and comorbidities. future health policies should focus on screening programs and prevention for the more vulnerable groups.

PMC4451559

pubmed-425

natural killer t (nkt) cells are innate-like lymphocytes typified by coexpression of receptors characteristic of natural killer and conventional t cells. as such, murine nkt cells generally bear ly49 receptors, nkg2 family of receptors, cd94, and nk1.1 (the latter only being expressed in specific strains, including the commonly used c57bl/6). human nkt cells often express similar surface molecules including cd56, cd161, cd94, nkg2d, and nkg2a. both human and mouse nkt cells display a variety of stimulatory and inhibitory t cell-associated receptors and ligands (e.g., cd28 and cd154), whose expression depends on the activation status of the cell. finally, both human and murine nkt populations include cd4 and cd4cd8 (double negative; dn) subpopulations; while cd8 nkt cells are found in humans, they are rare in mice. the t cell receptors (tcrs) expressed by nkt cells recognize the conserved and nonpolymorphic mhc class i-like molecule, cd1d. unlike classical mhc class i-like molecules, the expression of cd1d is largely restricted to cells of bone marrow origin including antigen presenting cells (apcs) such as dendritic cells (dcs), macrophages, and b cells. furthermore, the cd1d molecule (via heterodimerization with 2-microglobulin) specializes in displaying lipid moieties rather than protein polypeptides. importantly, intact expression of cd1d is critical for the development of nkt cell populations, as cd1d/ mice are devoid of these cells. nkt cells are further subclassified into type i or ii lineages, depending on the composition of their tcr and the cd1d-presented glycolipid antigens to which they respond. type i or invariant nkt (inkt) cells express canonical tcr chains comprised of specific gene segments (v14-j18 in mice and v24-j18 in humans) that preferentially pair with specific tcr chains (v8, v7, or v2 in mice and v11 in humans). these invariant tcr pairings confer reactivity to cd1d and a restricted array of presented glycolipid antigens. the dependence of inkt cells on the v14-j18-comprised tcr is demonstrated by v14 tcr transgenic mice, in which a higher frequency and number of inkt cells are observed, and also j18/ mice, in which no mature inkt cells develop. despite the conserved use of the invariant tcr, inkt cell populations are phenotypically (e.g., presence or absence of cd4 expression) and functionally (e.g., preferential production of certain cytokines, such as il-17) diverse. the prototypical (and first discovered) inkt cell stimulatory glycolipid, alpha-galactosylceramide (-galcer), was identified during a screening for compounds from marine sponges (agelas species) with antitumor activity. since this initial discovery, a number of naturally occurring and synthetic lipid antigens have been described to bind cd1d and activate inkt cells. these cells are now typically identified using cd1d tetramers loaded with -galcer or its synthetic analogs (e.g., pbs-57;). in contrast, type ii or variant nkt (vnkt) cells bear a more diverse array of tcr and chains and have been shown to recognize sulfatide moieties presented by cd1d. more recently, type ii nkt cells have also begun to be better characterized through development of cd1d tetramers loaded with sulfatide [9, 10], but these cells are still less well characterized than their invariant brethren. given that far more is known regarding the antitumor activity of inkt cells, we will predominantly focus our attention on these cells. inkt cells develop in the thymus, by originating from cd4cd8 double positive (dp) thymocytes. positive selection of inkt cells is mediated by homotypic interactions of dp cells and recognition of glycolipid antigen-cd1d complexes [1114]; however, the nature of the self-antigens involved in this process remains somewhat elusive. like conventional t cells, maturation of inkt cells at the dp stage and beyond depends on the ability to construct a functional tcr and intact signaling. as such, inkt cells are profoundly diminished or absent in mice lacking expression of rag, cd3, lck, zap-70, slp-76, itk, lat, or vav [1521]. transcriptionally, development of inkt cells at the dp stage is regulated by the transcription factor rort, which prolongs the survival of dp thymocytes by upregulating bcl-xl, to allow sufficient time for distal tcr gene segment rearrangements to occur [22, 23]. more recent studies have shown that heb, the e protein family of basic helix-loop-helix transcription factors, regulates inkt cell development by regulating rort and bcl-xl mrna. finally, the absence of the transcription factor runx1 also blocks inkt cell development at the earliest detectable inkt cell-committed subset. inkt cell development at the dp stage also critically depends on the signals generated by engagement of the signaling lymphocyte activation molecule (slam) family of surface receptors, which are expressed on developing inkt cells, as well as conventional dp thymocytes. slam family receptor signaling is transduced by the adaptor molecule sap (slam-associated protein), which in turn binds to the tyrosine kinase fyn, and results in propagation of a phosphorylation signal [25, 26]. accordingly, inkt cells fail to develop in mice and humans bearing mutations in the gene that encodes for sap [2729], in mice lacking fyn or expressing a mutant version of sap that can not bind fyn [23, 30], in mice in which both ly108 and slam signaling are simultaneously disrupted, and in those lacking the transcription factor cmyb (which is necessary for appropriate expression of sap and certain slam family members). taken together, these studies establish the importance of the slam-sap-fyn signaling axis in inkt cell development. following positive selection, inkt cells undergo distinct stages of maturation that are characterized by the sequential acquisition of cd24, cd44, and nk1.1: cd24cd44nk1.1 (stage 0), cd24cd44nk1.1 (stage 1), cd24cd44nk1.1 (stage 2), and finally cd24cd44nk1.1 (stage 3). as these cells progress through these developmental stages, they begin to upregulate nk cell markers (e.g., nkg2d and ly49 receptors), cd69, and cd122 and acquire distinct effector functions (e.g., production of il-4, ifn-, perforin, and granzymes). one of the key regulators of inkt cell development and acquisition of an effector/memory phenotype and functions is the broad complex tramtrack bric-a-brac-zinc finger transcription factor plzf, whose expression is highest in stage 0 and 1 populations [36, 37]. plzf-deficient animals exhibit a severe reduction in inkt cell number and plzf-deficient inkt cells fail to cosecrete th1 and th2 cytokines upon stimulation [36, 37]. recently, it was demonstrated that the lethal-7 microrna posttranscriptionally regulate plzf expression and inkt cell effector functions. the transcription factor t-bet is indispensable for the final maturation stages of inkt cells [39, 40] and absence of this transcription factor results in reduced inkt cell numbers due to developmental blockade at stage 2. t-bet-deficient inkt cells fail to proliferate in response to il-15 as they lack surface expression of cd122, a component of the il-15 receptor. in addition, t-bet-deficient inkt cells fail to produce ifn- in response to tcr stimulation and exhibit defective cytolytic activity [39, 40] as t-bet directly regulates the activation of genes associated with mature inkt cell functions, such as perforin, cd178, and ifn-. as inkt cells progress to stage 1, a proportion of cells downregulate cd4, giving rise to dn inkt cells. generation of the cd4 inkt cell lineage and production of th2-type cytokines is critically regulated by the transcription factor gata-3. similar to plzf-deficient inkt cells, gata-3 deficient inkt cells fail to produce th1 or th2 cytokines in response to -galcer. recent studies have identified a unique subpopulation of nk1.1cd4 inkt cells that are transcriptionally regulated by rort and capable of producing large quantities of il-17 upon stimulation. as such, inkt cells are also sometimes classified into nkt1, nkt2, and nkt17 based on their cytokine production profiles and respective expression of t-bet, gata-3, and rort [43, 44]. finally, mechanistic target of rapamycin (mtor) signaling has also been shown to be important for inkt cell lineage diversification and acquisition of effector functions [4548], and loss of mtor2 may result in loss of nkt17 cells. taken together, these recent studies provide new insights into the transcriptional regulation of inkt cell maturation and functional differentiation. the importance of inkt cells in mediating protection against tumors is highlighted by several findings. first, a number of independent studies have shown a decrease in the number of inkt cells in the peripheral blood of patients with a variety of cancers and even precancerous myelodysplastic syndromes [4951]. moreover, the inkt cells that persist appear to have decreased proliferative and functional responses [5254]. interestingly, an increased frequency of peripheral blood inkt cells in cancer patients portends a more favorable response to therapy [55, 56]. while these observations identify an association between inkt cell numbers and/or function and development of malignancy, they do not provide a direct causal link this link has been established in a number of mouse studies in which the biology of the host and initiation of tumors can be more systematically manipulated via gene knockouts, antibody depletion strategies, and adoptive transfer of various lymphocyte populations into cancer-predisposed or tumor-challenged hosts. in mice that are prone to development of tumors due to loss of one allele of a tumor suppressor (p53+/), absence of inkt cells (by virtue of genetic knockout of the j18 gene segment or cd1d) results in earlier and more frequent development of tumors and thus shorter survival, when compared to inkt-sufficient littermates. similarly, treatment of cd1d/ and j18/ mice with a carcinogen resulted in increased incidence and earlier onset of tumors in comparison to treated wild type mice. conversely, administration of -galcer to mice controlled the growth and metastasis of adoptively transferred [59, 60] or carcinogen-induced [61, 62] or spontaneous tumors. moreover, adoptive transfer of inkt cells into j18/ inkt cell-deficient mice prevented the growth of subcutaneous sarcomas. finally, adoptive transfer of small numbers of purified inkt cells into lymphocyte-deficient nod-scid-il2r/ (nsg) mice was sufficient to protect mice from challenge with a cd1d tumor. these findings collectively argue that inkt cells play a central and nonredundant role in the response to tumors. further studies would shed light on the mechanisms by which inkt cells exert these antitumor effects. engagement of the invariant tcr by cd1d/glycolipid antigen complexes results in inkt cell activation, an event that is typified by rapid and robust production of a variety of cytokines and chemokines, including but not limited to il-2, il-4, il-10, il-13 il-17, ifn-, tnf, tgf, gm-csf, rantes, eotaxin, mip-1, and mip-1 [65, 66]. the nature and magnitude of the inkt cell cytokine response depend on the glycolipid antigen; for example, -galcer-mediated inkt cell activation elicits a strong ifn--dominated cytokine response, while och (a synthetic analog of -galcer with a truncated lipid chain) elicits a response with significantly higher level of il-4 production. the rapidity of this cytokine response is attributed to the semiactivated state of inkt cells and the presence of preformed cytosolic mrna for a variety of cytokines. indeed, administration of -galcer to inkt cell-sufficient, but not inkt cell-deficient, mice results in polyclonal activation of conventional t, b, and nk cells within 3-4 hours and also eventually leads to the mobilization of macrophages and neutrophils. intriguingly, it was previously believed that mammalian species are incapable of producing glycolipids (such as -galcer), in which the sugar moiety is attached via an o-linkage to the ceramide backbone in an alpha-anomeric configuration. despite the absence of -glucosyl or -galactosyl transferases in mammals, recent findings indicate that a small percentage of the glycolipids that are constitutively presented by mammalian cd1d are indeed -anomeric. whether the percentage of cd1d-presented -anomeric glycolipids is altered in tumor tissues nonetheless, following encounter with cd1d/antigen complexes displayed by apcs, inkt cells not only produce cytokines but also upregulate surface expression of cd154 (see figure 1(a)). ligation of apc-expressed cd40 is especially important for mediating subsequent maturation and functional activation of dcs, subsequent upregulation of cd80 and cd86, and amplified production of ifn- [72, 73]. in addition, the ligation of the chemokine receptor cxcr6 on inkt cells by cxcl16 expressed on apcs also provides costimulatory signals resulting in robust -galcer-induced inkt cell activation. importantly, matured dcs are potent producers of il-12, which induces sustained ifn- production by inkt cells [7577]. the importance of inkt cells in il-12-mediated tumor rejection was effectively demonstrated by the defective clearance of a variety of tumors in j18/ mice. mature dcs also support the priming and activation of cd8 t cells, culminating in optimal effector and memory cell formation [72, 78]. finally, the sustained release of ifn- by inkt cells leads to activation and proliferation of nk cells and nk cell secretion of ifn-. the combination of cytokines (e.g., il-2, il-12, and ifn-) as a result of inkt cell activation also leads to upregulation of death-inducing ligands (e.g., cd178 or cd253) on nk cells and cd8 t cells [79, 80]. these sequential activation events are believed to be critical for the -galcer-induced inkt cell-mediated antitumor effects [76, 81, 82]. as such, inkt cells not only bridge the activation of innate and adaptive immunity, but also indirectly potentiate the antitumor activity of other cytotoxic effector lymphocytes. tumor establishment and growth are believed to be intricately modulated by a myriad of soluble and contact-derived signals obtained from the tumor microenvironment (tme), which consists of the tumor cells themselves, tumor-infiltrating lymphocytes (tils), and stromal cells that communicate in a dynamic and bidirectional manner. in addition to their indirect modulation of other effector lymphocyte populations, inkt cells may also regulate tumor growth via their effects on the tme (see figure 1(b)). indeed following intravenous administration, inkt cells were shown to represent a significant percentage of the tils in patients with head and neck carcinomas [83, 84]. importantly, higher frequency of tumor-infiltrating inkt cells correlated with overall and disease-free survival as an independent prognostic factor in primary colorectal cancer patients and with tumor regression in head and neck carcinomas. conversely, in patients with primary hepatocellular or metastatic cancer, cd4 inkt cells that produced high levels of th2-type cytokines and had low cytolytic activity were enriched within the tumor and appeared to inhibit the expansion of antigen-specific cd8 t cells, suggesting that these particular inkt cells may contribute to generate an immunosuppressive microenvironment. in experimental studies, cotransfer of human monocytes and inkt cells to tumor-bearing nod-scid mice suppressed tumor growth when compared with mice that received monocytes alone. importantly, inkt cells can target tumor supportive cells such as tumor-associated macrophages (tams), a highly plastic monocyte-derived subset of inflammatory cells that can exert immunosuppressive functions, and promote tumor proliferation and matrix turnover [88, 89]. indeed tams are known to produce il-6, a cytokine that appears to promote the proliferation of many solid tumors, including neuroblastomas and breast and prostate carcinomas. found that macrophage density correlated positively with microvessel counts and negatively with patient relapse-free survival. since tams cross-present neuroblastomaderived endogenous cd1d ligand(s), they can be specifically recognized and killed by inkt cells in an il-15-dependent process. other potential inkt cell tme targets include myeloid-derived suppressor cells (mdscs). mdscs have been found to accumulate in the blood, lymph nodes, and bone marrow and at tumor sites in most patients and experimental animals with cancer and inhibit both adaptive and innate immunity. the absence of inkt cells in mice during influenza virus infection resulted in the expansion of mdscs, high viral titer, and increased mortality. the adoptive transfer of inkt cells abolished the suppressive activity of mdscs and restored virus-specific immune responses, resulting in reduced viral titers and increased rates of host survival. thus, certain populations of inkt cells may help alter the tme via their effects on tams and mdscs, to help create a tumor-suppressive or immune-permissive milieu. in addition to their indirect control of tumor growth, inkt cells can mediate direct killing of tumor targets (see figure 1(c)). inkt cells alone, or in combination with nk cells, have been shown to kill a variety of tumor targets in vitro [6, 93, 94]. while this mechanism of killing appears to be dependent on the presence of stimulatory glycolipids and cd1d [95, 96], inkt cell cytotoxicity also appears to be triggered via ligation of nkg2d by target-expressed stress ligands. it remains to be seen whether mult1, the newly identified shed form of high affinity nkg2d ligand that triggers nk-mediated tumor rejection in mice, also activates inkt cells. consistent with their direct cytotoxic capacity, inkt cells express perforin and granzymes, as well as cd178 [34, 96, 99, 100]. in our hands, blockade of cd1d-mediated lipid antigen presentation, disruption of t cell receptor (tcr) signaling, or loss of perforin expression was found to significantly reduce inkt cell killing in vitro. moreover, we demonstrated that inkt cells alone were sufficient for control of the growth of a t cell lymphoma in vivo that preferentially relies on perforin and the adaptor protein sap [64, 69]. mechanistically, inkt cells rely on sap for formation of stable conjugates with the tumor targets as well as proper orientation of the lytic machinery at the immunological synapse. despite the majority of studies implicating inkt cells as having an antitumor role, a limited number of studies also implicate inkt cells as suppressing antitumor responses, but these paradoxic responses may be related to the level of tumor cd1d expression [102, 103]. alternatively, these differences may stem from the fact that contrary to the use of c57bl/6 mice in the previously discussed studies these last two studies were performed in balb/c mice, in which there is a predominance of il-4-producing th2 phenotype inkt cells. interestingly, the antitumor responses of inkt cells may be regulated by the activity of type ii nkt cells. demonstrated that type ii variant nkt (vnkt) cells were sufficient for the downregulation of tumor immunosurveillance and relapse growth of a model fibrosarcoma in an antigen-dependent manner, while a second study found that activation of vnkt cells with sulfatide antigen could suppress the activation of inkt cells. conversely, type ii vnkt cells were, in at least one study, suggested to promote the antitumor activity of cpg oligodeoxynucleotides. this suppression appears to be mediated through a contact- and il-10-dependent mechanism [109, 110]. indeed, induction of treg cells suppressed the protective effect of adoptive transfer of inkt cells into j18/ mice. consistent with these findings, depletion of treg cells or short-term elimination of their suppressive activity results in enhanced inkt cell-mediated antitumor responses and increased nk and cd8 t cell activation and ifn- production. interestingly, the ability of treg cells to suppress inkt cell proliferation depends on the degree of invariant tcr agonism, such that responses to weak (e.g., och), but not strong (e.g., -galcer), agonists were effectively suppressed. when viewed collectively, these findings suggest that inkt cells possess inherent capacity for direct cytotoxicity but their antigenic exposure may modulate whether their antitumor effects can be suppressed by treg and vnkt cells. given the preponderance of evidence suggesting that the activation of inkt cells provides protection against the growth and metastasis of a variety of tumors, safety of -galcer administration was examined in a phase i trial. while administration of -galcer was well tolerated at a range of doses, no clinical responses were observed in patients with advanced solid tumors. on the heels of this study, nieda et al. showed that treatment of metastatic cancer patients with -galcer-pulsed immature monocyte-derived dcs resulted in dramatic increases in serum ifn- and il-12 and activation of nk and t cells in the majority of subjects. importantly, this phase i trial also documented reduction in tumor biomarkers and tumor necrosis in several patients. these findings were extended in a study of a small number of patients, in which the -galcer-pulsed dcs were matured prior to adoptive transfer. this study demonstrated a>100-fold increase in blood inkt cell numbers in all patients, and this increase was long-lived (> 6 months). a number of subsequent clinical trials, all with limited number of patients with advanced head and neck or non-small cell lung cancers, have since employed similar strategies of adoptive transfer of -galcer-pulsed apcs [115118]. collectively, these studies demonstrate increases in blood ifn- levels and inkt cells in some but not all patients, stabilization of disease in a few of the subjects, and absence of severe treatment-related toxicities. in a different approach, chemotherapy-refractory 5 lymphoma patients were treated with autologous peripheral blood mononuclear cells (pbmcs) stimulated with anti-cd3, il-2, and ifn-. this ex vivo stimulation resulted in enrichment of nkt cells (to ~20% on average), and this cell fraction was shown to possess the highest cytotoxic capacity in vitro. of the nine patients who received adoptive transfer of these cells, two showed partial responses and two others had stabilization of disease. two subsequent studies by motohashi et al. evaluated the adoptive transfer of ex vivo expanded inkt cell-enriched cells to patients with advanced cancer. in the first, 6 patients with advanced non-small cell lung cancer were treated with either a low or a high dose of ex vivo expanded inkt cells. of the 3 patients treated with the high dose, all had an increase in the frequency of ifn--producing pbmcs and 2 showed expansion of inkt cells. although no clinical responses were observed in this study, a follow-up trial of 17 patients with advanced head and neck cancers treated with a high dose of inkt cell-enriched autologous pbmcs showed a significant increase in ifn--producing pbmcs in 10 of 17 patients. importantly, while none of these patients displayed tumor regression, 5 had disease stabilization and the mean survival time for the subjects with higher frequencies of ifn--producing pbmcs was tripled above those with low percentages of ifn--producing pbmcs (29.3 versus 9.7 months). administered both in vitro expanded inkt cells and -galcer-pulsed apcs to patients with advanced head and neck squamous cell carcinomas. treatment increased the frequencies of inkt cells and ifn--producing pbmcs, and a partial clinical response or disease stabilization was observed in 7 of 8 patients. although the responses in these studies have not been profound, it must be noted that these inkt cell-based immunotherapies have all been conducted on patients with advanced malignancies often those in whom standard chemotherapy, irradiation, and/or surgical excision treatments had failed. future studies of inkt cell-based immunotherapy may be able to take advantage of two recent technologies. as mentioned previously, many malignancies are associated with a decrease in the numbers and proliferative capacity of peripheral blood inkt cells. in order to circumvent the difficulty of being able to expand these infrequent and potentially defective cells from patients, watarai et al. generated induced pluripotent stem (ips) cells from mature inkt cells and then expanded large numbers of inkt cells from these established ips cells. ips-nkt cells generated in this fashion were demonstrated to be able to activate and expand antigen-specific cd8 t cell responses to limit the growth of leukemia in mice without inducing graft versus host disease (gvhd). described inkt cells engineered to express cars bearing specificity for gd2, a highly expressed moiety on neuroblastoma cells. in their studies, they showed that inkt cells expanded from the pbmcs of healthy human donors and transduced with retroviral car constructs could protect humanized nsg mice against metastatic neuroblastoma without inducing gvhd. whether these two technologies could be combined to generate functional car-bearing ips-nkt cells remains to be seen. inkt cells are innate-like effector lymphocytes that not only are directly cytotoxic, but also possess the unique ability to nucleate the antitumor responses of other effector lymphocytes and alter the cellular and angiogenic makeup of the tumor microenvironment. as such, the promise of an effective inkt cell-based immunotherapy can only be realized by devising and evaluating strategies that simultaneously maximize each of these antitumor effector mechanisms. the challenge for the future will thus be to identify these strategies and apply them to tumors against which inkt cells wield the most optimal responses.

natural killer t (nkt) cells are innate-like lymphocytes that were first described in the late 1980s. since their initial description, numerous studies have collectively shed light on their development and effector function. these studies have highlighted the unique requirements for the activation of these lymphocytes and the functional responses that distinguish these cells from other effector lymphocyte populations such as conventional t cells and nk cells. this body of literature suggests that nkt cells play diverse nonredundant roles in a number of disease processes, including the initiation and propagation of airway hyperreactivity, protection against a variety of pathogens, development of autoimmunity, and mediation of allograft responses. in this review, however, we focus on the role of a specific lineage of nkt cells in antitumor immunity. specifically, we describe the development of invariant nkt (inkt) cells and the factors that are critical for their acquisition of effector function. next, we delineate the mechanisms by which inkt cells influence and modulate the activity of other immune cells to directly or indirectly affect tumor growth. finally, we review the successes and failures of clinical trials employing inkt cell-based immunotherapies and explore the future prospects for the use of such strategies.

PMC4620262

pubmed-426

the vast majority of cancers in the oral cavity and in the head and neck are squamous cell carcinomas (sccs). it is the sixth most common cancer worldwide, and its incidence is rising in industrialized nations [1, 2]. similarly, other benign lesions of the oral cavity such as lichen planus may have a prevalence of 0.52% in the general population and may have a risk of malignant transformation of 1%. many benign oral mucosal lesions are not cancerous which presents a clinical dilemma to the physician. furthermore, precancerous lesions such as leukoplakia may exhibit mild structural alterations in the mucosa that can be difficult to distinguish from normal healthy tissue. currently, obtaining histopathology via biopsy is the gold standard of diagnosis; however, this procedure can pose significant morbidity to the patient such as the risk of bleeding, wound infection, and potentially impairment of speech and swallowing if multiple biopsies are performed. moreover, it becomes a clinical challenge to monitor patients for progression of diffuse dysplasia or leukoplakia, and many of them may require multiple biopsies over many years. the discomfort of biopsy and compromisation of tissue integrity can lead to problems with future biopsy interpretation or in the case of laryngeal biopsy, considerable problems in individuals with high vocal demands. subsequently, any technique that can yield histopathological information without injuring tissue has obvious advantages over biopsy. detailed examinations of the texture, color, contour, and extent of mucosal lesions have been performed utilizing many instruments such as the hopkins ' rod-lens scopes, flexible endoscopes, direct laryngoscopes, and advances in microlaryngoscopic visualization techniques. however, these methods are limited by their inability to provide histopathological data during the clinical examination. as a result, over the last decade, technological advances in optical imaging detection techniques have emerged with a variety of methods employed to facilitate detailed examination and provision of histopathological information of mucosal lesions. examples of such novel optical techniques include: aminolevulinic acid-induced fluorecence, autofluorescence, confocal endomicroscopy, and contact endoscopy. aminolevulinic acid-induced fluorescence is a technique whereby neoplastic cells undergo preferential fluorescence after aminolevulinic acid (ala) has been applied to the mucosa surface. in the presence of ala, once this dye-like substance has been applied, mucosa containing neoplastic cells will fluoresce orange red and normal mucosa will retain the normal green fluorescence. coupled with autofluorescence, several authors have noted that these techniques can diagnose laryngeal carcinoma and dysplasia with good accuracy. autofluorescence was first described in identification of neoplastic cells of the larynx by harris et al.. certain molecules then transform into photonic energy, which is emitted as long-wave scattered light which can be detected. the autofluorescence imaging method detects the fluorecence given off by the different concentrations of fluorophores seen in normal and neoplastic mucosa. thus, autofluorescence videoendoscopy for photodiagnosis of head and neck squamous cell carcinomas has been described as being quite accurate with good sensitivity and specificity in several studies [616]. unlike ala and autofluorescence where histological detail is not appreciated, other optical techniques such as narrow-band imaging endoscopy (nbie) nbie uses filtered light with wavelengths preferentially corresponding to peaks of absorption of hemoglobin to enhance superficial neoplasms based on their neoangiogenic pattern. these light wavelengths penetrate superficial mucosal and deep submucosal layers to enhance capillary and submucosal vasculature. the obtained image is further enhanced by using high-definition television (hdtv). carcinomas can then be identified based on the changes in the microvascular pattern of the mucosal lesion. several studies have shown good sensitivity, specificity, negative and positive predictive value, and accuracy in detection of squamous cell carcinoma of the oral cavity, oropharynx, larynx, and esophagus [1720]. however, instead of solely relying on neoangiogenic patterns for diagnosis of carcinoma, further histological detail can be obtained with the use of confocal endoscopy which is an in vivo optical imaging method whereby mucosal lesions can undergo significant magnification to allow examination of cellular histology. this technique also allows reconstruction of three-dimensional structures based on the acquired images. utilization of various stains to help highlight cellular structures has been tried by some authors to distinguish normal from invasive carcinoma cells. the utility of this new technology is highlighted in its capability to distinguish between benign or low-grade mucosal dysplasia thereby potentially reducing unnecessary biopsies. contact endoscopy is another novel noninvasive optical diagnostic imaging method that allows in vivo and in situ examination of the cellular architecture of the superficial layers of the mucosal epithelium. magnified images are obtained using hopkins ' rod-lens endoscope placed on the surface of the dye stained mucosal tissue. this technique allows assessment of precancerous and cancerous lesions in vivo and has significant potential in the histopathologic diagnosis of many suspicious head and neck mucosal lesions without tissue biopsy. ce was originally described and used by hamou in 1979 as a technique for visualization of cervical and uterine epithelial cells for screening and diagnosis of cervical and uterine pathology. the first reported use of ce in otolaryngology head and neck surgery was by andrea et al. as a diagnostic tool in the evaluation of various pathologies in the larynx in the 1990s [4, 2330]. they were able to visualize and diagnose laryngeal mucosal pathology from the magnification of vocal fold epithelium and microvasculature during microlaryngoscopy after staining the vocal cords with methylene blue dye. diameters, of these scopes come as either 4 mm or 5.5 mm and lengths of 23 cm and 18 cm. straight forward (o) and forward-oblique telescopes (30) are also available, and all are capable of 1x, 60x, and 150x magnification. these endoscopes require a high intensity xenon light source, and images can be digitally captured for real-time photographic and video documentation, figures 1 and 2. the most basic technique of ce involves staining of the superficial cells of the mucosa with a contrast dye, 1% methylene blue (mb) after which the magnifying endoscope (karl storz 8715 aa, tuttlingen, germany) 0 is then placed in contact against the mucosal surface, and the documented magnified cytological images (at 60x or 150x) are then recorded, figure 3. both a cytopathologist and an otolaryngologist can then assess these images, comparable to histology, figure 4. contact endoscopy and its efficacy in head and neck oncology, advantages, limitations, and future potential diagnostic utility will be briefly reviewed in this article. the literature search was conducted using the following key terms: contact endoscopy, contact microlaryngoscopy, aminolevulinic acid induced fluorecence, autofluorescence, confocal endomicroscopy, oral mucosa, oral cavity, larynx, oropharynx, hypopharynx, head and neck carcinoma, leukoplakia, and lichen planus. relevant search terms and combinations using boolean operators were performed, and relevant article selection was limited to the prospective, human and english studies without restriction to year of publication. examined 42 consecutive patients at a tertiary care center with suspicious lesions of larynx, pharynx, and esophagus under general anesthesia. the results obtained by the cytopathologist and otolaryngologist were based on images generated from the ce. they found that the more experienced the examiner, the higher the sensitivity of ce was in the diagnostic differentiation of benign versus malignant mucosal lesions. they included 142 patients undergoing microlaryngoscopy at their institution with various laryngeal diseases all underwent ce and subsequent biopsy for histopathological diagnosis. all malignant lesions identified by ce was confirmed by histopathology, but ce did not identify malignancy in 10 patients diagnosed histopathologically thus giving ce a sensitivity of 79.6%, specificity of 100%, and accuracy of 93%. their results were based on computer-assisted analysis of all ce images based certain nuclear morphometric parameters to determine benign from malignant lesions. thus, based on their computer-assisted analysis of ce images, their sensitivity was 91% and specificity 81%. all patients were examined with contact rhinoscopes under local anesthesia and biopsy of the area under examination was done. in all 5 cases of malignancy, ce and histological diagnosis directly correlated with each other. most significantly, for the prediction of persistent and recurrent disease, sensitivity and specificity for ce was 100% with an accuracy of 92.1%. pilot study examined 83 patients using both autofluorescence and contact endoscopy during microlaryngoscopy. for contact endoscopy, the calculated sensitivity was 94.7, specificity of 95.5 and an accuracy of 94%. in summary, authors of the above prospective trials have obtained the following results: a sensitivity of 79100%, a specificity of 81100%, and an accuracy of 8894%. overall, it appears that sensitivity, specificity, and accuracy of ce are similar across the trials. since the development of contact endoscopy, this technology has been used successfully by several authors in analyzing and diagnosing various pathologies of the larynx, oral cavity, oropharynx, and nasopharynx via real-time examination of mucosal cytological detail [4, 2631, 3336]. despite its introduction into otolaryngology, ce has yet to find a place in routine clinical practice despite its potential advantages. from the above clinical trials, ce appears to have good sensitivity, specificity, and accuracy as a noninvasive method for distinguishing between benign and malignant mucosal lesions in the head and neck. however, some authors state that it may be difficult for ce to detect mild (grade i) mucosal dysplasia because most of the cellular anomalies occur in at the level of the basal epithelium, and this technique can only examine cellular architecture found at the superficial epithelial layers [4, 21]. despite this limitation, most significantly, ce accurate ability to diagnose and tease out the histological differences between squamous metaplasia, atypia, and carcinoma even in the presence of irradiated mucosa was highlighted by the study performed by pak et al.. at present, most authors seem to agree that it has significant potential as a noninvasive detection method that could play a role as a future substitute for histological examination. most significantly, it offers a noninvasive, rapid, and repeatable in vivo assessment of the cytological architecture while avoiding the need for an invasive biopsy and its associated risks. ce provides immediate results, with the possibility of examining multiple mucosal areas in a short time. ce can also assess a wider surface mucosal area, providing more information than a selected histological section taken by biopsy. it also avoids tissue damage and alteration of cellular architecture which may occur in the biopsy and histological preparation. this noninvasive technique also helps to direct the site of biopsy by identifying areas with cellular atypia and thus avoiding the need for multiple biopsies. subsequently, this results in a dramatic improvement of the diagnostic yield of the biopsy. other potential roles of ce include the rapid diagnosis of benign and malignant mucosal lesions in an outpatient or operating room setting, surveillance, guided biopsies, and intraoperative evaluation of tumor resection margins. despite its advantages most notably, ce can only evaluate the most superficial cell layer of the mucosal epithelium. this is most likely due to a number of factors including (i) poor penetration of methylene blue which only stains a few superficial layers, (ii) short focal distance of the scope (i.e., ce can only assess to a depth of 80 um at 60x magnification and 30 um at 150x magnification), and (iii) optical artifact at high magnification due to glare from light reflected from cells not in focus. subsequently, assessment of submucosal lesions or lesions occupying deeper cell layers becomes more difficult [4, 27, 28, 30, 32, 33]. the lack of depth of penetration prevents the evaluation of important histological information especially when vertical extent of dysplasia is crucial in distinguishing the different grades of dysplasia from carcinoma in situ and invasive carcinoma. as a result, these factors could affect the sensitivity of ce, thus accounting for some of the false negative diagnostic results noted by authors. the potential impact of ce missing a malignant lesion needs to be taken into consideration if this technology is to one day substitute histopathology. future investigation into better penetrating dyes, advances in digital optics, and image enhancements will eventually allow better vertical staining and increased resolution of the deeper cell layers which would translate ce in becoming a much more sensitive and accurate diagnostic tool. a pilot study conducted at our institution also investigated some of the limitations and potential advantages of ce in the evaluation of head and neck mucosal lesions. from our preliminary experience, technical difficulties with line of sight, access difficulties to mucosal surfaces, scope positioning, and problems with consistent image quality due to artifact were consistent with those found by previous authors [30, 33]. our pilot study also demonstrated that although ce is a simple, rapid, repeatable, noninvasive examination performed with standard equipment, there is a learning curve associated with its use. however, once one is accustomed with this detection system, ce can be performed almost as quickly as an outpatient flexible fiberoptic nasopharyngoscopic examination. in conclusion, in vivo assessment of head and neck mucosal pathology may be applied to (i) early detection of premalignant and malignant lesions, (ii) serial follow-up examinations of suspicious lesions such as leukoplakia and lichen planus, and (iii) assessment of resection margins. despite its limitations, ce represents a promising optical technology that may afford reliable, accurate, and noninvasive in vivo assessment of cytological pathology. prospective investigation with ce we hypothesize that future study will demonstrate improved sensitivity, specificity, and accuracy of contact endoscopy in the diagnosis of head and neck tumors.

background. there are a variety of described noninvasive optical detection techniques for evaluation of head and neck mucosal lesions. contact endoscopy is a promising method of in vivo microscopic examination whereby a rigid telescope is placed on a previously dye-stained mucosa allowing evaluation of the superficial cell layers of the epithelium. this technique produces real-time, magnified images of cellular architecture of surface mucosa comparable to histology without the need for biopsy. in this review, we will briefly summarize the efficacy of ce in the detection of precancerous and cancerous mucosal lesions and its potential as a novel technique in early diagnosis, monitoring, and preoperative assessment of mucosal lesions of the head and neck. methods. pubmed, medline, and cochrane search revealed five prospective articles on contact endoscopy for the diagnosis of mucosal lesions in the head and neck. results. the literature search yielded five prospective studies examining contact endoscopy for the diagnosis of benign versus malignant head and neck mucosal lesions. these reported a sensitivity and specificity of 77100%, specificity of 66100% and an accuracy of 7292%. conclusion. contact endoscopy is a promising optical technology that may be a useful adjunct in the evaluation and diagnosis of benign and malignant head and neck mucosal lesions. future prospective randomized double-blind studies of this detection method are required.

PMC3010668

pubmed-427

the first historical study of quality assessment in the medical field was reported by ernest amory codman, md, of massachusetts general hospital in 1920. to support his end results theory, he made public the results of the review of his own hospital in a privately published book, a study in hospital efficiency, in which he emphasized the importance of patient follow-up and quality assessment. he helped to found the hospital standardization program, which eventually became the joint commission on accreditation of healthcare organizations in 1987, and the joint commission in 2007, with the motto helping health care organizations help patients. from the 1970s through the 1980s, the rapid increase in medical lawsuits and the medical malpractice insurance crisis promoted risk management of medical practices in the united states. in 1989, the society for thoracic surgeons (sts) started to establish national databases as an initiative to improve quality and patient safety among cardiothoracic surgeons and to respond to strong public opinion about the importance of accountability. in 1997, an initiative was begun to improve data quality and auditing, and staff were hired to support these efforts. in the sts congenital heart surgery database data specification, (http://www.sts.org/sites/default/files/documents/congenitaldataspecsv3_22.pdf), the patient national identification (social security number) is listed, but this field should be collected in compliance with state/local privacy laws. the sts national database complies with the health insurance portability and accountability act, and the federal government protects the sts national database. in 1998, the sts contracted with the duke clinical research institute (dcri) for data warehousing and data analysis. in 1999, the institute of medicine published a report titled to err is human: building a safe health system, which stated that 44,000 to 98,000 persons die in hospitals as a result of medical errors that could have been prevented. today, the management of the national database is one of the most important tasks of the sts. the database contains three components: adult cardiac surgery, general thoracic surgery and congenital heart surgery (fig. the sts was the first professional organization to seek approval for its measures from the national quality forum (nqf), a multi-stakeholder health policy organization head-quartered in washington, dc. in this manner, the sts has gained a positive reputation with the government and with health policy organizations. in addition, in 2010, the sts started to publicly report isolated coronary artery bypass grafting (cabg) composite star ratings not only on its own website but also on a consumer report website (www.consumerreportshealth.org). later, public reporting of aortic valve surgery (avr) and cabg+avr began, and this year, will be extended to congenital heart surgery. the nqf has been releasing quality indicators in medical fields under the rubric of nqf-endorsed standards (http://www.quality-forum.org/home.aspx). participation in the sts national database, operative mortality stratified by the five sts-eacts (european association for cardiothoracic surgery) mortality categories, and risk adjustment in congenital heart surgery (rachs-1) pediatric heart surgery mortality. the sts states on its website that sts believes the public has a right to know the quality of the surgical outcomes, and considers public reporting an ethical responsibility of the specialty. in turn, in 1998, at the 7th annual meeting of the asian society for cardiovascular and thoracic surgery in singapore, the need for an asian cardiovascular surgery database was discussed. first, a database ad hoc committee was formed by the japanese society for cardiovascular surgery (jscvs) and the japanese association for thoracic surgery (jats) (table 1). moreover, quality improvement of cardiovascular surgery has been discussed by the members of the board of jscvs and jats since early 2000. in pursuit of this goal, three committees were organized by the jscvs and jats among its academic groups: 1) a board certification committee, 2) a center aggregation committee, and 3) a nurse practitioner and physician assistant committee. in 2000, before this movement, the japan cardiovascular surgery database (jcvsd) was established with close ties to the jscvs and jats. the jcvsd and jscvs invited the founder of the sts national database to discuss starting the construction of the database. the jcvsd established input items comparable to those of the sts national database. in the congenital heart surgery database, the common terminologies and the definitions of congenital heart diseases published in the annals of thoracic surgery were adopted, and 193 input items were established in the japan congenital cardiovascular surgery database (jccvsd). thus, the congenital heart surgery databases in the united states, europe, and japan were integrated by using common language in these databases. as a result, although the results were not reported, for example, the discharge mortality in the jccvsd was 0.2%, 0.7%, 3.6%, 7%, and 17.6% for rachs-1 categories 1, 2, 3, 4, and 5/6, respectively, during 2008 to 2010 (fig. this result is comparable to that reported from the sts congenital heart surgery database. unlike the sts national database, the jcvsd employed web-based data collection. data on adult cardiac surgery (japan adult cardiovascular surgery database [jacvsd ]) were collected beginning in 2001 by five participating units and data on congenital heart surgery (jccvsd) were collected beginning in 2008 by seven units. jcvsd required informed consent from each patient according to the opt-in rule to comply with the private information protection law. for web-data transmission, high level secure socket layer was adopted for coding of the individual patient s information. the jacvsd and jccvsd grew to become national databases by the end of 2013 (fig. the most recent annual number of submitted procedures are 49,507 in jacvsd and 10,835 in jccvsd. twenty frequently cited papers dealing with topics such as risk models of isolated coronary bypass surgery, thoracic aortic surgery, and valve surgery have been published in indexed international journals. the performance of the congenital heart surgery risk model as measured by the c-index is over 0.8. on the basis of these risk models,, adult cardiac surgeons can estimate the 30-day mortality rate, in-hospital mortality rate, and major complication rate after inputting the patient s covariates before the surgical procedure. japanscore contributes to obtaining adequate informed consent from the patients and the families, leading to increased satisfaction. in addition, benchmark reports have been released as support tools for quality improvement of participating institutions. in japan, many adult cardiac surgeons learn about the risks faced by their patients, as well as their own performance as a surgeon, through the risk-adjusted mortality and benchmark report. to ensure fairness and transparency in evidence-based medicine (ebm), the jcvsd organized a data access and usage working group. this working group meets twice a year, and requests 100% of their data during at least for the immediate 2-years. after the working group accepts an application, the department of health quality assessment (hqa) of the university of tokyo analyzes the newly submitted data. the role of the hqa is similar to that of the dcri for the sts national database. the members of the working group include two to three adult cardiac surgeons; visits to 70 sites have been carried out so far. recently the hqa reported the details and outcomes of the site visits to the jccvsd. in 2011, the jcvsd started to collect a participation fee of 10,000 yen per year for each section in the jacvsd and the jccvsd. the total number of sections was 658 (541 in the jacvsd, 117 in the jccvsd) by the end of april 2014. this participation fee is much lower than that required by the sts national database; however, it is an important financial resource, especially for site visits. in 2011, the japanese board of cardiovascular surgery (jbcvs) decided to adopt the data of the jacvsd and the jccvsd for board certification. in 2013, there were 162 new applicants and 1,003 renewals. the jbcvs held its first web-based and paperless review in september 2013. compared with the previously employed review method that relied on the submission of operation records, the web-based and paperless review method had higher quality, lower cost, and required less time in 2010, the jcvsd served as the basis for the establishment of the national clinical database (ncd) in japan, which includes clinician-initiated databases reflecting all surgical fields. data collection with the same security level of jcvsd as mentioned above to protect the individual patient s information. through the central institutional review board in the university of tokyo, an opt-out rule was adopted, and informed consent became unnecessary. the ncd is governed by a committee whose members are representatives of medical associations related to surgery, such as the japan surgical society (jss), jscvs, jats, the japanese association for chest surgery, the japanese society of gastroenterological surgery, the japanese society of pediatric surgeons, the japanese society of vascular surgery, the japanese society of endocrine surgery, the japanese society for mammary cancer, and the japanese thyroid association. the ncd establishes the surgical board certification system for the jss, which requires 13 input items at the first level in the hierarchy of specialties. six board certification systems, including the jbcvs and the databases of nine academic associations, are set at the second level as subspecialties. the main server was transferred from the hqa to the university hospital medical information network (umin) with a mirror-image backup. the hqa focused on data analysis and site visits, whereas the umin is responsible for data warehousing. the ncd uses cutting-edge statistical techniques to detect any trace of data inconsistency. the participating associations have supported the ncd financially and the database has grown rapidly; the total number of participating hospitals is 4105, and the number of cumulative procedures was 4,138,000 at the end of april 2014. the participating associations will release, or have released, their own risk models [1618], and papers have been published based on data from the ncd. the administrative database, diagnosis procedure combination (dpc)/par-diem payment system (pdps) was introduced in japan by the japanese ministry of health, labour and welfare (mhlw, a government agency) in april 2003 to comprehensively assess fixed daily payments and to control medical expenditure in the acute setting based on the quality assessment. the number of participating hospitals by the end of april 2014 was 1,585, including all advanced-treatment hospitals, that is, university hospitals. in japan, total health care expenditures have been increasing by 1 trillion yen annually, and health care expenditures make up 9.5% of the gross domestic product, which puts japan in the 16th position of the 34 member countries of the organization for economic cooperation and development. on the other hand, the population aging rate in japan is over 24%, which is the highest rate in the world. changes in population makeup and the growing proportion of elderly persons are the underlying issues relating to rising health care expenditures, and successive cabinet office members and the mhlw have set policy directions to address this national issue. quality improvement, quality assessment, and the pay-for-performance system provide methods to control medical expenditures. the quality and outcomes framework (qof), a system for the performance management and payment of genaral practitioners (gps) in the national health service in england, wales, scotland and northern ireland was introduced as part of the new general medical services contract in april 2004. in contrast, in the united states, the agency of healthcare research and quality (ahrq) has defined never events or errors of medical care for which medicare, the government healthcare insurance for aged and disabled persons, does not pay. in the c. walton lillehei lecture of the 49th sts annual meeting, the director of the ahrq emphasized that the federal government will pay for the quality, not for the volume. in japan, the ncd and dpc/pdps could play complementary functions for quality assessment through adequate risk adjustment and the complete enumeration of procedures in various surgical fields. in the future, balancing professional autonomy and administrative leadership might be a recurrent issue for quality assessment and quality improvement in japan. recently, the japanese association of cardiovascular intervention and therapeutics proposed to the ncd a comparative study between percutaneous coronary intervention and cabg that would use well-tested statistical methods such as propensity score matching. thus, the participation of units from nonsurgical fields, such as medical therapy, intervention, radiation therapy, and chemotherapy, will facilitate risk stratification of each treatment modality, and will contribute to the search for the best management of diseases and patients. a longitudinal follow-up database is needed for the design of such studies, and it is under construction. recently, the pharmaceutical and medical device agency (pmda), a consultative organization of the mhlw, suggested to enroll in the jacvsd and perform follow-ups on the use of artificial valves for trans-aortic valve implantation. the pmda recognized the completeness and reliability of the data of the jacvsd, and from a cost-performance point of view, the pmda decided to outsource the post-market surveillance of newly covered medical devices in the cardiovascular surgical field. this demonstrates how the national database could contribute to the post-marketing surveillance of drugs and medical devices, and could help control randomized trials and multicenter studies. the ncd will start to collect fees from participating hospitals according to the total number of enrolled surgical procedures. clerical assistants have been widely employed throughout the country, which has gradually lightened the data input workload of young surgeons. governmental support and some government funds clinicians are responsible for patient safety and quality improvement, and the database will aid in achieving these goals. as reinertsen stated, to truly improve quality, the system must, 1) eliminate unnecessary variation (standardize processes), and 2) achieve and document continuous improvement (in care processes and outcomes). in recent years, the importance of certainty, not excellence of operations, and that of the concept of structure, process and outcome have been emphasized, and multiple approaches, for instance, postgraduate education systems, reporting systems of malpractice to prevent recurrence, introduction of information technology, introduction of simulators, ebm, and other techniques, have been used for patient safety. since it is methodologically based on the jcvsd, the ncd represents an interface between medical databases and board certification systems, which is its point of difference from the sts national database. in 2014, a new organization for medical board certification was established in japan that, beginning in 2017, will certify all medical boards in close collaboration with medical associations. this new organization will adopt the standards of the jcvsd and the ncd for evaluating the clinical practices of applicants. for the assessment of medical outcomes and quality, the jcvsd and the ncd will continue to be the sole reliable data source for surgical fields in japan, where medical system reform will be implemented quickly and based on professional autonomy. the national database is fundamental for quality improvement, patient safety, and the adequate control of medical expenditures in the country.

the jcvsd (japan cardiovascular surgery database) was organized in 2000 to improve the quality of cardiovascular surgery in japan. web-based data harvesting on adult cardiac surgery was started (japan adult cardiovascular surgery database, jacvsd) in 2001, and on congenital heart surgery (japan congenital cardiovascular surgery database, jccvsd) in 2008. both databases grew to become national databases by the end of 2013. this was influenced by the success of the society for thoracic surgeons national database, which contains comparable input items. in 2011, the japanese board of cardiovascular surgery announced that the jacvsd and jccvsd data are to be used for board certification, which improved the quality of the first paperless and web-based board certification review undertaken in 2013. these changes led to a further step. in 2011, the national clinical database (ncd) was organized to investigate the feasibility of clinical databases in other medical fields, especially surgery. in the ncd, the board certification system of the japan surgical society, the basic association of surgery was set as the first level in the hierarchy of specialties, and nine associations and six board certification systems were set at the second level as subspecialties. the ncd grew rapidly, and now covers 95% of total surgical procedures. the participating associations will release or have released risk models, and studies that use big data from these databases have been published. the national databases have contributed to evidence-based medicine, to the accountability of medical professionals, and to quality assessment and quality improvement of surgery in japan.

PMC4207111

pubmed-428

migraine is a disabling neurological disorder considered by the world health organization as the 19th leading cause of all years lived with disability among both males and females of all ages, and as the 12th leading cause of years lived with disability among females of all ages. apart from pain, the disability caused by migraine is aggravated by accompanying symptomatology, such as gastro-intestinal symptoms, the most common being nausea and vomiting, to such an extent that their presence is one of the diagnostic criteria for migraine. a telephone interview survey of 500 self-reported migraine sufferers found that nausea occurred in more than 90% of all migraineurs; nearly one-third of these had nausea during every attack. vomiting occurred in almost 70% of all migraineurs; nearly one-third of these vomited in the majority of attacks. indeed, 30.5% of those who had nausea, reported that it interfered with their ability to take their oral migraine medication. the american study ii stated that 73% of the migraineurs studied reported to have suffered from nausea during attacks and that 29% had vomited. one of the most interesting approaches to nausea adopted by traditional chinese medicine and, in particular, by acupuncture is the stimulation of the acupoint pc6 neiguan. indeed, there is documented evidence as to the efficacy of stimulating this point to alleviate chemotherapy-induced nausea and vomiting (cinv), postoperative nausea and vomiting (ponv) and motion sickness, both with acupuncture and acupressure. however, to the best of our knowledge, there are no studies in indexed medical literature as to the efficacy of treating pc6 acupoint for gastrointestinal symptoms in migraine attacks and particularly for nausea. therefore, our preliminary study aimed at verifying if pressure applied to the point pc6 was effective on the presence of nausea during migraine attacks. a total of 40 female patients were enrolled into this study, after having given their informed consent, and all were suffering from migraine without aura, diagnosed according to the criteria established by the international classification of headache disorders (ichd-ii). the patients were examined at the women s headache center, department of gynaecology and obstetrics of turin university. inclusion criteria were: at least two migraine attacks per month for a 1-year period before enrollment; no more than 15 days of pain per month. none of the patients were on prophylactic therapy, but were allowed to continue taking their usual symptomatic treatment. the patients medical history had to include the presence of nausea as accompanying symptomatology of their migraine, documented by a diary noting at least 1 month of attacks with nausea, prior to the inclusion in the study. subjects taking antiemetics to control their nausea, whether as a single product or present as a compound in a combination product for the control of migraine, were excluded from the study. the patients enrolled were asked to fill in a dedicated diary recording the details of the length and intensity of the migraine attacks along with the accompanying symptomatology, paying particular attention to the presence of nausea. a device known as the sea-band the sea-bands are elastic wristbands with a 1 cm protruding round plastic button; these devices apply continual pressure to the pc6 acupuncture point with the aim of decreasing, or completely eliminating nausea (fig. 1). the pc6 point, also called neiguan, is located on the anterior surface of the forearm, 3 fingers widths up from the first wrist crease and between the tendons of the flexor carpi radialis and palmaris longus. the sea-bands were applied bilaterally on both wrists on the neiguan point, starting from the onset of the migraine attack and left in place for no less than 4 h, or for the whole attack period. fig. 1the localization of the point pc6 neiguan and the correct positioning of the sea-bands the localization of the point pc6 neiguan and the correct positioning of the sea-bands the patients were asked to document a total of six migraine attacks: three without the use of the sea-band wristbands (phase c, control) and three with the application of the sea-bands (phase sb). the sequence of the treatment given for the attacks (with, or without sea-bands) was chosen at random according to a scheme provided by the computer and was applied to each single patient. the section of the diary provided that covered the symptom of nausea was detailed to include information as to the time of symptom onset and symptom resolution, the intensity of nausea at the onset (t0), at 30 (t1), 60 (t2), 120 (t3) and 240 (t4) minutes evaluated on a scale from 0 to 10, where 0 indicated no nausea and 10 the maximum sensation of nausea. diary analysis was carried out by an impartial operator who did not know in which attacks the sea-bands were used or not. in this preliminary study, the average values of nausea in phases c and sb were calculated at different times throughout the study and a statistical evaluation of the differences between the values obtained in t0, t1, t2, t3 and t4 in the two groups studied was performed using a non-parametric friedman test for repeated measures. moreover, a non-parametric wilcoxon test for paired data was always performed for each level of the variable time to evaluate the difference between phase c and phase sb. this test also took into consideration the average intensity of the three attacks in each of the two phases. all values given in the following text are reported as arithmetic means (sem). all analyses were performed using the statistical package for the social sciences (spss) software program. only 32 patients (mean age 39.65 years, range 1961) completed the study. four patients were lost to follow-up, three handed over a diary with incomplete, unreliable data and one patient did not suffer from any migraine attacks in the 3-month observation period. the friedman test for repeated measures showed a highly statistically significant reduction in the intensity of nausea in the sb group (p<0.001) during treatment (at t1, t2, t3 and t4). the wilcoxon test for paired data showed that the nausea intensities were significantly higher in phase c than in phase sb (fig. 2): after 30 min (t1 c 5.55 0.36 vs. t1 sb 4.6 0.40, p=0.006), 60 min (t2 c 4.93 0.33 vs. t2 sb 3.11 0.40 p<0.001), 120 min (t3 c 3.48 0.35 vs. t3 sb 1.89 0.31 p<0.001) and 240 min (t4 c 2.05 0.28 vs. t4 sb 0.93 0.23 p<0.001). there was no difference between groups at t0 (t0 c 5.96 0.38 vs. t0 sb 6.36 0.35; p=0.276). fig. 2average values of nausea score before treatment (t0), after 30 min (t1), after 60 min (t2), after 120 min (t3), after 240 min (t4), in phase sb (black columns) and in phase c (white columns). non-parametric wilcoxon test for paired data at t0, t1, t2, t3 and t4: at t0 p=0.276, n.s.; at t1*p=0.006; at t2, t3 and t4** p<0.001 average values of nausea score before treatment (t0), after 30 min (t1), after 60 min (t2), after 120 min (t3), after 240 min (t4), in phase sb (black columns) and in phase c (white columns). non-parametric wilcoxon test for paired data at t0, t1, t2, t3 and t4: at t0 p=0.276, n.s.; at t1*p=0.006; at t2, t3 and t4** p<0.001 the number of patients who reported having had at least a 50% reduction in the nausea score was: 0/32 at 30 min in phase c and 7/32 in phase sb (chi square test: p=0.16 rr 0.43; ci 95% 0.320.58); 1/32 at 60 min in phase c and 15/32 in phase sb (chi square test: p<0.001 rr 0.37, ci 95% 0.250.56); 11/32 at 120 min in phase c and 23/32 in phase sb (chi square test: p=0.003 rr 0.44, ci 95% 0.240.80); 21/32 at 240 min in phase c and 27/32 in phase sb (chi square test: p=0.083 rr 0.55, ci 95% 0.251.1). moreover, when the consistency of the treatment (response in at least two out of three treated attacks) is taken into consideration, it was reached: in 9 patients (28 %) at 60 min; in 13 (40 %) at 120 min and in 19 (59 %) at 240 min. noteworthy, the nausea was significantly reduced by acupressure in 3/3 attacks: in 5/32 patients (15 %) at 60 min; in 10/32 (31 %) at 120 min and in 17/32 (53 %) at 240 min. some studies have reported that nausea was present in 73 to more than 90% of the subjects studied and that almost one-third of these experienced nausea during every attack. moreover, 30.5% of the subjects who reported nausea indicated that its severity even interfered with their ability to take their oral migraine medication [3, 4]. traditional chinese medicine and especially acupuncture, stimulates some points that can be considered extremely valid from the point of view of nausea and/or vomiting control. in particular, the treatment of the acupoint pc6 neiguan may be applied to this aim, even with the application of acupressure alone, as has been validated by various studies. international literature reports numerous studies on the efficacy of stimulating the acupuncture point pc6 and its capacity to reduce nausea under various clinical conditions. a cochrane review on ponv concluded by stating that, compared with sham treatment, acupoint stimulation significantly reduces nausea (rr 0.71, 95% ci 0.610.83) and the need for rescue antiemetics (rr 0.69, 95% ci 0.570.83). from a cochrane review on cinv, it emerged that acupressure is effective for both mean and worst acute nausea severity, and, therefore, acupressure is able to offer a no-cost, convenient, self-administered intervention for chemotherapy patients to reduce acute nausea. on the basis of the data obtained in this study, the application of acupressure for the control of nausea during a migraine attack seems to be justified. indeed, the application of the sea-bands on the acupoint pc6 neiguan was observed to be effective in the control of nausea. the average nausea scores drop in the sb phase from 6.36 0.35 in t0, to 4.60 0.39 in t1, to 3.11 0.40 in t2, to 1.88 0.31 in t3 and to 0.92 0.22 in t4. at each time step taken into consideration after the application of the sea-bands, there was a statistically significant improvement over the non-treated phases. moreover, there was a high percentage of responders to the treatment: i.e. 46.8% at 60 min; 71.8% at 120 min; 84.3% at 240 min with a consistent response over time. even when the fact that our study is both preliminary and open is taken into consideration, the results obtained seem to be encouraging and advocate the continuous application of pc6 acupressure in all migraine attacks with the accompanying symptom of nausea. further controlled studies are, of course, required to validate the findings of this study.

migraine is a disabling neurological disorder, aggravated by accompanying symptomatology, such as nausea. one of the most interesting approaches to nausea adopted by traditional chinese medicine is the stimulation of the acupoint pc6 neiguan. actually there are no studies in medical literature as to the efficacy of treating pc6 acupoint for gastrointestinal symptoms in migraine attacks. our study aimed at verifying if pressure applied to the acupoint pc6 was effective on nausea during migraine. forty female patients suffering from migraine without aura were enrolled, if nausea was always present as accompanying symptomatology of their migraine. the patients were treated randomly for a total of six migraine attacks: three with the application of a device, the sea-band wristband, which applies continual pressure to the pc6 acupoint (phase sb), and three without it (phase c). the intensities of nausea at the onset, at 30, 60, 120 and 240 min were evaluated on a scale from 0 to 10. the values were always significantly lower in phase sb than in phase c. also the number of patients who reported at least a 50% reduction in the nausea score was significantly higher in phase sb than in phase c at 30, 60 and 120 min. moreover, the consistency of the treatment (response in at least two out of three treated attacks) was reached in 28% patients at 60 min; in 40% at 120 min and 59% at 240 min. our results encourage the application of pc6 acupressure for the treatment of migraine-associated nausea.

PMC3362706

pubmed-429

my experience in state government has been that after natural or manmade point-source disasters, a governor reflexively turns to the office of emergency management, the department of public safety (or homeland security), or the national guard for advice and counsel. the staffs of these agencies, however, have neither the expertise necessary to guide the response to an epidemic nor an established, ongoing communications and surveillance system with hospitals, laboratories, and medical providers. it is essential, therefore, to establish a formal process that allows public health and medical experts to assist elected officials in analyzing and interpreting information about the outbreak and in coordinating the public health response to the outbreak. guidance for fiscal year 2002 supplemental funds for public health preparedness and response for bioterrorism issued by the centers for disease control and prevention (cdc) requires that a state establish an advisory committee that includes representatives from health departments, first responders, hospitals, and voluntary organizations such as the red cross (11). in colorado this advisory committee includes not only the nine groups listed in the cdc announcement but also the presidents of the state board of health, state medical society, and state hospital association; the state veterinarian; a wildlife disease specialist; a medical examiner; a specialist in posttraumatic stress management; a pharmacist member of the board of pharmacy; the attorney general; the chief public information officer for the state health department; and, as an ex-officio member, the chief of the colorado national guard (3). these persons were named to the committee because they possess useful expertise or connections to the community. the statute authorizing the formation of the committee provided legal immunity to members for their advice (4), and the members pledged that they would attend the committee meetings during a bioterrorist attack rather than report to their regular jobs. by meeting regularly the committee members learn about each other s skills, experience, and roles and develop a working relationship that, by itself, can be extremely valuable during a crisis. one notable absence in the composition of the advisory committee is representation from federal agencies, such as cdc, the federal emergency management agency, the environmental protection agency, and the federal bureau of investigation. although these agencies can not, as a practical matter, attend meetings in every state and large municipality, during a crisis they will have an integral role, and disputes are more likely if the leaders are meeting for the first time in a highly stressful situation. for example, local-state-federal disagreements occurred in the management of the pneumonic plague epidemic in los angeles in 1924, the last instance of person-to-person transmission of plague in the united states, as well as during the anthrax outbreak in 2001 (12,13). some existing state regulations, which in normal times are intended to ensure quality medical care, could hinder community efforts during a bioterrorist attack. for example, consideration should be given to modifying, for a limited period through executive orders, the regulations that control the prescription and dispensing of medicine, licensing of physicians and nurses, and transfer of patients between hospitals. providing antibiotics or vaccinations in mass clinics and obtaining the services of retired or out-of-state physicians and nurses may be necessary. in colorado, executive orders that address these concerns have been drafted by the governor s technical advisory committee. the orders would permit a) health-care providers other than pharmacists and physicians, such as nurses and emergency management technicians, to dispense medications, b) medicines to be distributed without an identified patient s name on the packet or bottle, c) practice of medicine and nursing by professionals who are not currently licensed in colorado, provided the practice is restricted to caring for epidemic-associated illnesses and the persons are working under the supervision of a licensed practitioner (who is given legal immunity for the supervisee s work), and d) persons seeking medical care at one facility to be redirected to another facility without initial assessment or stabilization attempt if the initial hospital is unable to care for any more persons or if a specific facility (established or temporary) has been directed to receive epidemic patients, e.g., those with smallpox. these draft orders must still be tailored to the actual emergency and signed by the governor, but the background legal work can be completed ahead of time. two additional features of the colorado bioterrorism statute exist; these features were designed to encourage volunteers and remove legal barriers to cooperation among institutions and agencies. first, the statutory definition of civil defense worker was modified to include a physician, health care provider, public health worker, or emergency medical service provider who is ordered by the governor to provide specific medical or public health services during and related to an emergency epidemic and who complies with this order without pay or other consideration (7). with this amendment, civil defense workers may receive compensation for injury, including illness caused by bioterrorism, which is suffered as a result of civil defense service. second, the statute provides that persons and entities [including hospitals] that in good faith comply completely with board of health rules regarding the emergency epidemic and executive orders shall be immune from civil or criminal liability for any action taken to comply with the executive order or rule and that the state shall provide compensation for property if the property was commandeered or otherwise used in coping with an emergency epidemic (4). to ensure that a sufficient number of health-care providers, laboratory technicians, public health epidemiologists, and administrative support workers show up for work during a bioterrorist attack, appropriate personal protection (e.g., respiratory protection, vaccination, or chemoprophylaxis) for the worker and, probably, for household members of the worker are essential. when performing nonstandard work, the worker may also need legal protection, as discussed above. plans for a bioterrorist attack should include these factors and be written by the employer who knows how the agency operates and is staffed because people work for an agency, hospital, or institution, not a region. nonetheless, coordination of resources should develop mutual aid agreements with neighboring jurisdictions and integrate single institution or agency plans into community, regional, or statewide plans. in the 2000 colorado bioterrorism statute, the state board of health was given the new authority to promulgate rules requiring each state and local health department, general or critical access hospital, and managed-care organization to write a plan for responding to bioterrorism (7). while hospitals and health departments may have previously written plans for managing mass casualties resulting from aircraft, bus, or train crashes or natural disasters, such plans need to be modified to include consideration of the special circumstances of bioterrorism (e.g., chemoprophylaxis and personal protective equipment for workers, infection control, and handling of laboratory specimens). because pandemic influenza may pose challenges to the medical and public health systems similar to those of bioterrorism, a single plan for both types of epidemics should be drafted. during typical outbreaks of communicable diseases, clear and timely communication by the state health department with multiple local health departments and hospitals can be a challenge. in a bioterrorist attack, the communications challenge will likely be greater because many more persons and agencies will be involved. the telephone system may not have sufficient capacity for the increased demand or it may be damaged and disorganized, as happened during the response to the attacks on the world trade centers in new york city in september 2001 (14). furthermore, a large, sometimes overwhelming, number of inquiries made by members of the public to the public health agency usually occur during public health crises, and therefore, administrative plans for a bioterrorist event should include consideration of this workload. rather than relying on hospital personnel, public health agencies may find it advantageous to station their own personnel with mobile telephone or radio communications equipment in individual hospitals to assure that public health agencies get the information they need as rapidly as possible. accomplishing this may require an executive order of the governor that commandeers two-way radios. in colorado, board of health regulations require the state and local health departments to include assignment of employees to hospitals in the agency s emergency plan (8). disease reporting requires specification of what to report in what manner and timeframe to which parties. a first legal step in this process is to require immediate reporting of any suspected or confirmed illness, syndrome, or outbreak caused by any potential bioterrorist agent. for example, colorado regulations were modified in 1999 so that cases of plague, which had been required to be reported within 24 hours of diagnosis by telephone, fax, or through a web-based system, were to be reported immediately only by telephone to an on-call person if the physician or hospital suspected the case was related to a bioterrorist event (9). disease surveillance systems are critical not only for the initial detection of an outbreak but also for monitoring the extent and spread of the outbreak and for determining when it is over. managing a large outbreak would require gathering information from contact tracing and source-of-exposure investigations as well as information about the availability of critical medicine, medical equipment, and the handling of corpses. these information needs are much different than those needed for early detection of an attack. therefore, legal authority for surveillance should be modified as necessary to ensure collection of all information that could be needed by the public health agency to fulfill its duties throughout the epidemic. this legal authority may include requirements for groups that do not commonly report information, such as pharmacists, to provide it. administrative public health orders restricting personal behavior of persons with certain diseases, such as tuberculosis, are relatively common in this country (15). such orders are usually hand-delivered to a specific person(s), and the restrictions are removed after a specified period, such as after one incubation period or when an ill person is no longer infectious. another type of public health order might involve work restriction, e.g., health-care providers who can not demonstrate evidence of immunity to a vaccine-preventable disease are not permitted to work during an outbreak of such disease. few, if any states, however, have experience issuing and enforcing large-scale quarantine orders that last more than 12 days. orders restricting large numbers of contacts of cases of plague to home were issued in florence, italy, in 1630 and described in the 1999 book, galileo s daughter (16). the enforcement of orders restricting the movement of residents of an entire town in which there was an outbreak of viral hemorrhagic fever was depicted in the 1996 movie, outbreak. the images of severe disease and enforced quarantine are similar in the book and movie and are plausible and disturbing to lay audiences. a more recent, well-documented example of a large-scale movement restriction was the british epidemic of foot-and-mouth disease of 2001, which affected many farms and businesses and led to the quarantine and slaughter of 4 million sheep, cattle, and pigs for disease control purposes (17). in all three examples, a decentralized quarantine was imposed. in general, the advantage of a decentralized strategy (e.g., persons are restricted to home) is that it may reduce the risk for transmission of disease because fewer persons congregate. alternatively, the centralized strategy (e.g., restricted persons are taken to a sports arena, auditorium, theater, school, or hospital) is seemingly easier for the government to care for restricted persons and to enforce the order but could allow contagious and noncontagious persons to come into contact with each other. another example of large-scale quarantine occurred in los angeles in 1924 during the last epidemic of pneumonic plague in this country (12). three days after the first 15 cases in this outbreak became known to public health officials, eight city blocks that housed approximately 2,500 mexicans were placed in quarantine. public health nurses were sent to the area to make house-to-house inspections to identify new cases, and all patients with suspected cases in the area were examined by physicians at the patient s home and then sent to the county hospital. all persons who lived at addresses where cases had occurred were quarantined in the county general hospital, and a spanish-speaking priest and social workers were placed in the area to reassure and calm the residents. the quarantine actions taken in this outbreak were a combination of centralized and decentralized strategies. as has been discussed by barbera et al. (18), numerous concerns regarding large-scale quarantine exist. all states currently have in place varying degrees of legal authority enabling isolation, quarantine, or travel restrictions if needed to maintain the welfare and safety of the public. drafting restrictive orders in advance is less helpful than with the other types of orders discussed above because restrictive orders require more tailoring to the specific circumstances and parameters of an outbreak. factors such as duration and location of restriction are dependent on what the bioterrorist agent is, how it is transmitted, how widely the agent has been disseminated, whether exposed persons can be personally identified, and what resources are available to care for restricted persons. not drafting such orders in advance, however, means that they may be written during the turmoil of multiple agencies trying to control an outbreak. authorities should never hesitate to revise the orders on the basis of on updated information. at the end of the operation topoff exercise, for instance, when the governor had issued a travel restriction order for all of metropolitan denver and cdc had quarantined the entire state of colorado, such orders created many unforeseen problems, including how to enforce the orders, maintain essential community services, and distribute foods and prescription medicines. accurate and substantive information given to the public by credible public health and medical experts can do much to allay the fears of the public and encourage their cooperation and participation in constructive, organized community response efforts (19,20). i have discussed a number of ideas about legal and administrative preparations for a bioterrorist attack, but more work can be done, including development of strategies addressing issues related to mental health, disposal of corpses, performing forensic autopsies, signing death certificates, and managing potential animal vectors of disease. i have not discussed the sharing of medical and epidemiologic information between public health agencies and law enforcement agencies, such as the federal bureau of investigation. under normal circumstances, public health officials typically argue that release of disease surveillance information to the criminal justice system will discourage persons with reportable conditions from disclosing to public health officials where they have been and with whom they have had contact. however, a bioterrorist attack is not a routine event, and i recommend that state and local public health agencies review the laws and regulations governing the confidentiality of disease surveillance records and develop a legal and administrative protocol for sharing pertinent and relevant information with law enforcement agencies during a bioterrorist attack (21). finally, i have not discussed the protection of civil liberties and due process for persons affected by executive orders of the governor and public health officials. this is an important and difficult issue, especially when well persons are quarantined solely on the basis of their having visited, worked, or resided in a particular location at a particular time, as opposed to having had face-to-face contact with a known contagious person. public health officials and attorneys general should review existing safeguards for the protection of civil liberties and determine whether modifications need to be made for the special circumstances created by a bioterrorist attack.

this article proposes and discusses legal and administrative preparations for a bioterrorist attack. to perform the duties expected of public health agencies during a disease outbreak caused by bioterrorism, an agency must have a sufficient number of employees and providers at work and a good communications system between staff in the central offices of the public health agency and those in outlying or neighboring agencies and hospitals. the article proposes strategies for achieving these objectives as well as for removing legal barriers that discourage agencies, institutions, and persons from working together for the overall good of the community. issues related to disease surveillance and special considerations regarding public health restrictive orders are discussed.

PMC2901954

pubmed-430

the name of mucosa-associated lymphoid tissue (malt) lymphoma was first established in 1983 by isaacson and du. from the beginning, it was adopted well and is still used in an unchanged form. marginal zone lymphoma of malt is, apart from diffuse large b-cell lymphoma, the most frequent type of lymphoma that occurs in the stomach. what is important is that it can develop in almost every organ and tissue, for instance lungs, breast, thyroid gland, bladder, skin, or orbital adnexa. it is an indolent type, but clinical outcomes and response to treatment vary among patients. malt lymphoma arises from the extranodal sites reach in b-lymphocytes, which appears in response to chronic antigenic stimulation caused by infection (helicobacter pylori) or autoimmune process (hashimoto disease). this disorder is the best example of how infectious pathogens and genetic abnormalities lead to malignant transformation. gastric malt lymphoma pathogenesis is a complex process including many gene alternations that result in cancer appearance. better understanding of the background of the disease is crucial for discovering new prognostic factors, helpful in deciding when more aggressive treatment should be employed. the incidence of malignant lymphomas is at the rate of 3%-4% of all malignancy worldwide and has been increasing during the last 50 years. lately, some stabilization in the number of diagnosis was observed, but only in developed countries. malignant lymphomas are observed to be more frequent in north america, australia, and europe than in asia and africa. malt lymphomas determine almost 7% of all non-hodgkin's lymphoma, and at least 40% is primarily located in stomach. it is confirmed that gastric malt lymphoma occurs in younger patients than the rest of malignant lymphomas. the malt lymphoma is mainly a disease of older adults, with a median age of 60 years., there is much higher proportions of malt lymphomas, which can be caused by more frequent prevalence of helicobacter pylori in this region of the world. although 90% of population worldwide have confirmed bacteria colonization, only 2% will develop malignant lymphoma. it was confirmed by weber et al. that almost 90% of patients with gastric malt lymphoma are infected with helicobacter pylori. this curved bacillus, previously called campylobacter pyloridis, is a gram negative pathogen found in the stomach. although over 80% of people are asymptomatic, chronic infection can lead to gastritis, gastric and duodenal ulcer, gastric adenocarcinoma, and malt lymphoma [5, 6]. nowadays, it is widely accepted that helicobacter pylori gastritis is crucial in an evolution of malt lymphoma localized in stomach. it was confirmed by several studies that chronic gastric inflammation causes constant antigenic stimulation, which leads to clonal expansion of b-cell lymphocytes [7, 8]. in the gastric mucosal cells, there are elevated levels of some cytokines, including proliferation-inducing ligand (april), which belongs to the tumour necrosis factor (tnf) family. april is produced by macrophages present in the gastric malt infiltrate, located close to the neoplastic cells. april may also induce b-cells transformation and the progression to the diffuse large b-cell lymphoma (figure 1). they come either from t cells or directly by the antigenic autostimulation of lymphoma cells. gastric inflammation causes the appearance of a large number of macrophages, which, under a helicobacter pylori infection, release large amounts of april. importantly, a number of april-producing macrophages significantly decrease in complete remission after eradication therapy. other pathogens, are also suspected to play an important role in malt lymphoma pathogenesis. there are bacteria such as campylobacter jejuni, borrelia burgdorferi, and chlamydia psittaci and viruses like hepatitis c virus (hcv) that are potentially responsible for oncogenesis. these pathogens were found in histological material, but so far no strong evidences were established. patients with autoimmune disease have for sure higher risk of developing malt lymphoma. autoreactive b cells infiltrate the healthy organs and create lymphoid infiltrate similar to normal malt tissue with huge amount of reactive clonal b lymphocytes. this situation is observed in salivary gland in patients with diagnosis of sjgren syndrome and in the thyroid gland in hashimoto disease. sjgren syndrome is associated with 44 times increased risk of lymphoma, whereas hashimoto's thyroiditis causes 70 times increased risk of thyroid lymphoma. some of them are proven to be strongly associated with the disease, but some are still not confirmed. it is believed, that on the background of chronic inflammation not only reactive b-cells are stimulated but also activated neutrophils which can lead to production of oxygen species. as a result, this genotoxins provoke dna damages, which are responsible for mutations and transformations of genetic material. it originates from a fusion of two proteins: apoptosis inhibitor 2 (api2) and paracaspase malt lymphoma-translocation gene 1 (malt1). what is more important is that while t(11;18)(q21;q21) is detected, no other chromosome abnormality can be found. unfortunately, positive cases do not response to helicobacter pylori eradication, but, in contrast, they do not transform to more aggressive diffusive large b-cell lymphoma. it is known that complete remission can be seen in at least 20% of patients with t(11;18)(q21;q21). the incidence of positivity for this translocation malt lymphoma is at approximately 20% in europe [16, 17] but is not as common in the united states where only 5% are positive. moreover, it is usually connected with an advanced stage of disease and poor outcomes. bcl-10 gene is relocated from chromosome 1 to 14, which in consequence triggers overexpression of bcl-10 protein also known as ciper, carmen, or me10. in healthy organisms, higher expression is observed in lymph nodes, spleen, and testis. so far, it is believed that bcl-10 protein expression is responsible for proliferative effects [19, 20]. the t(14;18)(q32;q21)(igh-bcl-2) is commonly present in follicular lymphoma, in about 20% of diffuse large b-cell lymphoma and sometimes in chronic lymphocytic leukemia. although this aberration is extremely rare in other types of lymphomas, it can be found in some cases of gastric malt lymphoma. bcl-2 is an antiapoptotic protein, which helps in survival and expansion of clonal b cells. so far, the role of t(14;18) in gastric malt lymphoma is not fully understood. overexpression of bcl-2 is found not only in translocation positive patients but also in the negative ones. it is believed that similar to other types of lymphomas, t(14;18)(igh-bcl-2) must coexist with other genetic abnormalities in order to develop neoplasm. t(3;14)(p14;q32)(igh-foxp1) is a newly described abnormality present in patients with malt lymphoma. the first study showed that positivity for this translocation is approximately 10% of all malt lymphoma patients. the most recent studies described the presence of t(3;14)(p14;q32) in diffuse large b-cell lymphoma, outside the lymph nodes especially [23, 24]. only one study, so far, confirmed the existence of this translocation in gastric malt lymphoma which involved bad clinical outcomes. in pathogenesis of malt lymphoma, the above described translocation promotes oncogenesis by similar well-known mechanism. the majority of them involve the same pathway, which leads to antigen receptor-mediated activation of nfb. this is a crucial transcript factor which plays a key role in malt lymphogenesis [26, 27]. it regulates processes connected with b-cell development, growth, and survival by production of cytokines and growth factors, for example, tnf- family (baff). latest studies have shown that b-cell activation in malt lymphoma can be strictly connected with tnf family. it can be also responsible for activation of cell apoptosis [28, 29]. it is observed that in patients with higher baff levels in serum, the prognosis and survival are much worse. based on recent knowledge about genetic abnormalities in gastric malt lymphoma, there is a model of multistep pathogenesis. on the background of chronic inflammation and antigenic stimulation occurs genetic instability. as a result, many possible translocation and unbalanced aberrations are observed. gastric malt lymphoma can be long-time asymptomatic or associated with dyspepsia, abdominal pain, vomiting, diarrhea, obstruction, and nausea. sometimes bleeding from gastrointestinal tract or even perforation may occur while extensive lesions are present. as a result, symptoms of anemia like paleness, weakness, or easy fatigue can be observed. b symptoms (weight loss, unexplained fever, and night sweats) in gastric malt lymphoma are very rare, but the most common of the above is weight loss. a prompt diagnosis is crucial, but, unfortunately, it is usually made by incidence. patients with early stage of disease have usually low tumor growth and minimal possibility to spread. in contrast, patients with advanced stage of disease can undergo transformation to more aggressive lymphoma and may become resistant to treatment. not only symptoms but also endoscopic picture can be inconclusive. difficulties often arise to differentiate between chronic gastritis or ulcer from an early-stage lymphoma. in order to confirm the diagnosis, there always must be made pcr or fish analysis for t(11;18), which is important to separate groups that will not respond to standard treatment. characteristic for gastric malt lymphoma are lymphoepithelial lesions (lel) with the presence of mainly two types of cells: neoplastic centrocyte-like or small lymphoid. there is no specific immunohistochemical profile typical for gastric malt lymphoma diagnosis. in 50% of patients, there is coexpression of cd43/bcl2. neoplastic cells are positive for cd-20 and negative for cd-10, cd-23, and cyclin d1. if it is negative in histochemistry, rapid urea breath test or fecal antigen test have to be made. another analysis to prove absence of helicobacter pylori infection is serological test for caga antibodies and helicobacter pylori-igg antibodies. sometimes, there is possibility to detect other helicobacter species, for example, heilmannii or felis. before taking any decision on how aggressive the treatment should be, it is extremely important to perform a complete staging of the disease. what is more important is that risk factors and individual parameters, which can affect later therapy, are crucial. medical history must include information about the age, time of the first symptoms, the family history, and medical condition. the most important factor that we rely on during choosing method of treatment is clinical stage of the patient. during physical examination, it is important to remember about waldeyer's ring, which is mandatory in every gastric lymphoma patient. staging in gastric malt lymphoma is similar to that in other types of lymphomas. according to recent european society for medical oncology (esmo) recommendations, it should include morphology with basic biochemical studies. if the blood cell count is lower, it can be caused by infiltration of bone marrow. biochemical tests can detect liver or kidney problems, which can be important before the beginning of a chemotherapy. lactate dehydrogenase (ldh) and 2-mikroglobulin are prognostic factors and will be abnormally high in patients with fast-growing tumor. every newly diagnosed patient should be examined in case of certain viral infections that can affect treatment, such as hepatitis b and c or human immunodeficiency virus (hiv). in every case, computed tomography (ct) scans of neck, chest, abdomen, and pelvis, which are crucial to evaluate enlarged lymph nodes, should be performed. core needle biopsy of bone marrow is made to diagnose possible infiltration of neoplastic cells. it was confirmed that 15% of gastric malt lymphoma patients have lymphoma cells in bone marrow. positron emission tomography (pet) has still not confirmed clinical necessity, but it can be extremely helpful in controversial cases. moreover, during staging procedures of gastric malt lymphoma, gastroduodenal endoscopy must be made. biopsies are taken from different sites of gastrointestinal tract (e.g., stomach, duodenum, and gastroesophageal junction) and every location that looks suspicious. most often, ann arbour staging is employed, which describe the extend of all types of non-hodgkin lymphoma in adults. thus, staging of gastric lymphoma based upon the ann arbor system includes stage i e, which is disease limited to the stomach without nodal spread. stage ii e1 is tumor in the stomach with spread to adjacent contiguous lymph nodes. stage ii e2 is tumor in the stomach with spread to lymph nodes that are noncontiguous with the primary tumor. moreover, if the spleen is affected, we add s. if the person has any of the b symptoms, we add letter b, and if is asymptomatic, we assign a (table 1). prognostic factors in gastric malt lymphoma are similar to the value for non-hodgkin b-cells lymphoma. factors that determine poor outcome are age, high level of ldh in serum, higher ecog performance status, stages iii and iv in ann-arbour scale, white blood count, and more than one extranodal site. it was observed that patients with nodal invasion has difficulty with complete remission after eradication treatment. for instance patients, with t(11;18)(q21;q21) especially, are resistant to the first line therapy, and remission rate was lower than that in patients of api2-malt1 negative (78% versus 22.2%; p=0.0001). only one study so far proved that the presence of t(3;14)(p14;q32) is connected with poor clinical outcomes of patients with gastric malt lymphoma. while helicobacter pylori plays a main role in the pathogenesis of malt lymphoma, it is also crucial in approach to the treatment. according to current international guidelines, first line treatment for localized helicobacter the treatment may be used with every highly effective antibiotics against helicobacter pylori, taking into consideration the locally expected antibiotic resistance. if there is no response to the therapy above, second line triple or quadruple therapy is used. it was reported that after two lines of treatment, 99.8% of patients were cured from gastritis. in a large study of 1408 patients, remission after eradication treatment in early stage unfortunately, in 5%10% of gastric malt lymphoma patients, we can not confirm helicobacter pylori infection. moreover, more than 30% patients are resistant to first line treatment, and 30% of them have t(11;18)(q21;q21). treatment for this patients should be chosen individually depending on the clinical stage of disease. for those who have stable disease without any symptoms, radiotherapy, chemotherapy, and/or surgery can be considered after unsuccessful eradication treatment. further recommended surgery is considered to be a standard therapy in therapy of patients with gastric malt lymphomas, but, recently, the value of this therapy has been not confirmed. even if the lymphoma is localized at early stage, the gastrectomy should be rather extensive due to the nature of the disease. moreover, it is a major surgery and can be associated with serious complications and worsen a quality of life. german multicenter study group (gmsg) presented no difference between survival in patients treated with gastrectomy compared to eradication (overall survival rate 82% to 84%). what is more important is that there were observed 50% long-term complications were observed after surgery. in few studies the use of a modest dose of involved fields was performed on resistant-to-eradication therapy patients with early-stage disease. the dose was 2535 gy to the stomach and perigastric nodes for the period of 4 weeks [42, 43]. compared to surgery, no serious long-term complications and toxicity were observed. only nausea and anorexia were present during the time of radiotherapy. for a long time it was believed that gastric malt lymphoma is just a localized disease and that surgery and radiotherapy are the best treatment strategy. now, when it is well known that it is disseminated disorder chemotherapy, it became more important. still, there are no standard recommendations for relapse or progressive patients after therapy and for those with late stage of the disease from the beginning. it was observed that chemotherapy alone is more effective than surgery apart from some cases with gastric obstruction. complete remission (cr) after oral monochemotherapy with cyclophosphamide was 83% in a study by nakamura and coworkers. unfortunately, patients with positive translocation t(11; 18) are resistant to second line therapy with oral monochemotherapy with alkylating agents. nucleoside analogs are confirmed to be effective in treatment of different kinds of indolent lymphomas. a polychemotherapy with fludarabine and mitoxantrone (fm) has a very good effect on patients with gastric malt lymphoma in both first and second line treatment. the complete remission after 4 cycles achieved 84% of investigated and all of them reacted to the treatment. after 2-cda, there were observed complications such as toxicities of 3 and 4 grade of who, mainly leukopenia, infections, and secondary neoplastic disease. nowadays, immunotherapy became an extremely important part of treatment of non-hodgkin lymphomas. it is a chimeric mouse/human monoclonal antibody specified to cd20 antigen expressed on the surface of b lymphocytes. now it is widely used alone or in combination with chemotherapeutic drugs in many types of b-cell non-hodgkin lymphomas. rituximab binds to cd20 antigen and activates the lysis of b cells by mediating cytotoxicity of complement dependent (cdc) and cell-mediated cytotoxicity antibody dependent (adcc). the role of this drug is still not clear in gastric malt lymphoma. in 2003, there was a first-phase study by conconi et al. the cr was observed in 29% and overall response rate (orr) was 64%. the toxicity of this treatment was moderate or even mild, but the relapse rate was 36%. an important fact is that patients with translocation t(11;18) are responsive to rituximab treatment [58, 59]. what is more important is that in a study by the international extranodal lymphoma study group (ielsg), it was confirmed that chlorambucil in combination with rituximab was more effective than chlorambucil alone. the conclusion is that rituximab may a benefit in individual patients, but for the majority it is not sufficient when used alone. the efficacy of the combination of rituximab with chlorambucil was evaluated in a randomized study (comparator was chlorambucil alone) by the international extranodal lymphoma study group (ielsg) in gastric malt lymphomas that had failed antibiotics and in nongastric malt lymphomas. the preliminary report showed that the 5-year event-free survival was significantly better for patients treated with chlorambucil plus rituximab. there were also studies by raderer et al. with cycles generally used in more aggressive lymphomas. twenty-six patients were administrated rituximab plus cyclophosphamide, doxorubicin or mitoxantrone, vincristine, and prednisone. lately, bortezomib, the first therapeutic proteasome inhibitor, was examined by kiesewetter et al. in 2012 with cr in 33% and pr in 27.8%. the results on phase ii studies with chemotherapy and immunotherapy are shown in table 2. outcomes in gastric malt lymphoma patients with progressive, disseminated disease are very comparable with outcomes in follicular lymphoma. although gastric malt lymphoma has a very favorable outcome, it is still important to have a proper followup. it is possible that the disease will return even after 5 years of complete remission. the relapse can be due to reinfection of helicobcater pylori. in a study by zullo et al. the followup is obligatory in patients with gastric malt lymphoma to identify early phase of the recurrence of the disease. to confirm a complete remission, although, there are no specified recommendations for a followup, the biopsy of gastric sites should be made every 6 months in first two years, and later once a year for the next five years. systemic followup consist of blood tests and minimal adequate radiological and ultrasound and should be made at least once a year in the first 5 years. the transformation in more aggressive lymphoma is low at the level of 0.05%, but there is a higher risk of occurrence of secondary neoplasm and gastric cancer. these studies confirm that patients with gastric malt lymphoma need a long-term followup not only to detect early recurrence but also to find secondary disease. recently, enormous progress has been made in better understanding of pathogenesis of gastric malt lymphoma. it has a great influence on the development of new and more effective treatment strategy. still, not enough clinical trials are performed due to rare expression and high effectiveness of first line treatment of gastric malt lymphoma. what is more important is that early diagnosis of gastric malt lymphoma is extremely important. while the symptoms are unspecific or not, always during the endoscopic exam the complete histological biopsies must be taken to make diagnosis correctly. the less advanced the stage of the disease, the bigger the chances to achieve complete remission

nowadays, it is believed that the main role in the development of gastric mucosa-associated lymphoid tissue (malt) lymphoma plays helicobacter pylori infection. this world-wide distributed bacteria is in charge of most cases of not only upper gastrointestinal tract disorders but also some of extragastric problems. constant stimulation of the immune system causes a b-lymphocytes proliferation, which is considered to be responsible for the neoplastic transformation. on the other hand, there are 10%20% of patients who do not respond to helicobacter pylori eradication treatment. this group has often a chromosome translocation, which suggests that there is another unknown, so far, pathogenetic mechanism of malt lymphoma. majority of genetic abnormalities are connected with nuclear factor-b (nf-b) pathway, which activates the uncontrolled proliferation of neoplastic cells. translocations already described in studies are t(11;18)(q21;q21), which is the most common, t(14;18)(q32;q21), t(14;18)(q32;q21), and t(3;14)(p14.1;q32). this non-hodgkin's lymphoma is an indolent type originated outside lymph nodes. in more than 50% of cases, it occurs in the stomach. occasionally, it can be found in salivary and thyroid gland, lung, breast, bladder, skin, or any other place in the human body. this paper is a review of the current knowledge on etiology, pathogenesis, treatment, and follow-up of gastric malt lymphoma.

PMC3625579

pubmed-431

the adventitia is more than just the outermost layer of the artery; it is now known to play a critical role in vascular remodeling and other important processes of the artery [13]. recent attention to the role of the adventitia in vascular remodeling has increased reporting of common carotid artery interadventitial diameter (iad), a noninvasive measure of vascular geometry and health. the results from one of these studies, an ancillary of the study of women's health across the nation (swan) called swan heart, suggest that declining endogenous estrogen that accompanies the menopausal transition has a direct effect on the peripheral vasculature. the study found lower levels of estradiol were significantly associated with larger common carotid artery iad even after adjustment for cardiovascular risk factors. other studies have shown that larger iad is associated with increasing age [57], cardiovascular risk factors [46, 813], prevalent cardiovascular disease [12, 14, 15], and incident cardiovascular events. thus, the increase in iad observed with declining endogenous estrogen suggests that lower levels of endogenous estrogens are associated with a less healthy vasculature. the strong association between iad and endogenous estrogen suggests that a similar association may exist with the use of exogenous estrogen. the purpose of this study was to determine whether postmenopausal current ht users had significantly different iad than those who were former users of ht in the women on the move through activity and nutrition (woman) randomized trial. we also wanted to determine if there were differences between other measures of vascular health. this study evaluates cross-sectional associations using measurements from the baseline visit of the clinical trial (clinical trials registry number: nct 00023543). the woman trial tested the ability of nonpharmacological lifestyle intervention to modify cardiovascular risk factors in postmenopausal women. the study recruited 508 eligible african american and caucasian women from allegheny county, pa, between april 2002 and october 2003 through direct mailings. eligible women were postmenopausal, between 52 and 62 years of age, able to walk, currently using ht, and willing to participate in either intervention group regardless of assignment and had a waist circumference 80 cm, a body mass index (bmi) between 25.0 and 39.9 kg/m, blood pressure<160/95 mmhg, and low density lipoprotein (ldl) cholesterol between 100 and 160 mg/dl. women were ineligible if they were taking medication for cholesterol, diagnosed with or on medication for diabetes, diagnosed with a psychotic disorder, or suffering from depression. the results of the women's health initiative estrogen/progestin arm were published in the middle of recruitment; as a result the eligibility criterion of current use of ht was modified to current or recent history of hormone use. recent history of hormone use was defined as prior use of at least 2 years within 6 months of randomization. the decision to remain on ht was determined by the participant and her physician. at baseline, 40% of the women had discontinued use of ht (these women will be referred to as former ht users) and 60% remained on ht (these women will be referred to as current ht users). for those who discontinued use of ht the median time off therapy was 7 months prior to study randomization. common carotid artery intima media thickness (imt), iad, lumen diameter (ld), and plaque were assessed by b-mode ultrasound using a toshiba ssa-270a duplex scanner (toshiba american medical systems, tustin, ca, usa) with a 5 mhz-linear array transducer. right and left carotid images were taken of the near and far walls of the distal common carotid artery 1 cm proximal to the carotid bulb. imt was defined as the distance from the lumen-intimal interface to the medial-adventitial interface (figure 1). iad was defined as the distance from the adventitial-medial interface on the near wall to the medial-adventitial interface on the far wall (figure 1). ld was defined as the distance from the intima-lumen interface of the near wall to the lumen-intima interface of the far wall (figure 1). using a semiautomated edge detection software, the interfaces were traced electronically over the distal cca and a computer generated measurement was obtained for each pixel in the area of interest; these measurements were averaged to determine imt, iad, and ld used for this analysis. a reproducibility study, conducted in 20 women who were similar to the women in the current study, provided an intraclass correlation of 0.98 for imt and 0.99 for iad. the reproducibility study took place at the same lab and used the same equipment and readers as the current study. the presence of plaque was determined for each of the 5 segments of the left and right carotid artery (distal and proximal cca, carotid bulb, and proximal internal and external carotid artery). plaque was defined as a distinct area protruding into the vessel lumen at least 50% thicker than the adjacent imt. the first of two prerandomization screening visits included a 12-hour fasting blood draw, physical measures of height, weight, waist circumference, blood pressure, the long distance corridor walk, medical, physical activity, and weight history. conventional enzymatic methods were used to obtain total cholesterol, high density lipoprotein (hdl) cholesterol, and triglyceride concentrations from the blood samples. low density lipoprotein (ldl) cholesterol was estimated using the friedewald equation. medical history included history of drug, vitamin/mineral supplement, and alcohol use. common carotid artery iad, ld, imt and plaque were measured at the second screening visit. seventeen women had incomplete data for the calculation of iad or imt and were excluded leaving 491 women for analysis. means and standard deviations are presented for normally distributed variables and medians and 25th and 75th percentiles are provided for nonparametric variables; dichotomous variables are presented as percents. differences between the current ht users and the former ht users were determined using chi-square analyses for categorical variables and t-tests and wilcoxon-rank sum tests for continuous variables. simple linear regression was used to assess univariate associations between iad and ld with ht and the following cardiovascular risk factors: age, race, systolic blood pressure, diastolic blood pressure, pulse pressure, bmi, weight, height, waist circumference, total cholesterol, ldl and hdl cholesterol, triglycerides, glucose, insulin, smoking status, and antihypertensive medication use. when collinearity between covariates was suspected (r>0.4), the variable most strongly correlated to iad or ld was selected for the analysis. the following variables were collinear: glucose and insulin; systolic blood pressure and pulse pressure; bmi, weight, and waist circumference; weight and height; total cholesterol and ldl. based on spearman correlation results glucose, pulse pressure, weight, and ldl were chosen for the multivariable models. multivariable linear regression was used to test for the following predetermined covariates: age, race, pulse pressure, and smoking status. in addition, any statistically significant variable in the univariate analysis and any variable that differed by ht use status were also tested. total cholesterol, hdl, ldl, glucose, and insulin differed by ht use status; addition of these variables did not alter the regression model so they are not presented in the results. the median (25th, 75th percentiles) age of the women was 57 (55, 60) years, median bmi was 30 (28, 34) kg/m; 11% were african american and 6% were current smokers. there were 197 former ht users and 294 current ht users at the time of the baseline carotid ultrasound scan. former ht users were older and had a higher percent of african americans (table 1). overall former ht users had a significantly worse cardiovascular disease risk profile than current ht users: higher total cholesterol, higher ldl cholesterol, higher glucose and insulin, and lower hdl cholesterol (table 1). there were, however, no differences by ht status in blood pressure, measures of general or central obesity, and smoking status (table 1). the mean iad was 6.94 mm for former ht users and 6.79 mm for current ht users (p=0.001, table 2). ld was also significantly larger in the former ht users than in the current ht users (5.44 mm versus 5.31 mm, p=0.002, table 2). however, imt and presence of plaque were not different between the two groups (table 2). simple linear regression showed that in addition to former ht use, larger iad was significantly associated with greater systolic blood pressure, pulse pressure, bmi, weight, height, waist circumference (all p<0.0001), glucose, insulin (both p=0.001), age, caucasian race, current nonsmoking status and use of antihypertensive medications (p<0.05) (table 3). the most parsimonious model in the multivariate analysis revealed that higher pulse pressure, higher weight and former ht use were the key factors independently associated with larger iad. the model was also run forcing age, race, and smoking status (table 4). in this model, hormone therapy, pulse pressure, and weight remained significantly associated with iad (all p<0.01, table 4). the model was also run controlling for antihypertensive medication use, but this variable fell out of the multivariable model when pulse pressure was added. current ht use was associated with a 0.14 mm smaller iad (table 4). african american women had a 0.05 mm smaller iad than the caucasian women, although this was not significant (table 4). current cigarette smokers had 0.18 mm smaller iad than current nonsmokers, with borderline significance (table 4). former ht use, greater bmi, weight, height, waist circumference, insulin (all p<0.01), glucose, and pulse pressure (both p<0.05) were associated with larger ld in univariate linear regression (table 3). the variables that yielded the best multivariate model to explain larger ld were former ht use, higher pulse pressure, and higher weight. when age, race and smoking were forced in the model only ht use and weight remained significantly associated with ld (table 4); the same results were seen when antihypertensive medication was added to the model (data not shown). current ht use was associated with a 0.13 mm smaller ld (table 4). each kg of weight was associated with 0.009 mm larger ld (table 4). postmenopausal current ht users had statistically significant smaller iad than the former ht users; this relationship remained significant after adjustment for known cardiovascular risk factors. the current ht users also had statistically significant smaller ld than the former ht users. in contrast, imt and plaque were not statistically different between current ht users and former ht users. it also demonstrates the value of measuring iad and ld in this type of study. the adventitia, the most outer layer of the artery, is composed of supportive connective tissue, fibroblasts, collagen, and elastin fibers. estrogen is known to preserve arterial structure by slowing elastin and connective tissue degradation, and by slowing age- and estrogen-related increases in collagen which lead to increased vascular stiffening. a small diameter reflects a healthy vasculature that is able to maintain an optimal balance of shear and tensile stress [1921]. this can make the artery vulnerable to injury and atherosclerotic development [1, 11]. the results of the current study, specifically the association of current exogenous estrogen use with smaller iad, is in line with the swan heart study that showed an association between higher levels of endogenous estrogen and smaller iad. the current study observed a 0.15 mm difference in iad between the current and former ht users. a longitudinal study observed 0.03 mm increase in iad each year for women (with similar mean age and mean height as the women in the current study). so the difference in iad observed in the current study translates to the change in iad expected over 5 years (0.03 mm/year 5 years=0.15 mm). these findings agree with the results of a cross-sectional study that found smaller ld among non-oral (percutaneous gel or transdermal patch) ht users compared to ht non-users. together, the findings from iad and ld may suggest the positive effect of estrogen on the vasculature through maintenance of vascular structure and function. both the current study and the swan heart study found that larger diameter was associated with older age, higher systolic blood pressure, higher glucose, and higher insulin: all risk factors for cvd. additional supporting evidence that enlarged diameter is an indicator of poor vascular health come from several studies showing enlarged iad is associated with cardiovascular disease risk factors [46, 813], increased imt [5, 12], plaque [5, 12, 23], and prevalent [12, 14, 15] and incident cvd. polak et al. recently published an article that identified a positive relationship between iad and left ventricular mass, an indicator of left ventricular hypertrophy. each 1 gram difference in left ventricular mass was associated with 0.006 mm larger iad in a multiethnic population of women after adjustment for height, weight, and imt. arterial diameter differences in current ht users and former ht users were observed in this study but differences in imt were not. consistent with our findings, a cross-sectional study of an american cohort from the atherosclerosis risk in communities (aric) study, found no significant difference in imt by ht use. the women in the aric study were of similar age to the women in this study and also had an undefined ht regimen that was predetermined by the woman and her physician prior to the study. selection bias may be present in both studies since women who chose to go on ht or women who chose to continue ht may have been different from the women who did not chose ht. a longitudinal study of oral therapy with one year of follow-up did not find a difference in imt progression in ht users and non-users. three studies evaluated differences in imt by ht and age [22, 26, 27]. the women were dichotomized into younger versus older (using 55 or 60 as the age cut-point). significant differences in imt were observed only in the older women who had longer use of ht than the younger women [22, 26, 27]. this may suggest that the effects of estrogen on imt are evident after long-term use. other explanations are that the differences observed are attributed to differing vascular effects of oral ht compared to transdermal ht, and fewer years on ht in the negative studies than the positive studies. one study that compared oral and non-oral therapy found transdermal ht had greater statistically significant effects on imt than oral ht. the aric study and this study participants used oral ht, were relatively younger and had fewer years on ht compared to the positive studies. a limitation of this study is that the ht regimen was varied since the dose, hormone composition (estrogen only or estrogen plus progestin), and form were chosen prior to the study by the participant and her health care provider. a standard dose and regimen of ht would be easier to evaluate and compare this study to previous studies. this would likely be especially true for the imt results that were not significant in this study. another limitation is that the adherence to ht and the level of estrogen or estradiol in the current users and former users was not assessed in this study. although the women reported ht use we do not know their adherence rates or the level of estrogen metabolites present during the ultrasound measurements. in the future, assessment of estradiol levels should be included to improve our understanding of carotid diameter associations and dose-related effects. strengths of this study are that it fills a gap in the literature, the methods used are valid and reliable, the lab that performed the ultrasound measures has high quality control, and it is one of the first to evaluate iad and ht. this study demonstrates the importance of iad and ld as more sensitive indicators of vascular health than imt. high resolution b-mode ultrasound is a valid and reliable detector of structural atherosclerotic changes of the arterial walls. the ultrasound measures in this study were performed with excellent reproducibility (class intra correlations of 0.98 for imt and 0.99 for iad) and continuous quality control to ensure reliable and valid data. in conclusion, these data suggest that current ht use is associated with vascular geometry in the postmenopausal women independent of cardiovascular risk factors. it also demonstrates the importance of measuring iad and ld in postmenopausal women with differing ht use. these measures should be included in addition to imt to provide a more complete story of vascular response and health.

arterial diameter is an underutilized indicator of vascular health. we hypothesized that interadventitial and lumen diameter of the common carotid artery would be better indicators of vascular health than carotid plaque or intima media thickness (imt). participants were 491 overweight or obese, postmenopausal women who were former or current hormone therapy (ht) users, 5262 years, with waist circumference>80 cm. we evaluated cross-sectional associations of cardiovascular risk factors with carotid measures, by ht status. former ht users had a worse cardiovascular profile than current ht users: larger adventitial (6.94 mm versus 6.79 mm) and lumen diameter (5.44 mm versus 5.31 mm, both p<0.01) independent of cardiovascular risk factors; imt and plaque were similar. larger diameters were best explained by former ht use, higher pulse pressure, and greater weight. independent of potential confounders, overweight and obese postmenopausal former ht users had larger carotid diameters than current ht users. carotid diameter should be considered in studies of ht.

PMC3432379

pubmed-432

prostate cancer (pca) is the most common solid organ malignancy in american men with global statistics mirroring those found in the united states. screening with prostate specific antigen (psa) has resulted in a significant stage migration such that the majority of new cases of pca are now detected while the disease is still clinically localized. these patients can choose from several treatment options and must weigh the potential morbidity of each treatment modality on their quality of life. in the vast majority of cases, urologists are the primary physicians that diagnose these patients with pca and are typically involved in the initial work up, discussion of all treatment options, and counseling of patients. urologists are not only intimately involved with the treatment of the primary disease but also the consequential treatment-related complications while overseeing the long-term followup of these patients. several centers have been recently established where urologists partner with radiation oncologists acquire ownership interest in intensity-modulated radiation therapy (imrt) equipment and provide integrated prostate cancer care. although not yet validated in the literature, this may allow for improved quality of care and decreased cost. unfortunately, these efforts have been much maligned in both the media and radiation oncology literature as conduits to increased revenue for the urologists with only debatable patient benefit [3, 4]. these reports have not yet been supported by data. after acquiring financial interest in an integrated prostate cancer center, we sought to evaluate whether our investment in imrt resulted in an increased utilization of radiation therapy in our patients with newly diagnosed prostate cancer. in september of 2008, we acquired financial interest in an integrated pca center offering imrt. following institutional board approval, we identified all patients who were diagnosed with pca in the 12 months before and after the center became operational. newly diagnosed cases of pca were identified by searching our electronic medical record using both prostate biopsy cpt codes (transrectal ultrasound-guided [trus] needle biopsy of the prostate, 55700/76942/76872) and the icd-9 prostate cancer diagnostic code (prostate cancer, 185.0). all men were diagnosed with pca after pathologic review of biopsy needle cores obtained after trus. indications for biopsy included elevated psa, abnormal digital rectal exam, abnormal pca 3 test, and/or strong family history of pca. prostate biopsies were performed utilizing a routine sextant pattern with at least 12 cores obtained. in cases where clinically indicated, the medical records of these patients were retrospectively reviewed and the data pertaining to the patients ' demographics, cancer parameters, and initial pca treatment modality were extracted. patients were assigned to two discrete groups based on the date of their pca diagnosis as it related to the date of the first availability of imrt at our integrated prostate center. all consecutive patients diagnosed with pca on a biopsy within 12 months prior to availability of imrt constituted the pre-investment group and all consecutive patients diagnosed with prostate cancer on a biopsy in the 12 months following initiation of imrt services constituted the post-investment group. the primary treatments received were designated as active surveillance (as), brachytherapy (bt), radiation therapy (xrt), radical prostatectomy (rp), and androgen deprivation therapy (adt). treatment data were available for all patients and were stratified by the patient's age and gleason score. the age of 70 years old served as a cut-off point as in our clinical practice most of these patients are deemed suboptimal surgical candidates because of increased risk of postoperative urinary incontinence and erectile dysfunction. our integrated pca center was established in collaboration with an academic radiation oncology department from a national cancer institute (nci) designated cancer center with a nationwide reputation for clinical and academic excellence. the radiation oncologists that treat our patients are not employed by the cancer center, but serve as full time academic faculty and have no financial interest in imrt. radiation oncology residents have the opportunity to participate in all aspects of planning and delivery of radiation therapy. the center also employs a nurse-practitioner charged exclusively with the coordination and support of on-going clinical research programs. patients who were referred to us for second opinion with biopsy-proven pca as well as men referred to us specifically for robotic-assisted laparoscopic radical prostatectomy were excluded to eliminate the potential biases resulting from the treatment recommendations rendered by an outside urologist. the overwhelming majority of these patients choose surgical therapy and their inclusion would skew the results toward lower utilization rates of xrt. unpaired t-tests, chi-squared tests, and fisher's exact tests were implemented as appropriate. a total of 344 patients were diagnosed with pca on trus biopsy over the designated 24-month time period. of the total patient population, 198 men were diagnosed with pca in the 12 months preceding availability of imrt, while 146 men constituted the post-investment group. patient and cancer characteristics were similar between the two groups (table 1 and figure 1). the percentage of patients with gleason 7 pca was higher in the post-investment group but did not reach statistical significance, p=0.073 (figure 1). overall, the use of radiation therapy for those patients with newly diagnosed pca following investment in imrt (20.55% versus 20.71%, p=1.00) was similar between the two patient populations (table 2 and figure 2). the number of patients treated with rp (67.81% versus 71.72%, p=0.729), as (9.59% versus 4.55%, p=0.177), adt (1.37% versus 2.02%, p=0.999), and bt (0.68% versus 1.01%, p=0.999) were not significantly different between post- and pre-investment groups. while overall treatment trends afford succinct analysis, clinical decisions regarding treatment of pca are often driven by a multitude of patient-specific factors. as such, the data was analyzed stratifying both gleason score and age (70 years old serving as a cutoff point). treatments stratified by patient age are shown in table 3 and figure 3. despite the increased incidence of gleason score 7 disease in the post-investment group, there was no significant difference between the groups in all treatment patterns in men less than 70 years of age (table 3). an increase was found in the use of xrt in men older than 70 years of age in the pre-investment group (45.45%) as compared to men following acquisition of imrt (55.32%), but this did not reach statistical significance (p=0.355). analyzed by age and gleason score simultaneously, there was no difference between the treatment groups in patients younger than 70 regardless of the gleason score, table 4(a) and figure 4(a). for men 70 years or older with gleason 6 disease, there was a trend toward increased use of as (34.78% versus 15.79%) and decreased use of rp (21.74% versus 31.58%), but did not reach statistical significance (table 4(b)). there was no difference (43.48% versus 42.11%) in the use of xrt for men over 70 with gleason 6 disease. for patients with gleason 7 disease, there was a statistically significant increase in the utilization of xrt (pre-investment 41.38% versus post-investment 68.42%, p=0.035) and decrease in the use of rp in the post-investment group (15.79% versus 55.17%, p=0.006) seen in table 4(b) and figure 4(b). there was no difference between treatment groups in men over 70 with gleason 8 disease. external beam radiation therapy is widely used and is an effective treatment option for localized pca. there is now level i evidence that high-dose radiation therapy decreases the risk of biochemical failure in men with clinically localized prostate cancer as compared to conventional dose conformal radiation [57]. this improvement, however, comes at a cost of increased gastrointestinal (gi) and genitourinary (gu) toxicity. observed that 2% of men receiving high-dose radiation experienced acute urinary or rectal morbidity of radiation therapy oncology group (rtog) grade 3 or greater. intensity modulated radiation therapy allows for delivery of radiation with greater conformality to the target volume compared with traditional 3 d technique. several randomized trials have shown that imrt reduced gi and gu toxicity compared with 3 d conformal radiation [810]. were able to deliver 81 gy with less than 2% of grade 2 rectal morbidity and no grade 4 or greater rectal complications in patients with clinically localized pca. furthermore, imrt has been shown to reduce the acute and late gi toxicity of patients treated with high-dose radiation therapy and adjuvant androgen deprivation as compared to 3 d conformal radiotherapy. this reduction in gu and gi morbidity has made imrt extremely popular in the delivery of high-dose external beam radiation for patients with clinically localized pca in the united states. since 2004 several large urology groups in partnership with radiation and medical oncologists have established centers of integrated prostate cancer care. the integrated care model is patient-centered and disease specific, where the equipment and the staff are dedicated to the treatment of pca and no other disease entity. although yet to be validated, this model may potentially result in better recognition and management of treatment-related complications, improved access to care, and increased experience with each treatment modality and thus better clinical outcomes. recently, these centers have become the targets of intense criticism [3, 4]. the detractor's claim that integrated pca care centers lead to self-referral by financially motivated urologists and radiation oncologists and result in over-utilization of imrt contributing to the increased cost of health care. they further claim that these centers have a negative impact on residency training in radiation oncology by shifting patients away from the academic radiation oncology training programs. unfortunately, these claims are not substantiated by data, but rather rely on indirect analysis of medicare claims and a 12% negative impact report from a single 3-point questionnaire survey of 81 radiation oncology training programs [3, 4]. to our knowledge, this is a first study conducted to directly evaluate whether financial interest in imrt as part of the integrated prostate cancer care model changed treatment recommendations for newly diagnosed patients with prostate cancer. we compared the distribution of treatments choices of all consecutive patients diagnosed with prostate cancer on a biopsy in our practice during a 12-month period prior to acquiring financial interest in imrt to a 12-month period following that acquisition. our analysis revealed that overall there was a small, but statistically insignificant decrease in the use of radiation therapy and radical prostatectomy and a small increase in the use of active surveillance following investment in imrt. the increased use of active surveillance is likely due to the emergence of data from several large trials supporting the safety and efficacy of this approach in appropriately selected patients [1113]. once the data were stratified by gleason score and patient age, a statistically significant increase in the use of radiation was found in men over 70 with gleason 7 disease (41.3% versus 68.3%). however, because of the overall low number of patients in this subgroup, this increase was due to a single patient difference between the groups (12 versus 13). these findings are not surprising as we believe that several important attributes of our integrated prostate cancer program provide for many patient benefits without the recently theorized, yet unsubstantiated risks of overutilization of imrt. as previously described, our center was established in collaboration with an academic radiation oncology department from an nci designated cancer center with a nationwide reputation for clinical and academic excellence. the radiation oncologists that treat our patients are not employed by the integrated prostate cancer center, but rather serve as full-time academic faculty and have no financial interest in imrt. furthermore, the final determination on whether a patient is an appropriate candidate for primary or adjuvant radiation therapy is made entirely by the treating radiation oncologist. radiation oncology residents have the opportunity to participate in all aspects of planning and delivery of imrt, thus deriving an educational benefit from this partnership. the center employs a nurse-practitioner charged exclusively with coordination and support of the clinical research program. additionally, we are privileged to have cme accreditation by our state medical society and conduct regularly scheduled multidisciplinary morbidity and mortality conferences and discussions of challenging cases. finally, we maintain a very high volume surgical program that ranks second in the number of radical prostatectomies performed annually in the greater philadelphia, pa, usa. first, this study is underpowered due to a fairly low number of patients and therefore the results of our statistical analysis must withstand the test of a larger trial for our conclusions to be validated. second, even though we attempted to minimize limitations of the retrospective study design by including all consecutive patients diagnosed with prostate cancer within the 24-month period, the selection bias inherent in retrospective study design was not completely eliminated. third, we did not include patients undergoing adjuvant or salvage radiation therapy in the trial and therefore did not ascertain the effect of financial interest in imrt on utilization of radiation in these patients. finally, our findings may not be applicable to other integrated prostate cancer centers because of the unique structure of our specific program. financial interest in imrt does not result in an increased utilization of radiation therapy in the treatment of newly diagnosed patients with clinically localized prostate cancer in our integrated prostate cancer center.

objective. as recent participants in an integrated prostate cancer (pca) care center, we sought to evaluate whether financial investment in an intensity-modulated radiation therapy (imrt) center resulted in an increased utilization of radiation therapy in our patients with newly diagnosed pca. materials&methods. following institutional review board approval, we retrospectively reviewed the records of all consecutive patients who were diagnosed with prostate cancer in the 12 months prior to and after investment in imrt. primary treatment modalities included active surveillance (as), brachytherapy (bt), radiation therapy (xrt), radical prostatectomy (rp), and androgen deprivation therapy (adt). treatment data were available for all patients and were compared between the two groups. results. a total of 344 patients with newly diagnosed pca were evaluated over the designated time period. the pre-investment group totaled 198 patients, while 146 patients constituted the post-investment group. among all patients evaluated, there was a similar rate in the use of xrt (20.71% versus 20.55%, p=1.000) pre- and post-investment in imrt. conclusions. financial interest in imrt by urologists does not impact overall utilization rates among patients with newly diagnosed pca at our center.

PMC3329792

pubmed-433

neurofibromatosis type-1 (nf-1) is a multisystem, autosomal dominant disorder of peripheral nerves affecting nearly 1/3000 individuals worldwide.1 it was first described by a german pathologist, friedrich daniel von recklinghausen. inherited or spontaneous mutation of the neurofibromin gene located on chromosome 17 is responsible for this diverse disorder. common skeletal manifestations of nf-1 include spinal deformities, congenital tibial dysplasia (congenital bowing and pseudarthrosis), sphenoidal dysplasia and cystic lesions in bones. pathological fracture of the acetabulum with anterior dislocation of hip secondary to osseous involvement of the acetabulum, femoral head, and pubic rami has never been documented in a case of nf-1. a 16-year-old boy presented with the complaints of pain in the left hip associated with the inability to bear weight following a trivial fall. on examination, the affected limb was 1.5 cm short, abducted and externally rotated. on general examination, patient had 8 caf au lait spots over the body, bilateral axillary freckles and multiple palpable neurofibromas in the subcutaneous tissues of forearm, thighs and back [figure 1]. patient met three out of seven criteria described for the diagnosis of nf-1 [table 1].2 plain radiograph and computed tomography scan of pelvis revealed an ill-defined lytic lesion causing pathological fracture-dislocation of the left hip [figure 2]. magnetic resonance imaging (mri) showed additional soft tissue involvement and joint effusion [figure 3]. ultrasound guided fine-needle aspiration cytology showed scanty cellularity with round to oval cells having minimal pleomorphism; hyperchromatic nucleus and moderate cytoplasm with spindle cells and osteoblasts. clinical photograph showing skin lesions-caf au lait spots (black arrows) and axillary freckling (white arrow) criteria for diagnosis of nf-1 (at least 2 or more features) preoperative radiograph anteroposterior view (a) and computed tomography scan (b) of pelvis showing an ill-defined lytic lesion destroying anterior column of acetabulum, pubic rami and part of the femoral head coronal (a) and axial (b) sections of magnetic resonance imaging of pelvis showing expansile lytic lesion of acetabulum and pubic rami with soft tissue involvement and joint effusion a wide local excision followed by arthrodesis of the joint was planned. considering the extent of bony and soft tissue involvement, we used a modification of the ilioinguinal and iliofemoral approach to have a wide exposure. we used the conventional ilioinguinal incision and combined it with femoral part of the iliofemoral incision [figure 4a]. on exposing the pelvis, anatomy was distorted. the deformed femur head and acetabulum with deficient pubic rami there was extensive soft tissue involvement adjacent to the acetabulum and lower part of the ilium. the entire acetabulum with 2-3 cm clear margin of the ilium was resected along with the abnormal soft tissue. iliofemoral arthrodesis was done using a 14 hole stainless steel dynamic compression plate [figures 4c d, and 5]. intraoperative photographs showing (a) skin incision (b) deformed femoral head (c) 14 hole dcp (d) iliofemoral arthrodesis postoperative radiograph after tumor resection and iliofemoral arthrodesis histopathology revealed dense collagenous tissue cores with spindle cells having blunt nuclei with minimal atypia and no mitosis or necrosis [figure 6a]. on immunohistochemistry, cells were s-100 positive and of neural origin [figure 6b]. partial weight bearing was allowed at 6 weeks and full weight bearing at 10 weeks. at 1-year followup, the patient was comfortable, pain free, able to ambulate unassisted, stand on one limb, sit and climb stairs without any difficulty [figure 7]. (a) histopathological photomicrograph showing dense collagenous tissue cores and spindle cells with blunt nuclei. (b) immunohistochemistry showing neural marker s-100 positivity clinical photographs showing functional outcome at 1-year followup type 1 neurofibromatosis or von recklinghausen disease, is a multisystem disorder that primarily affects the cell growth of neural tissue and characterized by involvement of skin, peripheral nerves, subcutaneous tissue, eyes, and skeletal system. although involvement of the musculoskeletal system is common, there have been only a few cases of subluxation/dislocation of hip in patients with nf [table 3].34567891011121314 on reviewing the literature, the etiology of hip instability leading to pathological subluxation/dislocation in patients with nf-1 can be classified as local and remote. most of the cases are secondary to local (intra and peri-articular) neurofibromas, which can result in mass effect, bony erosions (ilium, acetabulum, and femoral neck), acetabular dysplasia, narrowing of the femoral neck, coxa valga, increased femoral neck offset, capsuloligamentous laxity, and synovial membrane proliferation.3458101113 remote causes of hip instability include intra spinal neurofibromas/schwannomas leading to motor deficit (hip abductor weakness) or sensory deficit (charcot's neuropathic arthropathy), limb length discrepancies secondary to hemi-hypertrophy of lower limb and abnormal biomechanical alteration in the spinopelvic alignment due to scoliosis.6791011 endo et al. described anterior subluxation of hip secondary to decreased femoral head coverage resulting from decreased lumbosacral lordosis and posterior pelvic inclination following scoliosis correction.11 until date, there has been no case of nf-1 reported in the literature with pathological fracture of the acetabulum with anterior dislocation of hip attributable to a neurofibroma involving the acetabulum, pubic rami and femoral head. orthopedic manifestations of nf-1 comprehensive literature review on published cases of hip dislocation/subluxation in nf-1 the various treatment options described for pathological hip dislocations in nf-1 include closed reduction, open reduction, shelf operation with fascia lata tenorraphy, rotational acetabular osteotomy with femoral varus osteotomy, girdle stone resection, total hip replacement with the trochanteric distalisation.791114 the rate of re dislocation is very high in most of the cases, subsequently requiring a secondary surgical procedure for stabilization. since only a handful of cases have been described in the literature, it is difficult to comment upon the best line of management. in our case, arthrodesis was the best possible option since the bone stock after tumor resection was so inadequate that none of the above mentioned procedures could be tried. moreover, the fear of redislocation, which might necessitate repeated surgeries, was negated. a combination of ilioinguinal and iliofemoral approach was employed to have a better exposure of the hip joint. the neurofibromatous tumors associated with nf-1 are usually benign; however, there is a 2-5% chance of malignant transformation, especially with plexiform neurofibromas.15 plexiform neurofibromas are diffuse, poorly defined nerve sheath tumors arising from multiple nerve fascicles and surrounding tissues. they are more prone for hemorrhage, dysfunction, pain, disfigurement, and malignant transformation.16 there was no clinical or radiological evidence of recurrence or malignant transformation in our patient at 1-year followup. subtle clinical signs such as skin patches, axillary freckling, and subcutaneous neurofibromas can be easily missed. orthopedic surgeons must be aware about the various management options available and tailor them as per the needs of their patient. iliofemoral arthrodesis offered a good functional outcome with improved quality of life in our case.

skeletal neurofibromatosis (nf) commonly manifests as scoliosis and tibial dysplasias. nf affecting the pelvic girdle is extremely rare. pathological fracture of the acetabulum leading to anterior hip dislocation in a patient with nf-1 has never been reported in the literature. the paper presents the clinical symptomatology, the course of management and the successful outcome of such a rare case of nf-1. histopathological and immunohistochemistry studies showing abundant spindle cells, which are s-100 positive and of neural origin are the classical hallmarks of neurofibromatous lesions. tumor resection and iliofemoral arthrodesis can be considered as a valid option in young patients with pathological fracture dislocation of the acetabulum.

PMC4759865

pubmed-434

they form an integral part of indian diet, especially for those who have type 2 diabetes for whom white rice is considered less desirable because of its high gi. chapattis and other flatbreads are popular in europe also where they form a part of daily diet among members of ethnic minority groups who follow traditional dietary patterns. chapattis are made from whole-wheat flour and cooked on hot flat open griddles. they can also be prepared by substituting wheat flour with other cereal or legume flours at different levels. incorporation of cereal brans at proportions up to 10% has resulted in good quality chapattis. the color and appearance of chapattis were found to be good with substitution of wheat flour with up to 10% cereal brans. generally, chapatti is prepared from whole-wheat flour obtained by grinding wheat in a disk mill (locally known as chakki). chapatti quality can be assessed from its softness and flexibility which may be affected by flour protein quantity and quality. the chapatti quality is also influenced by the dough consistency, which in turn depends mainly on the quantity of water added. bran is the hard outer layer of cereal grains, rich in a myriad of healthy phytochemicals, namely, phenolics, flavonoids, glucans, and pigments. unfortunately, these nutrition-rich components are often discarded during milling out of ignorance, organoleptic reasons, and rancidity problems. knowing the phytochemical constituents and pharmacological profile of bran is expected to give insight to their potential application in promotion of health. cereal brans, the by-products obtained in large amounts in grain milling industry, considered as inedible material for humans, is mostly used as animal feed. however, brans are concentrated source of dietary fibre and other nutrients (proteins, b-vitamins, and minerals). brans are generally composed mainly of insoluble cellulose and hemicellulose, with only about 5 percent soluble fibre, and have little hypercholesterolemia effect. bran contributes a pleasing, sweet, nutty flavour when added as a flavour enhancer in a variety of food products. commercial wheat flour and oat bran (baggry's india ltd., new delhi, india) were purchased from local market. chappati was prepared by the addition of cereal brans (wheat, oat, and rice) singly and in combination (w: r: o:: 2: 1.5: 1.5) to wheat flour at 5 and 10% bran supplementation. chapatti. cereal brans singly and in combination at 5 and 10% level were added to wheat flour and required quantity of water which were mixed manually to obtain dough of suitable consistency. the dough was divided into four equal parts and moulded into circular chapattis of 15.0 cm in diameter with rolling pin and board. traditional home baking procedure was followed to bake chapattis on iron plate (tawa). chapattis were cooled and comparative evaluation was done using the following criteria which also included observations on dough handling properties. characteristic score grade dough handling nonsticky sticky slightly sticky very sticky puffing of chapatti full partial nil. the instrument was calibrated with the user supplied black plate calibration standard that was used for zero setting. the instruments were placed on the plate and three exposures at different places were conducted. readings were displayed as a, b, and l color parameters according to the cielab system of color measurement. the a value ranges from 100 (redness) to+100 (greenness) and the b value ranges from 100 (blueness) to+100 (yellowness), while the l value, indicating the measure of lightness, ranges from 0 (black) to 100 (white). was evaluated by using texture analyser (stable micro systems, model ta-hdi, uk). one strip at a time was placed on the centre of the sample holder and the blade was allowed to cut the chapatti strip. the force (n) required to cut chapatti strip into two pieces was recorded. bran enriched products such as extruded snacks, breakfast cereal-porridge, and chapatti were evaluated for sensory attributes (appearance, colour, texture, flavor, and overall acceptability) through a panel of semi-trained judges using 9-point hedonic scale. water activity of bran enriched products was estimated using water activity meter having hygrolab 3 bench-top indicator (rotrogenic company). standard aoac procedure was followed for free fatty acids determination in cereal bran enriched products. product sample (5 g) was taken in flask and 50 ml benzene was added and kept for 30 min for extraction of free fatty acids. after extraction, 5 ml extract, 5 ml benzene, 10 ml alcohol, and phenolphthalein as indicator were taken in flask and titrated against 0.02 n koh till light pink colour disappeared: (1)%ffa (% oleic acid) =2820.02 n kohml of alkali useddilution factor1000wt of sample taken 100, moisture content by method of aacc 2000, total plate count by method of maturin and peeler. moisture content by method of aacc 2000, total plate count by method of maturin and peeler. data collected from the aforesaid experiments was subjected to statistical analysis for standard error and duncan's multiple range test using minitab software. the quality evaluation of chapattis prepared by different bran enriched levels is mentioned in table 1. the pooled scores obtained by the various bran enriched levels of chapattis for appearance, color, texture, and flavor were 7.92, 7.18, 7.68, and 8.10 for wheat bran, rice bran, oat bran, and bran in combination enriched chapattis, respectively. the overall acceptability at 5 and 10 percent level of supplementation was 7.65 and 7.80, respectively. reported that chapattis, prepared by the addition of 10% bran, showed better performance and were quite comparable with whole-wheat flour regarding the proximate components and sensory attributes. dough handling characteristics of bran enriched chapattis do not show much variation with respect to type of bran used. except for rice bran incorporated dough for chapatti (slightly sticky), all others showed nonsticky behavior during dough development. all types of bran enriched chapattis showed full puffing except 10% rice bran enriched chapattis in which partial puffing during chapatti preparation was visualised. the data presented in table 2 depicted color and texture analysis of bran enriched chapatti. statistically significant (p 0.05) difference was observed in l value of bran enriched chapatti. l value of various cereal bran enriched chapattis was 64.37, 59.12, 60.04, and 61.92 for wheat, rice, oat, and bran in combination, respectively. l value showed decreasing trend with increase in level of supplementation of cereal brans in chapattis. the l value of 66.83, 61.59, and 61.14 was observed at 0, 5, and 10% level of supplementation, which means slightly lower brightness at higher levels of supplementation. a value of wheat, rice, oat, and bran in combination was 4.18, 5.24, 4.71, and 4.28, respectively. with increase in level of supplementation, a value (redness) increased from 3.14 at 0% level of supplementation to 5.11 at 10% level. altan et al. stated that, among the color parameters, the l and a values showed marked changes due to addition of tomato pomace. an increase in tomato pomace level decreased the l value of the sample and increased the a value of samples. also, increasing bran level supplementation resulted in a decrease in the b value of chapattis. a negative correlation was found between a value and b value of the enriched chapattis. cutting force (n) reflects the texture of the chapattis and it stimulates the biting action of the human teeth on chapattis. cutting force (n) of various bran enriched chapattis varied as 6.56 n, 5.92 n, 5.30 n, and 5.99 cutting force increased due to presence of more fibres at higher enrichment levels. at 0, 5, and 10 percent level of supplementation, the corresponding cutting force (n) was 5.25, 5.91, and 5.97 n, respectively. manu and prasada rao reported that cutting force of chapattis prepared from different wheat varieties ranged from 4.22 to 6.96 n. hemalatha et al. also reported that the cutting force (n) for chapattis made from different wheat varieties ranged between 4.22 and 6.67. the variation in cutting force might be because of variation in protein and fibre content of brans which determine the resistance offered by the samples. increase in fibre content might have increased the water holding capacity of chapattis and hence increased moisture content (%) with addition of bran. reported that moisture content (%) of bran enriched chapattis was 31.0 percent while control had moisture content of 30.2 percent. maximum water activity was observed in rice bran enriched chapattis (0.462) which was statistically at par with water activity of bran in combination enriched chapattis (0.455). water activity of oat and wheat bran enriched chapattis was 0.429 and 0.406, respectively. it was observed from data that water activity of samples was positively correlated with moisture content and followed the same pattern. manthey et al. reported that water activity of bran/fibre enriched pasta increased with bran supplementation over control. the increase in water activity is correlated with increase in moisture content at higher levels of bran supplementation. the data pertaining to free fatty acids (%) is presented in table 3. the free fatty acids (%) of bran enriched chapattis ranged from 0.057 to 0.085. the highest free fatty acids were recorded in rice bran enriched chapattis (0.085%), being the lowest in wheat bran enriched chapattis (0.057). it is also evident from the table that, with increase in level of bran supplementation, free fatty acids increased significantly. the free fatty acids (%) at 5 and 10 percent level of supplementation were 0.067 and 0.079 percent, respectively. khan et al. reported similar results regarding free fatty acids while studying development and evaluation of long shelf life ambient stable chapattis. a significant variation (p 0.05) was observed in total plate count of bran extruded chapattis. total plate count of enriched chapattis varied from 6.5 to 17 10 cfu/g. it is also evident from the table that with increase in bran supplementation level, a slight increase in total plate content was observed. the mean value of total plate content for 5 and 10% level of supplementation was 10.25 10 and 12.5 10 cfu/g, respectively. frazier and westhoff reported that total plate content increased from 2.3 10 to 3.4 10 cfu/g for flour. quality characteristics for chapatti revealed that dough handling and puffing of bran enriched chapattis prepared by 5 and 10% level of bran supplementation did not vary significantly. all types of bran enriched chapattis except rice bran enriched chapattis showed nonsticky behavior during dough handling. rice bran enriched chapatti recorded maximum moisture (%), water activity, and free fatty acids (%). with increase in level of supplementation, moisture, water activity, and free fatty acids increased. the future emphasis can be given on development of functional flatbreads which has got increased demand due to increase in health conscious consumer base.

cereal brans singly and in combination were blended at varying levels (5 and 10%) for development of chapattis. cereal bran enriched chapattis were assessed for quality and physicochemical characteristics. on the basis of quality assessment, 10% enrichment level for chapatti was the best. moisture content, water activity, and free fatty acids remained stable during the study period. quality assessment and physicochemical characteristics of bran enriched chapattis carried out revealed that dough handling and puffing of bran enriched chapattis prepared by 5 and 10% level of bran supplementation did not vary significantly. all types of bran enriched chapattis except rice bran enriched chapattis showed nonsticky behavior during dough handling. bran enriched chapattis exhibited full puffing character during preparation. the sensory attributes showed that both 5 and 10% bran supplemented chapattis were acceptable.

PMC4745543

pubmed-435

these misconceptions have a significant influence on the day-to-day life including the search for treatment in times of illness. a number of studies have reported that misconceptions and inadequate knowledge present significant barriers to effective management of diabetes. it is imperative for physicians to understand myths and misconceptions in a particular community about a disease to improve patient care, especially when dealing with chronic diseases like diabetes. diabetes mellitus (dm) is one of the most common noncommunicable diseases in the world and its prevalence is increasing dramatically. currently, there are around 285 million diabetic patients around the world, and the numbers are predicted to rise to 439 million by 2030, with the largest increase in the developing rather than in the developed world. its dramatic increase began a few decades ago with the rapid urbanization and development in the country. studies in the 1980s showed a trend towards an increase among adult saudis especially females. a large study of saudi patients from 1995 to 2000 revealed prevalence of 23.7%. however, a study in 2011 showed a significant rise in prevalence reaching 34.1% in men and 27.6% in women. correct knowledge about diabetes and its management has an enormous impact on attitude and practice of diabetic patients. a study of diabetic patients in new york, reported that patients with diabetes frequently had beliefs of the disease and medication that were false and even dangerous. it is imperative that awareness of diabetes and its correct management be created and various myths and misconceptions surrounding its course and management be removed. a few excellent studies about prevailing misconceptions about diabetes have already been carried out in the saudi population, more specifically in female diabetic teachers, in the eastern region, western region and qasim region. however, no studies have examined in detail the determinants of the misconceptions of diabetic patients. therefore, this study was undertaken to identify the determinants of prevailing misconceptions on diabetes and the management of diabetic patients registered at a diabetes clinic of a tertiary care hospital in the eastern region of saudi arabia. this cross-sectional study was carried out at the diabetes clinic of a tertiary care hospital, in eastern saudi arabia. this clinic has a registered patient population of 2000 diabetic patients. at a confidence level of 95%, response distribution of 50% and accepted margin of error of 10%, we calculated a sample size of 200. a table of random numbers was used to select patients from the medical record numbers of the registered patients of the diabetes clinic. the inclusion criteria were all saudi patients who had been registered at the diabetes clinic of the tertiary care hospital in saudi arabia for 6 months or more since diagnosis and on continuous treatment. no freshly diagnosed patient was included. in the same vein uncooperative or nonconsenting patients, as well as patients with any illness (physical or psychological) that was likely to influence reliable, valid responses to the interviewer's questions were excluded. the same interviewer spoke to all the selected patients on their scheduled follow-up visits after taking their informed consent. when a prospective participant refused to take part, the next patient on the list of random numbers was asked until the sample size was completed. all attendees were interviewed in standardized conditions with prior information to ensure valid reliable responses. the data collection instrument was an interviewer-filled questionnaire prepared in accordance with brief illness perception questionnaire and belief about medicines questionnaire. the demographic and classification data included age categories (< 20, 21-40, 41-60 or>60 years), gender (male and female), area of residence (urban or rural), education (primary, middle, high school, graduate and above), family history of diabetes (present or absent), type of diabetes (type 1 or type 2), time since diagnosis (< 5, 6-10, 11-15 or>15 years), type of treatment (oral hypoglycemic or insulin), self-monitoring (yes or no) and diet control as per doctor's advice (yes or no). in order to establish validity and reliability of the questionnaire, a pilot study was conducted in a sub-sample of attendees who were not included in the study proper. necessary changes were made in the questionnaire as well as the interview style as necessary. questions on the commonest reported misconceptions relating to etiology, types, pathogenesis, day-to-day life, diet and treatment of diabetes were included after a review of studies from saudi arabia, usa, india, nepal, and pakistan. an answer in yes to a misconception question was considered a misconception and a score of one was given. the total number of misconception questions was 36; therefore, the possible maximum score for any patient was 0-36. low (scores 0-12), moderate (scores 13-24), and high (scores of 24-36). chi-square test was used to determine the association of the socio-demographic variables with the three categories of misconception scores. stepwise logistic regression analysis was applied to the socio-demographic variables showing significant association with the misconception score by chi-square test and values of -coefficient, chi-square, odds ratio, and confidence interval were reported. a total of 200 responses for each item in the questionnaire were recorded from 200 subjects. the distribution of subjects in the age groups was 32 (16%) in<20 years, 62 (31%) in 21-40 years, 81 (40.5%) between 41 and 60 years and 25 (12.5%) in>60 years. the subjects from urban areas were 168 (84%) and those from rural areas were 32 (16%). those with the highest level of education were 7 (3.5%) who had up to postgraduation, 36 (18%) were university graduates, 76 (38%) high school, 43 (21.5%) middle, 29 (14.5%) primary and 9 (4.5%) illiterate. there was a family history of diabetes in 147 (73.5%) and no history in 53 (26.5%). those with type 1 diabetics were 78 (39%) while 122 (61%) had type 2 diabetes. the time since diagnosis was<5 years in 73 (36.5%), 6-10 years in 72 (36%), 11-15 years in 21 (10.5%) and>15 years in 34 (17%) subjects. the type of treatment was insulin in 105 (52.5%) and oral hypoglycemics in 95 (47.5%) indicating that many type 2 diabetic patients were also on insulin. self-monitoring of blood glucose was done by 124 (62%) of the subjects while 76 (38%) did not. diet control was used by 112 (56%) subjects while 88 (44%) did not. a total of 167 (83.5%) had received formal diabetes awareness education while 33 (16.5%) had not. the total misconception score was low (0-12) in 115 (57.5%), moderate (13-24) in 77 (38.5%) and high (24-36) in 8 (4%) respondents (n=200). table 1 demonstrates the frequency distribution of respondents according to misconceptions about etiology, types and pathogenesis of diabetes. the most common misconceptions identified on etiology, types and pathogenesis were that overweight causes diabetes (89%), diabetes is only a hereditary disease (80.5%) and eating too much sugar causes diabetes (69%). in addition, this table shows the distribution of subjects with reference to misconceptions about daily life. the most frequent misconceptions about the daily life of diabetics were that diabetics can not lead a normal social life frequency of respondents with misconceptions about etiology, types, pathogenesis and day-to-day life of diabetes (n=200) table 2 illustrates the distribution of respondents according to misconceptions about diet. the second misconception on treatment was that there is no need to take medicines when blood glucose is normal table 3 shows the association of the selected socio-demographic variables on the misconception score. it shows that female gender, living in a rural area, little or no education,<5 or>15 years since diagnosis, lack of self-monitoring, poor compliance with dietary control and no diabetes education were significantly (p<0.05) associated with moderate or high misconception score. however, age, family history of diabetes, type of diabetes and type of treatment were not significantly associated with the misconception score. frequency of respondents with misconceptions about diet and treatment (n=200) association of misconception score category with sociodemographic variables table 4 gives stepwise logistic regression to determine the predictive value of independent variables [showing significant association as per table 3] with the misconception score (dependent variable). it indicates that diabetes education, gender, education and time since diagnosis are good predictors of misconception score. every chronic disease necessitates long-term commitment from the patient, family and health care professionals. health care is a full package that includes the proper education of society in general and the patient in particular, about all aspects of the disease in question. a label of no definitive cure on chronic diseases generates many myths and misconceptions. these misconceptions are affected by socio-demographic factors and are propagated by opportunists who take advantage of patients to market their products. understanding the myths and misconceptions about a disease, like dm, is important for the provision of excellent care and health education to both patients and healthy individuals. these myths and misconceptions are generally about forbidden foods, the use of herbs, life-style changes, side-effects of treatment and so-called dependence on medicines. they usually interfere with self-management plans for diabetic patients leading to undesirable complications. the frequency of common misconceptions reported by other studies is more or less similar to what our report presents. a previous study in eastern saudi arabia that was carried out on adult male attendees of primary health centers found misconceptions about the etiology of diabetes in 21.2% of those studied, about general concepts of diabetes in 13.8% and diet in 10.7% of the patients. however, our study population was different, in that all of them were diabetics. a study in western saudi arabia reported that the top misconceptions that diabetic patients had were that oral medications might be more effective than insulin, medications might cause habituation and serious complications, the efficacy of medications depends on their cost, cure is expected following a short course of treatment and a diabetic could eat anything as long as medications were taken. the prevailing myths reported in the population of qassim region of saudi arabia were that consuming sugar results in diabetes, diabetics should avoid sweets, some type of dates do not increase sugar level, honey intake does n't increase sugar level, and diabetes in its early stages can affect sexual performance. determinants of myths and misconceptions are the factors that are directly related to generation or propagation of these wrong beliefs. knowledge of the determinants of any misconception, attitude or behavior is very important to the management of disease. our results indicate that around 42.5% of the subjects had a moderate to high misconception score, indicating that there was much room for improvement in our patients ' education. the mean misconception score was 10.29 4.92, while 38.5% had moderate (1324) and 4% high (24-36) misconception scores. the factors which had a significant association with high misconception scores were female gender, rural residence, little or no education,<5 years or>15 years since diagnosis, no self-monitoring, poor diet control and no prior education about diabetes. the relationships of different determinants of misconceptions studied in our study are discussed below. female gender was found to be significantly (p<0.05) associated with moderate to high misconception score. this was not a totally unexpected finding due to social norms of a conservative society with significantly less exposure of women to information. this finding gave us a clear task to target our female patients to provide them with the correct knowledge about diabetes and its management. a study carried out on female school teachers of alkhobar showed that their understanding of diabetes was inadequate. similarly, a sudanese study also found poor compliance to therapy, poor glycemic control and poor knowledge about diabetes in women as compared to men. this association was also reported in misconception studies carried out in new york and in india. however, a study carried out in qasim did not find gender to be associated with myths and misconceptions. the proportion of patients from rural areas was very low in our study as a result of the saudi government's policy of ensuring that patients access health facilities nearest to their homes. however, the proportion of patients with moderate to high misconception scores was higher in patients from rural areas. this shows that certain socio-demographic factors render people from those areas more vulnerable to misconceptions. however, there are other associated factors that can modify beliefs of urban populations as well. in a study carried out in a low income population of new york, a survey carried out in the 4 largest city of india, chennai, on around 26000 subjects concluded that even though there had been diabetes education campaigns from as far back as 1948, the level of lack of knowledge and misconceptions was unacceptable. as expected, the proportion of subjects with moderate to high misconception was highest in the illiterate group, and this decreased as the level of education rose. the study by sabra et al. in eastern saudi arabia found high misconception scores in 23.5% of illiterate or those who could only read-and-write. a study in new york found that participants with less than a high school education were more likely to have misconceptions. a study of myths and misconceptions in the qasim region also found significant differences in responses according to educational status. this necessitates the identification of the less educated from the beginning in order to design special educational programs that suit their respective levels of education. time since diagnosis was found to be significantly associated (p<0.05) with misconception scores. it also had a high predictive value for the misconception score. patients who had been diagnosed with diabetes>15 years before were more prone to have a moderate to high misconception score, and the chances of having misconceptions decreased in categories as times since diagnosis lessened. this might be due to better current diabetes awareness programs as well as the ease of access to information for freshly diagnosed diabetics who are curious. the knowledge of the subjects presenting for the 1 time has generally been reported to be inadequate and other studies have indicated this relationship. the patients who reported that they were controlling their diabetes by self-monitoring of blood glucose had low misconception scores. this group comprised 62% of the total number of patients, about two-thirds of whom had low misconception scores. on the other hand, two-thirds of those who were not self-monitoring had moderate to high misconception scores. this shows that making the patient take charge of his or her diabetes control develops an interested attitude. in addition, a clear picture of alterations in blood sugar by medicines, diet control and life-style changes help in removing of unfounded baseless misconceptions. no other study has reported this association. however, a study from western saudi arabia has shown an association of discontinuity of treatment with a high level of misconception. noncompliance to the recommended diet control was found to be a major determinant of misconceptions. surprisingly, most of patients with the highest misconception scores (7 out of 8) reported to have little or no diet control. this reflects a general carefree attitude of these patients to all aspects of life-style changes associated with the management of diabetes. however, most of the patients using the recommended diet control were found to have less misconception scores. this suggests that we must identify patients with a carefree attitude from the beginning and design proper education programs, with psychotherapy sessions if possible for them. the majority of the patients (83.5%) reported some formal instruction by health care professionals about diabetes and its management. as expected most of the subjects who had undergone a proper education about diabetes (presentation, management and life-style changes) had low misconception scores. not surprisingly, this was the most significant single determinant of removing misconceptions about diabetes. the results also indicated that there was room for improvement in the education of the large proportion of patients with moderate to high misconception scores. in our study, family history of diabetes, type of diabetes and type of treatment were not found to have any significant association with misconception scores. as in our study similarly, the age of our subjects did not show a significant association with misconception score. however, the proportion of subjects with moderate to high misconception scores was highest in the categories of the youngest and the oldest patients (< 20 and>60 years). though not statistically significant, it still shows that we must make an extra effort to educate these two vulnerable age groups about their disease. suboptimal knowledge and beliefs are potentially modifiable and are logical targets for educational interventions to improve diabetes self-management. knowledge of all the identified determinants of moderate to high misconception scores will help in streamlining the awareness programs for patients in accordance with these factors. similarly, it will act as a guideline for other units in our region to develop better patient education programs. if the patients are given proper guidance and education on diabetes care, there would be a significant improvement in their life-style which would in turn help in producing good glycemic control. we conclude that myths and misconceptions about diabetes and its management are common in our patients. the strongest determinants of the misconceptions in our study population are female gender, rural area of residence, illiteracy or little education,<5 or>15 years since diagnosis, no self-monitoring of blood glucose, poor diet control and no education about diabetes. therefore, diabetes educational programs should focus on individuals with one or more of these predictors.

objective: to identify the determinants of misconceptions about diabetes in patients registered with a diabetes clinic at a tertiary care hospital in eastern saudi arabia. materials and methods: this cross-sectional survey was carried out at a diabetes clinic of a tertiary care hospital in eastern saudi arabia, from january to december 2012. a total of 200 diabetic patients were interviewed using a questionnaire comprising 36 popular misconceptions. the total misconception score was calculated and categorized into low (0-12), moderate (13-24) and high (25-36) scores. the association of misconception score with various potential determinants was calculated using chi-square test. step-wise logistic regression was applied to the variables showing significant association with the misconception score in order to identify the determinants of misconceptions. results:the mean age was 39.62 16.7 and 112 (56%) subjects were females. type 1 diabetics were 78 (39%), while 122 (61%) had type 2 diabetes. insulin was being used by 105 (52.5%), 124 (62%) were self-monitoring blood glucose and 112 (56%) were using diet control. formal education on diabetes awareness had been received by 167 (83.5%) before the interview. the mean misconception score was 10.29 4.92 with 115 (57.5%) subjects had low misconception scores (< 12/36). on the chi-square test, female gender, rural area of residence, little or no education,<5 or>15 years since diagnosis, no self-monitoring, no dietary control and no diabetes education were all significantly (p<0.05) associated with higher misconception scores. step-wise logistic regression suggested that diabetes education, gender, education and time since diagnosis were significant (p<0.05) predictors of misconception scores. conclusions:the strongest determinants of misconceptions about diabetes in our study population were female gender, rural area of residence, illiteracy or little education,<5 or>15 years since diagnosis, no self-monitoring, no diet control and no education about diabetes.

PMC4073566

pubmed-436

thoracic injury can result in a wide range of clinical manifestations depending on the structures involved like chest wall, diaphragm, mediastinum, trachea, lungs parenchyma etc. the non penetrating trauma to the lungs manifests as contusions, lacerations of pulmonary parenchyma, pneumatocele formation, hematomas, and fractures of trachea and bronchi. while pulmonary contusion is commonly associated with blunt thoracic trauma, appearance of cavitary lesions variably described as traumatic pulmonary pesudocysts, (tpp), traumatic pneumatoceles, traumatic lung cysts are rare, developing in less than 3% of patients with pulmonary parenchymal injuries. a 26 year old unmarried male was referred to us with history of left sided chest pain for two days. he had consulted at local hospital immediately after trauma. a skiagram chest taken there [figure 1] showed non homogenous opacity with central lucency in the region of left hilum. on examination, the patient gave history of trauma-a tractor wheel hitting him on the anterior chest wall, while he was lying down; following which he developed the pain. there was no history of breathlessness, fever, cough, hemoptysis, cough or expectoration. there was no history suggestive of bronchial asthma or exposure to pets at home or work place. the margins of the lesion were now thinned out and smooth [figure 2]. chest radiograph on day 1-pa view showing non homogenous opacity with central translucency near left hilum day 3-chest radiograph pa view showing multiple cavities near left hilum his sputum for afb was negative. other lab investigation revealed haemoglobin 14.4, leukocyte count 7900, and differential count of 78% polymorphs and 19% lymphocytes. his serum was non reactive for hiv antibodies. a ct scan of thorax done on 03-09-2011 showed a single smooth walled cavity abutting left chest wall with surrounding areas of ground glass haziness, probably suggestive of lung contusion [figure 3]. computed tomogram of chest showing smooth walled cavity in left upper lobe with surrounding ground glass opacity of lung contusion fibre optic bronchoscopy was performed to achieve a microbiological diagnosis and to inspect the trachea-bronchial tree in view of previous reports being inconclusive. bronchoscopy revealed a bleeding spot in left main bronchus, but no active bleeding was seen. bronchial brushings taken from left upper lobe were negative for malignant cells and afb, and the lavage fluid also was sterile. in view of the patient's history of ancedent recent trauma, negative past and present history for any infectious process, and negative microbiological reports, a diagnosis of post traumatic pulmonary pseudo cyst (tpp) was made. serial follow up chest skiagram showed rapid resolution of the cavities [figure 4] without any antibiotics, thus confirming our diagnosis of tpp. among the various names given to the cavitary appearance of pulmonary lacerations, the term traumatic pulmonary pseudo cysts (tpp) appears to be the best nomenclature because the wall of these lesions are formed by the inter lobar interstitial connective tissue and shows no epithelial lining or bronchial wall elements. the rarity of the entity can be estimated by the fact that there have been only around 10 large case series with 8 or more patients published since 1967. tpp following blunt or non penetrating trauma develops through a mechanism that allows transmission of high compressive forces to the lung parenchyma. retraction of normal lung elastic tissue from contusion induced cavities permits the escape of air and fluid into it. laceration of pulmonary parenchyma and appearance of cavity may also occur if there is closure of the glottis at the moment of injury which may prevent fast exit of the air from compressed lung segment. the parenchyma and/or interstitium get lacerated in a bursting manner resulting in a cavity formation. resolution of a pulmonary hematoma or drainage into a bronchus may result in development of tpp at a later stage and it is called secondary tpp. tpp can occur at any age, but they are more often seen in children and young adults, probably because of greater compliance of chest wall which permits a larger transmission of force of impact to the parenchyma. sorsdahl and powell reported that 85% of the patients with tpp were under the age of 30 years with male predominance. impact velocity and degree of chest wall displacement may play an important role in the development of tpp after blunt trauma to the chest. a high velocity impact causes peripheral tpps while a low velocity high displacement impact produces central parenchymal and major bronchial disruption. tpp is a clinical entity that manifests itself with minor clinical and major radiological signs. the usual clinical manifestations include hemoptysis, occurring in about half of the cases, cough, dyspnoea, chest pain, fever and leucocytosis. tpps may appear immediately or within a few hours after injury and their sizes range from 2 to 14 cm in diameter tpp can be differentiated from cavitating hematomas on the basis of radiographic appearance of air within the tpp's within 48 hours. tpps may be identifiable on chest radiography but ct scans are superior for detecting them. unlike other cystic lesions and cavities, the size, shape, and nature of wall of tpps change relatively quickly. therefore, serial skiagrams of chest, done over several days can help to differentiate pseudo cysts from other lesions. found a greater resolution time in blood filled tpps and those with>2 cm in size. they may be seen on the site of injury or on the opposite side secondary to countre coup effects. the majority of tpps are found in the lower lobes, the ct appearance of single or multiple thin walled cystic lesions with air space consolidation of the surrounding lung parenchyma in the backdrop of antecedent trauma is diagnostic. differential diagnosis includes ruptured oesophagus, or herniation of viscera, post pneumonia pneumatocele, tuberculosis cavity, cavitating bronchial carcinoma, lung abscess, bronchogenic cysts, and pulmonary sequestration. a history of trauma, rapid sequential changes over days on chest skiagram, and presence of contusion on the base of lesion usually delineates any confusion. if the cavitary lesion does not decrease with time other aetiology must be considered. conservative management is recommended as long as evidence of a decrease in size of the lesion occurs within 6 weeks after trauma in adults and 3-4 months in children. the use of antibiotics is controversial and may be used more to provide simple reassurance that the pseudo cyst wo nt get infected. tpp's can be complicated and may require surgery. they may rupture and cause a secondary pneumothorax that may require tube thoracostomy. the indications for diagnostic and therapeutic bronchoscopy are endobronchial bleeding, thick sputum, large air leak, mediastinal emphysema and lobar collapse. the approach to an infected pseudo cyst is similar to that for a lung abscess. if an infected pseudo cyst is larger than 2 cm, or there are unremitting signs of sepsis after 72 hours of antibiotics, the pseudo cysts may be drained percutaneously. early lobectomy may be considered for complex tpp's with extensive lung abscess surrounded by necrotic parenchyma, failure of bronchoscopic treatment of massive airway bleeding, infected pseudo cyst more than 6 cm in size, or no response to more conservative treatment. video assisted thoracoscopic surgery (vats) may be considered for managing a persistent air leak, hemothorax due to pseudo cyst rupture, failure of lung expansion, progressive enlargement of pseudo cyst and compression of lung parenchyma. late thoracotomy has been reported (lobectomy, cystotomy, capitonnage) up to 6 months after trauma because of pneumonic infiltration and persistent cavitary size. our patient presented with few abrasions on anterior chest wall and history of blunt trauma to chest wall of one day duration. there was no previous history of any respiratory illness or unconsciousness during or after trauma. the immediate chest skiagram revealed an area of consolidation with central area of lucency around left hilar region. there was no associated cough, fever, expectoration, hemoptysis or breathlessness which clinically ruled out an infective process. serial skiagrams over next 24 hours showed a rapid development of a cavity which regressed spontaneously and significantly over next 7 days without any antibiotics. ct thorax done was also suggestive of tpp, in view of a cavity adjacent to anterior chest wall with surrounding contusion. exclusion of other infectious diseases like tuberculosis, lung abscess, by absence of relevant clinical history, sputum negativity on zn staining, sterile pyogenic culture, negative mantoux test, negative microbiology of bal, and cytology of bronchial brushing led us to the diagnosis of tpp which was well supported by the available literature. it is generally a self limiting benign condition, diagnosed by excluding other conditions which may present with similar radiological manifestations. a typical history of blunt trauma to chest, rapid radiological changes of the cavity and exclusion of other cavitary pulmonary diseases by adequate laboratory work helps in diagnosing this rare condition. the disease at times might become complicated and becomes life threatening; hence, should be closely monitored and followed up.

blunt thoracic trauma manifests in various ways, depending on the structures injured and type of injury. commonly manifested as parenchymal contusion, at times, pseudacavitation may also been seen on the chest x ray. they are to be differentiated from other causes of pulmonary cavitations which are often done based on history. the so called pulmonary pseudo cysts usually have a benign course and needs only observation.

PMC3743341

pubmed-437

. however, cerebral phaeohyphomycosis (cp) caused by darkly pigmented fungi appears to be a common exception to this rule because about one-half of this fungal infection occurred in patients with no underlying disease or risk factors. cp is a very rare cause of brain abscess, but is often a fatal disease regardless of immune status14,17,23). the authors illustrate a 75-year-old, immunocompetent male patient who had a single brain abscess from dematiaceous fungi. to the authors ' knowledge, this is the first case of cp in korea. a 75-year-old male, a resident of a rural area and a farmer by occupation, visited our outpatient clinic with the symptoms of poor cognition and memory decline over 2 weeks. he denied any history of fever, headache, blurred vision, vomiting or seizure. there were no laboratory abnormalities including leukocytosis or c-reactive protein rising. upon the neurologic examination, he was conscious and there were no neurologic deficits except intermittent expressive dysphasia and disorientation. brain magnetic resonance imaging (mri) was performed because of suspicion of some type of dementia. it showed a 20 mm sized nodular enhancing mass with peritumoral edema in the left frontal lobe. high-grade glioma or metastatic tumor was initially presumed based on his age and progressive symptoms. the lesion appeared to be white and took the form of a relatively hard mass with a clear boundary, permitting radical excision of the mass (fig.. septated hyphae and melanin pigments were confirmed at fontana-masson stain consistent with cp (fig. the patient was started on intravenous amphotericin b at a dose of 68 mg daily. after 10 days, he was switched to 270 mg of intravenous voriconazole twice a day because of the elevation of serum creatinine. he took the injection for 8 weeks, followed by oral voriconazole 200 mg twice a day for 2 months. a follow-up brain mri 3 weeks after surgical excision demonstrated a significant resolution of the edema. ongoing resolution of the lesion was found on the latest follow-up mri (fig. he showed dramatic improvement in his symptoms including disorientation and memory disturbance after completion of surgery and antifungal therapy. cp is a rare infection caused by darkly pigmented fungi, namely dematiaceous fungi23). dematiaceous fungi represent a group of filamentous molds that contain melanin pigment in their cell walls3,6,14,17). rhinocladiella mackenziei (formerly ramichloridium mackenziei) is the second most common cause of cp, which is exclusively endemic in the middle east area17,22). because of this, occupational predisposition has been reported in agricultural workers, especially farmers due to risk of soil exposure17,23). cp commonly occurs in the second and third decades of life with male predominance, except rhinocladiella mackenziei which affects adults with a median age of 62 years without male predominance3,13,23). the most unique characteristic of cp is its occurrence irrelevant to the immune status of the host15,17,19,23). even though immunodeficiency may play a role as a risk factor, there are many reports of this infection in immunocompetent individuals similar to the patient in this report12,15,17,19,25). the portal entry to brain is unclear, although several possible routes have been suggested, such as hematogenous dissemination of inhaled spores or accidental skin inoculation as well as direct extension from adjacent paranasal sinuses or ears2,6,10,14,15,19,22,23). pathogenesis of cp is associated with the presence of melanin as a virulence factor that provides advantages in evading host defense and crossing the blood-brain barrier by binding to hydrolytic enzyme14,16,17). clinical spectrum of phaeohyphomycosis was listed as a variable, ranging from solitary subcutaneous nodules to a life-threatening infection5,11,16,17,18). in the central nervous system (cns) about 70-80% of cases typically manifest as a single brain abscess particularly on the frontal lobe (52%) like in our case, while multiple brain abscesses can be seen in immunocompromised patients3,6,17,19). the diagnosis of cp can be difficult because dematiaceous fungi are often considered contaminants when identified in culture. furthermore, the pathogen can not always be cultured and isolated from the serum or cerebrospinal fluid (csf)12,21,24). no molecular techniques are available to speedily identify these fungi even to the genus level17). only the tissue examination can be useful to identify irregularly swollen hyphae with yeast-like structure and to confirm the presence of dematiaceous hyphae in melanin-specific fontana-masson stain14,17). unfortunately in this case, fungus was not identified in the culture of surgical specimen, therefore, the species that causes cp could not be detected. meanwhile, the brain mri reveals a ring-enhancing lesion with a low-attenuation core, suggesting the presence of necrosis or pus10,19). in cases where high-grade glioma or metastasis is mimicked by irregular and variably contrast-enhancing lesions, imaging findings of this patient were more suggestive of a glioma than an abscess, because nodular heterogeneity on contrast injection mimicked the images seen in high-grade tumors. consequently, surgical biopsy is essential for the diagnosis of cp. because of the rarity of the cases complete excision of brain lesions may provide better results than simple aspiration unless the lesion is multiple or is located within the eloquent area of the brain3,17). antifungal agents are generally used in combination of amphotericin b, 5-flucytosine and itraconazole because it is associated with improved survival rates3,14,17,20). voriconazole can be used as alternative to itraconazole because of its good penetration into both csf and brain tissue17,22). duration of taking the medications is still unknown because most reported patients expired during treatment except a few survivors who received voriconazole for about 12 months8,19). in addition, posaconazole may be a potent drug when pathogen is rhinocladiella mackenziei1,3,8). in this case, amphotericin b has fatal side effects such as nephrotoxicity, therefore, close observation on kidney function is needed. mortality rate approaches 100% in untreated patients, while that of treated cases as high as 65% to 73% despite the aggressive treatment6,9,10,17,19,23). interestingly, mortality rate did not differ significantly between immunocompromised and immunocompetent patients (75% vs. 71%)12,17). fortunately, the patient reported here had a good response to surgery and chemotherapy and showed fine recovery without any sequela. solitary lesion and the good general condition of the patient, together with an aggressive therapeutic approach, are therefore inferred to contribute to a favorable outcome. further studies are necessary to find more potentially useful antifungal regimen for these refractory infections and to investigate more detailed pathophysiology and prognostic factors to increase the survival rate. cp is rare disease, but challenging one with high mortality rate, particularly when the cns is affected. as shown in this report, complete resection and adequate antifungal therapy are the most recommended modality for patients with cp-related abscess to this time.

cerebral phaeohyphomycosis (cp) is a very rare but serious form of central nervous system fungal infection that is caused by dematiaceous fungi. it is commonly associated with poor prognosis irrespective of the immune status of the patient. in this study, the authors describe the first case of cp in korea that occurred in a 75-year-old man without immunodeficiency and showed favorable outcome after surgical excision and antifungal therapy. in addition, the authors herein review the literature regarding characteristics of this rare clinical entity with previously reported cases.

PMC4273007

pubmed-438

cephalopelvic disproportion (cpd) in labour occurs when there is a mismatch between the size of the fetus and the dimensions of the maternal pelvis. the factors which mainly influence the outcome of the delivery can be summarised as the three ps of the labour: passageway, passenger, and power of the uterus. the passageway component of this trinity has been investigated by pelvimetry which measures the maternal bony pelvic dimensions, with very little emphasis on its shape or pelvic floor muscles. during the last decades, the use of pelvimetry has been discouraged, but at present, no replacing methods to evaluate the maternal pelvis have been introduced. the benefits of vaginal deliveries are well known when no risk factors are present even after previous cesarean section (cs) [5, 6]. on the other hand, unplanned interventions during labour such as acute or emergency cesarean sections as well as operative vaginal delivery increase both maternal and fetal morbidities as does a prolonged second stage of the delivery. the safety and the accuracy of the measurements obtained in pelvimetry have improved in the era of the mri technology [9, 10]. it is also in the interest of the mother and her physician to minimize the number of unplanned interventions during labour. the purpose of this observational cohort study was to evaluate whether pelvic measurements, especially pelvic outlet, displayed any association with operative vaginal deliveries and the duration of the second stage of the delivery. this retrospective study was approved by the ethical committee of north-carelian central hospital. it investigated caucasian women, that had been examined by x-ray or mri pelvimetry during 20002008 in north-carelian central hospital. eligibility criteria included that pelvimetric and fetal measurements had been recorded. in the operative delivery group, there were no signs of fetal distress in cardiotocography, inertia was not diagnosed, and there was no malpresentation. originally, 915 women were screened for possible inclusion, but 429 women were excluded because of breech presentation. a total of 486 patients with the fetus in the cephalic presentation were screened in the study, but those 234 women that went through elective or acute cesarean section were excluded from the analysis. the clinical indication for pelvimetry was breech presentation, or if the fetus was in cephalic presentation, the indication was suspected cephalopelvic disproportion in clinical examination. the findings that referred to cpd in clinical examination were clinically small pelvis, unengaged presentation, or suspected macrosomia. pelvimetric measurements were found in all patients, as required by the inclusion criteria. there were 252 participants with fetal cephalic presentation delivered vaginally, of whom 184 women delivered spontaneously and 68 women went through operative vaginal delivery with vacuum extraction. of this latter group of women, in 26 patients, the vacuum extraction was undertaken primarily because of fetal distress and inertia, and these patients were excluded from the final analysis, leaving 42 women in the operative vaginal delivery group. thus, the total number of participants evaluated in the final stage of this study was 226. the obstetric and radiologic data were collected from patients ' medical records by the author (uk) and transferred into a commercially available worksheet (excel, microsoft 2003, ireland). the following pelvimetric parameters were recorded: in the pelvic inlet, anteroposterior (conjugata vera) and transverse diameters and in outlet, interspinous diameter and sagittal diameter from the surface of the pubic symphysis to the surface of the sacrum measured at the spinous level. pelvic inlet and outlet circumferences were calculated from the pelvic anteroposterior and transverse diameters using the formula (ap+dt 1.57). until the year of 2003, all pelvimetries were performed with an x-ray technique, and from the year 2004 onwards, they were performed with magnetic resonance imaging (mri). during the transition period, both x-ray and mri pelvimetries were performed to verify the repeatability of the measurement results. already at the beginning of 1990, in order to minimize the variability in pelvimetric measurements, they were centralized so that instead of being conducted by several radiologists, they were conducted by trained obstetricians. when the mri pelvimetry was taken into clinical practice, there was one radiologist with previous experience of mri pelvimetry, and during a two-year period (20042006), three radiologists and further three obstetricians were also trained to measure the images. in this evaluation of the diagnostic accuracy of the pelvimetry in vaginal deliveries, patients were divided into subgroups according to the size of the fetus and also by the parity to evaluate the variability reflecting differences in patient groups. for statistical analysis, we used spss 17.0 (spss inc., 2009, receiver operating characteristic (roc) curves were established, and the area under curve (auc) values with significances were calculated. pelvimetric measurements were found in all patients, as required by the inclusion criteria. in the spontaneous vaginal delivery group, most of the nulliparous patients were sent to maternity clinics consultation because of suspected disproportion. in the multiparous group, 24% had delivered by cs and 37% by operative vaginal delivery. in the operative vaginal delivery group, of the nine multiparous patients, six had delivered by cs in their previous pregnancy and two had had previous vaginal operative delivery. the demographic data of these 226 patients subdivided according to the route of delivery are shown in table 1. patients were further subdivided into two subgroups according to the infant's weight and the mode of delivery. the maternal pelvic inlet and outlet sizes and duration of the first and second stages of the delivery by the mode of delivery in infant weight subgroups are shown in table 2. the mean maternal outlet (sd) was 3613 (20) mm in all, 351 (17) mm in infant weight<3700 g, and 369.5 (17.7) mm in infant weight 3700 g groups. no clinically or statistically significant differences in the pelvic sizes were found between the modes of delivery within the subgroups. between the subgroups, the size of the maternal pelvic size was 4%-5% larger in the mothers with infant weight 3700 g. the duration of the second stage of the delivery was 54 minutes longer (p<0.01) in the operative vaginal delivery group amounting to a 45-minute longer duration (p=0.01) in infant weight<3700 g and 62 minutes longer (p<0.01) in infant weight 3700 g group. the one-minute apgar scores were above 8 in all groups with the exception of those with infant weight less than 3700 g in the operative vaginal group, where the mean of apgar score at one minute was 7.8 1.8. the receiver operating characteristic curve analysis for pelvic inlet and outlet as a diagnostic test for the mode of vaginal delivery is shown in figures 2(a) and 2(b). the area under the curve (auc) for the pelvic inlet was 0.566 with the p value of 0.18 and 95% confidence interval (ci) of 0.4650.667. for pelvic outlet, the main finding of this study was that the maternal bony pelvic dimensions displayed virtually no correlation to the need for operative vaginal deliveries. the indications for intervention in vaginal deliveries were chosen on clinical grounds as evidenced by the fact that there was an association between the duration of the second stage of the delivery and the size of the pelvic outlet. if the delivery had reached the second stage, it was probable that the uterine power played a more significant role in the overall outcome than either the passageway or the passenger. on the other hand, pelvic floor muscles, the three-dimensional shape of the bony pelvis, or other soft tissues the pelvimetry was performed in most of the patients because of suspected disproportion, or an intervention had been required in a previous labour. of those patients that had previous cs and were now exposed to the trial of labour, over 80% delivered spontaneously, and less than 20% required an operative vaginal delivery. this is in agreement with previous studies [6, 13]. of those women that had had a previous operative vaginal labour, only 5% underwent repeated vaginally assisted delivery. this may have been due to the fact that the patients were chosen for the trial of labour correctly irrespective of the previous operative delivery. there were no statistically significant differences between the size of the maternal inlet or outlet in the spontaneous and the operative vaginal delivery groups. when patients were divided into subgroups according to the infant weight, the maternal inlet was 4.7% and the outlet was 5.1% larger in the infant weight 3700 g subgroup among those who delivered spontaneously compared to those vaginally assisted. the duration of the first stage of the delivery was longer in the smaller infant group, whereas the second stage was shorter than in the larger infant group. in the two delivery subgroups, the duration of the second stage of the delivery was significantly longer in operative vaginal delivery group than in spontaneous vaginal delivery group. the apgar scores were acceptable in all delivery groups referring to the fact that both spontaneous and operative vaginal deliveries were uncomplicated and severe shoulder dystocia was not present. the apgar 1-minute scores were lower in the operative vaginal delivery group than in spontaneous vaginal delivery group when infant weight was<3700 g. these results refer to the fact that operative vaginal delivery increases the time of the second stage of the delivery and decreases the apgar 1-minute scores. the roc curve analysis for maternal pelvic inlet and outlet revealed that both inlet and outlet had only a fair prognostic value in predicting the mode of the vaginal delivery. the poor predictive value of pelvimetry to predict protracted labor is a well-known fact from previous studies, whereas the evidence on the need of vaginal operative deliveries is less extensively evaluated. the data did not reveal in detail the fluency of the operative deliveries. as mentioned earlier, therefore, it was not possible to evaluate the influence of the pelvic dimensions on the severe dystocia. for that kind of study, the cohort examined here study is too small due to the rare incidence of severe dystocia. accordingly, due to the retrospective nature of the study, no blinding was present, and the caregivers were aware of the pelvimetric measurements during the labour. furthermore, the study women were at high risk of operative deliveries due to the inclusion criteria, but in clinical care, this would be the group eligible for pelvic assessment before delivery. in conclusion, our study revealed that maternal bony pelvic dimensions, either pelvic inlet or outlet, were not associated with the need for operative vaginal deliveries. it was more likely that other factors related to the maternal perineal soft tissue, maternal resources, and the passenger were the reasons leading to operative vaginal deliveries. subsequently, we can not recommend that caregivers use pelvimetric measurements to predict the outcome of the second stage of the labour. observational studies with larger cohorts would be needed, if one wished to investigate whether the maternal bony pelvic size has any effect on severe dystocia. in addition, the three-dimensional shape of the bony pelvis and the soft tissues are worth considering in future studies.

objective. to evaluate whether pelvic measurements have any association with operative vaginal deliveries and the duration of the second stage of the delivery. study design. a retrospective study of pregnant women at an increased risk of fetal-pelvic disproportion during 20002008 in north-carelian central hospital. the mode of the vaginal delivery was chosen to represent the reference standard. the target condition was spontaneous vaginal delivery. patients were divided into subgroups according to the size of the fetus and also by the parity to evaluate the variability reflecting differences in patient groups. receiver operating characteristic (roc) curves were established. results. a total of 226 participants with fetal cephalic presentation delivered vaginally; of these, 184 women delivered spontaneously, and 42 women required operative vaginal delivery with vacuum extraction. there were no clinically or statistically significant differences between the size of the maternal pelvic outlet and the different modes of delivery types within these subgroups. with respect to the pelvic inlet and outlet, the areas under the curve in roc were 0.566 with the p value of 0.18 and 95% confidence interval (ci) of 0.4650.667 and 0.573 (95% ci: 0.4840.622; p=0.14). conclusions. the maternal bony pelvic dimensions exhibited virtually no correlation with the need for operative vaginal deliveries.

PMC3649162

pubmed-439

visfatin, also known as nicotinamide phosphoribosyltransferase (nampt) as well as pre-b-cell colony-enhancing factor, is a multifaceted protein with suggested enzymatic, immunological, and metabolic properties. visfatin has been analyzed in hypo- and hyperthyroidism in in vitro and in vivo studies, but results are inconclusive. in addition, nampt level was found to be elevated in many autoimmune diseases, that is, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel diseases, and psoriasis [25]. visfatin also positively correlates with activity and severity of rheumatoid arthritis and psoriasis [2, 5]. we have recently found an overexpression of nampt in leukocytes of patients with graves ' ophthalmopathy with corresponding increased serum concentration (accepted manuscript). our findings suggest that visfatin might be involved in autoimmune processes in thyroid diseases. in our opinion, the controversial findings of visfatin in thyroid hormone deficiency may arise from the heterogeneity of thyroid dysfunction. we hypothesized that regulation of visfatin in hypothyroidism might be altered by coexisting chronic autoimmune thyroiditis, since high visfatin levels were observed in other autoimmune diseases. to answer the question about the influence of coexisting chronic autoimmune inflammation on visfatin level, we analyzed its serum concentration among hypothyroid patients with chronic autoimmune thyroiditis and in patients after thyroidectomy, who were negative for thyroid antibodies. this is a prospective case-control study of 118 subjects. the autoimmune study group (ait) consisted of 39 patients newly diagnosed with hypothyroidism in a course of chronic autoimmune thyroiditis. the nonautoimmune study group (tt) consisted of 40 patients thyroidectomized due to the differentiated thyroid cancer staged pt1. tt patients were evaluated five years after radioiodine remnant ablation and were negative for thyroglobulin and radioiodine uptake in a whole body scintigraphy (wbs). they achieved endogenous tsh stimulation and became hypothyroid after l-t4 withdrawal for at least 4 weeks. the control group comprised 39 healthy volunteers adjusted for age, sex, and bmi with normal thyroid function and negative thyroid antibodies. exclusion criteria consisted of other autoimmune diseases, active neoplastic disease, diabetes mellitus, and infection, which were reported to alter visfatin level. fasting blood samples were taken for visfatin, tsh, free thyroxine (ft4), free triiodothyronine (ft3), antithyroperoxidase antibodies (tpoab), antithyroglobulin antibodies (tgab), glucose, and insulin levels. in tt group tsh, ft4, and ft3 were measured using the electrochemiluminescence technique in cobas e 601 (norm ranges: tsh 0.274.2 tpoab and tgab were measured by radioimmunoassay (norm range:<34 iu/ml and<115 iu/ml, resp.). glucose level was assessed with the use of hitachi cobas e601 chemiluminescent analyzer (roche diagnostics) and insulin concentration was assessed using elisa kit from phoenix pharmaceuticals. the estimate of insulin resistance by homeostasis model assessment (homa-ir) was calculated. the study was approved by the local ethics committee, and informed consent was signed by every subject. statistical analysis was performed with medcalc version 12.1.3.0 (medcalc software, mariakerke, belgium). variables with normal distribution were compared between three groups with one-way analysis of variance. if data did not follow normal distribution, comparison of the analyzed parameters between three groups was performed with the kruskal-wallis test. simple regression analysis was used to test for the relationships between them. before inclusion to this statistical analysis, furthermore, stepwise multiple regression analysis was employed to investigate the influence of various parameters on visfatin serum concentration [age, bmi, ft3, autoimmunity (yes/no), homa-ir]. variables were entered into the model if their associated p-values were less than 0.05 and then sequentially removed if their associated p-values became greater than 0.2. tests were considered to be statistically significant if p-value was lower than 0.05. clinical and laboratory data of the study groups and the control group are shown in table 1. the highest visfatin serum concentration was in ait group, and healthy controls had visfatin level higher than tt (p=0.0001) (figure 1). three groups did not differ in age, sex, bmi, fasting glucose, and insulin levels, homa-ir. they had statistically different tsh, ft4, ft3, and tgab levels (table 1). simple linear regression analysis revealed that visfatin serum concentration was significantly associated with autoimmunity (= 0.1014; p=0.003), ft4 (= 0.05412; p=0.048), ft3 (= 0.05242; p=0.038), and tpoab (= 0.0002; p=0.0025) (figure 2). there was no association between visfatin and age, sex, bmi, tsh, tgab, fasting insulin and glucose levels, and homa-ir (table 2). in the stepwise multiple regression analysis we confirmed the association between serum visfatin level and autoimmunity (coefficient=3.8461; p=0.0001), and ft3 (coefficient=0.4198; p=0.0441), whereas age, bmi, and homa-ir did not contribute significantly. in separate stepwise multiple regression analysis we confirmed the association of serum visfatin concentration with autoimmunity (coefficient=4.1105; p=0.0001) and ft4 (coefficient=0.1397; p=0.038), whereas age, bmi, and homa-ir did not enter the model. similarly, association of visfatin with tpoab (coefficient=0.0057; p=0.0163) was observed with adjustment for age, bmi, ft3, and homa-ir in multivariate regression analysis. to date, visfatin serum concentration in hypothyroidism has been analyzed in a few studies. reported elevated level of this adipocytokine in hypothyroidism with further increase after restoration of thyroid function. ozkaya et al. observed that visfatin level decreased after recovery. in those articles the etiology of hypothyroidism varied from chronic autoimmune thyroiditis, postpartum thyroiditis to thyroid function insufficiency after radioiodine treatment or after thyroidectomy. hence, to date autoimmune status of studied patients has not been taken into consideration. we hypothesized that these controversial findings might result from heterogeneity of study groups. to answer the question, whether coexisting autoimmune inflammation influences visfatin level in hypothyroid patients, we analyzed its serum concentration in chronic autoimmune thyroiditis and thyroidectomized patients negative for thyroid antibodies. since we have previously proved that visfatin mrna expression is increased in thyroid malignancies and is correlated with tumor stage, we recruited only those patients who did not have any features of active neoplastic disease. we also recruited the control group adjusted for age, sex, and bmi. to the best of our knowledge, this is the first study addressing the changes in the release of visfatin in thyroid autoimmunity. we came to interesting results indicating that visfatin serum concentration in hypothyroid patients is associated with both autoimmunity and free thyroid hormones level (ft4, ft3). visfatin has been recognized as a cytokine with a broad range of immune and inflammatory activities, including induction of inflammatory cytokines, and regulation of macrophage and lymphocyte proliferation. visfatin stimulates the production of proinflammatory cytokines (il-6, tnf-, and il-1) and potentially acts as a chemotactic factor for monocytes. furthermore, its expression is upregulated by il-6, tnf-, and il-1 [1012]. enhanced mrna expression of visfatin was observed in inflamed mucosa of patients with inflammatory bowel disease (ibd). further analysis identified that antigen presenting cells (i.e., macrophages, dendritic cells) might be a main source of this protein in ibd. visfatin has also potency for activation of t cells by upregulation of costimulatory molecules (cd40, cd54, and cd80) on monocytes. we observed the positive association between visfatin and tpoab, and the latter is considered the best serological marker of chronic autoimmune thyroiditis. furthermore, tpoab contribute to thyroid destruction through antibody- and complement-dependent cell-cytotoxicity [13, 14]. the first mechanism is associated with mononuclear cell infiltration of thyroid stroma. in addition, th1-derived cytokines (il-2, tnf-, and inf) were found to be elevated in patients with chronic autoimmune thyroiditis. also, il-1 and tnf- have been recently reported to discriminate chronic autoimmune hypothyroid children from healthy controls. altogether, association of visfatin with tpoab observed in our study further supports our hypothesis that visfatin might be involved in the pathogenesis of chronic autoimmune thyroiditis. our findings about the association of visfatin with ft3 levels are in accordance with the results of in vitro and in vivo studies. however, controversial results whether t3 stimulates or downregulates the production of visfatin were found. in contrast, caixs et al. did not find any relationship between visfatin and free thyroid hormones. in vitro experiment showed the nonlinear regulation of visfatin mrna expression in the 3t3-l1 cell culture model affected by t3. since our study groups significantly differed with free thyroid hormones levels, we were able to analyze visfatin concentration in a broad spectrum of ft3 and ft4. therefore, we might suggest that pattern of visfatin changes varies in different ft3 concentration. according to our observations, as well as other authors, visfatin did not reflect insulin resistance assessed by homa-ir [18, 19]. our study might then prove that visfatin in hypothyroidism depends on thyroid hormones level and coexisting autoimmunity. we may assume that these two factors should be taken into consideration to assess visfatin level in patients with thyroid dysfunction. in addition, the possible involvement of visfatin in pathogenesis of chronic autoimmune thyroiditis needs further research. the main limitation of our study is its cross-sectional design that does not enable us to reveal the causal pathways of relationship between visfatin and autoimmune thyroiditis. however, we used very strict criteria of exclusion to limit the possible influence of other known factors such as diabetes mellitus, other autoimmune processes, infection, and active neoplastic disease. our nonautoimmune group with hypothyroidism had been thyroidectomized at least 5 years earlier and did not have any clinical nor laboratory features of active thyroid cancer.

we hypothesized that regulation of visfatin in hypothyroidism might be altered by coexisting chronic autoimmune thyroiditis. this is a prospective case-control study of 118 subjects. the autoimmune study group (ait) consisted of 39 patients newly diagnosed with hypothyroidism in a course of chronic autoimmune thyroiditis. the nonautoimmune study group (tt) consisted of 40 patients thyroidectomized due to the differentiated thyroid cancer staged pt1. the control group comprised 39 healthy volunteers adjusted for age, sex, and bmi with normal thyroid function and negative thyroid antibodies. exclusion criteria consisted of other autoimmune diseases, active neoplastic disease, diabetes mellitus, and infection, which were reported to alter visfatin level. fasting blood samples were taken for visfatin, tsh, free thyroxine (ft4), free triiodothyronine (ft3), antithyroperoxidase antibodies (tpoab), antithyroglobulin antibodies (tgab), glucose, and insulin levels. the highest visfatin serum concentration was in ait group, and healthy controls had visfatin level higher than tt (p=0.0001). simple linear regression analysis revealed that visfatin serum concentration was significantly associated with autoimmunity (= 0.1014; p=0.003), ft4 (= 0.05412; p=0.048), ft3 (= 0.05242; p=0.038), and tpoab (= 0.0002; p=0.0025), and the relationships were further confirmed in the multivariate regression analysis.

PMC4739229

pubmed-440

bladder cancer (bc) is the ninth most common malignancy in the united states with 74,000 new cases and 16,000 deaths estimated for 2015. since radical cystectomy (rc) is the standard treatment for clinically localized muscle-invasive bladder cancer many patients undergo surgery with curative intention. unfortunately, though the majority of patients are rendered disease-free after surgery, a significant proportion go on to develop bc recurrence and to ultimately succumb to the disease. for patients undergoing cancer surgery, there has been recent interest in identifying perioperative factors that may modulate recurrence and cancer-specific survival after surgery. it has been suggested that perioperative blood transfusion (bt) may be one such factor [35]. blood transfusions represent the top five most frequently overused therapeutic procedures in the united states [6, 7]. unfortunately, a clinically significant number of patients (3075%) with bc receive blood products during and after rc [810]. although bts can be life-saving in some clinical perioperative circumstances, there are adverse events associated with their administration including transfusion-related immune suppression (trim). trim is one proposed mechanism by which bts may be linked to poor oncologic outcomes. several retrospective studies have demonstrated that perioperative bts are independently predictive of poor survival in patients with bladder cancer [1214]. a meta-analysis by wang and colleagues demonstrated the association between bts and decreased recurrence-free survival (rfs) and overall survival (os). however, three recent studies that included more than 6,500 patients in aggregate were recently published and are not part of that meta-analysis [1618]. we sought to assess the impact of bt on cancer-related outcomes and mortality in patients who had rc for muscle-invasive bladder cancer. we conducted a systematic review of the literature and meta-analysis to test for an association between perioperative bts and recurrence-free, cancer-specific, and overall survival in patients undergoing rc. we searched ovid medline and embase, pubmed, cochrane library, and the clinicaltrials.gov databases from inception to june 2015, with no limits of language or publication type. to identify additional studies, we also searched the 20102015 meeting abstracts of the american society of clinical oncology, the american urological association, and the european association of urology. database search strategies included controlled vocabulary (e.g., medical subject headings) and keyword terms to find studies addressing perioperative transfusions or related procedures (such as blood salvage or hemodilution) of whole blood or blood components in bladder cancer patients. outcomes sought by the search strategies included blood loss (intraoperatively or postoperatively), cancer-specific outcomes (e.g., recurrence, metastasis, and disease progression), and survival. all searches were performed by a medical librarian (greg pratt) who has contributed to more than 50 systematic reviews and meta-analyses. the primary outcomes of interest were recurrence-free survival, cancer-specific survival, and overall survival. we defined a perioperative bt as any amount of prbc within one month before and one month after rc. we included randomized controlled trials (rcts), prospective cohorts, and retrospective studies that evaluated the impact on any (allogeneic versus autologous versus intraoperative recovered cell saved) packed red blood cells (prbcs) in patients with bc who underwent rc. we excluded studies considering patients with distant metastases at surgery; those in which recurrence-free survival, cancer-specific survival, or overall survival were not indicated; and abstracts or poster presentations. studies with a score of 6 or lower in the ottawa-newcastle scale were also excluded from any statistical analysis. for studies with overlapping patient populations, we calculated the pooled hazard ratio (hr) estimates and 95% confidence intervals by random effects model using the method of dersimonian and laird (d+l). to derive pooled estimates, the d+l method calculates weights by taking the inverse of a combination of within-study and between-study variability, which provides a larger variance compared with the variance produced from fixed effects analyses and thus wider confidence intervals. cochran's q-test was used to test the null hypothesis of no significant heterogeneity across studies. cochran's q-statistic follows distribution with (k 1) degrees of freedom, where k is the number of studies. i or the percentage of variation in the measures of association across studies due to heterogeneity was also calculated. i is the equivalent to the quantity of cochran's q minus its degrees of freedom divided by cochran's q, or i=(q df)/q. the value of i ranges between 0% and 100%, where 0% indicates no observed heterogeneity and larger values indicate increasing heterogeneity. the summary effect measure on hazard ratio for intraoperative transfusion on the time-to-event endpoints (overall survival, cancer-specific survival, and recurrence-free survival) was obtained. lastly, a sensitivity analysis was conducted to test whether the results of the meta-analysis were sensitive to restrictions on any of the included studies. all statistical analyses were performed using r software (version 3.0.2, the r foundation for statistical computing). the initial search identified 14 potential studies that underwent full review (figure 1). of these, 6 studies were excluded and 8 studies were included in the analysis. abel's study included data from 2 different institutions; thus the 2 substudies were considered separately for statistical analysis. only 5 studies clearly stated that patients were transfused with allogeneic blood; the remaining studies did not specify the type of blood. the leukoreduced status of the blood units was not clarified in any of the included studies. two studies differentiated between intra- and postoperative blood transfusion and found that patients transfused intraoperatively but not postoperatively had worse survival [10, 17]. eight studies including a total of 15,655 patients reported overall mortality as an outcome (table 1(a)). of those patients, the 2 studies that did not identify bt as an independent risk factor of os did observe an important trend to worse os [9, 10]. as shown in figure 2, perioperative bts were associated with a 27% (or [95% ci]: 1.27 [1.151.40], p<0.05) increased risk in mortality (figure 2(a)). the i test demonstrated moderate to substantial heterogeneity (68.3%, p=0.0014) across the studies. seven studies including a total of 14,878 patients estimated cancer-specific survival in the statistical analysis (table 1(b)). the rate of transfusion in this pool of patients was 38% (n=5,618). five of the 7 studies (n=6,521) demonstrated a negative impact of bt. as shown in figure 3, the risk of dying from cancer after perioperative bt was 29% (or [95% ci]: 1.29 [1.131.46], p<0.05) (figure 2(b)). the i test demonstrated moderate heterogeneity (60%, p=0.012) across the studies. five studies including a total of 8,778 patients estimated recurrence-free survival (table 1(c)). three of the 5 studies (n=4,910) showed a significant association between perioperative blood transfusions and poor survival [10, 17, 20]. in abel's study patients the association was present for the mayo clinic's population of patients but not for university of wisconsin's patients. as shown in figure 2, perioperative bts were associated with a significant increased risk in reduced rfs (or [95% ci]: 1.12 [1.121.31], p<0.05) (figure 2(c)). the i test demonstrated low heterogeneity (0%, p=0.549) across the studies. the sensitivity analysis demonstrated that none of the studies included in the meta-meta-analysis was very influential, as the hr ranged from 1.20 to 1.30, 1.24 to 1.34, and 1.18 to 1.26 for os, css, and rfs, respectively, for the pooled meta-analysis and all omitted meta-analyses (table 2). both the true nature of an association between bt and cancer recurrence and the biologic mechanism to explain this association are still very much unanswered research questions. the most commonly cited and investigated mechanism is the one that involves immune suppression or trim. however, it has been speculated that the infusion of growth factors (vascular endothelial growth factor and transforming growth factor-b) and an enhanced inflammatory response as a result of the exposure of the recipient immune system to donor microparticles could also stimulate spread and proliferation of cancer cells [24, 25]. the present meta-analysis was not designed to investigate these possibilities; however, our results support the hypothesis that the perioperative administration of prbcs is an independent risk factor for reduced rfs, css, and os after rc for bladder cancer similar to what has been reported for other cancers such as colon, lung, and esophagus [12, 2628]. although a recent meta-analysis conducted by wang and colleagues showed similar results to ours, we consider the findings of the present study clinically relevant because first we included data from two recently published cohort studies with relatively large sample size. therefore, a larger number of transfused and not transfused patients were part of the pooled analysis of the present meta-analysis. and second we conducted a different analysis (random effects and fixed effect models) in comparison to that published by wang and colleagues who used a fixed model paradigm. we believe that a random effects model strengthens the analysis and adds significant information to the current evidence because this model assumes that the pooled studies are not functionally equivalent as they were conducted by researchers operating independently. sources of variation among the studies used in the meta-analysis are, for instance, time of transfusion (intra- versus post- versus intra- and postoperative) and trigger of transfusions. therefore, our analysis can be generalized to different clinical scenarios of bladder cancer surgery [29, 30]. it is worth mentioning that two studies did try to evaluate the impact of time of transfusion on outcomes and found that intraoperative bts are an independent risk factor for poor survival while postoperative bts do not show an association with worse outcomes [10, 17]. our meta-analysis shows significant heterogeneity or high degree of dissimilarity among studies for css and os but not for rfs. although the high level of heterogeneity between studies for css and os tempers the strength of any conclusions that can be made about the effect of bt on these two survival outcomes, the low heterogeneity and identical estimated hrs for rfs using both random effects and fixed effect models suggest a strong association between perioperative bt and bc recurrence after rc. in this meta-analysis, patients who received a perioperative bt had a 21% higher risk of bc recurrence than patients who did not receive bt. the present study has the limitations inherent to any study level meta-analysis of cohort studies. although we used the ottawa-newcastle score to grade study quality, all of the included studies were retrospective; the possibility exists that confounding variables (i.e., staging and tumor volume) may have influenced the individual study results and by extension the findings of this meta-analysis. furthermore, the results of the present study can not be extrapolated to the use of autologous blood transfusion since it is assumed that all studies included in the meta-analysis reported outcomes in patients transfused with mainly allogeneic blood. in conclusion, perioperative bt may be associated with reduced rfs, css, and os in patients undergoing rc for bc. a well-designed prospective rct is needed in this population to provide the high level evidence necessary for answering this question.

background. perioperative blood transfusions are associated with poor survival in patients with solid tumors including bladder cancer. objective. to investigate the impact of perioperative blood transfusions on oncological outcomes after radical cystectomy. design. systematic review and meta-analysis. setting and participants. adult patients who underwent radical cystectomy for bladder cancer. intervention. packed red blood cells transfusion during or after radical cystectomy for bladder cancer. outcome measurements and statistical analysis. recurrence-free survival (rfs), cancer-specific survival (css), and overall survival (os). we calculated the pooled hazard ratio (hr) estimates and 95% confidence intervals by random and fixed effects models. results and limitation. eight, seven, and five studies were included in the os, css, and rfs analysis, respectively. blood transfusions were associated with 27%, 29%, and 12% reduction in os, css, and rfs, respectively. a sensitivity analysis supported the association. this study has several limitations; however the main problem is that it included only retrospective studies. conclusions. perioperative bt may be associated with reduced rfs, css, and os in patients undergoing rc for bc. a randomized controlled study is needed to determine the causality between the administration of blood transfusions and bladder cancer recurrence.

PMC4752988

pubmed-441

chronic rhinosinusitis (crs) is characterized by severe inflammation of the sinus mucosa leading to blockage of the nasal passageway and the accumulation of mucus and pathogens in the nose and paranasal sinuses [1, 2]. crs affects around 1.9 million australians and puts a large financial burden on health care systems. crs is subdivided in crs with nasal polyps (crswnp) and crs without nasal polyps (crssnp) based on the presence or absence of polyps in the sinonasal cavities. crswnp patients typically display a t helper 2 (th2) polarization, whereas patients without nasal polyps (crssnp) are often characterized by a th1 polarization with high levels of interferon-. cytokines regulate innate and acquired immunity and can disrupt mucosal barrier function by altering tight junction (tj) composition and structure. this occurs through signalling pathways independent of cell death and the effect is cell type specific, pleiotropic, and time and dose-dependent. relatively few studies have demonstrated cytokine effects on nasal epithelial tissue or barrier function [5, 6, 9, 10]. th1 cytokines such as interleukin-2 (il-2), interferon- (ifn-), and tumour necrosis factor alpha (tnf-) are the primary source for proinflammatory th1 responses, in which they are effective in controlling infection with intracellular pathogens and for perpetuating autoimmune responses. in contrast, th2 immune responses are characterized by the production of the interleukins il-4, il-5, and il-13 that are associated with the promotion of eosinophil recruitment and activation, and inhibition of several macrophage functions, thus providing phagocyte-independent protective responses. th17 cells are a subset of activated cd4 t cells and are characterized by the production of the interleukins il-17a, il-17f, il-22, and il-26. th17 cells act as a bridge between adaptive and innate immunity where they play crucial roles in the development of autoimmunity, inflammation, and allergic reactions. here, we tested the effect of interferon proteins and of th1, th2, and th17 cytokines on the mucosal barrier structure and function of primary nasal epithelial cells harvested from nasal polyps of crs patients. ethical approval for nasal brushing from crs patients was granted from the queen elizabeth hospital human ethics committee and only consented patients were included in the study. exclusion criteria included active smoking, age less than 18 years, and systemic disease. primary human nasal epithelial cells (hnecs) were harvested from nasal polyps by gentle brushing in a method as described in. extracted cells were suspended in bronchial epithelial growth media (begm, cc-3170, lonza, walkersvill, md, usa), supplemented with 2% ultroser g (pall corporation, port washington, ny, usa). the cell suspension was depleted of monocytes using anti-cd68 (dako, glostrup, denmark) coated culture dishes, and hnecs expanded in routine cell culture conditions of 37c humidified air with 5% co2 in collagen coated flasks (thermo scientific, walthman, ma, usa). hnecs were tested at passage two and confirmed to be of epithelial lineage via reactivity to pan-cytokeratin and cd45 antibodies (both from abcam, cambridge, ma, usa), and a diff-quick staining method used in the assessment of cell morphology by professional cytologists (imvs, the queen elizabeth hospital, woodville, australia). hnec were maintained at an air liquid interface (ali) medium, following the lonza ali culture method (lonza, walkersville, usa). briefly, transwells (bd biosciences, san jose, california, usa) were treated with collagen (stemcell technologies, australia). 70,000 cells were seeded in a volume of 100 l b-ali medium into the apical chamber of the transwell plate and 500 l of b-ali growth medium was added to the basal chamber in all wells containing the inserts. cells were incubated at 37c. on day 3 after seeding, b-ali growth medium was removed from the apical and basal chambers and 500 l b-ali differentiation medium was added to the basal chamber only, exposing the apical cell surface to the atmosphere. cytokines were added to the basal transwell chamber at the following final concentrations: recombinant human interferon- (500 ng/ml, sigma, saint louis, usa), interferon 1a (50 ng/ml, sigma, saint louis, usa), interferon- (500 ng/ml, sigma, saint louis, usa), tumour necrosis factor- (500 ng/ml, sigma, saint louis, usa), il-1b (500 ng/ml, sigma, saint louis, usa), il-4 (50 ng/ml, gibco, life technology, usa), il-5 (50 ng/ml, gibco, life technology, usa), il-13 (50 ng/ml, gibco, life technology, usa), il-17a (50 ng/ml, gibco, life technology, usa), recombinant human il-22 (50 ng/ml, sigma, saint louis, usa), and recombinant human il-26 (50 ng/ml, abnova taiwan corp). paraffin-embedded tissue samples were cut in 4 m thick sections on a microtome (thermo scientific hm 325 rotary microtome). slides were then stained with routine hematoxylin and eosin (h&e) staining using mayer's hematoxylin and eosin (lillie's modification, dako, thermo fisher scientific, waltham, ma, usa). all slides were then scanned using digital whole-slide imaging technology (wsi) on the nanozoomer digital pathology system (hamamatsu photonics, hamamatsu city, japan) under high resolution (40x objective magnification power). transepithelial electrical resistance (teer) was measured by using an evom volt-ohmmeter (world precision instruments, sarasota, fl, usa). briefly, 100 l of b-ali medium was added to the apical chamber of ali cultures to form an electrical circuit across the cell monolayer and into the basal chamber. cultures were maintained at 37c during the measurement period using a heating platform. only wells displaying baseline resistance readings greater than 500 /cm were used for the experiments. cytokines and control (b-ali medium for the negative control and 2% triton 100 for the positive control) were added to the bottom chamber of each well, and teer measurements were obtained at time 0, 4 h, and 24 h. paracellular permeability was studied by measuring the apical-to-basolateral flux of fitc dextran 4 kda (sigma, saint louis, usa). briefly, after treating the cells for 24 h, the upper chambers were filled with 3 mg/ml of fitc-dextran and incubated for 2 h at 37c. 40 l samples were recovered from the bottom chamber and serially diluted on a 96-well plate (corning costar cell culture plates (96 wells)), and the fluorescence was measured with a microplate fluorometer (fluostar optima, bmg labtech, ortenberg, germany). the amount of lactate dehydrogenase (ldh) in the medium was measured at 24 hours using the cytotox homogeneous membrane integrity assay (promega, australia). briefly, 50 ml of the media from each well was transferred to a new plate, and 50 ml of ldh reagent was added to the supernatant and incubated for 30 minutes in the dark at room temperature. the od was measured at 490 nm on a fluostar optima plate reader (bmg labtech, ortenberg, germany). after cell incubation with the cytokines, the culture medium was discarded and washed with pbs, and 100 l of trypsin was added to each well. the plate was incubated for 5 minutes, and then 250 l of supplemented culture medium was added. the contents of each well were aspirated, placed into labeled microtubes, and centrifuged at 1000 rpm for 5 minutes. the supernatant was discarded, and the cells were suspended again in 100 l of culture medium. an aliquot of 10 l of cell suspension was removed and mixed with 10 l of trypan blue. after homogenization, the live and dead cells were counted and the percentage of viable cells was calculated. cells were fixed with 2.5% formalin in phosphate-buffered saline (pbs) for 10 min, and then the cells were rinsed with tris-buffered saline-0.5% tween (tbst) four times, permeabilized with 1% sds in pbs, and blocked with serum free blocker (sfb; dako, glostrup, denmark) for 60 minutes, at room temperature. mouse monoclonal anti-human zo-1 (invitrogen, carlsbad, ca, usa), diluted to 5 g/ml in tbst-10% sfb, was added to the excised culture support membranes and allowed to incubate for 1 hour at room temperature. excess primary antibody was removed with tbst, and 2 g/ml anti-mouse alexa-594 conjugated secondary antibody (jackson immunoresearch labs inc., west grove, pa, usa) the filters were rinsed in tbst, and after the third wash 200 ng/ml of 4,6-diamidino-2-phenylindole (dapi; sigma, aldrich) was added to resolve nuclei. membranes were rinsed once with ultrapure water, and 95% ice cold ethanol was added for 1 hour at 4c. membranes were transferred to a glass slide and a drop of anti-fade mounting medium (dako, glostrup, denmark) was added before cover-slipping. samples were visualized by using a lsm700 confocal laser scanning microscope (zeiss microscopy, germany). the teer experiment was performed using three replicates from four crswnp patients with values normalised against the mean value from the patient at time 0. statistical analyses of all data were carried out using anova, followed by tukey hsd post hoc test. ali cultures were established from 4 independent crs patient donors (2 males and 2 females, aged 4560 years). two patients were diagnosed with grass-pollen allergy, one had aspirin-exacerbated respiratory disease (aerd), and two were asthmatic. eosinophil and neutrophil counts [11.1 (4.621.3) and 0.8 (02.4)] per high power field (hpf) were not different between the different patients (p>0.05). the effect of interferons and of th1, th2, and th17 cytokines was examined by measuring the teer across hnec monolayers from crs patients at different time points. all th17 cytokines tested (il-17, il-22 and il-26) caused a significant reduction in teer (average of 1.9 times; 1.7 times; 1.61 times for il-17, il-22, and il-26 resp.) after 24 h of incubation. th1 and th2 cytokines or interferon,, or did not show any significant effect on teer (figure 1). all il-17 family cytokines (il-17, il-22, and il-26) led to a significant enhancement of paracellular permeability (p<0.05) (figure 2). il-17 had the strongest effect, with 89.33% of the fluorescent dextran crossing the hnec monolayer whereas il-22 and il-26 increased paracellular permeability with 49.85% and 53.92%, respectively. th1 and th2 cytokines and interferons,, and did not show any significant effect on the paracellular permeability in crs patients (figure 2). the effect of interferons and of th1, th2, and th17 cytokines on cellular toxicity was examined by measuring ldh release from hnecs. there was no statistically significant increase in ldh release after 24 h incubation with any of the cytokines (figure 3). in addition, the cell density estimated by the trypan blue assay did not show any significant differences in cell density or cell viability in cytokine treated cells compared to control cells (results not shown). the effect of interferon proteins and of th1, th2, and th17 cytokines on the localization of zona occludens-1 (zo-1) was examined by using immunofluorescence staining and confocal laser scanning microscopy, 24 hours after application of the cytokines. in untreated cells, zo-1 was located at the periphery of the apical side of the monolayer, as expected. similarly, interferons,, and and th1 and th2 cytokines, which had no effect on either teer or paracellular permeability, led to no alterations in the localization of zo-1. in contrast, application of th17 cytokines, which significantly altered epithelial barrier function, resulted in profound disruption of zo-1 immunolocalisation evidenced by faint or discontinuous regions of fluorescence (figure 4). cytokine mediated insult on mucosal membranes, causing disruption of tight junctions and increased paracellular permeability, contributes to a multitude of pathologic conditions in inflammatory diseases of the upper airways [1820]. in this study, we compared the effect of interferons and of signature th1, th2, and th17 cytokines on the barrier function of primary nasal epithelial cells harvested from crs patients with nasal polyps. immunolocalisation of the tight junction protein zo-1 was used to analyse tight junction integrity to gain insights into mechanisms of cytokines dependent disruption of the airway epithelial barrier. our study indicates that, in crswnp patients, il-17 family cytokines (il-17a, il-22, and il-26) can significantly disrupt epithelial barrier function in association with a disruption of tight junction integrity and without causing cellular toxicity. in contrast, th1 and th2 cytokines or interferons showed no significant difference on either teer or paracellular permeability of hnecs. it has been well established that different cytokines cause different, often opposing effects on epithelial barrier function depending on the cell type used and that any observed effect is dose and time-dependent (reviewed in). our results indicate that application of th1 cytokines such as ifn- and tnf- does not have detrimental effects on epithelial barrier function. rather, application of these cytokines appeared to slightly enhance the teer of human nasal epithelial monolayers derived from some of the crswnp patients 24 hours after application. whereas ifn- and tnf- generally decrease barrier function in different cell lines [21, 22], in airway epithelial cells, ifn- has been reported to decrease barrier function by soyka et al. and promote epithelial barrier function by ahdieh et al.. these differences in the response to ifn- could be attributed to many factors including interindividual variability in response to cytokines, different origin of cells (mucosa or polyps), and differences in experimental techniques. in the experiments by soyka et al., for example, teer changes in response to ifn- treatment from crswnp, crssnp and controls were pooled while in our studies, only cells from crswnp were used. given the small number of samples used in most studies, further experiments using a larger number of donors will need to address the cause of these discrepancies. tnf- can increase teer in mammalian uterine cell monolayers in a dose-dependent manner. interestingly, a recent study revealed that patients had developed a recurrence of crs after the start of tnf- inhibitor administration with a remission of the disease only after cessation of tnf- inhibitor treatment. moreover, we demonstrated no significant reduction of teer by th2 family cytokines (il-4, il-5, and il-13) after 4 h and 24 h in our experiments. using airway epithelial cells, saatian et al. showed that il-4 and il-13 caused a reduction in teer 72 h after challenge but not after 24 h. also used airway epithelial cells from crswnp patients (n=2) and controls (n=2) and similarly showed significantly decreased teer after il-4 challenge; however, this effect was already evident after 12 and 24 hours. the reason for these discrepancies is not clear and can be dependent on numerous factors. while physiologically relevant, it is well known that experiments using primary cells have limitations due to inherent interindividual differences of age, genetic make-up and medical history this is particularly important in crs, a multifactorial disease that can be associated with th1 or th2 responses. also ethnicity may play a role as caucasian crswnp patients are often characterised by a predominant th2 type eosinophilic inflammation with high level of il-5, whereas asian crswnp patients preferentially have a th1/th17 polarization signature. th17 cytokines also affect the gut mucosal barrier function by promoting the amplification of the host response to secrete neutrophil chemoattractants and antimicrobial peptides such as lipocalin-2 and calprotectin. in addition, th17 cells can expand within mucous layers in association with the presence of pathogens that are resistant to some of the induced antimicrobial responses. the expression level of il-17 was also shown to be significantly higher in recalcitrant crswnp compared to controls. in the present study, we found that il-17 induced barrier dysfunction as assessed by reduction in teer and enhanced macromolecular permeability, whereas soyka et al. we also demonstrated significant reductions in teer and enhanced macromolecular permeability of il-22 and il-26 which is the first such analysis using human nasal epithelial cells. in tight junction formation, zo-1 plays an essential role, by linking the transmembrane proteins occludin, claudin, and junction adhesion molecule (jam) cytoplasmic components of the tight junctions to the actin cytoskeleton. disruption of the actin-myosin structure has been understood to modulate paracellular permeability. we observed a loss of normal zo-1 immunolocalisation in hnec monolayers of crswnp patients secondary to challenge with th17 (il-17, il-22, and il-26) cytokines, in association with disruption of barrier function. we also observed that il-17, il-22, and il-26 treated cells appeared in higher cell densities than cells treated with other th1 or th2 family cytokines. it is known that tjs regulate epithelial proliferation by different molecular mechanisms, which generally suppress proliferation as cell density (and hence tj assembly) increases (reviewed in). changes in expression of zo-1 and zo-1-associated nucleic acid binding protein (zonab), a y-box transcription factor, affect cell proliferation; however, these effects take place at least 48 hours after changes in gene expression. given that the duration of exposure to the treatments in our experiments was only 24 hours and that cell counts did not show significant differences, we believe that tj disruption secondary to il-17, il-22, and il-26 exposure might render the pseudostratified layer of cells into a monolayer which might appear relatively overcrowded. in summary, in patients with crswnp, th1 and th2 in contrast, the th17 cytokines family (il-17, il-22, and il-26) showed significant disruption of the epithelial barrier, leading to increased paracellular permeability associated with reduced tight junctionintegrity. in future studies, it will be important to determine the cellular mechanism of the effect of th17 cytokines on the mucosal barrier in crs patients to provide an opportunity for therapeutic modulation in inflammatory stress.

cytokine mediated changes in paracellular permeability contribute to a multitude of pathological conditions including chronic rhinosinusitis (crs). the purpose of this study was to investigate the effect of interferons and of th1, th2, and th17 cytokines on respiratory epithelium barrier function. cytokines and interferons were applied to the basolateral side of air-liquid interface (ali) cultures of primary human nasal epithelial cells (hnecs) from crs with nasal polyp patients. transepithelial electrical resistance (teer) and permeability of fitc-conjugated dextrans were measured over time. additionally, the expression of the tight junction protein zona occludens-1 (zo-1) was examined via immunofluorescence. data was analysed using anova, followed by tukey hsd post hoc test. our results showed that application of interferons and of th1 or th2 cytokines did not affect the mucosal barrier function. in contrast, the th17 cytokines il-17, il-22, and il-26 showed a significant disruption of the epithelial barrier, evidenced by a loss of teer, increased paracellular permeability of fitc-dextrans, and discontinuous zo-1 immunolocalisation. these results indicate that th17 cytokines may contribute to the development of crswnp by promoting a leaky mucosal barrier.

PMC4745600

pubmed-442

positron emission tomography-computed tomography is useful for diagnosing primary and metastatic lesions of esophageal cancer. the sensitivity and specificity of a pet/ct are also relatively preferable to other diagnostic imaging tools. although the number of cases is few, false positives exist in which an inflammatory tumor mass and a nonspecific tumor mass are difficult to differentiate between, even when combined with other image findings. in cases of esophageal cancer, the types of treatment and their prognosis are greatly affected if there is a metastasis or recurrence. positron emission tomography-computed tomographic false positives can not simply be ignored even if they appear to be due to inflammation or some other factors. this case report discusses the reasons to conduct a pet/ct test and its limitations in diagnosing esophageal cancer. the subjects of this investigation had suspected metastasis from a pet/ct which occurred before surgery of the 129 resected cases of esophageal cancer at our department from june 2010 to march 2015. however, after surgery, biopsy and/or follow-up observation, only the cases that were confirmed as metastasis negative were considered to be pet/ct false positives for this assessment. there were 3 cases of preoperative pet/ct false positives (table 1). two cases in which bone metastasis was suspected revealed almost normal range of squamous cell carcinoma (scc)associated antigen, alkaline phosphatase, and serum calcium levels. abbreviations: ln, lymph node; m, metastasis; mt, middle thoracic lesion of esophagus; n, node; t, tumor; ut, upper thoracic lesion of esophagus. in case 1, a 59-year-old man, without particular medical history, had a diagnosis of esophageal carcinoma by endoscopy. multiple bone metastases were suspected by a pet/ct in the spine, sternum, ribs, and pelvic bone (figure 1). after conducting a ct-guided biopsy of the right iliac bone, the patient is alive with no recurrence 4 years and 10 months after surgery, although the false-positive lesions in pelvic bone are still detected by pet/ct. in the case 1, multiple bone metastases were suspected by a positron emission tomography-computed tomography in the pelvic bone (suvmax=2.94). in case 2, a 71 year-old man, without particular medical history, abnormal accumulations of fluorodeoxyglucose (fdg) in the manubrium sterni, vertebra th5, and the pedicle of vertebral arch were found by pet/ct. therefore, after the sufficient informed consent, the patient underwent a laryngopharyngeal esophagectomy, a transhiatal esophagectomy without thoracotomy, and reconstruction with the gastric tube through the posterior mediastinal route. pathological finding showed poorly differentiated scc of esophagus and moderately differentiated scc of hypopharynx with a cervical lymph node metastasis. the patient is alive with no recurrence 3 years and 11 months after the surgery. in case 3, a 76-year-old man, without particular medical history, had a diagnosis of a superficial esophageal cancer by endoscopy. he was suspected of lymph node metastasis of right recurrent nerve lymph node (figure 2) by preoperative pet/ct.. scattered silicotic nodules and inflammatory lymphadenopathy might be caused by pneumoconiosis (figure 3). case 3 was suspected of right recurrent nerve lymph node metastasis by preoperative positron emission tomography-computed tomography (suvmax=3.00). pathological finding of case 3 revealed scattered silicotic nodules in resected lymph nodes with infiltration of histiocytosis to sinusoid. in esophageal cancer treatment, fdg-pet/ct is also used to diagnose metastases and to judge the effectiveness of treatment. especially, pretherapeutic there are cases when the surgery may not apply to a patient if remote metastasis or high-grade lymph node metastasis is found. many cases have already been reported claiming that fdg-pet is a useful modality for a pretherapeutic staging. esophageal cancer causes 30% to 40% of lymph node metastases among cancers that spread to the submucosa, and the prognosis of esophageal cancer is known to be defined by the presence of lymph node metastasis, how many metastases are present, and how much the metastasis spreads. therefore, evaluating lymph node metastasis is a very important factor in deciding the course of treatment. the diagnosability of lymph node metastasis using a fdg-pet for esophageal cancer is generally reported to have a sensitivity of 32% to 51.9%, a specificity of 94.2% to 100%, and an accuracy of 48% to 93%. in addition, fdg-pet/ct, which is a combination of a fdg-pet and a ct, improves the diagnosability even more. yuan et al have reported that they evaluated the diagnosability of lymph node metastasis using fdg-pet/ct for esophageal squamous cancer and found a sensitivity of 94%, a specificity of 92%, and an accuracy of 92%. however, the size of a lymph node that can be detected by pet/ct is said to be only 6 to 8 mm, thus it is difficult to detect micrometastasis. also, detecting metastasis to neighboring lymph nodes could be difficult in cases with a lot of fdg accumulation in the primary lesion, or accumulation in the intestinal tract such as in the stomach, or in the heart. the fdg accumulates in inflamed lymph nodes as well, so false positives often appear in hilar lymph nodes or in patients who have chronic lung disease. lymph node metastasis was suspected after a preoperative pet/ct for the case 3, as described above, but it was actually an inflammatory lymphadenopathy caused by pneumoconiosis. dual-time-point fdg-pet has been reported to be useful in reducing this kind of false positive. it uses the time differences of peak fdg accumulation for inflammation versus for tumors. after injecting the fdg, pet has revealed occult distant metastases at nodal and non-nodal sites in 5% to 40% of patients. distant metastases of esophageal squamous cancer are mainly found in the lungs, liver, and bones. kato et al reported the diagnosability of fdg-pet for esophageal cancer as having a sensitivity of 92%, a specificity of 94%, and an accuracy of 93%. when comparing those numbers to bone scintigraphy, whose diagnosabilities are 77%, 84%, and 82%, respectively, the diagnosability of fdg-pet is superior, especially for osteolytic lesions. but 2 false positives out of 44 cases came from pet, showing that fdg-pet is not perfect. the suvmax of metastic tumor might be helpful to increase the accuracy of pet/ct. our previous study demonstrated that the suvmax of the primary tumor was positively correlated with tumor size and vessel invasion and was positively related to the suvmax of metastic tumor. in the report, the diagnostic accuracy of pet/ct (87.3%) was higher than that of conventional ct scans. when diagnosing metastatic bone tumors, the positive predictive value of bone metastasis is known to be quite high if the findings for both pet and ct match. therefore, even when bone metastasis is suspected due to abnormal fdg accumulation after a fdg-pet, yet no bone destruction image is found in the same site by ct, then interventional radiology or a surgical biopsy should be conducted to obtain a pathological diagnosis. this case report was on 3 patients with suspected metastasis due to a false-positive pet/ct who were later confirmed to not have metastasis. the metastases were originally suspected after conducting a pet/ct before surgery to treat esophageal cancer or during the postoperative course. conducting a pet/ct is useful when diagnosing esophageal cancer metastasis, but there should be an awareness of the possibility of false positives. therapeutic decisions should be made based on appropriate and accurate diagnoses, and a pathological diagnosis should be actively introduced if necessary.

of 129 esophagectomies at our institute from june 2010 to march 2015, we experienced three preoperative positron emission tomography-computed tomographic (pet/ct) false positives. bone metastasis was originally suspected in 2 cases, but they were later found to be bone metastasis negative after a preoperative bone biopsy and clinical course observation. the other cases suspected of mediastinal lymph node metastasis were diagnosed as inflammatory lymphadenopathy by a pathological examination of the removed lymph nodes. conducting a pet/ct is useful when diagnosing esophageal cancer metastasis, but we need to be aware of the possibility of false positives. therapeutic decisions should be made based on appropriate and accurate diagnoses, with pathological diagnosis actively introduced if necessary.

PMC5398648

pubmed-443

prostate cancer (pca) is one of the major causes of cancer death in men worldwide. the molecular basis of the disease involves an irregular behavior of the functions mediated by the androgen receptor (ar). human ar belongs to the nuclear receptor (nr) superfamily of transcription factors, which regulate gene transcription upon ligand binding. the structure of nrs is extensively documented in the literature, and in general, nrs share the following common organization: a variable amino-terminal activation function domain (af-1), a highly conserved dna-binding domain (dbd), a hinge region that contains the nuclear localization signal, a conserved c-terminal ligand-binding domain (lbd) comprising a 12 helical structure that encloses a central ligand binding pocket (lbp), and a second activation function domain (af-2) that is located at the carboxy-terminal end of the lbd and mediates ligand-dependent transactivation. ar is activated by the endogenous hormone testosterone (tes) and its more potent metabolite dihydrotestosterone (dht), both of which bind in the lbp. the binding of these endogenous modulators induces a reorganization of helix 12 to the so-called agonist conformation, generating a structured hydrophobic surface (af-2) suitable for the recruitment of tissue-specific nr coactivators. such nr coactivators can be thought of as master switches, directing and amplifying the subsequent transcriptional activity of the target nr. in a recent work, an additional secondary function site called binding function 3 (bf-3) has been reported on the surface of the ar that could also play a relevant role in the allosteric modulation of the af-2. nr drug development has traditionally focused on advancing full or partial agonists/antagonists interacting within the lbp of the lbd. pca has been treated by intervention at the early stages through utility of classical antiandrogens, which act by displacing the natural hormones from the pocket and inducing a conformational change of the helix 12 so that coactivators can not be recruited. tissue specificity, detrimental side effects, and a loss of the pharmacological effect (acquired drug resistance) over time are major and ongoing concerns with such lbp targeting treatment regimes. it has been demonstrated that it is possible to inhibit the transcriptional activity of the nrs by directly blocking the critical receptor: coactivator interaction. this alternative approach to traditional nr modulation may furnish greater pharmacological insight and afford opportunities to modulate not only under tissue specific circumstances but without adversely affecting natural ligand binding and so preserving the beneficial/nondisease linked functions of the receptors. specifically, the steroid receptor coactivator (src) family has been postulated as a feasible target for pharmacological intervention. the viability of targeting ar coactivator interaction using small molecules has been recently demonstrated. moreover, it has been postulated that circumventing the lbp will overcome the problem of drug resistance in pca. here we describe the discovery and characterization of a novel class of selective non-lbp true antiandrogens, characterized by full ar antagonism in inhibiting the recruitment of coactivators and lacking intrinsic partial agonistic properties. mechanistically, these compounds are totally differentiated from the recent description of true lbp antiandrogens like mdv3100 and rd162, while their selectivity and druglike nature underpin the potential of a non-lbp intervention strategy in advanced prostate cancer resistant to classical therapy, first described for the true non-lbp targeting antiandrogens pyrvinium pamoate (pp) and harmol hydrochloride (hh). the biological data obtained both on target with time-resolved fluorescence resonance energy transfer (tr-fret)/fluorescence polarization (fp) assays and in cellular pca models demonstrate the non-lbp antagonist activity of the series and an alternative mechanism of inhibition, furnishing a new class of nonpeptidic, small molecule ar: coactivator selective disruptors as leads for the development of novel treatments for prostate cancer. a virtual (computational) screen of six vendor compound databases (see experimental section) was performed through a combination of 3d pharmacophore generation and docking. seven x-ray structures of coactivator peptide bound ar were used to define key ligand-derived pharmacophoric features of the most represented motifs occurring in known ar coactivators. initially, common key interaction motifs within the peptide of the form fxxlf, lxxll, or fxxlw were considered to generate a consensus af-2 pharmacophore. subsequently, a second site-derived pharmacophore model was advanced based on the specific characteristics of the androgen receptor af-2 region, which demonstrates known selectivity toward the fxxlf coactivator motif (figure 1b). the cocrystallization of the ar lbd bound with dht in the presence of the fxxlf peptide (pdb i d 1t7r) provided the structural basis of the af-2 interaction for docking studies. virtual screening and identification of diarylhydrazide scaffolds. (a) a series of coactivator peptides cocrystallized in the af-2 groove was employed; for illustrative purposes we present the fxxlf coactivator motif from pdb entry 1t7r. the af-2 groove is represented in dark gray. for clarity reasons, only lys720 and glu897 are shown and dht is not illustrated; (b) a 3d pharmacophore model was derived containing the common features between ar coactivators and the two aromatic features of the fxxlf motif. pharmacophores were used to screen vendor compound databases and to guide the docking of putative hits into the af-2 site. (c, d) two first round actives 1 (mdg173) and 2 (mdg15) docked poses in the af-2 site, with the surface rendered and only key amino acids shown. partial mapping of initial hits to the pharmacophore suggested additional virtual screening to identify more potent family members. images were generated with molecular operating environment (moe) and pymol. from the virtual screen, a first series of compounds with predicted target affinity was selected from commercially available databases (see experimental section) and evaluated for biological activity using tr-fret and fp techniques. this initial screen (figures 1c, d and 2) identified two small molecules, 1 and 2, both diarylhydrazides, as possible non-lbp ar antagonists. non-lbp modulatory activity was evidenced by demonstration of an ic50 in the range of 50100 m in ar tr-fret coactivator displacement assay and their inability to displace bound fluorescently labeled ligand from the lbp through an fp assay. these first round hit molecules map only partially to the screening pharmacophore (figure 1c, d). accordingly, an optimization round of screening was initiated to explore the utility of the scaffold for more effective disruption of ar: coactivator interaction. from these initial data, a simple molecular similarity search was performed (tanimoto coefficient>70%) to furnish a new screening series of 37 compounds bearing the desired diarylhydrazide scaffold. this second round screen identified four small molecules (figure 2), 3 (mdg 483), 4 (mdg 292), 5 (mdg 506), and 6 (mdg 508), with improved activity (ic50<50 m in an ar tr-fret assay). these ligands were taken forward for additional investigation and characterization. the series of diaryl-substituted hydrazides identified through the vs process (figure 2) inhibited the recruitment of the fluorescent labeled d11-fxxlf coactivator peptide in the presence of an agonist (dht) concentration equal to ec80 using time-resolved fret assays. d11-fxxlf is a peptide developed from random phage display technology that resembles the src family of coactivator proteins in its flanking sequence but that also has an ar n-terminal interaction domain. thus, it is a biological mimic of the n-terminal and the src coactivator interactions with the lbd. a 12-point dose response curve was determined for those compounds that inhibited coactivator binding in the micromolar range, acting as full ar antagonists, 36 (figure 3a and table 1). the background signal, representing diffusion-enhanced fret in the absence of ar, was subtracted from the fret value of each compound and from the maximal signal, representing fxxlf-bound ar in presence of dht. diarylhydrazides inhibit the ar recruitment of a fluorescent-labeled d11-fxxlf peptide but do not displace a potent fluorescent ligand from the ar-lbp. (a and b) compounds were tested in a tr-fret assay across a concentration range from 100 m to 45 nm in the presence of a concentration of dht=ec80 in ar-lbd wt (a) and ar-lbd t877a (b). error bars represent the standard error of the mean (sem) for n=6 values. data was fitted using log antagonist concentration vs response (variable slope) with graphpad prism 5 (see experimental section for details). (c) fluorescence polarization data is plotted as percent maximal activity represented by ar-lbd and fluorophore complex (0% inhibition). the minimum control value represents free fluorophore (free f) in solution (100% inhibition). the tr-fret assay can not differentiate between direct coactivator antagonists acting on the lbd surface and classical ar antagonists, which also functionally disrupt coactivator recruitment by displacing dht from the ligand binding pocket. to characterize the nature of the antagonist effect, compounds were tested for their ability to displace a potent fluorescent ligand (fluorophore) from the ar lbp through a fluorescence polarization (fp) assay at a single point concentration (50 m), using cyproterone acetate (cpa) at the same concentration as a reference, a known ar lbp-mediated antagonist. all compounds tested showed 0% inhibition of the ar-lbd and fluorophore complex, indicating a non-lbp-mediated mechanism of ar transactivation inhibition (figure 3c). compound 3 gave an unusually high value of millipolarization units (mp), 20% higher than the maximal control (figure 3c). this could be indicative of solubility issues in the assay buffer and therefore could generate a false negative result. it is known that fp assay outcomes can be influenced by intrinsic fluorescence of the test compounds and/or light scattering phenomena due to poor solubility and precipitation. to minimize the possibility of such false negative or positive reporting, none of the compounds tested showed competing autofluorescence in the assay conditions or was shown to be a false negative. results are shown in the supporting information (supplementary figure 2). to further validate the utility of these ligands in pca, on-target binding experiments were also performed using the recombinant t877a ar mutant characteristic of advanced stage androgen-independent pca. in tr-fret, the compounds demonstrated similar activity to that observed in the wild type assays, indicating their potential in advanced phases of prostate cancer (figure 3b). activity data are in agreement for 4 and 5 in both ar wt and art877a. the higher confidence mp values and experimental reproducibility obtained for 4 and 5 in coactivator studies were used as the basis to advance these compounds to cellular characterization and receptor subtype selectivity evaluations. we undertook to profile the selectivity of these compounds for ar over other members of the same phylogenetic branch of the steroidal nuclear receptor subfamily. compound binding affinities for progesterone receptor (pr), glucocorticoid receptor (gr), estrogen receptor (er-), and estrogen receptor (er-) were determined using tr-fret (table 2). er- and estradiol er- complex and do not displace fluorescent-labeled coactivator src1-4 from dexamethasone gr complex at concentrations up to 100 m (supporting information, supplementary figures 35). compound 5 binds pr with comparable affinity to that observed for ar, while 4 demonstrates approximately 2-fold binding selectivity for the ar over pr. in functional evaluation we determined that the diarylhydrazide compounds are full ar antagonists, with a partial antagonistic profile demonstrated in pr, displacing src14 from progesterone the non-lbp nature of this interaction was confirmed by an fp assay (supporting information, supplementary figure 6). compounds were tested at a concentration range from 100 to 1 m in the presence of a concentration of progesterone=ec80. error bars represent the standard error of the mean (sem) for n=6 values. data was fitted using log antagonist concentration vs response (variable slope) with graphpad prism 5 (see experimental section for details). ic50 values are shown in table 2. to ascertain the translational (clinical) potential of these ligands, compounds were evaluated in cellular models of prostate cancer (lncap, an androgen-dependent cell line and pc-3, an androgen-independent cell line) and in normal prostatic epithelia cell line pwr-1e. cell viability was assessed after 24 h of incubation with the test compounds at three different concentrations (figure 5). the classical antiandrogen cpa was used as a reference, showing a minor effect at 50 m in the androgen independent cell line pc-3. at 50 m 4 reduces cell viability to a 5060%, whereas 5 acts consistently across the three cell lines, retaining cell viability at around 80%. compounds were tested at 5 10, 1 10, and 5 10 m. error bars represent the sem of two independent experiments done in triplicate (n=6). the diarylhydrazides were evaluated for their effects on the ar signaling pathway and on hormone-dependent cellular growth of lncap cells. compound 5 was well-tolerated after 5 days of treatment at 10 and 20 m concentrations and enabled observation of a specific reduction in dht-treated cell count (figure 6a). prostate specific antigen (psa) is a serine protease normally secreted by the prostate epithelia. psa is widely used as a marker for pca, as its serum levels are increased in this condition. compound 5 was shown to reduce dht-induced psa secretion in a dose response fashion as quantified by an elisa experiment in lncap cells (figure 6b). it is well-documented that classical antiandrogens (i.e., those binding within the lbp/competing with endogenous ligands) have partial agonistic properties, which make them less useful in the management of advanced prostate cancer. arising from this inherent agonism, in an androgen-deprived lncap cell line, antiandrogens such as cpa can actually activate the ar pathway and stimulate cell growth. in direct contrast to the behavior of traditional antagonists, 5 shows no detectable agonist or partial agonist activity at tested concentrations, consistent with an alternative mechanism to that of the classical antiandrogens. finally, treatment with 5 at 10 m was found to antagonize cpa partial agonist activity (measured as secreted psa levels in the cellular media in an elisa experiment), suggesting its potential benefit in combination therapy for advanced stages of prostate cancer (figure 6c). to further challenge this hypothesis, the compounds also demonstrated similar effects in this alternate system, supporting the hypothesis of their functioning as true antiandrogens (supporting information, supplementary figure 7). (a and b) 5 reduces androgen-stimulated cell growth and dht-dependent ar signaling measured as psa levels secreted in the cellular media in a dose-dependent fashion. (c) 5 at 10 m reduces cpa-induced ar signaling (in absence of androgens) measured as psa levels secreted in the cellular media in a dose-dependent fashion. data are presented as the mean of two independent experiments, and bars show sem for n= 6 values (a). secreted psa (ng/ml) was measured considering the optical density at 450 nm minus the optical density at 540 nm and interpolating the values from the standard curve. data are presented as mean of two independent experiments, and bars show sem for n=4 values (b and c). classical antiandrogen therapy is known to have limited beneficial effects in hormone-insensitive pca. alternative ar inhibitors are therefore needed in the treatment of pca. in this study, we demonstrate the successful implementation of a virtual screening approach in the identification of small molecule ar modulators, where the structural motif of ar coactivators was included in a 3d pharmacophore. we report the discovery, identification, and characterization of a novel series of diarylhydrazide non-lbp-binding antiandrogen compounds, with demonstrated ability to displace ar coactivators and with established potency in ar-dependent prostate cancer cell lines. activity was measured with a tr-fret assay and a non-lbp-mediated mechanism of inhibition was confirmed by fp assay. these compounds are shown to function without any demonstrated intrinsic or partial agonist activity in ar and therefore can be classified as true non-lbp antiandrogens. the nature of nr coactivators and the high homology of nr coactivator binding sites are such that, to more fully profile the potential utility of these ligands, their selectivity was evaluated across members of the subclass of steroid receptors, including er- and er-, gr, ar, and pr. the selectivity of the diarylhydrazide scaffold for the ar was demonstrated through tr-fret evaluation in the estrogen and glucocorticoid receptors, where agonist bound receptor recruitment of coactivator was unimpaired at screening concentrations up to 100 m. we additionally investigated the potential cytotoxicity of the diarylhydrazides in three different cell lines, selecting 5 for its favorable cytotoxic profile (cell viability was retained at around 80% in different prostatic cellular models). unmodified diarylhydrazide screening hits were also shown to have 2-fold selectivity for ar over pr, with partial antagonist activity demonstrated for the scaffolds in a pr functional assay, remarkable given the high (> 60%) homology of these nr family members. futhermore, given the established utility of mifepristone (a pr modulator which also has antiandrogenic activity) in the treatment of castration resistant prostate cancer, the narrower selectivity window observed for these ar ligands in pr over the other nr s assessed is not a significant concern in the context of the therapeutic area under consideration. classical antiandrogens can be also distinguished for their different behaviors at a cellular level. save for two recent examples, all lbp antiandrogens described to date have also intrinsic partial agonist activity, demonstrated by induction of psa in the absence of hormone stimulation in lncap cells. in this study, the novel non-lbp diarylhydrazide antiandrogen 5 did not induce psa expression in absence of hormone stimulation when compared to cpa. in androgen-deprived lncap cells, 5 reduces psa expression in combination with cpa, antagonizing its partial agonist activity in a dose responsive fashion. this result supports the hypothesis of a nonclassical mechanism of ar inhibition for these diarylhydrazide ligands and it also demonstrates the potential application of these and other non-lbp antiandrogen small molecules targeting alternative ar sites in combination with existing prostate cancer therapy. through application of virtual screening methodologies, we present and characterize novel diarylhydrazide scaffolds as true antiandrogens displacing ar coactivator interaction and having a full antagonistic profile on ar (both wt and t877a), partial antagonistic profile for pr, and selectivity for the other members of the nr-3 family (gr, er-, and er-). the initial small molecule non-lbp true ar modulators provided by this study will be used to further characterize the ar coactivator interface, to understand the basis of selectivity, and to further guide rational drug design in the search of other novel scaffolds directed at this interface. black, low volume, 384-well assay plates (corning, ny, cat. no. 3676) were used to perform the assay (total volume 20 l), and tr-fret signal was measured with pherastar equipment (bmg labtech) using a lanthascreen optic module (excitation, 335 nm; emission, 520 nm channel a and 495 nm channel b). tr-fret values were calculated at 10 flashes per well, using a delay time of 100 s and integration time 200 s as recommended by the invitrogen assay guidelines. a serial dilution of compounds was first prepared in 100 dmso (sigma-aldrich) starting from the maximum desired concentration to achieve a 12 point range concentration using 96-well polypropylene plates (nalgene nunc, rochester, ny). each 100 solution was diluted to 2 concentration with tr-fret coregulator buffer a (invitrogen proprietary buffer), yielding a final concentration of 1% dmso in each well. ten microliters of 2 solution was then added to the 384-well plate, following addition of 5 l of 4 ar-lbd and 5 l of d11-fxxlf/tb anti-gst antibody in agonist mode and 5 l of d11-fxxlf/tb anti-gst antibody/dht (included at a concentration equal to ec80 as determined by running the assay in agonist mode first).d11-fxxlf and tb antibody were premixed in light protecting vials prior to use. a final concentration of 5 mm dtt was used in the assay buffer in order to prevent protein degradation. all plates (agonist and antagonist mode) were incubated between 2 and 4 h at room temperature protected from light prior to tr-fret measurement. ic50 values were determined by testing each ligand at concentrations ranging from 100 m to 45 nm using 2- and 3-fold dilutions to generate a 12 point dose data was fitted using the sigmoidal dose response (variable slope) available from graphpad prism 5.the z factor for these assays was>0.5, as calculated by the equation provided by zhang et al. in line with the assay protocol, a known agonist, dihydrotestosterone (dht, cat no. a8380, sigma), and a known antagonist, cyproterone acetate (cat no. a control with no ar-lbd present was included to account for diffusion-enhanced fret or ligand-independent coactivator recruitment. a negative control with 2 dmso was present to account for any solvent vehicle effects. the assay was adapted to exclude possible nonspecific aggregation mechanism of inhibition by adding very low concentration of detergent triton x-100 (0.01%) to the assay buffer following the shoichet review guidelines (supporting information, supplementary figure 1). p3018) was used to investigate the binding of the test compound to the lbp site, occupied by a high-affinity fluorophore ligand (fluormone). the 100 test compound solutions in dmso were diluted in ar green buffer (invitrogen) to achieve 2 concentrations and placed in a 384-well plate (corning, cat no. ar-lbd and fluormone (2) mix were prepared separately and then added to each compound dilution to achieve a final concentration lbd-fluormone of 50 and 2 nm, respectively. plates were incubated protected from light for at least 4 h. controls included a maximum mp positive control, which consists of the ar-lbd and fluormone mix (2), and a minimum mp control, containing only fluormone (2). a vehicle control was added to account for dmso effect, and a blank control containing buffer only. fluorescence polarization was measured with pherastar equipment (bmg labtech) using an optic module with excitation at 485 nm and emission at 530 nm. lncap cells (androgen-dependent), pc-3 (androgen-independent), and pwr-1e (normal prostatic epithelia) were cultured in rpmi-1640 glutamax (invitrogen), f12k (invitrogen), and k-sfm media (invitrogen). the first two were supplemented with 10% fetal bovine serum (fbs), penicillin (100 units/ml), and streptomycin (100 g/ml). k-sfm was supplemented with 5 ng/ml epidermal growth factor (egf) and 0.05 mg/ml bovine pituitary extract (bpe). cells were propagated at 1:3 or 1:6 dilutions at 37 c in 5% co2. for cell viability (end point) assays lncap, pc-3, and pwr-1e cells were seeded at 2.5 10/ml density in 200 l volume of a 96-well plate in triplicate and incubated for 24 h prior testing. test compounds were included at different concentrations to achieve a final concentration of 0.5% dmso in each well. cell viability was assessed after 24 h of treatment using 10% alamarblue reagent (invitrogen) for each well. cell viability was monitored by the reduction of resazurin, a blue, cell-permeable, nontoxic compound, to resorufin, a red and highly fluorescent product. viable cells continuously convert resazurin to resorufin, increasing the overall color and fluorescence of the media surrounding cells. fluorescence intensity can be quantitatively determined with a fluorescence microplate reader at excitation/emission 544 nm/590 nm (spectramax gemini). for hormone-dependent cell proliferation assays in androgen-deprived lncap cells, cells were seeded at 2 10 cells/ml in a 24-well plate in triplicate. cells were plated in phenol red free rpmi glutamax (invitrogen) supplemented with 10% charcoal-stripped fbs to deplete endogenous steroids 48 h prior to the assay, as described in previous reports. the optimal condition for the treatment was found to be 5 days and the concentration of dht included to stimulate the cells was 0.1 nm. cells were treated with different concentrations of test compounds with or without 0.1 nm dht to achieve a final concentration of 0.1% dmso in each well. a control for the vehicle was included to ensure that no effect on viability could be detected. media and treatments were replaced every second day, after washing the cells twice with 1 pbs. supernatants were collected after 5 days for evaluation of secreted psa levels, and cell proliferation was assessed for the same plate using alamarblue in order to exclude nonspecific effects due to toxicity issues. secreted levels of prostate specific antigen were evaluated with a commercially available kit (quantikine human kallikrein 3/psa immunoassay, r&d systems)., 50 l of standards and cell culture samples were added to precoated wells containing assay diluent rd1w (r&d systems) and incubated for 2 h at room temperature. unbound material was washed several times and 200 l of horseradish peroxidase (hrp) labeled psa conjugate antibody was added to each well and further incubated for 2 h at room temperature. wells were washed and treated with colored substrate (tetramethylbenzidine) for an additional 30 min, after which 50 l of stop solution (2 n sulfuric acid) was added per well and optical density (450 nm with correction at 540 nm) was read with a plate reader within 30 min (versamax). a virtual screen was designed to select compounds mapping onto the peptide binding surface (af2) of the ar receptor, based on an ensemble of documented x-ray crystal structures (pdb i d 1t73, 1t74, 1t76, 1t79, 1t7f, 1t7 m, 1t7r, and 1t7 t). molecular operating environment (moe) software was employed to preprocess the proteins and to remove the coactivator peptides from the complexes. an initial pharmacophore was generated using the moe pharmacophore elucidator and considering the most significant features, which involved hydrophobic, donor, and acceptor features. a second pharmacophore was developed including two additional hydrophobic/aromatic features to represent the phe side chains present in the fxxlf coactivator motif (1t7r), so as to increase the selectivity for ar over other families of nuclear receptor. these pharmacophore models were then applied for in silico screens of small-molecule commercial libraries to identify compounds that resemble the active principle of the starting peptides. a number of vendor databases were selected for screening of ligands, including amsterdam (5389 compounds), peakdale (8188), asinex platinum collection (75 258), specs (175 800), maybridge (56 870), and zinc (4.6 million) compounds. a bayesian analysis was performed on the peptide structures to estimate parameters of an underlying distribution based on the observed distribution. the above databases were then filtered for those compounds with properties similar to the peptides, thus focusing the search on the ar ligand chemical space. all molecules were standardized for stereochemistry and charges and ionized at a ph of 7.4 and all calculable tautomers were enumerated. at this stage the conformational flexibility of the screening compounds was explored using the omega software (openeye scientific package). a maximum of 50 conformations were generated for each molecule in the data set. the virtual molecules were overlaid on and compared to the generated pharmacophore of the active ligands, and those molecules that compared favorably were advanced for additional virtual screening and scoring. the fast rigid exhaustive docking (fred) software as implemented in openeye scientific s package was used to exhaustively examine all possible poses within the protein site, filtering for shape complementarity and scoring. the smaller databases (amsterdam and peakdale) were screened on all 13 crystal structures and only ligands scoring well on more than one crystal structure were considered. the larger databases specs, asinex, maybridge, and zinc were screened on the 1t7r crystal structure. a structural similarity search was conducted on 1 and 2 using a tanimoto coefficient of>70% on the specs compound database. thirty-seven compounds were purchased and four small molecules were selected for optimization and characterization studies based on their improved on-target activity determined by tr-fret. all screening compounds described in this work were purchased as commercial samples from specs nv. compound purity in all instances was greater than 95% as determined by lcms and nmr.

prostate cancer (pca) therapy typically involves administration of classical antiandrogens, competitive inhibitors of androgen receptor (ar) ligands, dihydrotestosterone (dht) and testosterone (tes), for the ligand-binding pocket (lbp) in the ligand-binding domain (lbd) of ar. prolonged lbp-targeting leads to resistance, and alternative therapies are urgently required. we report the identification and characterization of a novel series of diarylhydrazides as selective disruptors of ar interaction with coactivators through application of structure and ligand-based virtual screening. compounds demonstrate full (true) antagonism in ar with low micromolar potency, selectivity over estrogen receptors and and glucocorticoid receptor, and partial antagonism of the progesterone receptor. mdg506 (5) demonstrates low cellular toxicity in pca models and dose responsive reduction of classical antiandrogen-induced prostate specific antigen expression. these data provide compelling evidence for such non-lbp intervention as an alternative approach or in combination with classical pca therapy.

PMC3295204

pubmed-444

pregnancy-associated plasma protein-a (papp-a) is a metzincin metalloproteinase primarily produced by the placental syncytiotrophoblast during pregnancy. papp-a is also synthesized by fibroblasts, osteoblasts, vascular smooth muscle cells (vsmcs), and endothelial cells (ecs). in vitro, papp-a functions to cleave insulin-like growth factor-binding protein 4 (igfbp-4), an inhibitory igfbp, consequently increasing igf bioavailability for receptor activation [14]. in vivo, several studies have shown a similar role for papp-a in modulating site- and event-specific igf signaling during injury repair processes. recent studies have indicated that papp-a is a novel biomarker for plaque instability and inflammation useful in early diagnosis, risk stratification, and prognostic prediction in patients with acute coronary syndrome (acs) [5, 6]. papp-a was found abundantly expressed in ruptured and eroded human atherosclerotic plaques, colocalized with activated smooth muscle cells and macrophages [7, 8]. since plaque-derived papp-a is being considered as a new biomarker that may potentially play a role in the development of atherosclerotic lesions [9, 10]. a better understanding of its cellular source and regulation is important to the future development and implementation of therapeutics utilizing this biomarker. previous studies have indicated that proinflammatory cytokines, interleukin- (il-) 1, and tumor necrosis factor- (tnf-) were potent stimulators of papp-a expression in cultured human fibroblasts, osteoblasts, coronary artery smooth muscle cells, and endothelial cells (ecs) [9, 11]. despite these significant findings, little is known about the effect of c-reactive protein (crp) and tnf- on papp-a expression in human peripheral blood monocytes (pbmcs). the current study investigates the ability of crp and tnf- to induce papp-a expression in the pbmcs of healthy volunteers. furthermore, inhibitor experiments have been designed to explore the underlying intracellular signaling pathways involved in papp-a expression. the focus of these studies is nuclear factor- (nf-) b pathways, a major pathway associated with cytokine stimulation in various cell types. peripheral blood was collected from the forearm vein of healthy volunteers enrolled in the current study. for each experiment, 30 ml samples were freshly collected from 6 healthy subjects for use in pbmc preparations. each of these 6 subjects provided 4 blood donations over the course of the study. the study was approved by the ethics committee of the beijing friendship hospital and conforms to the principles outlined in the declaration of helsinki. human pbmcs were isolated from 30 ml fresh blood samples obtained from healthy volunteers by ficoll-paque (amersham bioscience, uppsala, sweden) centrifugation. resultant cells were washed 3 times with pbs and subsequently resuspended in rpmi 1640 (gibcobrl, grand island, ny, usa) supplemented with 10% fetal calf serum (gibcobrl, grand island, ny, usa), penicillin (100 u/ml), and streptomycin (100 g/ml). cells were then cultured for 24 hours in plastic dishes at 37c in a humidified atmosphere of 5% co2. upon observation of subconfluent growth, the medium was replaced with fresh medium. all nonadherent lymphocytes were discarded during the medium change, reserving only healthy adherent monocytes. after 24 hours, trypan blue exclusion indicated that 95% of cultured pbmcs were living. the pharmacological agents recombinant human tnf- (perprotech, rocky hill, ct, usa), crp, actinomycin d, and bay11-7082 (sigma chemicals, deisenhofen, germany) were dissolved into solution according to the manufacturer's instructions. resultant solutions were added to cultured pmbcs at defined time intervals (2, 8, 16, 24 hours) and concentrations (crp: 5, 10, or 20 mg/l; tnf-: 25, 50, or 100 ng/ml; bay11-7082: 20 m) in the absence or presence of actinomycin d (1 g/ml), as further described in the following sections. individual controls were determined for each experiment, as described in figures 14. briefly, total rna was isolated using trizol (invitrogen, calsbad, ca, usa) according to the manufacturer's instructions. reverse transcription-generating cdna was performed using the superscript iii first-strand synthesis system (invitrogen, calsbad, ca, usa). papp-a cdna was amplified using forward (5-ata tct cac gtg acc gag ga-3) and reverse (5-aga tga tgg tgc tgg aag tc-3) primers, which produce a 529 bp product. amplification was performed at 94c for 2 min for preheating, followed by 30 cycles of 94c for 45 s, 65c for 45 s, 72c for 60 s, and a final extension of 72c for 10 min. -actin was amplified using forward (5-gca tgg agt cct gtg gca t-3) and reverse (5-cta gaa gca ttt gcg gtg g-3) primers, which produce a 320 bp product. amplification was performed at 94c for 2 min for preheating, followed by 28 cycles of 94c for 30 s, 60c for 30 s, 72c for 30 s, and a final extension of 72c for 20 min. the resulting bands were photographed under ultraviolet light and analyzed using a gel imaging system (gel doc2000, bio-rad, hercules, ca, usa). the relative intensity of bands of interest was expressed as the ratio to -actin mrna bands. for cell lysates, cells were washed twice with ice-cold pbs and lysed in ripa buffer. total protein was quantified using the bca assay (pierce, rockford, il, usa). equal amounts of protein (40 g) were separated by sds-page in 14% tris-glycine gels (tefco, tokyo, japan). after electrophoresis, the proteins were blotted onto a nitrocellulose membrane and blocked with 5% skim milk powder diluted in tris-buffered saline (tbs) with 0.05% tween 20. rabbit polyclonal antibodies against human papp-a (1: 1000, abcam systems, cambridge, usa) were used as the primary antibody. membranes were incubated with diluted antibody preparations overnight at 4c. after washing the next day, membranes were incubated with horseradish peroxidase- (hrp-) conjugated affinity-purified goat anti-rabbit igg antibody (1: 3000, santa cruz. papp-a levels were determined using the ultrasensitive elisa kit (diagnostic systems laboratories, webster, tx). the assay was calibrated using recombinant papp-a calibrated against the world health organization's international reference preparation 78/610 for pregnancy-associated proteins, by definition containing a papp-a concentration of 100 minimum sensitivity was 0.24 miu/l with intra- and interassay coefficients of variation of 4.7% and 4.2%, respectively. all statistical analyses were carried out using the spss statistical package, version 13.0 (spss inc., p-values less than 0.05 were considered statistically significant (p<0.05). the time course of papp-a mrna expression in pbmcs under basal and cytokine-stimulated conditions is presented in figure 1(a). little papp-a expression was observed in pbmc cultures under basal conditions after 24 hours. treatment with crp (20 mg/l) or tnf- (100 ng/ml) significantly increased paap-a mrna expression at all time points (2, 8, 16, 24 hours). papp-a mrna levels increased 2 hours after stimulation with crp (20 mg/l) and remained elevated by approximately 3.7-fold up to 24 hours. pappp-a mrna expression, however, rapidly increased and peaked at approximately 6.8-fold 2 hours after tnf- (100 ng/ml) stimulation. a subsequent decrease was then observed, though levels remained elevated at approximately 4.5-fold up to 24 hours. maximal papp-a protein expression in pbmcs and concentrations in culture supernatants were achieved with crp stimulation by 24 hours and tnf- stimulation by 8 hours (figures 1(b) and 1(c)), reflecting the changes in papp-a mrna expression. as shown in figure 2, dose-response experiments confirmed crp or tnf- treatment elicited dose-dependent increases in papp-a mrna expression, protein expression in pbmcs, and secretion in the supernatant after 24 hours. crp showed half-maximal effectiveness at approximately 5 mg/l, with maximal effectiveness at approximately 20 mg/l (figure 2). tnf- showed half-maximal effectiveness at approximately 25 ng/ml, with maximal effectiveness at approximately 100 ng/ml. the dependence of papp-a expression on mrna synthesis was explored in the following three experiments. figure 3 showed that the effects of these proinflammatory cytokines appeared to be at the level of transcription, as the dna-directed rna polymerase inhibitor, actinomycin d, completely prevented crp or tnf- induction of papp-a mrna expression, protein expression, and concentrations in culture supernatants. these results showed that crp or tnf- was responsible for new protein synthesis of the papp-a protein. furthermore, papp-a protein was actively secreted into the supernatant. as indicated in our previous experiments, treatment of human pbmcs with crp (20 mg/l) or tnf- (100 ng/ml) significantly increased papp-a mrna expression, protein expression, and concentrations in culture supernatants. to confirm the role of nfb activation, bay11-7082, which inhibits inducible phosphorylation of ib, was shown to effectively inhibited crp and tnf--stimulated papp-a expression (figures 4(a), 4(b), and 4(c)). it is well known that the nfb pathway is the critical mediator of prooxidant stimuli, such as inflammatory cytokines. the basic mechanism by which nfb is activated is through phosphorylation of intrinsic inhibitors, with the ib, subsequently freeing nfb to translocate into the nucleus where it regulates gene expression. novel markers of coronary artery disease progression have been confirmed in recent years, with circulating levels of papp-a standing out as one of the most prominent indicators of this profile. the current study indicates that papp-a expression in human pbmcs may be regulated by crp and tnf- through the nf-b pathway, a mechanism that may play a critical role in increases in serum papp-a levels during acute coronary syndrome (acs). these findings are consistent with previous reports indicating that papp-a is a marker of atheromatous plaque instability as well as extent and prognosis of cardiovascular disease [1315]. serum papp-a levels increase in patients with acs, indicating that papp-a may also be a marker of adverse events [1618]. moreover, in chronic stable angina (csa) patients, papp-a is an independent predictor for the extent of vessel stenosis, where it has been shown to correlate with the presence of vulnerable coronary artery stenosis [19, 20]. thus, papp-a levels have demonstrated a firm association with angiographic plaque complexity in csa patients. the stimulation of papp-a expression by tnf- has been observed previously in human fibroblasts, osteoblasts, vsmcs, and ecs [4, 11, 22]. using specific monoclonal antibodies, bayes-genis et al. reported that papp-a was abundantly expressed in both eroded and ruptured plaques, but was only minimally expressed in stable plaques. moreover, in plaques with large lipid cores and cap rupture, staining for papp-a occurred mostly in the inflammatory shoulder region, in areas surrounding the lipid core, and in areas with localized cd68-positive cells. thus, papp-a levels have been associated with inflammation in regions of atherosclerotic plaques, potentially contributing to progression and poor outcomes in patients. most evidence of monocyte involvement of papp-a expression has been completed through analysis of atherosclerotic plaques, where monocytes are the predominant leukocyte contributing to the development, progression, and instability of atherosclerotic lesions, they often contain high levels of papp-a. because samples of plaques may contain a mixture of leukocytes and circulating compounds, these tests have left the source of papp-a observed in these monocytes a point of debate among researchers. double immunofluorescence confocal microscopy (icm) has been used to characterize cell types expressing papp-a, suggesting that monocytes are the primary source of papp-a in plaques and the target cells for cytokines. it has also been speculated that papp-a may be produced by activated monocytes or macrophage cells in unstable plaques and released into the extracellular matrix and circulation. conversely, conover et al. suggested that in vivo macrophages actually failed to produce papp-a, but instead internalized the compound through their membranes, thus accounting for the accumulation of circulating papp-a produced by other sources in macrophage cells associated with plaques. the current study, however, indicates that the specific mrna expression associated with papp-a production increases in macrophages in vitro, resulting in expression and supernatant secretion of papp-a. these findings indicate that papp-a production occurs in macrophages rather than being internalized through the membrane, as suggested by conover et al. further in vivo studies will be required to assess the affect of circulating papp-a levels on macrophage papp-a expression and secretion, which may account for the discrepancies between these two studies. cumulatively, the findings of the current study indicate that macrophages in cultured human pbmcs can synthesize and secrete papp-a. furthermore, crp and tnf- were indicated to be potent stimulators of papp-a gene expression and protein secretion in human pbmcs, suggesting a link between the increase in local inflammatory cytokine production and papp-a during acs. while further studies of the in vivo effects in acs patients will be required to confirm these results, these findings suggest that serum papp-a, hscrp, and tnf- levels may be significantly higher in acs patients than in patients with stable angina pectoris and that increasing papp-a mrna levels in patients with acs may also have a positive association with serum hscrp and tnf- mrna expression. exploration of these effects is a topic being explored in our current research based on the initial positive findings of the current research. furthermore, the time course of stimulation by crp and tnf- differed, with papp-a expression increasing 2 hours after stimulation with crp, peaking at approximately 3.7-fold by 24 hours, whereas a rapid increase to approximately 6.8-fold was seen in only 2 hours with tnf- stimulation. the current study provides significant evidence for papp-a production in pmbcs and stimulation by the cytokines crp and tnf-; however, further in vivo studies will be required to verify these findings and assess the effects of circulating papp-a on pmbc papp-a production. actinomycin d was observed to complete block the induction of papp-a mrna expression by crp and tnf-, indicating potential regulation at the level of transcription. the rapid increase in papp-a mrna levels after treatment with these cytokines further confirms the hypothesis of transcriptional regulation. as expected, increases in papp-a protein expression and secretion into the supernatants were paralleled by increases in gene expression. based on this observation, it is possible that the biological consequence of crp- and tnf--induced papp-a expression in human pbmcs was enhanced igf-i bioactivity mediated by papp-a proteolysis of igfbp-4, thus contributing to the progression of both coronary atherosclerosis and restenosis. it is well known that the free fraction of circulating and locally synthesized igf-i stimulates vsmc proliferation, migration, and extracellular matrix synthesis. in macrophages, igf-i also promotes excess ldl cholesterol uptake, production of proinflammatory cytokines, and chemotaxis [26, 27]. crp and tnf- were also identified as potential regulators of papp-a expression, operating through a mechanism involving the activation of the nfb system in pbmcs of healthy volunteers. nfb is a ubiquitous transcription factor that is activated by inflammatory cytokines, infection, oxidative stress, and shear stress. ritchie reported that nfb was activated in peripheral monocytes using an electromobility shift assay in patients suffering from unstable angina. the nfb family of transcription factors plays a critical role in coordinating and regulating the expression of a wide variety of inflammatory genes that have been linked to the pathologies of acs. some studies have even demonstrated that crp induces nfb activity in various cell types, including peripheral monocytes, saphenous vein endothelial cells, and human aortic endothelial cells [23, 33, 34]. moreover, resch et al. reported that tnf- induced ib degradation and papp-a-regulated expression in human fibroblasts by tnf- was mediated by nfb activation. the current study also demonstrated that bay11-7082 was a potent inhibitor of both crp- and tnf--stimulated papp-a expression in human pbmcs. the view of nfb as a transcription factor for papp-a gene expression, however, will require further study to identify the promoter region and validate these findings. the current study provides a variety of evidence to support the expression on papp-a by leukocytes in pmbcs, including increased papp-a-associated mrna expression, papp-a expression, and papp-a secretion into the supernatant of fresh in vitro samples collected from healthy subjects. furthermore, the cytokines crp and tnf- were shown to stimulate papp-a expression in these cells. based on these findings and the previously observed association between pmbcs and acs, it is likely that activated monocytes or macrophages in pmbcs surrounding developing artherosclerotic plaques may secrete proinflammatory cytokines, thus stimulating the expression and secretion of papp-a. further studies will, however, be required to assess the effects of elevated papp-a concentrations on autocrine and paracrine mechanisms for the exacerbation of atherosclerosis procession and plaque rupture through igfbp-4 cleavage and enhanced local igf-i bioavailability. the current results, however, provide the fundamental mechanistic groundwork for further understanding of the entire mechanism associated with cytokine regulation of papp-a expression and igf bioavailability. this understanding may lead to the future development of novel therapeutic targets for the treatment of acs.

objective. the effects of c-reactive protein (crp) and tumor necrosis factor- (tnf-) on pregnancy-associated plasma protein-a (papp-a) expression in human peripheral blood mononuclear cells (pbmcs) require further investigation. methods. the papp-a levels in culture supernatants, papp-a mrna expression, and cellular papp-a expression were measured in human pbmcs isolated from fresh blood donations provided by 6 healthy volunteers (4 donations per volunteer). analyses were conducted by ultrasensitive elisa, western blotting, and rt-pcr following stimulation with crp or tnf- cytokines. results. papp-a mrna and protein levels after crp stimulation peaked at 24 hours, whereas peak papp-a mrna and protein levels were achieved after tnf- stimulation at only 2 and 8 hours, respectively. these findings indicate the dose-dependent effect of crp and tnf- stimulation. actinomycin d treatment completely prevented crp and tnf- induction of papp-a mrna and protein expression. additionally, nuclear factor- (nf-) b inhibitor (bay11-7082) potently inhibited both crp and tnf- stimulated papp-a mrna and protein expression. conclusions. human pbmcs are capable of expressing papp-a in vitro, expression that may be regulated by crp and tnf- through the nf-b pathway. this mechanism may play a significant role in the observed increase of serum papp-a levels in acute coronary syndrome (acs).

PMC3446755

pubmed-445

there is an increasing interest in the study of the consultation process and patients ' satisfaction with it. the core activity in primary care is the consultation irrespective of whether patients consult for cure, services, counseling, prevention, or care. a widely accepted model views the consultation as a dialogue involving elements of negotiation to create a common reality to which agenda setting is paramount. in the medical consultation the doctor and patient meet on common grounds with tolerance for each other's rights. this consultation by necessity requires a doctor who is expected to possess the requisite knowledge which will be useful in solving the problems the patient presents with the assumption that the doctor will act in the best interest of the patient. guided by rules of professional conduct, objectivity, and being emotionally detached the doctor is guaranteed the right to examine the patient physically and to enquire into intimate areas of the patient's physical and emotional life. during the consultation, the reason for attendance is defined and an appropriate action is chosen. this process aims at achieving a shared understanding, involving the patient in management and using time and resources appropriately. physicians have been noted to have fixed ideas about what is best for a patient, and this inflexibility leaves little room for negotiation. quite often animosity is expressed when the patient attempts to negotiate. however, as demonstrated by a study in the netherlands, interindividual and intra-individual variability does occur among physicians, who were noted to adjust their styles according to the situation. this study sought to identify the factors contributing to patients ' satisfaction, shed more light on the burden of patients ' dissatisfaction with the consultation in our environment, and help devise strategies for practicing physicians to strive for an improvement in the overall patient care in our cultural context. the findings would provide some of the information needed to further fill the knowledge gap about our patient needs in our environment and highlight the need to teach the consultation process at both undergraduate and postgraduate medical training. this study comes from a background where patient awareness of their opportunities in the patient doctor encounter is still in its early days, and thus this study brings to the literature a unique perspective of the patients ' views from this environment. this study was conducted in the general outpatient clinic (gopc) of the university of calabar teaching hospital, (ucth) calabar. the university of calabar teaching hospital is a tertiary hospital located within calabar metropolis, which lies along latitude 4, 58 north of the equator and longitude 820 east of the greenwich meridian. margaret's annex, maternity annex, the permanent site, and the comprehensive health centre (chc) okoyong. the general outpatient clinic (gopc) is situated at the permanent site and has fourteen outpatient consulting rooms in which about 1014 doctors (family physicians or resident doctors in family medicine) consult from 8 am4 pm on a daily basis. three consulting rooms are for consultants, three for doctors dedicated to the hiv clinic, one for consultancy patients, one for patients who are staff, and six consulting rooms are for outpatient consultation by other doctors. the department also had 16 nurses, 6 records ' personnel, 5 orderlies, 3 counselors, and 5 administrative staff who usually assisted the doctors during the consultation. all adults between the ages of 18 and 65 years who consented to participate in the study were recruited. informed consent was obtained from the patients before they were given a questionnaire to complete. the average attendance of patients at the ucth gopc in the three months preceding the study was 74 patients per day. therefore, the number of patients estimated to attend the clinic was 74 22=1628. from the calculated sample size of 430 subjects, a sampling interval of 4 was used to systematically select subjects who were recruited to participate in the study. the patients ' attendance register for each day was used as the sampling frame from which patients were selected. the first subject was chosen randomly from this sampling frame, and subsequently every fourth patient was selected and invited to participate. if a selected subject did not meet the inclusion criteria or refused to participate in the study, the next patient was approached until the recommended sample size was recruited. a self-administered, pretested questionnaire adapted from the general practice assessment questionnaire the general practice assessment questionnaire was developed in the united kingdom and used to study certain components of the consultation. the questionnaire consisted of 21 questions divided into five sections that investigated the proportion of patients that are satisfied with their patient-doctor encounter, which patient-factors are associated with patients ' satisfaction or lack of satisfaction with the consultation. some of the questions had options from which the patient selected the response while others made room for a narrative response. prior to the commencement of the consultation, all the patients waiting to be consulted were addressed on the possibility of being approached to join an ongoing study. selected patients on leaving the consulting rooms were approached by the trained assistants and requested to complete the questionnaire. data generated in the study was analyzed using the epi info software for analyzing medical data from the centre for disease control, atlanta, georgia, usa. privacy of the patients was maintained during the study, and all information provided by the patients was treated with utmost confidentiality. patients ' consents were sought and formally obtained after a detailed explanation of the intention of the author concerning the research findings. ethical approval for this study was sought and received from the ethical committee of the university of calabar teaching hospital. the age distribution of the respondents varied with the highest proportion being young adults aged 2640 years (44%), adolescents aged 1825 years (34%), middle-aged persons aged 4160 years (18%), and elderly patients aged 60+9 (4%), table 1. two hundred and fifty-five (59.3%) were satisfied with their patient-doctor encounter. the average age of the respondents was 29 years while the average age of all the patients who presented to the hospital during the study period was 31 years. the sex ratio was almost equal with males accounting for 201 (47%) and females 229 (53%) of the respondents (table 1). there was a wide variation among the occupational characteristics with patients who had any form of paid employment accounting for 27%, students 32%, retired persons 5%, unemployed 9%, housewives 7%, and others 22% (table 1). among the others were artisans, self-employed businessmen, and farmers. sex and occupational distribution of the respondents were shown to be similar to those of all the patients who presented in the clinic during the study period. majority of the patients 230 (53%) felt the time they spent with the physician was adequate or very adequate (table 2). only 26 (6%) respondents assessed the time they spent with the physician as inadequate (table 2). two hundred and twenty-five (52%) respondents felt they understood the illness much more than when they came to visit the doctor (table 2). seventy-eight percent of the patients who participated in the study and perceived that the encounter had made it possible to cope with the illness were satisfied with their encounter (table 2). a good majority of the patients perceived that their ability to cope with the illness after the visit influenced the patients ' satisfaction with the encounter (p<0.001). three hundred and fifty (81%) of patients who found an improvement in their ability to maintain their health were satisfied (table 2) with their encounter (p<0.001). table 3 shows that the frequency of visits did not statistically influence the patients ' satisfaction with the consultation (p>0.25). the patient's assessment of time spent in the consultation was shown to have a statistically significant influence on the patients satisfaction with the consultation (p<0.001). this table also shows that the patients preference for a particular physician did not statistically influence the patients satisfaction with the consultation (p>0.05). none of the sociodemographic variables studied were found to have any statistically significant relationship with a patient satisfaction in a consultation. this study could not demonstrate any statistical significance between a patient age and their satisfaction with the consultation. this agrees with some studies which demonstrated similar findings [8, 9] but differs from other studies which demonstrated that patients ' satisfaction rates usually improve with advancing age [10, 11]. the elderly patients included in this study were only 18, and perhaps with a larger elderly population the study could have demonstrated an age-related effect on satisfaction in the consultation. many elderly patients did not agree to complete their questionnaires, and this was probably due to the influence of the accompanying persons who often insisted they had to return to work as soon as possible. the living arrangements in our society possibly make us share similar illness perceptions of what is good or bad accompanied by a shared cultural understanding of wellness or illness. this study could not demonstrate any statistically significant influence of a patient's sex on his/her satisfaction with a medical encounter (table 1). the patients ' frequency of visits to the gopc was not found to statistically influence the patient's satisfaction with the consultation (table 3). it was believed that the higher the number of visits, the higher the level of dissatisfaction because this was thought to be related to the higher likelihood of social factors not being addressed in these frequent users of the hospital services.. patients often request to see particular doctors, but this was not shown to influence their satisfaction in this study (table 3) and is supported by a study among israeli patients. this finding differs from other studies which have shown that continuity and being seen by a particular doctor improve concordance and satisfaction [10, 13, 14]. the difference in this study may be accounted for by the fact that the patients were in a teaching hospital and many were usually referred to other clinics when the need arose. this may explain the fact that 95% of patients in this study did not insist on seeing a particular doctor as many patients often see the clinic as a transit route to other specialist clinics. only about 32% of patients had been to the clinic on at least three previous visits with the majority (44%) having attended just 1-2 times or with no previous visits (25%) in the last 12 months. it is possible they had not cumulatively spent enough time with the doctors to form an opinion. there is also the practice of doctors changing rooms, duties, and postings in between patients ' visits. this makes patients wary of requesting for a particular doctor who may not be on duty. usually in the study area clinic, patients are not given the choice of selecting a doctor and may be rebuked by the nurses who do the sorting if they request for a particular doctor. also patients probably did not request for a particular doctor because they did not know the doctors, were not aware if a particular doctor was on duty, or how long they needed to wait to see a preferred doctor. the use of time in the consultation has been shown to be crucial to consultation satisfaction ratings. this study demonstrates a statistical significance between patients ' perception of time spent in the consultation and satisfaction (table 2), but not all studies agree. fifty-three percent of the patients rated the time spent in their consultation as adequate or very adequate, with 47% describing the time spent as either fair or inadequate (table 2). patients ' assessment of the adequacy of time is crucial in gauging their satisfaction as it has been linked to satisfaction with psychosocial issues in the consultation. patients ' assessment of time spent in the consultation may be influenced by certain individual traits such as age. in this study, patients often confused the time spent in the consultation with the time spent in the waiting room, and throughout the data collection patients were encouraged to make this distinction as they completed their questionnaires. the patient perception of time is crucial in the consultation, and this influenced whether the patient was satisfied with the consultation or not. duration of a patient illness has been shown to have an influence on the consultation by a study of chronic illnesses while a patient satisfaction with a consultation can also affect the duration of his illness. however, this study could not demonstrate any statistically significant influence of chronic illness on patients ' satisfaction (table 3). patients with chronic illnesses are expected to know more about their illness than those with acute illness and are thought to require more attention. chronic illnesses are usually not what doctors expect to manage when they graduate, and their management may be a form of psychological burden to the physician. this study did not demonstrate any effect on satisfaction rates by the presence or absence of a chronic illness (table 3). this finding may be explained by the fact that many patients in our environment are not well informed about their illnesses, so their knowledge of the illnesses does not necessarily increase as the durations of their illnesses increase. this study could not demonstrate any significant influence of occupation on a patient's satisfaction with the consultation (table 1). students made up 32% of the respondents, employed persons 27%, and housewives 7%. despite this spread the patient's occupational status is closely linked to the person paying for their medical expenses. this was also found not to have any statistically significant influence on patients ' satisfaction with their consultation (table 1). fifty-two percent of the patients were paying for their medical expenses themselves while families were paying for 30%. ten percent of the patients including two males were being paid for by their spouses. however, 3% of the patients did not know who would pay and an equal number was being sponsored by their employers. the number being sponsored by their employers was unexpectedly low (2.6%) considering the nigerian government's efforts at promoting a national health insurance scheme (table 1). the effect of managed care in this study can not be discussed owing to the low number of patients who were using health care insurance, but studies in the united states have demonstrated that managed care affects neither the perception of time used in the consultation nor patients ' satisfaction with it. there was also a possibility that the number of students might be lower than the observed figures because some young people in calabar town often claimed to be students when they were not. this finding suggests that people from different occupational backgrounds in our practice environment may not bring their psychological expectations to influence their satisfaction with the consultation. the more a consultation contributes to a patient's understanding of his illness, the higher the likelihood for the patient to be satisfied with the consultation [18, 19] but very often patients get less information than they expect. fifty-nine percent of the patients had a satisfying consultation, while 52% had some improvement in the understanding of their illness. however, of the 41% of patients with unsatisfying consultations, 28% of them still had an improvement in their illness understanding (table 2). this suggests that, despite the lack of illness understanding there is still some satisfaction with the consultation. however this study clearly demonstrates that a patient understanding of his/her illness has a statistically significant effect on the patient's satisfaction with the consultation (table 2). it seems clear that the more informing a consultation is, the more likely a patient is to be satisfied with the consultation. this finding supports the call by one report for the patient to be more involved in decision making. many patients were observed in this study to have shown great interest when they found the doctor to be willing to provide some explanation about their illness. a patient's ability to cope with his illness can be helped or marred by a consultation, and this is more evident in chronic illnesses. patients ' abilities to cope with their illness based on the information received have been demonstrated by this study to statistically affect patients ' satisfaction with the consultation (table 2). it is increasingly clear that better informed patients have better outcomes, choose less risky procedures, and avoid equivocal treatments. a patient's ability to maintain health after a consultation would be addressing one of the core issues in family medicine by promoting prevention of illnesses. this study demonstrates a statistically significant effect of a patient's ability to cope with his illness on his/her satisfaction with the consultation (table 2). this is vital in our environment considering that the bulk of illnesses we manage is due to preventable diseases. in conclusion, factors influencing the patient-doctor consultation are numerous, and the exact influence of any of these factors is not easily isolated, but together these factors influence the interaction either positively or negatively. however, the study has shown that, despite the various factors that are considered to encourage client satisfaction at primary care consultation, a few of such factors contributed to end of consultation satisfaction in our environment. this calls for a refocusing if improvement in the overall patient care in our cultural context is to be achieved with the aim of meeting patient needs. teaching of consultation process must take these factors into consideration. to further address effort towards improving patient satisfaction rates in the study centre, it is recommended that the physician-related factors that influence the doctor-patient encounter should be further studied. in particular the effect of sociodemographic variables such as same-sex consultation, cultural/language diversity, and experience should also be further explored.

medical consultation is at the centre of clinical practice. satisfaction of a patient with this process is a major determinant of the clinical outcome. this study sought to determine the proportion of patients who were satisfied with their doctor-patient encounter and the patient-related factors that affected patients ' satisfaction with the consultation process. a clinic-based, cross-sectional study using a modified version of the general practice assessment questionnaire (gpaq), which employed a systematic sampling technique, was used. the questionnaires were administered on 430 patients within the ages of 18 years and 65 years. among the 430 subjects within the ages of 18 years and 65 years studied, 200 (46.5%) were males and 230 (53.5%) were females. only 59.3% were satisfied with their patient-doctor encounter. the patient's perception of time spent in the consultation, illness understanding after the visit, ability to cope with the illness after the visit, and ability to maintain health after visit were the only factors that affected patient's satisfaction with the consultation. in our environment, nonsatisfaction with the patient-doctor encounter is high. only few factors considered to encourage a patients satisfaction at primary care consultation contributed to end-of-consultation satisfaction. this calls for refocusing so as to improve the overall patient care in our cultural context and meet the patient needs in our environment.

PMC3363396

pubmed-446

acute pancreatitis is an inflammatory intra-abdominal process, which in approximately 1520% of patients presents in a severe form, with a gradual establishment of multiple organ dysfunction or local complications, including necrosis, pseudocyst, and abscess. severe acute pancreatitis is a condition associated with high mortality, which is characterized by a complex and incompletely understood pathophysiological mechanism [2, 3]. the deficit in our understanding of the mechanism driving the inflammatory process in acute pancreatitis is a reason why our therapeutic strategy has failed to reduce mortality, despite ongoing research. when acute pancreatitis leads to the establishment of acute kidney injury, there is a 5- to 10-fold rise in mortality, which can reach 70% [46]. the prevention of acute kidney injury can be a useful strategy in the prevention of the morbidity and mortality associated with acute pancreatitis. eugenol (1-allyl-4-hydroxy-3-methoxybenzene) is a naturally occurring substance, found in the essential oil of commonly consumed spices such as clove oil as well as cinnamon, basil, and nutmeg oils. it has many pharmacological properties which are mainly analgesic, anti-inflammatory, antioxidant, and vasodilatory action, while it has been shown to ameliorate kidney injury in a model of gentamycin-induced nephrotoxicity. the aim of this study is to assess the possible reduction in the extent of acute kidney injury after administration of eugenol in an experimental model of acute pancreatitis. 106 male wistar rats, aged 3-4 months and weighing 220350 gr, were used in this study. they were housed in cages under standard laboratory conditions (12 hr light-dark cycles, 2225c room temperature, and 5558% humidity), with free access to food and water. the animals were procured from the hellenic pasteur institute (athens, greece). the experiment took place at the elpen experimental research center (pikermi, greece), while the histological analysis was carried out at the lab of histology, embryology, medical school, democritus university of thrace. the experimental surgical procedures and the general handling of the animals conformed to the international guidelines of directive 86/609/eec on the protection of animals used for experimental and other scientific purposes. the animals were randomly assigned in 3 groups: sham (n=20), control (n=46), and eugenol (n=40). the animals were anaesthetized initially by being placed in a glass box containing isoflurane and then through administration of 0.25 ml of butorphanol (dolorex; intervet/schering/plough animal health, boxmeer, holland) by subcutaneous injection. the animals were intubated with a 16 g venous catheter, which was then connected to a ventilator set at 70 breaths/min and a tidal volume of 3 ml. after confirmation of the success of intubation, anaesthesia was maintained by a mixture of 93% o2, 5% co2, and 2% isoflurane. briefly, after induction of anaesthesia and preparation of the surgical site, the abdomen was entered via a 3 cm midline incision under sterile conditions. the biliopancreatic duct was identified and ligated near the duodenal wall with a 4-0 silk sutures (in the control and eugenol groups, but not in the sham group). 1 ml of normal saline and 1 ml of 5% d5w were instilled in the abdominal cavity. the abdomen was closed with vicryl 2-0 sutures. in the eugenol group, eugenol was administered by a nasogastric catheter in a dose of 15 mg/kg, while the sham and control groups received corn oil solution without eugenol. postoperatively, analgesia was maintained through subcutaneous administration of 2 ml/kg butorphanol (dolorex; intervet/schering/plough animal health, boxmeer, holland). euthanasia was performed at a predetermined time for each animal with the use of ketamine (narcetan; vetoquinol, buckingham, uk) 0.30.6 ml and xylazine (rompun; bayer, uxbridge, uk) 0.10.3 ml, followed by a midline laparotomy and exsanguination of the abdominal aorta. time points for analysis were 6, 12, 24, 48, and 72 hours postoperatively. serum samples for measurement of urea and creatinine as well as specimens from both kidneys for histopathological examination were acquired. louis, mo, usa) was purchased and prepared in an oily solution in the chemical laboratory of elpen pharmaceutical co. inc. this was achieved with the admixture of pure eugenol in a corn oil solution in a concentration of 1.5 mg eugenol/ml. samples were placed in 10% buffered formalin solution, and 4 m paraffin-embedded sections were stained with hematoxylin/eosin. all specimens were evaluated by a pathologist blinded to the sequence of the biopsy specimens. slides were evaluated with regard to 5 histopathological parameters and with the use of a semiquantitative scoring system as depicted on table 1. the scores of each individual parameter for each slide were added and a histopathological score was obtained for each specimen. immunohistochemical staining was applied to detect the possible expression of inflammatory cytokines like il-6, tnf-, and myeloperoxidase. the following antibodies were used: myeloperoxidase (rabbit polyclonal), dako (a 0398), diluted 1: 400 tnf- (rabbit polyclonal), abnova (pab8016), diluted 1: 1000, il-6 (rabbit polyclonal), and abcam (ab6672), diluted 1: 500. the buffers, blocking solutions, secondary antibodies, avidin-biotin complex reagents, and chromogen were supplied in a detection kit (envision hrp, mouse/rabbit detection system (k 5007), dako). to inhibit endogenous peroxidase, the specimens were incubated with 3% h2o2 (200 ml h2o and 6 ml h2o2) for 15 min in a dark room. before the primary antibody was applied, the sections were immersed in 10 mm citrate buffer (ph 6.0), rinsed in tris-buffered saline, and subsequently heated in a microwave oven (650800 w) for three cycles of 5 min. the slides were washed with tris-buffered saline before application of the primary antibody in order to reduce nonspecific binding of antisera. sections were then briefly counterstained with mayer's hematoxylin, mounted, and examined under a nikon eclipse 50i microscope (nikon instruments inc, ny, usa). the average labeling index was assessed according to the proportion of positive cells, after scanning the entire section of the specimen. the results were graded as negative (0) for<10% of stained cells, low (1) for>10% and<30% of cells stained, moderate (2) for>30% and<70% cells stained, and high expression (3) for>70% cells stained (table 1). the statistical analysis of the results was completed with the use of the 20th version of spss (statistical package for the social sciences, spss inc., we performed an analysis in which the data were treated as qualitative using fisher's exact test (this test was preferable to x because of the small number of animals in each subcategory/time point). evaluation of the different variables was performed to determine whether they were normally distributed (kolmogorov-smirnov are shapiro-wilk). the three different groups were then analyzed using the kruskal-wallis one-way analysis of variance test. finally, the mann-whitney u test was further used to compare the groups in pairs. these tests were applied to the overall sample and for each individual subgroup corresponding to individual time points (6, 12, 24, 48, and 72 hours postoperatively). the difference between the eugenol and control groups is apparent at 48 and 72 hours after induction of pancreatitis (figures 1 and 2). the histological score for these two groups is higher compared to the sham group at 48 and 72 hours and for the whole sample. eugenol administration lowers hyperemia and dilation of renal parenchyma capillaries and the difference was statistically significant for the 48 and 72 hour time points and for the whole sample. the eugenol group exhibited lower values than the control group and both exhibited higher values than the sham group. the same was true for hyperemia and dilation of renal corpuscles capillaries for the 48- and 72-hour time points, but not for the whole sample. there were no inflammatory infiltrations in any of the animals in our experimental model and measurement of this factor did not produce any results. edema was reduced through the administration of eugenol and, again, the difference to the control group was significant for the 48- and 72-hour time points and the whole sample. the control group had higher values than the sham group at 48 hours and also higher values than both the sham and eugenol groups at 72 hours. when values of the whole sample were considered, the control group had higher values than the eugenol group, which in turn had higher values than the sham group. analysis of the whole sample showed only higher values for the eugenol and control groups when compared to the sham group. there was no clear difference regarding il-6 expression between the different groups (figures 3 and 4). on the contrary there was a statistically significant difference between the eugenol and control groups 72 hours after induction of pancreatitis, while both groups exhibit higher tnf- expression than the sham group. there was no statistically significant difference between the eugenol and control groups for mpo expression, although there was a trend toward higher expression for the control group after 72 hours. eugenol administration resulted in lower serum levels of urea and creatinine especially at the 48- and 72-hour time points, compared to the control group. urea and creatinine levels were higher for both the eugenol and control groups, when they were compared to the sham group (figure 4). the results of this study suggest that eugenol attenuates the intensity of the histopathological changes and the expression of tnf- and mpo in the renal parenchyma, while lowering the values of serum urea and creatinine when administered in a rat acute pancreatitis experimental model. to evaluate the extent of kidney injury, we decided to evaluate serum urea and creatinine levels and the histopathological changes in the kidney, as well as the expression of tnf-, il-6, and mpo in the renal parenchyma. the role of cytokines, such as tnf- and il-6, in the pathophysiology of acute pancreatitis has been studied extensively and they have been found to contribute to the activation of the systematic inflammatory response process and multiorgan failure, which is a hallmark of severe acute pancreatitis and is, ultimately, correlated with the observed high mortality rates [10, 11]. the role of cytokines in acute kidney injury has been found to be equally important. the cytokine-mediated inflammatory response has a central role in the pathophysiology of acute renal failure irrespective of its cause. mpo has been used as a marker of neutrophil migration in acute pancreatitis studies and has been correlated to the severity of kidney injury [1214]. the histopathological evaluation showed that the histologic score was lower for the eugenol group in comparison to the control group at 48 and 72 hours from the initiation of the inflammatory process (means: 3.75/6.5 and 4.12/7.62, resp.) and this difference was statistically significant. this difference between the two groups was also present for individual histological changes such as hyperemia and dilation of renal parenchyma and renal corpuscles capillaries and edema. the difference observed in the degree of acute tubular necrosis and inflammatory infiltration was not statistically significant. regarding the expression of inflammatory mediators, tnf- levels were higher for the control group in comparison to the eugenol group with the difference reaching statistical significance at the 72-hour time point, while there was a trend for higher mpo expression in the control group at 72 hours, which was, however, not statistically significant. in contrast, il-6 levels did not show the same correlation and there were no statistically significant differences between the eugenol and control groups. we chose the bile-pancreatic duct ligation model as it is a well-characterized model of acute pancreatitis, which mimics acute pancreatitis caused by biliary obstruction, which is a frequent clinical scenario and results in multiorgan failure similar to that observed in humans [15, 16]. we have previously used this experimental model and we were able to show that it generates acute pancreatitis with histopathological changes in the pancreatic tissue including hemorrhage and necrosis. out of a total of 106 animals, 6 died and the fact that they were all in the control group could be seen as further evidence supporting the protective role of eugenol. it is possible that these animals would have exhibited signs of severe kidney injury, if they had survived until the predetermined time of euthanasia. however, since the distal bile-pancreatic duct ligation model is not usually fatal, we can not directly attribute the death of these animals to the severity of acute pancreatitis. eugenol has been shown to possess a multitude of pharmacological effects, some of which make it a likely candidate for use in the setting of acute pancreatitis and can explain the results observed in our study. the analgesic action of eugenol has been well documented and doses in the range of 40100 mg/kg have been shown to be effective in rat experimental models [1820]. in addition, eugenol acts as an anti-inflammatory substance inhibiting cyclooxygenase and reducing the release of proinflammatory mediators such as il-1, tnf-, and pge2 [2224]. the antioxidative potential of eugenol has been studied in a number of, mainly in vitro, studies where it has been shown to bind to free oxygen radicals and attenuate the action of oxidative substances [2528], while a recent study of gentamycin-induced nephrotoxicity offers insight into how eugenol can prevent kidney injury by reducing oxidative damage. these combined properties of eugenol can be used to explain the observed reduction in tnf- expression, as well as the reduction of kidney inflammation. eugenol administration causes a dose-dependent, reversible vasodilation through its effect on the endothelial cells [29, 30], which is comparable to nifedipine. the potential of eugenol to inhibit the vasoconstriction that is associated with kidney injury points to another potential mechanism for its effect in the model of acute pancreatitis. a number of authors have proposed strategies to reduce kidney injury caused by acute pancreatitis. zhang et al. have tried dexamethasone administration in an experimental model of retrograde injection of sodium taurocholate in the pancreatic duct. the dexamethasone group exhibited milder congestion of the glomerular capillary, swelling of the renal tubular epithelial cells, and less inflammatory cell infiltration than that of the control group, which was shown by the lower histological score at the 6- and 12-hour time points. the same authors found a significant difference in the serum levels of tnf- in favor of the dexamethasone group, while expression of nf-b in the renal tissue was more pronounced in the dexamethasone group. the same model has been used to study octreotide and baicalin (5,6,7-trihydroxyflavone-7-o-d-glucuronic acid). the administration of these substances had a protective effect on the kidney and both the histological score and renal parenchyma nf-b expression were lower in comparison to the control group. il-6 were reduced compared to the control group in another study with the same experimental protocol. there have been a number of studies of plant derived substances, used in traditional chinese medicine. ligustrazine proved to be protective for the kidney as was demonstrated by the lower creatinine levels and the milder histopathological changes in comparison to the control group. in another study, the administration of 3 traditional chinese medicine substances (ligustrazine, kakonein, and panax notoginsenosides) resulted in reduced mortality and milder histopathological changes in the rat kidney. finally, the model of induction of acute pancreatitis through sodium taurocholate administration was used for the study of poly(adp-ribose) polymerase inhibition, through 3-aminobenzamide (3-ab) administration. the administration of 3-ab resulted in reduced mortality and a reduction in the increase of creatinine, tnf-, il-1b, and il-6, milder histopathological changes, and reduced mpo expression in the kidney. the half life of eugenol in the rat has been determined to be 18,3 hours; therefore, at 72 hours, most of the initial dose would have been cleared from the circulation. it is possible that a repeat administration of eugenol could further increase the therapeutic result. moreover, the time frame of our protocol reached 72 hours, which was not adequate for the complete evaluation of the effect of eugenol. indeed, a difference in the extent of kidney injury between the eugenol and control groups is first observed 48 hours after the onset of acute pancreatitis and it is greater at 72 hours. in conclusion, the administration of eugenol in a rat model of acute pancreatitis was protective for the kidneys in our experimental model. further research is necessary to determine the possible role of eugenol in the management of acute pancreatitis.

aim. acute pancreatitis is an inflammatory intra-abdominal disease, which takes a severe form in 1520% of patients and can result in high mortality especially when complicated by acute renal failure. the aim of this study is to assess the possible reduction in the extent of acute kidney injury after administration of eugenol in an experimental model of acute pancreatitis. materials and methods. 106 male wistar rats weighing 220350 g were divided into 3 groups: (1) sham, with sham surgery; (2) control, with induction of acute pancreatitis, through ligation of the biliopancreatic duct; and (3) eugenol, with induction of acute pancreatitis and eugenol administration at a dose of 15 mg/kg. serum urea and creatinine, histopathological changes, tnf-, il-6, and mpo activity in the kidneys were evaluated at predetermined time intervals. results. the group that was administered eugenol showed milder histopathological changes than the control group, tnf- activity was milder in the eugenol group, and there was no difference in activity for mpo and il-6. serum urea and creatinine levels were lower in the eugenol group than in the control group. conclusions. eugenol administration was protective for the kidneys in an experimental model of acute pancreatitis in rats.

PMC4739212

pubmed-447

periodontal health can be described as a dynamic state where the activity of proinflammatory/antimicrobial cytokines to control infection is optimally balanced by anti-inflammatory mechanisms to prevent unwarranted inflammation. in subjects susceptible to periodontal disease (pd), an imbalance of the inflammatory response results in excessive production of proinflammatory cytokines and the subsequent loss of periodontal attachment. on the other hand, furthermore, the release of tissue regenerating factors may contribute to periodontal regeneration by regulating the function of periodontal ligament cells, endothelial cells, and cementoblasts. in this setting, neurotrophin brain-derived neurotrophic factor (bdnf) has been reported to enhance periodontal tissue regeneration [4, 5]. bdnf is a member of the neurotrophin family which is expressed by vascular endothelium and osteoblastic, immune, and neuronal cells. bdnf is reported to be involved in the joint inflammatory process and its production is increased in response to proinflammatory cytokines. although a role for bdnf in periodontal regeneration has been proposed, no information is available concerning bdnf and periodontal disease. the aim of this study was to measure the levels of bdnf in periodontal tissues from patients with chronic periodontitis. the presence of polymorphisms rs6265 and rs4923463 of the bdnf gene and its correlation with inflammatory and clinical parameters were also assessed. twenty-eight patients with cp, treated at the periodontal clinic, school of dentistry, at universidade federal de minas gerais (ufmg, brazil), were enrolled in this study. patients in this study met the following inclusion criteria: previous history of cp, diagnosed according to previously described criteria: (1) exhibiting more than one tooth with probing depth higher than 5 mm, (2) exhibiting more than two sites with clinical attachment loss deeper than 6 mm, and (3) exhibiting lesions distributed in more than two teeth in each quadrant. patients who met the following criteria were excluded: (1) having a history of smoking, (2) use of antibiotic, (3) usage of anti-inflammatory and/or immunosuppressive medications during the 6 preceding months, and (4) a history of any systemic diseases (i.e., immunologic and autoimmune disorders, diabetes mellitus). the control group (hc) comprised 29 age and gender matched periodontally healthy patients enrolled for third molar removal surgery. periodontal examination was performed in both groups of patients, cp and hc, at the initial visit to determine probing depth (pd), clinical attachment loss (cal), and bleeding on probing (bop). measurements were performed full-mouth at 6 sites per tooth (mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual, and distolingual). all the measurements were performed by the same examiner. at the time of the examination a peripheral blood sample periodontal tissue samples from periodontal pockets or healthy oral mucosa extracted during surgery of impacted third molars were fixed in 10% buffered formalin, embedded in paraffin wax, and cut longitudinally (3 m). the sections were deparaffinized, rehydrated, and stained with h&e for evaluation of the inflammatory infiltrate. inflammatory cells were counted in four fields in two independent sections, using a light microscope (axioskop 40 zeiss; carl zeiss, gottingen, germany) at 400x magnification. the concentrations of the il-17a, bdnf, il-10, and tnf- and the chemokine cxcl10 were measured in periodontal tissues by enzyme-linked immunosorbent assay (elisa) using commercially available kits (r&d systems, minneapolis, mn, usa). the lower limit of detection for each cytokine was 15 pg/ml, 3.9 pg/ml, 5.5 pg/ml, 20 pg/ml, and 4.5 pg/ml, respectively, for il-17a, il-10, tnf-, bdnf, and cxcl10. the data were determined using a standard curve prepared for each assay and expressed as picograms of cytokine/chemokine per 100 mg of tissue. periodontal tissue samples were also used for determination of myeloperoxidase (mpo) activity, a neutrophil enzyme marker, as described earlier. the mpo activity in homogenized periodontal tissues was evaluated by enzymatic reaction and absorbance was measured at 450 nm. the mpo content was expressed as relative units calculated from standard curves based on mpo activities from 5% casein peritoneal-induced neutrophils assayed in parallel. total genomic dna was extracted from blood samples using qiaamp dna blood mini kit (qiagen, valencia, ca, usa) according to manufacturer's instructions. quality, integrity, and quantity of dna were analyzed by nanodrop spectrophotometer (thermo scientific, wilmington, de, usa). all amplifications were carried out in an abi 7900h thermal cycler (applied biosystems, foster city, ca, usa) using taqman genotyping master mix and following manufacturer's recommended amplification conditions. chi-square test analysis was used to test for deviation of genotype frequencies from hardy-weinberg equilibrium. the levels of cytokines in periodontal tissues and the frequency of gene polymorphisms were compared by the student's t-test and chi-square tests. the sample included in the current study was composed by age and gender matched groups. the clinical features pd, cal, and bop were significantly higher in the cp than in the hc group (p<0.0001) (table 1). the levels of il-17a, cxcl10, il-10, tnf-, and bdnf in periodontal tissues were greater in cp patients than in controls (figure 1). moreover, the mpo activity and the inflammatory infiltrate in the periodontal tissues, characterized by polymorphonuclear and mononuclear leukocytes, were significantly higher in the cp than in the hc group (figure 2). the bdnf and il-10 levels in periodontal tissues were negatively correlated (r=0.691, p=0.002), whereas no correlation between bdnf and il-17a, tnf-, cxcl10, or clinical parameters was observed (pd, cal, and bop). following the clinical investigation, the frequencies of polymorphisms (bdnf) were assessed in blood samples of hc and cp subjects (table 2). the frequency of these genotypes agreed with the hardy-weinberg equilibrium (p>0.05). the distribution of the bdnf polymorphisms was similar between the groups (table 2). we also investigated whether some of these polymorphisms were associated with worse clinical periodontal parameters. as shown in table 3, no differences in clinical parameters were found when comparing the genotypes. the levels of bdnf and the inflammatory mediators cxcl10 and tnf- were increased in gg genotype of bdnf rs6265 polymorphism (figures 3(a), 3(b), and 3(c)), but mpo levels did not alter significantly (figure 3(d)). in bdnf rs4923463 polymorphism the levels of bdnf and mpo did not differ, but the levels of cxcl10 and tnf- were higher in patients with aa genotype (figures 3(e)3(h)). a wide range of nonneural cells in peripheral tissues or in the immune system expresses neurotrophins and their receptors. thus, the mitogenic and immune regulatory functions of neurotrophins have been discussed [6, 1012]. the neurotrophin bdnf is reported to be involved in inflammatory reactions, and its production is increased in response to proinflammatory cytokines. the present study is the first to demonstrate that bdnf levels were increased in periodontal tissues from chronic periodontitis compared to healthy subjects. in agreement with our findings, bdnf was found in high levels in the plasma of patients with osteoarthritis and in patients with rheumatoid arthritis. while some authors reported that bdnf levels were significantly correlated with self-reported pain, others did not find association between bdnf and clinical parameters of arthritis. several studies analyzed bdnf rs6265 polymorphisms in psychiatric disorders [1618]. to date, few studies examined the rs4923463 polymorphism [16, 1820]. while one study found correlation between snp rs4923463 and attention-deficit/hyperactivity disorder, schizophrenia, and risk of suicide in bipolar disorder, another study did not find any correlation between schizophrenia and rs4923463 polymorphism. previously, two studies have evaluated the effects of bdnf polymorphisms in bone [21, 22], but there are no available studies in periodontal disease. in the present study we did not find differences in bdnf genotype distribution between patients with cp and controls. nevertheless, we found that subjects with gg (rs6265) genotype expressed higher levels of bdnf in periodontal tissues, in agreement with a previous report showing that bdnf-m66 variant alters intracellular trafficking and impairs bdnf secretion. on the other hand, rs6265 polymorphism interestingly, the snp rs6265 was reported as a phossnp, which means this snp regulates protein phosphorylation. the snp rs6265 affects substrate-kinase interaction between bdnf protein and chek2 kinase and regulates bdnf phosphorylation at site t62. subjects aa genotype carriers exhibited lower bone mineral density compared to g carriers. specifically, bdnf-v66 (major allele g at rs6265) transfection significantly increases expression of osteoblast specific markers (opn, bmp2, and alp) and promotes osteoblast differentiation and maturation in cell culture. an association of rs6265 with bone metabolism was also suggested in the largest meta-analysis involving 32,961 individuals of european and east asian ancestry. they found that homozygous minor allele a carriers (aa) have significantly decreased bmd compared to major allele g carriers (ga and gg). it has been reported that bdnf is able to induce an increase in il-10 expression. however, a negative correlation between the production of bdnf and il-10 was observed in samples from patients with periodontitis. il-10 can inhibit the release of proinflammatory cytokines from monocytes/macrophages and can therefore inhibit the lipopolysaccharide- and ifn--induced secretion of inflammatory cytokines (e.g., tnf-, il-1, il-6, cxcl8, and others). in periodontal disease, il-10 is thought to be associated with lower disease severity. previous studies demonstrated that bdnf induces periodontal tissue regeneration by activation of cementoblasts differentiation, vascular endothelial cell migration, and also has a positive effect on bone remodeling [5, 28]. this data together suggested that bdnf has a role in bone remodeling and any change in this neurotrophin levels could have an impact in bone repair. finally, we observe that cp subjects with gg (rs6265) and aa (rs4923463) genotypes demonstrated increased levels of tnf- and cxcl10. cxcl10 has several roles, such as chemoattraction of macrophages, t cells, nk cells, and dendritic cells. in addition, previous studies showed that exposure to bdnf substantially and synergistically enhanced tnf- levels in vitro, and tnf- preconditioning increased proliferation, mobilization, and osteogenic differentiation in vitro. we can hypothesize that the concomitant increase of bdnf, tnf-, and cxcl10 in patients with the gg genotype may be an attempt of the host to induce periodontal healing. so, maybe if these patients were examined after periodontal treatment, they could display higher and better levels of tissue regeneration compared to patient who do not exhibit the gg genotype. in conclusion, bdnf seems to be related to periodontal pathogenesis and also involved in tissue repair. the results obtained here provide a benchmark for future studies with a large cohort of patients to help strengthen and understand the influence of neurotrophins in periodontal disease.

brain-derived neurotrophic factor (bdnf) is a member of the neurotrophic factor family. outside the nervous system, bdnf has been shown to be expressed in various nonneural tissues, such as periodontal ligament, dental pulp, and odontoblasts. although a role for bdnf in periodontal regeneration has been suggested, a function for bdnf in periodontal disease has not yet been studied. the aim of this study was to analyze the bdnf levels in periodontal tissues of patients with chronic periodontitis (cp) and periodontally healthy controls (hc). all subjects were genotyped for the rs4923463 and rs6265 bdnf polymorphisms. periodontal tissues were collected for elisa, myeloperoxidase (mpo), and microscopic analysis from 28 cp patients and 29 hc subjects. bdnf levels were increased in cp patients compared to hc subjects. a negative correlation was observed when analyzing concentration of bdnf and il-10 in inflamed periodontium. no differences in frequencies of bdnf genotypes between cp and hc subjects were observed. however, bdnf genotype gg was associated with increased levels of bdnf, tnf-, and cxcl10 in cp patients. in conclusion, bdnf seems to be associated with periodontal disease process, but the specific role of bdnf still needs to be clarified.

PMC4283396

pubmed-448

we analyzed information about 634 immigrants from latin america seen at the tropical medicine unit of the ramn y cajal hospital in madrid, spain, during april 1989june 2008. we used 5 strict criteria for diagnosing vlm: 1) positive serologic test for toxocara sp. roundworm infection, performed by using a commercial elisa toxocara immunoglobulin (ig) g ridascreen (r-biopharm gmbh, darmstadt, germany), following the manufacturer s recommendations; 2) absolute peripheral blood eosinophil count>500 cells/mm; 3) exclusion of other parasites causing eosinophilia, such as intestinal nematodes, particularly strongyloides stercoralis (excluded by larval culture and serology by elisa igg), schistosoma sp., fasciola hepatica, trichinella spiralis, taenia solium, echinococcus granulosus, and cutaneous and blood microfilariae; 4) symptoms associated with vlm (respiratory signs, such as asthma, dyspnea, and eosinophilic pneumonia; dermatologic symptoms, including pruritus and recurrent urticaria; and abdominal symptoms, including abdominal pain and hepatomegaly); and 5) response to treatment with albendazole (1015 mg/kg/d in 2 doses orally for 5 days) assessed 6 months after treatment, decreased titers to toxocara sp. the most frequent countries of origin for patients were ecuador 221/634 (34.9%), bolivia 176/634 (27.8%), peru 71/634 (11.2%), and colombia 56/634 (8.8%). median age was 32 years (range 440 years); 421 (66.4%) patients were male. the median number of months from arrival in spain to first consultation at the tropical medicine unit was 19 months. concomitant serologic results positive for toxocara sp. roundworm infection and eosinophilia were found in 28 (4.4%) patients; 606 patients were excluded. of these 28 patients, 11 were excluded because of other concomitant parasitic infections that also can cause eosinophilia: 8 patients had positive elisa results for s. stercoralis nematodes (not detected in fecal samples or larval culture); 1 had ascaris lumbricoides eggs in feces; 1 had a positive indirect hemagglutination result but negative elisa result for e. granulosus tapeworm; and 1 had a positive elisa serologic result for t. spiralis nematodes. only 4 of the 5 remaining cases fulfilled the strict inclusion criteria (table); 1 patient was asymptomatic. after 6 months of treatment with albendazole, titers for toxocara sp. roundworm infection and eosinophil count decreased, and symptoms improved or resolved for the 4 patients. all patients were treated with albendazole (1015 mg/kg/d in 2 doses orally for 5 days). clinical toxocariasis is rarely diagnosed in western countries as previously described despite evidence of environmental exposure (1). results of seroprevalence surveys performed in healthy adults in france were positive for 2%5% of persons in urban areas, compared with 14%37% in rural areas (2). in latin america, rates vary from 1.8% to 51.6% (3,4). however, literature references to vlm imported by immigrants are scarce (5), and the disease may be underdiagnosed in the immigrant population, partly because of nonspecific symptoms and the limitations of serologic diagnosis. in our study, toxocariasis is a common cause of eosinophilia in peripheral blood, although its absence does not exclude infection by toxocara sp. roundworm infection without eosinophilia (6); similarly, 27% of patients with high antibody titers had eosinophil counts within the reference range (7). by including only patients with eosinophilia, our study applied more stringent criteria. thus, 28 (90%) of 31 patients who had positive serologic results showed an elevated eosinophil count, in accordance with previously described high toxocara sp. roundworm seroprevalence (< 68%) in patients with eosinophilia of unknown cause (8). eleven of the 28 patients with positive serologic results for toxocara sp. roundworm and eosinophilia also had positive serologic results for other parasites that cause eosinophilia. one patient who was infected with a. lumbricoides roundworm had asthma, hepatomegaly, and pruritus. the latter is not usually associated with this parasite, which suggests possible co-infection. serologic tests for toxocara sp. roundworm infection should be interpreted with caution because commercial elisa kits that use excretory and secretory antigens derived from second-stage larvae of toxocara sp. roundworms exhibit a sensitivity of 91% and a specificity of 86%; cross-reactivity has also been described with other nematode infections. the positive serologic results for t. spiralis nematodes and e. granulosus tapeworms may have been caused by cross-reactivity (9). these patients had asthenia and asthma, respectively, and symptoms resolved after treatment with albendazole. eight patients with strongyloides antibodies were also excluded; however, this finding does not exclude co-infection by both parasites. finally, a limitation of the study was that we could not definitively exclude cryptic strongyloidiasis for 12 patients because of the difficulty in finding s. stercoralis threadworms in feces and because detection of strongyloides antibodies was not possible. this study illustrates the difficulties in diagnosing vlm in immigrants from tropical and subtropical areas of latin america because only a very small proportion of patients in the series (n=4) had vlm. the most common symptoms were respiratory (3/4); 2 patients had asthma-like syndrome and 1 had chest pain followed by abdominal pain (2/4). typical manifestations of vlm are abdominal symptoms (pain, hepatomegaly) and respiratory symptoms (severe asthma, eosinophilic infiltrates). in addition to this, evidence points to toxocara sp. roundworm infection as a risk factor for asthma in some populations (11,12). albendazole is the treatment of choice for vlm; for practical purposes, it could be recommended for presumptive treatment in immigrants from latin america with eosinophilia in whom strongyloidiasis is suspected (13). however, the superiority of ivermectin over albendazole has been documented in the treatment of chronic strongyloidiasis (14). vlm may be difficult to diagnose, especially in immigrants from regions in latin america where polyparasitism is endemic. positive serologic test results, marked eosinophilia, absence of other helminthic infections, compatible clinical signs, and disappearance of symptoms after specific treatment can help establish a vlm diagnosis, especially in areas of low parasitism. vlm should be included in the differential diagnosis of eosinophilia in immigrants (children and adults) from tropical areas if respiratory or abdominal symptoms are evident. albendazole is an effective and relatively safe drug that could be used to treat suspected vlm and other concomitant nematode infections, including cryptic s. stercoralis threadworm infections. empirically described treatment may lead to resolution of clinical symptoms, even though ivermectin is a better treatment for chronic strongyloidiasis.

to determine whether increased migration is associated with an increase in incidence of toxocariasis (visceral larva migrans), we analyzed clinical data obtained from immigrants from latin america. although infection with toxocara sp. roundworm larvae is distributed worldwide, seroprevalence is highest in tropical and subtropical areas.

PMC3381370

pubmed-449

protein phosphorylation and dephosphorylation are central events in cell recognition of external and internal signals, leading to specific responses. while protein kinases transfer a phosphate group from atp to a protein (i.e., phosphorylate), protein phosphatases catalyze the removal of phosphate groups from specific residues of proteins (i.e., dephosphorylate) [1, 2]. the balance between the antagonistic activities of protein kinases and phosphatases are responsible for many cellular functions, including metabolic pathways, cell-cell communication, proliferation, and gene transcription. the complete genome sequencing of various microorganisms made it possible to assemble the kinome and phosphatome of a few trypanosomatids [4, 5]. these strategies have brought new perspectives of researches in the areas of biochemistry, physiology, and genetics, providing knowledge about the microorganisms ' life cycles, as well as predicting diagnostic biomarkers, novel drug targets and vaccine candidates against parasitic infections. parasites engage a plethora of surface and secreted molecules in order to attach and enter mammalian cells. some of these molecules are involved in triggering specific signaling pathways both in the parasite and the host cell, which are critical for parasite entry and survival. plasma membranes of cells contain enzymes that are oriented with their active sites facing the external medium rather than the cytoplasm, which are important for host-parasite interactions [7, 8]. in the case of an ectoenzyme other criteria can be included as: (1) the enzyme has to act on extracellular substrate, (2) cellular integrity is maintained during enzyme activity, (3) the products are released extracellularly, (4) the enzyme is not released to the extracellular environment; and (5) the enzyme activity can be modified by nonpenetrating reagents [7, 8]. supporting this idea, the presence of surface-located phosphatases, called ecto or extracytoplasmic phosphatases have been characterized in several microorganisms. however, the physiological roles of these enzymes in these cells are not well established yet. in eukaryotes, thus, catalytic signature motifs and substrate preferences classified these proteins into four major groups: phosphoprotein phosphatases (ppps), metallo-dependent protein phosphatases (ppms), aspartate-based phosphatases with a dxdxt/v motif (the members of these three groups are ser/thr specific phosphatases) and the distinct group of protein tyrosine phosphatases (ptps). protein tyrosine phosphatases belong to three evolutionarily unrelated classes: protein tyrosine phosphatases (ptps), cdc25 and low molecular weight phosphatases (lmw-ptps), which have a common motif (cx5r) in their catalytic sites. the classical ptps are classified, depending on the presence or absence of transmembrane domains, into receptor or nonreceptor type phosphatase groups. the use of inhibitors, divalent cations, metal chelators and different ph range has also been an important tool for classification of these enzymes. likewise, phosphatases may be acid or alkaline according to their ph range for activity. the optimum ph for acid ectophosphatases lies on the acid range (ph values between 4.5 and 5.5), while the optimum ph for alkaline ectophosphatases lies on the alkaline range (ph values between 8.0 and 9.0) [9, 10]. the inhibitors classically used include: phosphotyrosine phosphatase inhibitors ammonium molybdate and sodium orthovanadate; acid phosphatase inhibitor sodium fluoride (naf); secreted phosphatase inhibitor sodium tartrate; alkaline phosphatase inhibitor levamisole and phosphoserine/threonine phosphatases inhibitors okadaic acid and microcystin-lr [1113]. these enzymes may provide microorganisms with a source of inorganic phosphate by hydrolyzing phosphomonoester metabolites [1315] protect them upon entering the macrophage by suppressing the respiratory burst, as well as play a role in cell differentiation, infection of host cells [1820] and protecting the cells from acidic conditions by buffering the periplasmic space with phosphate released from polyphosphates. some protein phosphatases have been described as being active towards low molecular weight nonproteic phosphoesters, such as alkyl and aryl phosphates, including the phosphotyrosine analog, p-nitrophenylphosphate. from a general standpoint, the surface accessibility of ectophosphatases, along with protein phosphorylated on serine/threonine/tyrosine residues at the cell surface make this set of enzymes a key tool for the survival of pathogens in hostile environments and escaping the host immune responses [19, 2224]. in this review, we describe the role of ectophosphatase activities in host-parasite interactions, particularly ectophosphatases in parasitic protozoa and fungi. little is still known about the physiological role of protein phosphatase activity in trypanosomatids, even though the first demonstration of this activity in trypanosoma brucei and t. cruzi took place in 1972. the kinetoplastid parasites have complex life cycles and some of their life forms are difficult to grow in culture, which may represent a problem for studying ectophosphatases. pathogenic trypanosomatids have at least two different host environments in their life cycles, an insect vector and a mammal. also, each trypanosomatid genus has different abilities to survive and reproduce in such hosts. t. cruzi invades and replicates in many cell-types, including macrophages, fibroblasts and myocytes. t. brucei is an exclusively extracellular parasite that resides in the bloodstream of the mammalian host. as the life cycles of these parasites take place through widely different environments, frequent and substantial adaptive changes are required in many cell processes, resulting in changes in gene expression, protein levels and protein modifications [26, 27]. along with those, cell surface components play a key role in the survival of protozoan parasites in hostile environments and in confrontation with host immune responses. since, these flagellates have an unusual composition of phosphatases with the ptp family being greatly reduced while the stp family is expanded by comparison with human phosphatases. the low similarity to their vertebrate counterparts indicates that these enzymes may be potentially suitable targets for development of potent inhibitors with minimal effects on the physiology of mammalian hosts. under these conditions, ectophosphatases play an important role in the interaction of cells with their surroundings, especially because their catalytic sites face the extracellular milieu. ecto-phosphatases has been reported in some protozoa parasites, such as t. rhodesiense, t. congolense, t. brucei [30, 31], t. cruzi, t. rangeli [13, 33], some leishmania species [11, 34], herptomonas muscarum muscarum, phytomonas spp. [36, 37], entamoeba histolytica, giardia lamblia and trichomonas vaginalis. in general, these ectoenzymes are usually reported to have optimum activities in the acidic ph range, and they are therefore also known as membrane-bound acid phosphatases [28, 29]. in trypanosomatids, the low optimum ph and the surface location of these enzymes suggest its role in an acidic microenvironment and/or a close relationship with lysosomal digestion, possibly reflecting an adaptation of the parasite to the intracellular or phagosomal environment [41, 42]. cloning and purification of an acidic phosphatase in t. brucei suggest that these enzymes may represent a new ectophosphatase class lacking homology to other known phosphatases. it seems that these proteins are related to the regulation of t. brucei development, since these acidic phosphatases are expressed in bloodstream forms, but not in the insect procyclic form. likewise, an ectophosphatase activity on the surface of intact procyclic and bloodstream forms of t. brucei was demonstrated by fernandes et al. [43, 44]. similarly, an ectophosphatase was also cloned and purified in l. mexicana, where it was located in the endosomal/lysosomal compartment between the flagellar pocket and the nucleus in wild-type promastigotes, and the overexpression of this protein leads to its abundant exposure on the cell surface [45, 46]. the same was seen with membrane-bound acid phosphatase from the bloodstream form of t. brucei, where the enzyme is supposed to participate in the maintenance of endocytosis/exocytosis and in differentiation to the insect stage. the wide distribution of acid phosphatases on the cell may reflect some physiological adaptation for parasite survival within the host. in this scenario, ectophosphatase activities were identified at the cell surface of all t. cruzi development stages: epimastigote, trypomastigote and amastigote forms [18, 32]. it seems that in amastigote forms these enzymes are magnesium-dependent and can hydrolyse phosphoaminoacids and phosphoproteins under physiological conditions [18, 32]. this behavior could facilitate the interaction between parasite and host cells, once t. cruzi phosphatases leads to dephosphorylation of proteins important in the signal transduction pathway or cycle regulation of this protozoan parasite. supporting this idea, y strain presents mg-dependent ectophosphatase activity, while colombiana strain expresses mg-independent activity. among other characteristics, members of these two groups parasites from the colombiana strain appeared to be more infective to myoblasts than those from the y strain, while the latter is more infective towards macrophages than the parasites of the colombiana strain. intriguingly, platelet-activating factor (paf), a phospholipid mediator involved in differentiation cellular in t. cruzi, induces the secretion of an ectophosphatase in these parasites, associating this event with the infectivity of the parasite. addition of sodium orthovanadate (a protein tyrosine phosphatase inhibitor) in the interaction medium from l. amazonensis and macrophages significantly increased parasite binding and internalization, suggesting that leishmania induces tyrosine phosphorylation [24, 51]. under these conditions, protein tyrosine kinase-linked pathways regulate the leishmania promastigote invasion, which ectophosphatase activity upregulate l. amazonensis binding ligands for macrophage receptors and intracellular survival within these cells [24, 51, 52]. it seems that during macrophage infection by leishmania the parasite attenuates map kinase signaling, as well as c-fos and inos expression in macrophages, stimulating the phosphotyrosine phosphatase activity in these cells [5355]. possibly by other intracellular pathogens as a strategy of the parasites to interact and survive within their hosts. in l. donovani tyrosine phosphatase activity was also detected, suggesting that tyrosine phosphorylation occurs, though not via receptor tyrosine kinase or tyrosine kinase-like activities but very likely due to the activity of atypical and/or dual specific kinases. futhermore, a membrane-bound ptp has been describe in l. major metacyclic promastigote forms, which is translocated to the cytoplasm in promastigotes. in spite of the increased level of the molecule in metacyclic promastigotes compared to the procyclic forms, the specific activity of the enzyme was lower in metacyclic than in procyclic promastigotes. interestingly, a protein tyrosine phosphatase, has been identified in l. major (lmptp1) that allows amastigotes forms to survive in mice. although its biological function is unclear, this may be an important factor in virulence, enabling the invading pathogen to survive in a host. ecto-phosphatase isolated from l. donovani promastigotes inhibits the production of superoxide anions in intact human neutrophils. this activity could contribute to the survival of the parasite within the host, we can hypothesize that parasites with greater ectophosphatase activity would be more resistant to oxidative bursts from the host's immune system. the role of ectophosphatases in invasive amoebiasis is still unknown, even though two acid phosphatases have been characterized in these parasites: a membrane-bound acid phosphatase (map) [58, 59] and a phosphatase that is secreted to the culture medium (sap), as well as to the cell interface in amoebic liver abscess [60, 61]. these enzymes may be associated with cellular adhesion processes, since the invasive e. histolytica showed much higher ectophosphatase activity when compared to the noninvasive counterpart and the free-living e. moshkovskii. the fungal cell wall is a compact albeit dynamic structure that plays important roles in several biological processes determining cell shape, morphogenesis, reproduction, cell-cell and cell-matrix interactions, osmotic and physical protection. several different cell wall components have been characterized such as specific enzymatic activities, heat-shock proteins, glycosphingolipids (gsl), melanin, histone and integrin-like proteins. even though the roles of ectophosphatases in fungi are still largely unknown, the cellular distribution of ectophosphatases, together with their ability to interfere with physiologic processes through the removal of phosphate groups of regulatory proteins, suggest a task for these molecules during the infection of host cells. the presence of surface-located acid phosphatases, called ecto or extracytoplasmic phosphatases has been demonstrated in nonpathogenic yeast saccharomyces cerevisiae and in pathogenic species such as candida albicans, candida parapsilosis [19, 65], sporothrix schenckii, aspergillus fumigatus, fonsecaea pedrosoi [22, 68], cryptococcus neoformans and pseudallescheria boydii. futhermore, most of the phosphatases synthesized under pi-limiting conditions are either located on the extracellular medium or are associated with the plasma membrane or cell wall [15, 22]. corroborating with this hypothesis, kneipp et al. demonstrated that conidial forms of f. pedrosoi has an ectophosphatase activity modulated by exogenous phosphate. it seems that in f. pedrosoi, conidial cells that were cultivated in a pi-depleted medium had an ectophosphatase activity significantly higher than that of fungal cells grown in the complete medium. these cells expressing high phosphatase activity were significantly more capable of adhering to epithelial cells and fibroblasts than fungi expressing basal levels of enzyme activity. it was then proposed that the removal of phosphate groups from surface proteins in host cells could result in conformational transitions and in an attenuated electrostatic repulsion between fungal and epithelial cells. probably, the removal of inorganic phosphate could therefore expose at the host surface additional sites for interaction with infectious agents. it seems that ectophosphatases may contain adhesive domains that could directly promote the attachment of fungal cells to their hosts, therefore functioning similarly to the well-characterized microbial adhesins. probably, they could regulate the functional activation of surface adhesins, which would be the key structures mediating fungal attachment. intriguingly, known activators of signaling pathways and cell differentiation, paf and propanolol, promoted an enhancement of f. pedrosoi ectophosphatase activity suggesting that f. pedrosoi ectophosphatase may be considered a surface marker for morphological transition and infection. in the fungus c. neoformans a thick capsule composed of neutral and charged polysaccharides, can be modulated by different environmental conditions, including the sites of fungal infection inside the host. it seems that the molecules coating the outer layer of the cell wall could be relevant during the interaction of poorly encapsulated cells with host tissues. ectoenzymes possibly have their accessibility to external receptors masked by the capsule polysaccharides of c. neoformans, diminishing the potential of these structures to be surface molecules influencing the interaction between fungal and host cells.. however, the levels of enzyme activity, varied considerably among the isolates and no correlation between enzyme activity and capsular size or serotype was observed. evidences show that isolates with capsular polysaccharides of the same serotype varied greatly in ectophosphatase activity. in addition, the strain, which is poorly encapsulated, removed phosphate groups much more efficiently than strain, which expresses a large capsule, indicating that the presence of the capsule impairs enzyme activity in this process. on the other hand, some encapsulated strains presented levels of ectophosphatase activity higher than that observed in the acapsular mutant. moreover, some strains that had very similar levels of enzyme activity, but differ greatly in capsule size were also found. taken together, these data indicate that differences observed in enzyme activity should be derived from natural variation of ectophosphatase expression in different c. noeformans strains. corroborating with the previous findings, kiffer-moreira et al. investigated three different isolates of c. parapsilosis, including a laboratory-adapted strain (cct 3834) and two recently isolated strains (rfo and h297). they observed that the rfo strain exhibits the highest levels of enzyme activity and adhesion to cho cells, followed by the h297 and the cct 3834 isolates. pretreatment of yeasts with the irreversible inhibitor sodium orthovanadate caused a significant reduction in the ability of these fungi to attach to epithelial cells. although sodium orthovanadate can affect different biological processes and inhibit atpases involved in cation transport [72, 73], its major biological activity in living cells occur on the cell surface, as the oxidation reduction reactions that take place in the cytoplasm diminish its inhibitory effect. similarly, c. albicans isolate from oral cavities of hiv-infected children (hiv) present an ectophosphatase activity significantly higher than the hiv-negative children (hiv). the c. albicans yeasts from hiv patients showed higher indices of adhesion to epithelial cells, which suggests that the activity of fungal acidic surface phosphatases may contribute to the early mechanisms required for disease establishment. it is reasonable the hypothesis that ectophosphatases represent a virulence marker, since these enzymes represent part of the outer layer and are linked to cell differentiation and host cell-pathogen interactions. the balance of phosphorylation-dephosphorylation of serine, threonine and tyrosine residues modulates signaling pathways critical for determining the outcome of multiple cellular functions. further studies are warranted to resolve the roles of ectophosphatases in host-pathogen interactions, as well as the possible correlations between the expression of these enzymes and the clinical manifestation of the diseases.

the interaction and survival of pathogens in hostile environments and in confrontation with host immune responses are important mechanisms for the establishment of infection. ectophosphatases are enzymes localized at the plasma membrane of cells, and their active sites face the external medium rather than the cytoplasm. once activated, these enzymes are able to hydrolyze phosphorylated substrates in the extracellular milieu. several studies demonstrated the presence of surface-located ecto-phosphatases in a vast number of pathogenic organisms, including bacteria, protozoa, and fungi. little is known about the role of ecto-phosphatases in host-pathogen interactions. the present paper provides an overview of recent findings related to the virulence induced by these surface molecules in protozoa and fungi.

PMC3095255

pubmed-450

human neurocysticercosis (ncc) is caused by larval stage of zoonotic tapeworm taenia solium (pork tapeworm) which remains a major public health problem in developing and some developed countries. porcine cysticercosis is the cause of human taeniasis and neurocysticercosis is a consequence of taeniasis. based on the available information, a very conservative and rough economic estimate indicates that the annual losses due to porcine cysticercosis in 10 west and central african countries amount to about 25 million euros.. also stated that, in china, the amount of pork discarded in the whole country due to cysticercosis annually has been estimated as 200,000,000 kg with a value of more than us $120,000,000. stated that notably data on myanmar are lacking, although there are several reports of porcine cysticercosis based on meat inspection in the abattoirs in neighboring countries, 9.3% in india, 32.5% in nepal, 5.4% in china, 0.022.63% in indonesia, and 0.04 to 0.9% in vietnam. although most of myanmar culinary habits are based on thorough cooking, new food style such as barbecue and dishes based on raw or undercooked pork or pork product becomes popular among customers. moreover, small-scale pig husbandry has become one of the major sources of income in myanmar farmers. so it may be high risk of getting food-borne zoonotic diseases according to the new food style and traditional husbandry method. due to lacking of information on porcine cysticercosis in myanmar up to now, it is important to investigate the prevalence and associated risk factors. nay pyi taw area, the capital of myanmar, has big population of pigs (about 200,000 pigs) to support the demand of pork consumption in this area. most of the pig farmers are smallholders and most of pig husbandry systems are free ranging or semi-intensive with lack of proper sanitation. one of the main obstacles to control the t. solium infections is the lack of adequate epidemiological data on cysticercosis/taeniasis. therefore, the objectives of this community-based study were to investigate the prevalence of porcine cysticercosis and associated risk factors in pigs within study area. moreover, findings of this study will assist to develop the control strategies of porcine cysticercosis for the public health aspect. the cross-sectional studies were conducted from january to march and june to july 2014, to investigate the prevalence of taenia solium cysticercosis in slaughtered and farmed pigs within pyinmana, lewe, and tatkon townships, nay pyi taw area. it is located between latitude 1945n and longitude 966e and with climate data; the altitude is 115 m above sea level, annual rainfall is 115 mm, and annual temperature is 21.232.5c. an expected prevalence of 30% with a confidence level of 95% was used in this unit. in this study, 300 slaughtered pigs and 364 farmed pigs from the study area were examined although calculated samples were 298 and 323, respectively (table 1). blood collected from the jugular vein of farmed pig was conducted for the seroprevalence and a structured questionnaire with both closed and open-ended questions was administered to owners to obtain management practices in pig husbandry. piglets younger than two months, pregnant sows, and nursing sows with litters less than two months old were excluded from this study to overcome the stress which causes adverse effect in animals. meat inspection was carried out as described by boa et al. in the three slaughterhouses of these townships. there were 300 randomly selected pigs recruited and 9 different muscles (tongue, masseter, brain, shoulder, diaphragmatic, heart, skeletal, fore limb, and hind limb muscle) from each pig in meat inspection. briefly, long and parallel incision into the masseter muscles on both sides of face in an upward direction was made. a deep longitudinal incision covering about 3/4 the thickness of the tongue and covering the whole length of the tongue was made to examine the cysts. after opening the pericardium, the heart was also visually examined for the presence of cysts. the heart was cut open and a deep (3/4 the thickness of septum) incision into the septum was made to expose any metacestodes. all the other muscles were viewed, palpated, incised by surgical blade, and visually examined. the pig was kept under restraint at standing position and blood samples were obtained from the external jugular vein by using sterile disposable syringes and put into vacutainers with clot activators. those vacutainers were kept in cold boxes with ice and transported to department of pharmacology and parasitology, university of veterinary science, nay pyi taw, and allowed overnight at 4c to clot. to obtain serum, the clear sera were transferred to 1.5 ml microvial tubes and stored in labeled wails and kept at 20c until analysis. detection of igg antibody of t. solium cysticerci was carried out by using antibody-elisa kit (novatec immundiagnostica gmbh co., belgium) according to manufacturer's instruction. briefly, all thawed samples were diluted as 1+100 with igg sample diluent (phosphate buffer) before assaying. the 100 l controls and diluted samples were dispensed into their respective wells and the foil was covered. after incubation for 1 hour at 37c and the foil being removed, the contents of the wells were aspirated and washed three times with washing solution. and the 100 l protein a conjugate (horseradish peroxidase) was dispensed into all wells except a1 and covered with foil and incubated for 30 min at room temperature. after washing three times, 100 l tmb (3,3,5,5-tetramethylbenzidine) substrate solution was dispensed into all wells and incubated for exactly 15 min at room temperature in the dark. the reaction was stopped by adding 100 l stop solution (0.2 m h2so4). the absorbance was determined at 450/620 nm using an elisa reader (stat fax). in each elisa kit testing, there are two cut-off controls (c1 and d1). the mean absorbance of these cut-off controls was used as cut-off value. samples are considered positive if the absorbance value is higher than 10% over the cut-off and samples are considered negative if the absorbance value is lower than 10% below the cut-off. the sensitivity and specificity of these kits to diagnose swine cysticercosis are 93.8% and>95%, respectively. a questionnaire was developed and used to collect information on hypothesized risk factors and other related pieces of information from sampled pig owners. households in each township were selected by using the snowballing technique from those farmers willing to participate in the study. it is a technique for developing a research sample where existing study subjects recruit future subjects from their acquaintances. the questionnaire interviewed data were analyzed for the relationship between the prevalence of t. solium cysticercosis and hypothesized risk variables such as age, gender of pigs, husbandry system, feed type, environment of pig farm (accessibility of human feces), personal hygiene of owners, pork consumption, cooking and eating habit of pork, use of anthelmintics in pigs and owners, and knowledge on taeniasis. they were examined for testing its significance by pearson chi-square test at =0.05. seroprevalence of porcine cysticercosis in farmed pigs was 15.93% (58/364) in the study area. prevalence of households with pigs infected with t. solium cysticerci by ab-elisa examination was 23.15% (47/203 households). the households with porcine cysticercosis in pyinmana, lewe, and tatkon were 0/12 (0%), 13/124 (10.48%), and 34/67 (50.75%), respectively. all the infected pigs presented parasites located in the tongue. only in one pig, the prevalence in slaughterhouses of pyinmana, lewe, and tatkon townships was 22% (44/200), 23.33% (7/30), and 28.57% (20/70), respectively. univariate analysis of hypothesized risk factors of gender (or=3.0; 95% ci=1.75.4), increased age (or=2.3; 95% ci=1.24.2), husbandry system (or=5.1; 95% ci=2.411.2), feed type (or=16.9; 95% ci=2.3124.3), no hand washing habit before feeding (or=31.5; 95% ci=4.3230.9), not using anthelmintic in pigs (or=11.9; 95% ci=5.028.5) and owner (or=2.5; 95% ci=1.44.4), and pork consumption of owner (or=37.4; 95% ci=9.0156.1) was significantly associated with cysticercus cellulosae infection (p<0.05). the distribution and odds ratio of significant risk factors concerning porcine cysticercosis are shown in table 2. in southeast asia, pigs are an important source of food and economic important for smallholder farmers. older pigs may be penned or tethered although common raising practice of pigs is freely roaming in the village. in myanmar, most of the pig farmers are smallholders and practice as free-range or backyard farming. in myanmar, most of the pig farmers usually keep the weaned pigs until six to eight months of age and then send to slaughterhouse. in the village, every household keeps at least one pig not only for table waste feeding to pigs but also for extra income. most farms are having the habit of feeding waste materials such as swill and kitchen leftover, broken rice, rice bran, groundnut meal, sesame meal and local forage, and poor sanitation. the present study is the first report of t. solium cysticercosis in pigs in myanmar. pigs in the study area positive for cysticercosis have been exposed to t. solium eggs. among the 17 hypothesized risk factors, the gender of pigs (being female) was significantly associated with porcine cysticercosis in this study. it can be explained that female pigs were for kept long time for breeding purpose than male and so they have more risk to get exposed to t. solium eggs. however, jayashi et al. reported that gender was not a significant risk factor for porcine cysticercosis. the present study demonstrated that the older the pigs, the greater the chance to get infection. these results are in agreement with those reported by pouedet et al., jayashi et al., sarti et al., garca et al., and pondja et al. older pigs might also have greater chance to get exposed to t. solium eggs than younger ones. they might have much time to develop cyst and trigger the production of circulating antibodies. besides, it could be possible that younger pigs are protected during their first months of life against parasite infection, due to the presence of maternal cysticercus antibodies and they become susceptible later after the slow clearance of those antibodies. the result showed that pigs from households practiced semi-intensive system (the pigs are allowed to roam freely in the environment and only panned or tethered at feeding time and night) were more likely to have porcine cysticercosis than intensive (the pigs are kept in the backyard or corral and not allowed to roam) pigs. therefore, semi-intensive management system represented as an important risk factor for porcine cysticercosis in the study area as the pigs in this practice could access the infected human faeces. accessibility of infected human faeces is the main source for porcine cysticercosis [17, 19, 20]. among the feed types used in pig farms, this might be contaminated with t. solium eggs from infected food preparers of swill collected houses. so the collected swill should be cooked thoroughly before feeding to prevent infection including cysticercosis. use of anthelmintic in pigs and owners was significantly associated in this study. by interviewing the farmers and township veterinary officers, although ivermectin can not kill any larvae of cestode, albendazole can kill these larvae. although all the farmers wash their hands after feeding the pigs, only 21.2% famers (43/203) wash their hands before feeding. all cysticercosis positive samples were from those who do not practice hand-washing habit. pork consumption of owners is also one of the risk factors in survey of porcine cysticercosis. nine hypothesized risk factors not included in analysis were breed of pigs, place of purchase, presence of latrine, hand-washing after feeding the pigs, source of water for pigs, cleanliness of water, knowledge on taeniasis and cysticercosis, and occurrence of cyst in pork. in this study, all pigs are indigenously bred. all farmers have latrines using water, but the children do not use latrine and are used for defecation out of latrine. some farmers washed the hands before feeding the pigs and all farmers washed their hands after feeding. all farmers did not have the knowledge on taeniasis and cysticercosis and they have never seen the cysts in the pork in the study area. the presence of zoonotic agent, cysticercus cellulosae, may depend on intrinsic factors: age, gender, and extrinsic factors: pig husbandry system, hand-washing habit of owner, use of kitchen waste as pig feed, not using anthelmintic in pigs and owners, and pork consumption of owner in the study area. presence of this infection is of public health importance because it may lead to the occurrence of neurocysticercosis in human. although the occurrence of human neurocysticercosis has not been reported yet in myanmar, all public should take awareness of potential risk factors due to the prevalence with high percentage observed in this study. myanmar has no national monitoring program for t. solium cysticercus spp. in these animals yet. therefore, it is advisable to monitor whether there is high or low prevalence of t. solium cysticercosis in the whole country. it could also be suggested that confinement housing system should be developed in pig industry of myanmar to efficiently prevent porcine cysticercosis. for practicing sanitary and culinary habit, thorough cooking education programs should also be implemented for both swine breeders and consumers so as to prevent taeniasis in human and porcine cysticercosis and also other zoonotic helminth diseases in myanmar. this prevalence with relatively high percentage of porcine cysticercosis (15.93%) in ab-elisa and 23.67% in slaughtered pigs indicates the presence of human taeniasis and it also leads to the associated risk of human cysticercosis and neurocysticercosis.

cross-sectional surveys were conducted to determine the prevalence and associated risk factors of taenia solium cysticercosis in pigs within nay pyi taw area, myanmar. meat inspection in three slaughterhouses, elisa test, and questionnaire surveys were conducted in this study. three hundred pigs were inspected in slaughterhouses and 364 pigs were randomly selected and examined from 203 households from three townships in nay pyi taw area. the prevalence of porcine cysticercosis in meat inspection was 23.67% (71/300). seroprevalence of t. solium cysticercosis in pigs in the study area was 15.93% (58/364). significant associated risk factors with t. solium cysticercosis were gender (or=3.0; 95% ci=1.75.4), increased age (or=2.3; 95% ci=1.24.2), husbandry system (or=5.1; 95% ci=2.411.2), feed type (or=16.9; 95% ci=2.3124.3), not using anthelmintics in pigs (or=11.9; 95% ci=5.028.5), not using anthelmintics in owner (or=2.5; 95% ci=1.44.4), no hand-washing before feeding (or=31.5; 95% ci=4.3230.9), and pork consumption of owner (or=37.4; 95% ci=9.0156.1) in the study area. this is the first report of porcine cysticercosis in myanmar.

PMC4590850

pubmed-451

organized by nei dan school, european school of taiji quan and by the tao and science studies centre, under the aegis of the provincia di bologna and the partnership of asi (alleanza sportiva italiana) and luni editrice. goodwill of the conference is to act as a starting point to develop a net of experts, doctors and scientists, who will investigate the dynamic interactions between spiritual insight and scientific analysis to come to the creation of a new paradigm of modern science. science, philosophy, medicine and body arts of the ancient east are reunited together to create a new ecological awareness of body and mind. modern science, which paved the way for an outlook of reality considering the universe as a whole, in which all parts and phenomena are connected among them, can be integrated to the ancient eastern wisdom for the control of the mind and to the body arts (taiji quan, qi gong, yoga) to develop a new ecological awareness, an awareness based on nature and on the dynamic relation among all living creatures. the conference was divided in two sections: a gathering of experiences, of paths where science meets metaphysics to have a new language born, made of images and movement, and a panel to understand how taiji quan, the arts of movement and meditation, can prolifically meet cognitive sciences and neurosciences. speakers during the tao and science conference were: andrea pezzi (presenter and tv author); professor edwin l. cooper professor, laboratory of comparative neuroimmunology, department of neurobiology, david geffen school of medicine, university of california los angeles, editor-in-chief, the journal: evidence based complementary and alternative medicine, oxford university press; professor carlo ventura (professor of molecular biology at the faculty of medicine, university of bologna; director of the laboratory of molecular biology and stem cell bioengineering, national institute of biostructures and biosystems by the institute of cardiology of santorsola malpighi hospital in bologna); professor angelo marzollo (professor of systems theory, faculty of sciences, university of udine; vice general secretary of the international centre for mechanical sciences; unesco ex-person in charge for mathematics and now consultant); professor giovanni sambin (professor of logic mathematics, university of padova); dottor matteo luteriani (publisher, journalist and master of martial arts), dottor massimo mori (doctor, poet and master of taiji quan) and eng. flavio daniele (writer and master of taiji quan) the following guests took part in the conference through their representatives: professor james k. gimzewski (department of chemistry and biochemistry, university of los angeles ucla; director of the pico lab laboratory at ucla); professor aldo stella (teacher of medical psychology, university of urbino; teacher of psychology of cognitive processes, university for foreigners of perugia); professor carmelo di stefano (teacher of teaching didactics of the adapted movement and sports activity, faculty of motor sciences, university of bologna). carlo ventura, md, phd: western science has long been entangled with increasing reductionism and the development of field restricted approaches to understand cell biology and the molecular basis of disease. it is now becoming increasingly evident that reductionism is a remarkable bias in pursuing some of the major goals of modern biology and medicine. complex problems, including cell growth and differentiation under normal or malignant conditions (cancer), and the adaptive mechanisms of humans to multiple changes in cell signaling networks now pose the need for holistic approaches both at the molecular biology and medical levels. such a requirement is even more urgent in spite of the emerging interest in stem cell biology, since taking a glimpse at the mechanisms underlying cell commitment and fate specification may hold promises for a revolutionary field, the so-called regenerative medicine. while moving from reductionism to holistic approaches, the cell is studied as an integrated system, behaving as a neural network with complex and sophisticated logics. awareness of these features has progressively led to wide-ranging strategies in the investigation of gene and protein expression. techniques such as the dna microarray and the serial analysis of gene expression (sage) are now able to follow the expression of thousands of genes and signaling molecules at a time, attempting to uncover the overall plans that underlie molecular patterning and cellular decisions. omics era (genomics, proteomics) and will hopefully form the scientific underpinning for moving from basic science to a clinical practice in which physicians will learn how to deal with illness rather than disease (or even worst, diseased organs). a major sign of these cultural changes is provided by the ongoing development of nanobiotechnologies. in both the philosophical and visual sense, seeing is believing does not apply to nanotechnology, for there is nothing even remotely visible to create proof of existence. on the atomic and molecular scale, data is recorded by sensing and probing in a very abstract manner, which requires complex and approximate interpretations. more than in any other science, visualization and creation of a narrative becomes necessary to describe what is sensed, not seen. we have growing needs for separating the informational content of life from its material substrate. information is thought to be the essence of life, as in the dna code (james k. gimzewski, university of california at los angeles, department of chemistry and biochemistry, director of the pico lab at ucla). edwin cooper highlighted how different alternative medicines if integrated can be useful to reclaim that holistic view of the diseased person, that an excessive specialization has made modern western medicine lose and he illustrated the work done in this direction by the biomedical journal ecam. disease has always been of enormous concern in human society. from prayers and spells to the birth of medicine as a rational science, man has developed all sorts of medical treatments to combat different illnesses and chronic ailments: according to the chinese proverb: life is worth more than a thousand gold pieces. the first objective in a serious approach to complementary and alternative medicine (cam) should be to obtain a broad understanding, with a minimum of detail, of how cam fits into the pattern of biology of the way in which the nervous, endocrine and immune systems coevolved, their function and coordination with other body systems, and their development from the embryo onwards including aging. at the same time, such an outline should provide an adequate background for easy application of cam ideas to the detail of practical cam work in public health, clinical and medical practice, and yet not stray far away from its essence, the very biology that under girds it. cam is organismic, considers the whole individual and is inclusive, not reductionist nor exclusive. concerning senescence and age-associated diseases, that accompany longer living populations, substantial attention is now focused on searching for: (i) mechanisms of aging and (ii) approaches to ameliorate or lessen the effects through the use of therapies, some of which utilize natural products from aquatic and terrestrial plants and animals. clearly there are numerous treatments to explore and to understand long past the anecdotal information that has been passed on through centuries. there is evidence for treatment of diseases of an ever-aging society especially in developed countries. it is of great interest that these remedies now being refined by ecam approaches derives almost entirely from primitive societies where there are minimal facilities essential for analyses by an evidence-based approach. massimo mori said: [] if the scientific research is free, the same is not valid for its technological applications, which have to serve an ecology of culture marked by wisdom, an ecology of the mind, as wrote gregory bateson, in harmony with nature; nature, where the uncertainty principle of werner heisenberg has scientific and philosophical value; harmony, comprising the clash and entropy of ilya prigogine as factors of transformation. a holistic view recomposing the divided self of ronald d. laing, giving deepness to the one-dimensional man of herbert marcuse. prof. angelo marzollo highlighted the importance of combining the art of to say with the art of to do, i.e., that the learned man, the intellectual is direct evidence of the ideas carried on by him, that the body does not contradict the mind, that the body-mind unit remains not only a theoretical statement. giovanni sambin said: although the theme is science, i can speak only about my specialization. i am fascinated by oriental wisdom, but i know almost nothing about it because of lack of time. i am here only because i am trying to learn tai ji quan, with master roberto benetti. mathematics is important in western science. galilei was the first to propose that the laws of nature are written in the language of mathematics. this has been the base for inventions characterizing industrial revolution in xixth century. by using mathematics so mathematics is at the base of present-day western technological supremacy. in modern times, debate concerning foundations of mathematics has been most lively at the beginning of xxth century. one of its outcomes was the invention of programmable computers, which speaks of its importance. i am not a believer, but i find constructivism much more convincing than the now dominating theory, which is called classical and is considered by most scientists as an absolute truth. to do modern mathematics one needs an abstract notion of concept, or set. in the classical approach, one has classical logic (by which all propositions are either true or false, any third possibility like abstinence is excluded) and axiomatic set theory (all sets are there already, a static universe containing now, and ever, all possible concept-sets). the mental scheme looks as something like: classical mathematics is at the base of western science, which is the reason for technological superiority, which makes western people the owners of the world. hence it must be that western mathematics, and all what follows from it, is an absolute truth, which can be imposed on others by force. needless to say, this view can be extremely dangerous. here and in all my life, my aim is to show that a different foundation of mathematics is possible, which has the same applications, but which avoids any form of fundamentalism. a way of doing mathematics is possible, in which the will of power is replaced by harmony with nature, control is replaced by knowledge, brute external force is replaced by internal energy, the search for the i want to emphasize that originally this was not motivated by some ethical or political principles, rather by the search for a better foundation of mathematics. master flavio daniele emphasized as most advanced research of the last decades of the past century in the field of cognitive sciences and neurosciences led to a new theory, revolutionizing the traditional cartesian concept of mind. this theory, known as the santiago theory of cognition, claims that mind can no longer be regarded as a thing but as a process. process, which is cognition to which belong perceptions, emotions and actions, the language, the conceptual thought and all the attributes of conscience, which is peculiar to man. this view, perfectly in line with the eastern traditional thought, entails that mind with its cognitive processes goes beyond the rational aspect as it includes the whole process of life. a further implication of this theory, which will show its vast potential, when it will be absorbed at the general cultural level, is that mind and matter are no longer regarded as separated dimensions, but as complementary aspects of the sole phenomenon of life: the process (the mind) and the structure (the brain). mind and matter, process and structure are indivisibly connected at all levels of life: from the simplest cell to the most complex organism. this connection is so deep, as most recent studies in the field of cognitive sciences has demonstrated, that we can state, conceptual thought, on the whole, is physically incarnated in the body and in the brain. this goes beyond the simple consideration that to think we need a brain and leads to state that human reason does not transcend body, but it is structurally shaped by our physicality and body experience. a further discovery of cognitive sciences, consequent to it that: (1) mind is deeply incarnated in the body, and the other that, (2) thought is mainly unconscious, is that (3) abstract concepts are to a large extent metaphorical. for the time being, present researchers have not explained in detail the neurophysiological dynamics underlying the formation of abstract concepts; however, the scientists lakoff and johnson state that the neural and cognitive mechanisms enabling us to sense and move are the same to create also our conceptual structures and our ways of reasoning. this statement that conceptual structures and ways of reasoning come from the same neural and cognitive structures of perception and movement, is extremely important for the practitioners of the arts of movement (taiji quan, yoga, sacred dances, ritual gestures or mudra), as it demonstrates and confirms the creative power of movement, which no longer acts as simple instrument at the service of mind, to play the fundamental role of shaper of the cognitive capabilities (conceptual thought, speech, conscience) of the human mind. by paraphrasing maturana and varela cognition, the process of knowledge, and it is identical to the process of life self. nevertheless, beyond the dialectics coming from the differences, it is more and more important to reintroduce a principle, acting as basis for the man universally, a principle which is internal and unconventional, an equal able to give the equal to all psycho-biological behaviors of the human being. in italy this kind of research has always taken place, and in recent years has made its way into the experimental evidence of a psychology able to touch this dimension.

the conference was organized and supported by: nei dan school (european school of internal martial arts), nib (laboratory of molecular biology and stem cell bioengineering, national institute of biostructures and biosystems, institute of cardiology, s.orsola-malpighi hospital, bologna), wacima (worldwide association chinese internal martial arts), arti doriente (magazine of eastern culture and traditions), nuovo orizzonte (taiji quan school in florence), samurai (journal on martial arts), and pinus (first national institute for the unification of medical strategies). nei dan school (www.taichineidan.com, [email protected]) was in charge of the organization. future meetings of the centro studi tao and science will take place in spring 2007 in firenze and in october 2007 in bologna. for information: e-mail: [email protected]; web site: www.taichineidan.com, www.taoandscience.com

PMC2249746

pubmed-452

cardiovascular disease is the most common cause of mortality in dialysis patients that is responsible for about 60% of their mortality and is also 30 times more common than in general population. different cardiovascular disorders, such as left ventricular hypertrophy (lvh), coronary artery diseases, congestive heart failure (chf), and arterial hypertension are commonly seen in these patients. in addition, calcification of cardiac valves are common and may cause valvular and annular thickening that in turn could lead to valvular stenosis or regurgitation. some predisposing factors of cardiac disorders in dialysis patients are secondary hyperparathyroidism, long term hypertension, and anemia. different electrocardiographic abnormality may be seen in dialysis patients, such as st, t change, ventricular and supra-ventricular arrhythmia and qt interval prolongation. in the electrocardiogram, qt interval is the time between the start of the q wave and the end of the t wave. when heart rate increased then qt interval was decreased, so qt interval could be corrected with heart rate. there are a number of different correction formulas, but the standard clinical correction is to use bazett's formula (qt/r-r, where r-r is the rr interval in seconds). the normal corrected qt (qtc) interval for males and females are 430 ms and 450 ms, respectively. the borderline qtc for a male is 431-450 and borderline qtc for a female is 451-470 and the abnormal qtc range for a male is>450 and for a female is>470. the qtc interval could be prolonged due to electrolyte abnormalities (hypomagnesaemia and hypokalemia), drug consumption (antihistaminic, antiarrhythmic, and antibiotics), brain trauma and genetic abnormalities (long qt syndrome). patients with chronic renal failure and dialysis patients had a greater qtc interval and qt dispersion (qtd) (qtd=qtmax qtmin) compared with the normal population. a single session of hd could be increased qtc in patients undergoing hd. moreover, qt interval may be a predictor of ventricular arrhythmia and cardiovascular mortality in chronic kidney disease and dialysis patients. although there are some studies about qtc and qtc dispersion in hemodialysis (hd) patients with different results; however, there are only a few studies concerning relationship of qtc internal and qtd with echocardiography findings, so the aim of this study was the evaluation of these relationship in dialysis patients. in a cross-sectional study, 60 hd patients with age>18 years and the dialysis duration>3 months were enrolled. exclusion criteria were: antiarrhythmic drugs consumption, history of cardiac diseases such as arrhythmia, heart block or chf. before dialysis session, electrocardiography (ecg) and echocardiography also, qtd was measured by mentioned formula (qtd=qtmax qtmin) in 12 leads ecg. the patients were on hd, by fresenius digital machine (4008b, germany) and gambro digital machine (ak95 and ak96, sweden), 2-3 times/week, as a regular method (4-4.5 h with blood flow of 250-350 ml/min, dialysate flow of 500 ml/min and ultra-filtration based on the patient's condition). the used buffer was bicarbonate powder, and the type of filters were intermediate and high efficient polysulfone membrane (r5, r6) made by soha factory under license of fresenius company. serum creatinine, hemoglobin, ca, parathyroid hormone (pth), na, k, hco3, and ph were checked by ra1000 machine (made in italy). all laboratory tests were done before the dialysis session and were checked in a single laboratory. echocardiography and ecg were done before the dialysis session and by single cardiologist and technician respectively. all data and information were confidential, and for ecg and echocardiography taking an informed consent was taken from each patient. in echocardiography, left ventricular ejection fraction (lvef), lvh, pulmonary artery pressure (pap) and valvular disorders were evaluated. at the end of the study, data were analyzed using spss software (version 19, ibm corporation). pearson correlation coefficient, two-independent samples t-test and anova were used for statistical analysis. this study was approved by ethical committee of shahrekord university of medical sciences with the grant number of 904. mean body mass indexes (bmis) (post dialysis) and duration of dialysis were 21.77 3.6 mean qtc interval of the patients was 0.441 0.056 s; however, qtc interval in men and women were 0.43 0.04 s and 0.45 0.07 s, respectively (p>0.05). qtd in all of the patients was 64.17 25.93 ms, however in men and women were 62.70 28.05 ms and 66.52 22.48 ms respectively (p=0.87). mean urea reduction ratio in men and women were 68.14% 9.34% and 68.22% 3.88%; however, kt/v was 1.45 0.17 and 1.49 0.16 among men and women, respectively. characteristics of patients and their association with qtc and qtd sd=standard deviation, bmi=body mass index, pth=parathyroid hormone, qtc=corrected qt, qtd=qt dispersion there was no statistically significant relationship between qtc interval or qtd with duration of dialysis, bmi, age, and gender (p>0.05). in ecg, lvh was seen in 23 (38.3%) patients and st change in 14 patients (23.3%). in echocardiography, mitral regurgitation (mr), tricuspid regurgitation (tr), and aortic insufficiency (ai) were found in 54, 47, and 11 patients respectively [table 2]. in addition, no significant relation was found between qtc interval and qtd with mr, tr, ai, lvh, septal thickness (st) and pap [table 3]. qtc interval and qtd has also no correlation with serum pth or ca, k or hco3. severity of valvular disorders in the patients mr=mitral regurgitation, tr=tricuspid regurgitation, ai=aortic insufficiency echocardiographic findings and their association with qtc and qtd qtc=corrected qt, qtd=qt dispersion, pap=pulmonary artery pressure, lvef=left ventricular ejection fraction, lvh=left ventricular hypertrophy, mr=mitral regurgitation, tr=tricuspid regurgitation, ai=aortic insufficiency, pe=pericardial effusion our findings revealed that, in hd patients, qtc interval and qtd had not any correlation with valvular disorders (mr, ai, and tr), lvh or other echocardiographic findings such as pap and st. furthermore, there was no relationship between qtc interval and qtd with serum ca, k, hco3, pth. in hd patients, cardiac abnormalities such as vascular calcification are common and are associated with the development of lvh, that may cause increased cardiac arrhythmias and mortality. elevation of calcium-phosphorus product can also cause valvular calcification and stenosis in these patients. mitral and aortic calcification and stenosis are common in these patients; however, tricuspid and pulmonic valve calcification is rare. qtc interval prolongation in chronic kidney disease and hd patients was shown in some studies such as covic et al. and ljutic et al. studies which postdialysis qtc interval was 434 29 ms and 445.7 36.9 ms respectively, while in our patients qtc interval was 444.6 54.5 ms. selby and mcintyre in a review article reported that hd can increase qtc interval and qtd and is also capable of inducing arrhythmias and increasing mortality especially in patients with ischemic heart disease. in nakamura study on 48 dialysis patients, throughout the follow-up period, there was a higher incidence of cardiovascular death in patients with prolongation of qtc dispersion after hd. showed that the serum potassium was significantly higher in hd patients when compared to continuous ambulatory peritoneal dialysis (capd) patients and rate of qt interval dispersions was significantly higher in hd and capd patients when compared with healthy controls. they concluded that there is a tendency to cardiac arrhythmias in hd patients during the postdialysis period. maule et al. showed that after the hd session, qtc increased in 56% and decreased in 43% of the patients after dialysis session, cell-associated mg levels and qtd increased significantly in averbukh et al. study, so he concluded that excess daily mg intake and increased concentrations of cell-associated mg could be responsible for qtc prolongation in these patients. change in serum electrolytes during hd may also be responsible at least partially, to increase qtc or qtd. for example, increase of qtc interval after a dialysis session, and its correlation with plasma calcium level, postdialysis blood pressure, lvef and st was shown in covic et al. showed that qtd increased during hd due to serum k depletion and then return to baseline 2 h after the end of dialysis. qt prolongation during dialysis may also predispose to arrhythmia especially in the cardiac disease patients, nppi et al. demonstrated that hd increases qtd if a low-calcium dialysate is used. therefore, use of a low-calcium dialysate may predispose hd patients to ventricular arrhythmias. 16 hd patients showed that qtc interval has a reverse correlation with k and ca concentrations of dialysate. however, in hd children, ozdemir et al. found that qtc interval has no correlation with the patients sex, age and presence of hypertension or lvh but patients with left ventricular systolic dysfunction had significantly greater qtc dispersion. similar to our study, the changes in serum ca or k of the patients during dialysis were not associated with the qtc interval in this study. moreover, in hekmat et al.s study on 49 hd patients, qtc interval had not correlation with serum electrolytes and blood gas findings. in voiculescu study, there was no statistically significant correlation between qt interval and serum concentrations of mg, po4 in hd patients too. in addition, qtc interval was not dependent from lvef, arrhythmias or sudden death. our results had some similarities with some of above-mentioned studies as well as some differences with the others. the observed controversy between our study and some of these studies might be due to the discrepancy in the number of cases or racial differences of the patients, and more importantly, the fact that we did not compare predialysis and postdialysis qtc interval. we could not find any study on the correlation of qtc interval with lvh or valvular heart disease in hd patients; however, there are some studies related to the correlation of qtc and lvh with other diseases or conditions. for example, mayet et al. reported that qtd has correlated with left ventricular mass index in hypertensive individuals. found that in athletes, lvh induced by physical training activity is not associated with an increase in qtd, whereas pathological increase in lvh secondary to hypertension could increase qtd. the study of dimopoulos et al. on the evaluation of qtd and left ventricular mass index in elderly hypertensive and normotensive patients reported that hypertensive patients had greater left ventricular mass index and higher qtd. the study had some limitations such as small sample size and lack of qtc measurement after hd session and comparison of it before and after procedure. by our knowledge, this is one of the first studies on the correlation of qtc or qtd with valvular abnormality and lvh in hd patients. there were a few studies about the relationship of qtc and lvh in nonrenal patients, so we could not compare our results with other studies. based on our results, in hd patients, qtc interval or qtd was not correlated with echocardiographic findings or laboratory exam results. therefore, it can be concluded that qtc interval prolongation probably has not any correlation with cardiac mortality of the hd patients.

introduction: cardiovascular disease is the most common cause of mortality in dialysis patients. chronic renal failure and hemodialysis (hd) patients may have longer corrected qt (qtc) interval compared with the normal population. long qtc interval may be a predictor of ventricular arrhythmia and cardiovascular mortality in these patients and hence the aim of this study was the evaluation of the relationship between qtc interval and some echocardiographic findings and laboratory exam results in hd patients. materials and methods: in a cross-sectional study, 60 hd patients with age>18 years and the dialysis duration>3 months were enrolled. blood samples were taken, and electrocardiography and echocardiography were done before the dialysis session in the patients. results:mean age of the patients was 56.15 14.6 years. qtc interval of the patients was 0.441 0.056 s and qt dispersion (qtd) was 64.17 25.93 ms. there was no statistically significant relationship between qtc interval and qtd with duration of dialysis, body mass index, age, and gender (p>0.05). there was also no significant relationship between qtc interval and qtd with mitral regurgitation, tricuspid regurgitation and aortic insufficiency (p>0.05). in addition, qtc interval and qtd of the patients had not any correlation with serum parathormon and serum ca, k, hco3 (p>0.05). conclusion: based on our results, in hd patients, qtc interval and qtd were not correlated with echocardiographic findings or laboratory exam results. therefore, it can be concluded that qtc interval prolongation probably has not any correlation with cardiac mortality of the hd patients.

PMC5353447

pubmed-453

from november 2008 through may 2010, m. tuberculosis isolates were cultured during routine care of adults>18 years of age with primary tb and no history of treatment. the patients were from 2 regional referral centers, the irkutsk regional tb-prevention dispensary and the research practice center for phthisiatry (yakutia); the study was approved by the institutional review boards at the university of virginia and irkutsk state medical university. initial pretreatment isolates were grown on lowenstein-jensen agar slants and identified to species in accordance with world health organization recommendations. drug susceptibility was tested by absolute concentration method on agar slants; drugs tested were rifampin (critical concentration 40 g/ml), isoniazid (1 g/ml and 10 g/ml), ethambutol (2 g/ml), streptomycin (10 g/ml), ethionamide (30 g/ml), and kanamycin (30 g/ml). dna extraction was performed on all isolates, followed by 12-loci mycobacterial interspersed repetitive unit variable number tandem repeat (miru-vntr) analysis (7) and further lineage definition by region of difference deletions, or for ural strains as described (5). phylogenetic tree construction was based on the miruvntrplus database (8), and vntr international type numbers were confirmed on the sitvit database (9). dna from mdr isolates was amplified and sequenced for the known drug-resistance determining regions katg, inha, rpob, embb, gyra, rrs, and eis by using methods described by the centers for disease control and prevention (10). sequences were compared with published sequences for m. tuberculosis h37rv by using genedoc version 2.7.0. among 235 patients with primary tb (130 from yakutia, 105 from irkutsk), isoniazid monoresistance was found in isolates from 16 (12%) from yakutia and 19 (18%) from irkutsk (p=0.27). multidrug resistance was found for 61 patients (36 [28%] from yakutia and 25 [24%] from irkutsk) (p=0.55). mean age (sd) for these 61 patients was 33 (12) years, 40 (66%) were male, and these characteristics did not differ significantly between patients from irkutsk and from yakutia. however, no hiv-infected patients were identified from yakutia compared with 11 (44% with mdr tb) from irkutsk (p<0.001). twelve mdr tb patients from irktutsk died (outcome unknown for the other 13 patients), including all with hiv, compared with 4 (11%) from yakutia who died (p=0.002). follow-up varied and was limited mostly to inpatients. among all 235 patients with primary tb, strains of the beijing family were significantly more common among those from irkutsk (70 [67% ]) than from yakutia (40 [31% ]) (p<0.001). however, strains found in yakutia (s 256 [11%], t 8 [7%], and ural 171 [5% ]) were not found in irkutsk (table 1). the cluster of s 256 (miru profile 233325153325) was the most common among primary mdr tb isolates from yakutia and was fully 86% mdr (table 1; technical appendix).*miru-vntr, mycobacterial interspersed repetitive unit variable number tandem repeat (original 12-loci profile). included genotypes found in>5 isolates only; mit, miru vntr international type; mdr, multidrug-resistant tuberculosis (conventional resistance to isoniazid and rifampin); na, not applicable. significance determined by analysis with yates correction or fisher exact test when appropriate. among isolates from patients with primary mdr tb, 51 (84%) were available for dna sequencing: 27 from yakutia and 24 from irkutsk (table 2). among isoniazid-resistant isolates, the mutation in codon 315 of katg was present in 91%. among rifampin-resistant isolates, mutations in the resistance-determining region of rpob (codons 511533) were present in only 79%. the pnca mutation was common across genotypes from both sites, occurring in 62% of isolates amplified. notably, both isolates with mutation in eis from yakutia occurred in mdr strains with the s 256 genotype and without rrs mutation.*all inha mutations were associated with a ser315thr mutation in katg except for 1 isolate in which katg did not amplify. excluding 25 silent pnca mutations (ser32ser most common, n=14). for all 3 mutations of eis associated with kanamycin resistance, rrs was wild type. in eastern siberia,>25% of primary tb was mdr, equivalent to the highest proportion reported from the russian federation (2). however, regionally specific genotypic patterns and resistance mutations were identified. as expected, in irkutsk primary mdr tb was driven by strains of beijing lineage (5,6). yet in the more geographically isolated population of yakutia, a strain previously unidentified in the russian federation, s 256, had a miru profile recently found among canadian aboriginal populations (11). in yakutia, s 256 was highly drug resistant and was the most common genotype among patients with primary mdr tb. although rpob mutations were found in only 79% of rifampin-resistant isolates, these findings are consistent with those in a recent report from novosibirsk oblast, which similarly included non-beijing and s-family strains and found a sensitivity of only 63% for the rpob mutation (12). lack of phenotypic correlation can result from alternate mechanisms of resistance or imperfect conventional susceptibilities in lowenstein-jensen medium or from use of old drug stock. such discrepancy necessitates urgent clarification because substitution of conventional susceptibility testing with molecular probe based methods such as genexpert mtb/rif (cepheid, ca, usa) has been strongly advocated but would lead to dramatically different results and treatment regimens (13). of note, isolates of the s 256 strain accounted for a proportion of the cases in which mutation in the promoter region of eis was associated with kanamycin resistance, but rrs was wild type. commercial assays have focused on the rrs locus, which has greater sensitivity for amikacin, as the sole target for the class of injectable agents (14), yet in eastern siberia, the injectable agent available is kanamycin. furthermore, we found a range of reported and unreported mutations across the entire pnca gene; most were point mutations resulting in amino acid substitution, but some strains had mutations that resulted in deletion or frameshift. phenotypic methods and assays of functional pyrazinamidase activity should be performed in this region because results might have major implications for novel mdr tb drugs that work best with pyrazinamide (15). we were unable to obtain detailed clinical information about all patients with primary tb, thus preventing adequate comparison of nongenotypic risk factors for mdr tb or establishment of definitive epidemiologic links among clustered isolates. furthermore, lack of conventional fluoroquinolone or pyrazinamide susceptibility testing limited comparison with gyra and pnca mutations, respectively. despite these limitations, this work characterizes severe isoniazid monoresistant and mdr tb in eastern siberia among patients with no history of tb treatment. the regionally distinct phylogenetic patterns and certain drug-resistance mutations necessitate careful application of novel diagnostics and empiric therapeutic strategies. phylogenetic trees of mycobacterium tuberculosis from patients with primary tuberculosis, yakutia and irkutsk, russian federation.

of 235 mycobacterium tuberculosis isolates from patients who had not received tuberculosis treatment in the irkutsk oblast and the sakha republic (yakutia), eastern siberia, 61 (26%) were multidrug resistant. a novel strain, s 256, clustered among these isolates and carried eis-related kanamycin resistance, indicating a need for locally informed diagnosis and treatment strategies.

PMC3810730

pubmed-454

the earliest mammals were shrew-like creatures who could search for food and hide in places that were too cool and dark for many of their lizard contemporaries (1). in addition to being warm blooded, mammals also were adept at performing adaptive thermogenesis, giving them the ability to further increase their metabolic rates when conditions would otherwise result in hypothermia. from the perspective of humans in 2015, it is easy to forget that thermoregulation, in all of its dimensions, was a critical part of mammalian history thermogenesis comes from chemical reactions in which the liberated free energy is not captured in other molecules (e.g., atp or creatine phosphate) or used for work. the best known example of a heat-generating pathway is the futile cycle of proton pumping in brown fat and beige fat through the actions of uncoupling protein 1 (ucp1) (2,3). in cells expressing ucp1, the oxidation of lipids and carbohydrates results in the extraction of high-energy electrons, which flow down the electron transport chain (etc) as protons are pumped across the inner mitochondrial membrane (fig. the resulting electrochemical gradient across the inner mitochondrial membrane, which is usually coupled to atp synthesis via complex v of the etc, is dissipated by a leak of protons back across the inner membrane by ucp1. thus, much of the chemical energy generated by fuel oxidation in brown fat cells powers a futile proton cycle, which does no work and is instead liberated as heat. this process is typically thought of as being indirectly activated by cold, via the sympathetic nervous system (sns). adaptive thermogenesis can also be activated directly by cold in beige adipocytes and by other stimuli that may signal independently from the -adrenergic receptors. the reducing equivalents generated by the tricarboxylic acid (tca) cycle enter the etc. this generates a proton gradient across the inner mitochondrial membrane. instead of linking this gradient to atp synthesis via complex v, it is also worth noting that, from a thermodynamic perspective, there is nothing special about proton cycling, and such a thermogenic machine could, in theory, be built from other components of cellular metabolism. indeed, some deep-diving fish have a heater organ, which is a specialized muscle that entirely lacks a myofibrillar apparatus. this organ, found between the eye and brain in deep-diving marlin and certain tuna, uses a futile cycle of calcium leaking from the sarcoplasmic reticulum and atp-dependent calcium uptake to raise the local temperature near the brain of the fish (4). in this case, the physiological goal is not to warm the whole body of the marlin, but to just make it a bit more astute than the other fish around it! is ucp1 the only thermogenic pathway of importance in mammals? while no other pathway of thermogenesis except shivering has been convincingly demonstrated, suggestive data from the scientific literature hints that such pathways probably exist. if mice lacking ucp1 are abruptly switched from ambient temperatures to cold (4c), they develop life-threatening hypothermia (5). on the other hand, if they are gradually exposed to lower temperatures, they can survive quite normally at 4c (6). this suggests that there are compensatory thermogenic programs that can be activated as long as thermal stress is applied more gradually. in addition, there are examples of experiments in mice where certain mutations have caused very robust increases in energy expenditure and thermogenesis, but where the levels of ucp1 mrna and protein are unchanged. for example, deletion of par-1b/mark2 in mice results in increased energy expenditure and brown fat activation, although ucp1 protein levels were unchanged (7). ucp1 brown fat has long been known to occur in two distinct anatomical locations in rodents. developmentally formed depots, best typified by the interscapular and perirenal regions, are composed of tightly packed brown fat cells of relatively uniform appearance. these depots exist under most physiological conditions, although they may change in color and lipid content. on the other hand, ucp1 cells can accumulate in small pockets in white fat depots, especially in the subcutaneous adipose tissues, particularly when mice are exposed to long-term cold or stimuli with hormones such as catecholamines and other -adrenergic agonists (8). this pathway of adaptive thermogenesis is robustly activated by cold via an indirect pathway mediated by the sympathetic nervous system. however, fat cells are also able to directly sense cold, though the mechanism is not well understood (9). in both anatomical situations, brown fat cells have multiple small lipid droplets, numerous mitochondria, and rich innervation and vascularization. despite these similarities, it is now clear that the classical brown fat cells and the inducible beige fat cells come from different developmental lineages and are, in fact, distinct cell types. the key finding in this regard was the observation that the classical developmentally formed brown fat arises from a myf5 lineage shared with skeletal muscle (10). consistent with this, primary cultures of classical brown fat express low levels of certain genes characteristic of skeletal muscle (11). subsequent work indicated that the brown fat/skeletal muscle decision occurs between 9.5 and 12.5 days of gestation in mice (12). conversely, the ucp1 cells induced in subcutaneous white fat depots by cold or -adrenergic agonists come from a myf5 lineage (10). these beige fat cells have now been cloned and express a relatively low level of thermogenic genes, such as ucp1 and type 2 deiodinase in the basal state, but can induce these genes to levels essentially equivalent to classical brown fat cells when given hormonal stimuli (13). moreover, the beige and brown fat cells have gene signatures that allow for distinction of these cells types; however, their comparative physiological properties and overall roles in metabolism are not completely understood. it is now clear that thermogenic adipocytes, taken as a whole, contribute very importantly to metabolic homeostasis in rodents. the partial ablation of ucp1 cells through the transgenic expression of a toxigene led to these animals being more susceptible to obesity and diabetes (14). similarly, mice lacking ucp1 through targeted mutation had an increased body weight and fat content, at least when raised at thermoneutrality (15). excessive shivering apparently prevented obesity when these experiments were performed at ambient temperatures. the first transgenic model with increased brown and beige fat was the adipose-selective expression of foxc2, a transcription factor that activated camp metabolism in recipient cells (16). these mice, which were resistant to diet-induced obesity and diabetes, had expansions of both the classical brown and beige fat. deciphering the individual roles of brown and beige fat cells prd1-bf-1-riz1 homologous domain-containing protein-16 (prdm16) is an important transcriptional coregulator in both brown and beige fat, but when its expression was elevated through a promoter expressed in all fat depots (ap2), phenotypic changes were observed in the erstwhile white fat depots, which developed copious pockets of beige fat cells (17,18). the classical brown fat, which expresses very high levels of prdm16 in the basal state, showed few or no changes with a further elevation in prdm16 expression. but this occurred only when the transgenic mice also received stimulation with a -adrenergic agonist. while studies in cultured cells had shown that classical brown fat cells require prdm16 to develop and maintain a thermogenic gene program, ablation of prdm16 from all fat cells in vivo was not sufficient to significantly alter the function of the classical brown fat. on the other hand, the development of beige fat cells was severely reduced, at both the histological and molecular level (19). these mice developed a moderate obesity, compared with control animals, with an unusual expansion of the subcutaneous fat depots. these beige fat deficient mice also showed a rather profound hepatic insulin resistance that was associated with liver steatosis. one last point of interest is that the subcutaneous fat from the prdm16-ablated mice showed the presence of more inflammatory macrophages and increased the expression of proinflammatory genes. the latter aspect was also observed with cultured subcutaneous cells lacking prdm16, indicating that this transcriptional coregulator plays a role in the relative resistance of subcutaneous fat to inflammation and is an important determinant of subcutaneous versus visceral fat phenotype. the role of prdm16 in the classical brown fat in vivo is interesting and somewhat more complex. ablation of this factor with a myf5-driven cre recombinase causes a loss of brown fat function and gene expression, but only at 6 months of age (20). however, ablation of prdm16 along with its closest homolog, prdm3, causes an early and severe loss of brown fat thermogenic gene expression and normal histology. thus, it appears that both thermogenic cell types depend on prdm16, but, at least in early life, the classical brown fat has a second factor that can support development, prdm3. harnessing the therapeutic potential of brown and beige fat requires a detailed understanding of the molecular mechanisms responsible for the determination and maintenance of each cell type. the nuclear hormone receptor peroxisome proliferator activated receptor (ppar) is necessary and sufficient for the development of all fat cells (21). however, white and brown adipocytes have drastically different phenotypes, indicating that other transcriptional regulators must be involved. a yeast two-hybrid screen identified ppar coactivator-1 (pgc-1) as a cold-inducible binding partner of ppar (22). in brown fat pgc-1 is now appreciated to regulate numerous physiological processes in a variety of metabolically important tissues. while brown and beige fat cells lacking pgc-1 have significantly blunted thermogenic gene expression, these cells still retain the molecular signature of brown fat cells (23,24). the transcriptional regulator prdm16 was found to be highly enriched in brown fat, whereas it is virtually undetectable in visceral white fat cells. forced expression of prdm16 in cultured white fat cells induces pgc-1 and thermogenic genes, as well as mitochondrial genes and brown fat identity genes (17). in addition to activating these pathways, prdm16 also binds ccaat/enhancer binding protein (c/ebp) and recruits the corepressor proteins ctbp1 and ctbp2 to repress white fat or muscle gene expression (25). importantly, the expression of prdm16 and its binding partner c/ebp in fibroblasts is sufficient to promote differentiation into functional brown fat cells, which can be detected by [f]-2-fluoro-2-deoxy-d-glucose ([ f]-fdg) positron emission tomography (pet) when transplanted into mouse models (26). an increasing number of transcriptional regulators has been identified as important in brown and beige fat biology. the transcription factor early b-cell factor-2 (ebf2) is enriched in brown relative to beige adipocytes. it functions upstream of prdm16 to promote binding of ppar to the promoters of brown-selective genes (27). ehmt1 is a histone lysine methyltransferase that purifies with the prdm16 transcriptional complex in brown fat. ehmt1 is required for brown fat lineage specification and for thermogenesis (28). presumably, there are also beige-selective factors upstream of prdm16 and beige-selective epigenetic regulators that modulate the phenotype of these cells. it is able to compete with prdm16 for binding to ppar, and thereby modulates white versus brown/beige phenotype (29,30). forced expression of tle3 in adipocytes results in impaired thermogenesis, while deletion enhances thermogenesis in brown and beige fat. a number of other factors have also been shown to play roles in this biology, but a comprehensive review is beyond the scope of this article. until fairly recently, brown fat was thought to be present in meaningful amounts only in human infants and small mammals. the combination of insufficient hair, fur, and insulation, along with a high body surface area-to-mass ratio make babies and small mammals particularly susceptible to hypothermia. as a result, they have developed significant interscapular brown fat, which defends body temperature by adaptive thermogenesis. this brown fat was thought to regress by adulthood, unless exposed to catecholamine excess (as in pheochromocytoma) or long-term cold exposure (as in outdoor workers in cold climates). in 2009, these studies made use of [f]-fdg pet to confirm that adults have glucose-avid tissue with imaging characteristics of adipose in deposits in the supraclavicular and spinal regions. as assayed by [f]-fdg pet, the amount of active tissue is inversely associated with bmi, and this activity is increased by cold exposure. follow-up studies (34) have shown that this tissue has the histological appearance of adipose tissue and expresses several molecular markers of brown fat including ucp1. current efforts are focused on developing optimal imaging modalities to detect this tissue and to quantify its absolute amounts and activity. this is particularly important since brown and beige fat also oxidize lipids, which would not be detectable by [f]-fdg pet technology. research has increasingly suggested that adult human brown fat shares features of the inducible beige fat described in rodents. brown and beige fat cell lines have now been cloned from mice, allowing for the identification of unique molecular markers of each cell type. multiple studies (13,35,36) have shown that beige-selective markers are expressed more in human brown fat samples than are markers of the classical brown fat of rodents. a very recent study (37) characterized a human brown fat cell line and showed that it actually shares more molecular features with beige than brown adipocytes. however, the brown fat in human infants, located in the interscapular region, seems most similar to the interscapular classical brown fat in rodents. in some adult humans, cells with both brown and beige characteristics have been identified, with the depth in the neck seeming to be a determinant of relative brown versus beige character (36,38,39). while further studies will clarify this question, this tissue may well contain both brown and beige cells in varying amounts in each individual. as a deeper understanding of brown and beige fat has emerged, researchers have turned to strategies to induce the activity of these tissues as a possible therapy for obesity and metabolic diseases. calculations have suggested that maximal cold-induced brown fat thermogenesis would be between 25 and 400 kcal/day in lean healthy volunteers (40). given the inducibility of beige fat, activating these cells could result in even more substantial effects. however, the greatest benefits may not relate to increased energy expenditure per se, but rather might result from enhanced glucose and lipid disposal. in addition to taking up and consuming glucose (3133), brown adipose tissue takes up free fatty acids from triglyceride-rich lipoproteins. cold exposure in mice upregulates this pathway, resulting in accelerated plasma triglyceride clearance (41). while compounds like dinitrophenol can result in impressive weight loss, they have also been associated with complications, including fatal hyperthermia. while this has understandably resulted in caution, it is possible that more specific uncouplers or targeted activators of ucp1 or other futile cycles may provide metabolic benefit with an acceptable safety profile. in that regard, a recent publication (42) described the development of controlled-release mitochondrial protonophores that can apparently safely uncouple in the liver, and in rats these compounds can improve insulin resistance, diabetes, hypertriglyceridemia, and hepatic steatosis. brown and beige fat activity is robustly induced by thyroid hormone and catecholamines, but side effects would almost certainly limit their use. the 3-selective adrenergic agonist cl 316,243 potently activates brown and beige fat in rodents (43). this drug was recently shown to activate human brown fat, which may renew interest in this pathway (44). since cold robustly activates brown and beige fat, some investigators have suggested that moderate cold exposure could be used as a therapeutic approach. at least in healthy subjects, daily exposure to 19c for 2 h was sufficient to activate brown fat, resulting in weight loss (45). in another small human study (46) while these studies suggest the potential of cold as a treatment modality, our societal preference for thermal comfort may make this unfeasible as a broad approach. thiazolidinediones (tzds) cause browning of the white fat and may do so by stabilizing prdm16, resulting in its accumulation (47). however, these drugs have been associated with weight gain, fluid retention, and cardiovascular events, which have diminished enthusiasm for their use. deacetylation of ppar by sirt1 promotes browning of the white fat, suggesting the possibility of developing compounds that selectively modulate this pathway (48). other regulators of interest include bone morphogenetic protein 7 (bmp7) and bmp8b, cyclooxygenase-2 (cox-2), and natriuretic peptides, though their pleiotropic actions might limit their potential as drug targets. fibroblast growth factor 21 (fgf21) is being examined as a therapeutic agent, but it also has diverse actions and may be associated with bone loss. translating these discoveries into drug candidates will ultimately require a more complete understanding of brown/beige fat biology in humans. mouse studies suggest that prdm16 is a key control point in brown and beige fat phenotype. an increasing understanding of the relevant modifications of prdm16 and the upstream signaling pathways involved may provide new opportunities for therapeutic targeting of brown and beige fat.

the epidemic of obesity and type 2 diabetes has increased interest in pathways that affect energy balance in mammalian systems. brown fat, in all of its dimensions, can increase energy expenditure through the dissipation of chemical energy in the form of heat, using mitochondrial uncoupling and perhaps other pathways. we discuss here some of the thermodynamic and cellular aspects of recent progress in brown fat research. this includes studies of developmental lineages of ucp1+adipocytes, including the discovery of beige fat cells, a new thermogenic cell type. we also discuss the physiology and transcriptional control of brown and beige cells in rodents and the state of current knowledge about human brown fat.

PMC4477363

pubmed-455

in central and northern europe as well as in north america a significant proportion of patients who suffer from birch pollen allergy develop intolerance to certain kinds of fruits and vegetables. such birch pollen-related food allergies are the result of initial sensitization to the major birch pollen allergen, bet v 1, and subsequent immunologic cross-reactivity of the bet v 1-specific ige antibodies with structurally homologous food proteins. among the most frequent triggers of birch pollen-related food allergies are apples, with>70% of all individuals that are sensitized to birch pollen developing allergic reactions when consuming apples. symptoms typically occur locally at the site of food contact and within minutes after apple consumption, including itching and swelling of the lips, tongue, and throat (oral allergic syndromes, oas). frequently, allergic patients can also exhibit symptoms of food-induced rhinoconjunctivitis and dyspnea. in apples (malus domestica), the major allergen that is responsible for birch pollen-related food allergies is the 17.5 kda protein mal d 1. mal d 1 belongs to group 10 of pathogenesis-related (pr) proteins that are activated in plants in response to different kinds of stress. the concentration of mal d 1 in apples is highly dependent on the cultivar and also influenced by various biotic and abiotic factors, storage conditions, and storage duration. typically, 130 g of mal d 1 per gram of fresh apple (accounting for up to 7% of total soluble protein) is present directly after harvest. after storage, these values can rise to values exceeding 100 g mal d 1 per gram of apple. although mal d 1 has been found in both the pulp and peel of apples, higher concentrations are present in the peel. on the basis of this observation, and because mal d 1 appears to be up-regulated upon biotic stress, it has been speculated that this protein may play a role in plant defense response to pathological situations. mal d 1 is encoded by a multigene family, and a number of isoforms of mal d 1 have been identified to date, which are clustered into four groups on the basis of their dna sequence similarities, that is, mal d 1.01, mal d 1.02, mal d 1.03, and mal d 1.04. pcr screening and mass spectrometric studies showed that mal d 1 isoforms are not cultivar specific and that mixtures of isoforms are present in apple fruits. along with mal d 1.02, and depending on the cultivar, isoforms from the mal d 1.01 cluster are by far the most abundant isoforms found in apples. within the mal d 1.01 cluster, immunologic investigations of naturally occurring mal d 1 isoforms revealed only small differences of their ige binding capacities and it appears that divergent allergenicities of apple strains are predominantly determined by different mal d 1 expression levels. whereas the immunological properties of mal d 1 suggest that this protein has a three-dimensional structure and ige binding epitopes that are similar to those of bet v 1 and other members of the pr-10 protein family, experimental structural data for mal d 1 have not been available to date. as a first step toward structural characterization, we recently assigned the nmr backbone and side chain h, c, and n chemical shifts of the isoform mal d 1.0101. golden delicious, were the first isoforms for which the dna sequence was determined and are identical at the amino acid level. here the dna of mal d 1.0101 (genbank nucleotide code x83672, protein code caa58646) was cloned into the expression vector pet28b by using the restriction sites ncoi and xhoi. construct integrity was ensured by dna sequencing (microsynth ag, balgach, switzerland), and the protein was expressed in escherichia coli bl21 star (de3). mal d 1.0101 was purified by anion exchange and size exclusion chromatography as described in detail elsewhere. the mass and the amino acid sequence of purified mal d 1.0101 were confirmed by mass spectrometry using a 7 t fourier transform ion cyclotron resonance (ft-icr) mass spectrometer (bruker daltonics, bremen, germany) with an attached electrospray ionization (esi) source. protein concentrations for nmr spectroscopic experiments for structure determination were 0.5 mm for n/c-labeled and 0.8 mm for n-labeled samples in 91% h2o/9% d2o (v/v) at ph 6.9, 10 mm sodium phosphate, and 7 or 11.2 mm l-ascorbic acid, respectively. all nmr experiments were carried out at 298 k, using either a 500 mhz agilent directdrive spectrometer (agilent technologies, santa clara, ca, usa) equipped with a room temperature probe or a 600 mhz bruker avance ii+ spectrometer (bruker biospin, karlsruhe, germany) equipped with a prodigy cryoprobe. nmr resonance assignments of mal d 1.0101 were made using standard triple-resonance methods and were deposited at the biological magnetic resonance data bank (bmrb) under accession no. three-dimensional n and c edited noesy-hsqc experiments (mixing times of 150 ms) were recorded for derivation of distance restraints. nmr data were processed using nmrpipe and analyzed with ccpnmr. for measuring protein translational diffusion, we employed a stimulated echo pulsed field gradient nmr experiment. experimental details were identical to those reported for bet v 1. for the determination of the hydrodynamic radius of mal d 1.0101, we used dioxane as a standard reference under identical buffer conditions, assuming a hydrodynamic radius of 2.12. structure calculations were performed with the program xplor-nih 2.42 using a simulated annealing protocol. an initial structural model was generated with cs-rosetta using the bmrb cs-rosetta server. a total of 2079 distance restraints were obtained from 3d n and c edited noesy-hsqc spectra. noe values were converted on the basis of peak intensities into distances with upper limits of 3.0 (strong), 4.0 (medium), 5.0 (weak), and 6.0 (very weak). dihedral angle restraints were predicted using talos+ and cs-rosetta. in all regular secondary structure elements hydrogen bonds were included for backbone amide protons, if the n edited noesy-hsqc spectra did not show a water exchange cross peak. of 100 generated structures, the 20 lowest energy structures were picked and further refined in explicit solvent with the amber14 simulation package using pmemd.cuda and the amber force field 99sb-ildn. each structure was soaked into a truncated octahedral solvent box of tip3p water molecules with a minimum wall distance of 10. for the refinement, hydrogen atoms and water molecules were minimized with fixed heavy atoms. the temperature was increased from 0 to 300 k, where the structures were simulated using the noe distance restraints, minimized again, and validated using the protein structure validation software (psvs) suite (table 1). the coordinates of the mal d 1.0101 structures were deposited in the protein data bank under the accession code number 5mmu. calculated for all residues, using sum over r. largest violation among all 20 reported structures. the three-dimensional structure of mal d 1.0101 consists of a curved, seven-stranded antiparallel -sheet (1-7) embracing a long helix at the c-terminus of the protein (3) and two consecutive short helices (1, 2) (figure 1). the edges of the -sheet are formed by strands 1 and 2, which are connected by helices 1 and 2 that form a v-shaped support for the c-terminal part of helix 3. in total, 35% -sheet and ca. 25% helical structure, agreeing well with secondary structure estimates from infrared and circular dichroism. as in other proteins from the pr-10 family, strands 2 and 3 are connected by a glycine-rich loop motif (gly46-asn47-gly48-gly49-pro50-gly51). together, these structural elements create the large internal cavity that is typical for the canonical pr-10 fold. from figure 1b it is evident that in our nmr structural ensemble of mal d 1, secondary structure elements are very well-defined and conformationally homogeneous in all 20 structural models. only slightly elevated levels of conformational heterogeneity are observed for some of the solvent-exposed loops that connect secondary structure elements and the c-terminus of the protein. nmr solution structure of the major apple allergen mal d 1.0101 (pdb accession code 5mmu). secondary structure elements are labeled 1 (val2ser11), 2 (gln40glu45), 3 (ile53thr57), 4 (tyr66ile74), 5 (ser80gly88), 6 (glu96val105), 7 (ser111thr121), 1 (pro15val23), 2 (ala26ile33), 3 (lys128asp152). (b) backbone overlay of the ensemble of the 20 lowest energy structures of mal d 1.0101. secondary structure elements are colored from red (n-terminus) to purple (c-terminus). a peculiar feature of the pr-10 fold is the large internal cavity. in mal d 1.0101, the volume of this cavity is ca. 2230, which is comparable in size to those of other pr-10 proteins. as found in the birch pollen allergen bet v 1 and other homologous food allergens, in mal d 1 the majority of amino acids that form the surface of the cavity are hydrophobic (figure 2). a large proportion of the inner cavity surface is formed by amino acid residues in the -sheet whose hydrophobic side chains are located at the protein interior (ile56 (3), val67 (4), ile71 (4), tyr81 (5), tyr83 (5), leu85 (5), ile98 (6), tyr100 (6), ile113 (7)) along with inward-pointing residues in the long amphiphilic helix 3 (val132, val134, ala139, leu142, phe143, ile146), the two short helices 1 (phe22, val23) and 3 (ala26, ile30), and loop regions (ile38, phe58, tyr64, ala90). in addition, a few polar and charged side chains are located at the inside of the molecule and form part of the cavity surface, such as asp27 (2), his69 (4), ser115 (7), and lys138 (3), so that the cavity itself is actually amphiphilic, as noted before for the major birch pollen allergen, bet v 1. in crystal structures of other pr-10 proteins the cavity is occupied by water, amphiphilic ligand molecules, or components of the crystallization buffer. for mal d 1, it is currently not known whether ligands bind specifically to the cavity or what the biological function of ligand binding could be. (a) internal cavity of mal d 1.0101, colored according to the lipophilic potential as implemented in moe, where hydrophilic regions are colored in blue and lipophilic regions are colored in yellow. (b) surface representation of the lowest energy solution structure of mal d 1.0101. the two amphiphilic entrances to the internal cavity are indicated as 1 (between the n-terminal end of helix 3 and the loops connecting strands 34 and 56) and 2 (between the edge of the -sheet and the c-terminal end of helix 3). the internal cavity in mal d 1 can be reached by two openings (figure 2). one entrance to the protein interior, 1, is shaped by residues in the n-terminal half of helix 3 (his131, val134) along with the loops connecting strands 34 (gln63, tyr64) and strands 56 (asp89). together, these amino acids create an amphiphilic access route to the protein interior. a second amphiphilic entrance, 2, is present at the edge of the -sheet between helix 3 (lys136, his140, lys144, and glu147) and strand 1 (asn7, phe9, and ser 11). in the nmr solution structures of mal d 1 this access route entries to the internal cavity at similar locations have also been described for other members of the pr-10 protein family. figure 3 shows a comparison of mal d 1 with bet v 1 and birch pollen-related food allergens from the pr-10 family whose structures have been determined so far. despite the fact that sequence identities between these proteins are only slightly higher than 50% in some cases, their three-dimensional structures are generally very similar, with backbone rmsd values for secondary structures typically below 2. in light of the observed immunologic cross-reactivity between mal d 1 and the major birch pollen allergen, bet v 1 the backbone rmsd between mal d 1.0101 and the hyperallergenic isoform bet v 1.0101 (61% sequence identity) of the birch pollen allergen is 2.13 (1.70 for secondary structure elements). of note, mal d 1 and bet v 1 differ in length by one amino acid, and divergent presumptions have been made about the location of the gap in mal d 1. on the basis of sequence alignments of pr-10 food allergens it has been proposed that either the loop right before or right after strand 7 is one residue shorter in mal d 1. our solution structure shows that the loop right before strand 7 is the one that is shorter in mal d 1.0101 (glu96val105 in both mal d 1.0101 and bet v 1.0101) and 7 (ser111thr121 in mal d 1.0101 and ser112thr122 in bet v 1.0101) occupy identical positions and have equal hydrogen bonding patterns in the antiparallel -sheets of these proteins. they are connected via loops consisting of four residues (cys-gly-ser-gly in mal d 1) and five residues (thr-pro-asp-gly-gly in bet v 1), respectively, which produces a small structural difference in these loop segments between the two proteins. (a) overlay of the lowest energy structure of mal d 1.0101 (green, pdb accession code 5mmu) with the structures of the major birch pollen allergen bet v 1.0101 (blue, 4a88), the carrot allergen dau c 1.0103 (orange, 2wql), the celery allergen api g 1.0101 (gray, 2bk0), the soybean allergen gly m 4.0101 (yellow, 2k7h), the strawberry allergen fra a 1e (red, 2lpx), and the cherry allergen pru av 1.0101 (purple, 1e09). amino acids are marked with asterisks (identical), colons (conserved), and dots (semiconserved). mal d 1 is known to have a tendency for cysteine-mediated dimerization, as shown for the isoform mal d 1.0108 by nonreducing gel electrophoresis and size exclusion chromatography. like mal d 1.0108, the isoform mal d 1.0101 contains a single cysteine residue, cys107. in the three-dimensional solution structure of mal d 1.0101 cys107 is located at the c-terminal tip of strand 7, with its side chain oriented toward the protein surface. to probe the oligomerization state of mal d 1.0101 under the conditions that we employed for nmr structure determination (ph 6.9, 10 mm sodium phosphate, 14 mol equiv of l-ascorbic acid, 298 k) we performed pulsed-field-gradient nmr diffusion experiments. we obtained a value of 21.6 0.8 for the hydrodynamic radius of mal d 1.0101, which is comparable to the hydrodynamic radius of monomeric bet v 1.0101 (20.1) under similar experimental conditions. this is consistent with our observation that, using the same buffer, mal d 1.0101 elutes from a size exclusion column with a retention time that is virtually identical to that of bet v 1.0101. these results were further verified by ft-icr mass spectrometry, which shows that mal d 1.0101 does not form dimers or higher order aggregates. the nmr solution structure of mal d 1 shows that this protein consists of a highly curved antiparallel -sheet and three -helices forming a large internal cavity, very similar in fashion to other pr-10 proteins. this is in agreement with the observed immunologic cross-reactivity between mal d 1 and the major birch pollen allergen, bet v 1, as well as other food allergens from the pr-10 protein family. in most patients bet v 1 is the sensitizing agent, whereas bet v 1-specific ige antibodies subsequently cross-react with mal d 1 and elicit an allergic response, as reflected by the clinical observation that apple allergy develops only after the onset of birch pollinosis. along these lines, cross-inhibition experiments of mal d 1 using sera from apple-allergic patients showed that mal d 1 shares ige epitopes with the major birch pollen allergen, bet v 1. from a structural perspective, limited information about the exact nature of binding epitopes of mal d 1 and bet v 1 is available. detailed structural information about a sequentially discontinuous (i.e., conformational) b-cell epitope in bet v 1 was obtained by cocrystallizing the particular isoform bet v 1.0112 with an antigen-binding fragment (fab) derived from the murine monoclonal igg antibody bv16. this epitope is formed by the segment between glu42 and thr52 (including the glycine-rich loop motif between strands 2 and 3), along with arg70, asp72, his76, ile86, and lys97 of bet v 1, covering approximately 10% (900) of the entire protein surface. binding of bv16 to this epitope measurably reduces serum ige interactions, indicating that ige and monoclonal igg bv16 compete for overlapping binding surfaces on bet v 1. moreover, mutation of a central residue (glu45ser) significantly reduced the ige binding capacity of bet v 1, confirming the significance of this particular epitope for interactions with ige. figure 4a shows the molecular interaction surface that corresponds with the bv16 epitope in the apple allergen. in mal d 1 these residues form a contiguous surface patch along with a somewhat distal residue (glu76), similar in shape and size to the bv16 epitope of bet v 1. moreover, the contributing amino acids are largely conserved between mal d 1 and bet v 1. thirteen of the 16 amino acids in the bv16 epitope are identical, whereas only 3 residues are different in mal d 1.0101 and bet v 1.0101 (figure 4b). these data thus provide a structural rationale for the observed allergic cross-reactivity between birch pollen and apple allergens. interestingly, mutational studies indicate that the ability of mal d 1 to bind serum ige from patients with birch pollen allergies can be increased by increasing the similarity of the bv16 epitope in mal d 1 to that of bet v 1, indicating that these amino acids are indeed involved in binding of bet v 1 specific to mal d 1. amino acid residues that correspond to the molecular interaction surface between monoclonal igg bv16 and bet v 1.0112 (residues glu42thr52, arg70, asp72, glu76, ile86, and lys97 in mal d 1.0101) are colored in blue. amino acid positions that were shown to be crucial for ige recognition of mal d 1 in mutational analyses (thr10, ile30, thr57, ser111, thr112, and ile113) are shown in green (ile30 and ile113 are located in the protein interior and do not contribute to the surface). (b) amino acid similarities between bet v 1.0101 and mal d 1.0101 using a color gradient from lilac (highly similar) to teal (highly dissimilar). epitope residues that are different between bet v 1.0101 and mal d 1.0101 are labeled. similarities were calculated on the basis of substitution matrix scores (blosum62) as implemented in moe. it is likely that mal d 1 contains more than a single conformational epitope. a number of amino acid positions that are relevant for ige recognition have been identified by mutational analysis. for a five-point mutant of mal d 1.0108 (thr10pro, ile30val, thr57 asn, thr112cys, and ile113val) a markedly reduced capacity for binding mal d 1-specific ige was found in vitro. skin prick tests in apple-allergic patients comparing wild-type mal d 1 with the five-point mutant further showed a significantly lower ability of the mutant protein to induce skin reactions in vivo. further experiments showed that the t-cell recognition level of wild-type mal d 1 is conserved in the five-point mutant. because these five amino acids are likely involved in ige interactions not only in mal d 1 but also in bet v 1, they could well be part of common cross-reacting epitopes in these two allergens. this is corroborated by mutational studies, which showed that peptide stretches encompassing these residues are indeed involved in immunological cross-reactivity between mal d 1 and bet v 1. in addition, in an independent study, ser111 was identified as being essential for ige binding to mal d 1, and a ser111cys mutation resulted in significantly reduced affinity for ige in immunoblotting experiments. figure 4a shows that these six residues are fairly dispersed on the protein surface of mal d 1 and that neither of these amino acids overlaps with the bv16 epitope. amino acids thr10, ser111, and thr112 form a common patch on the protein surface, whereas thr57 is located approximately 3739 away and close to the bv16 epitope. considering that an epitope of typical size (600900) and circular shape would have an arc length of 2834 on the mal d 1 surface, residues thr10, ser111, and thr112 are probably too far away from thr57 to be part of a common binding epitope. the remaining two residues, ile30 and ile113, do not reach the protein surface in mal d 1. whereas ile113 is close in space to the thr10-ser111-thr112 patch, its hydrophobic side-chain is pointing toward the interior of the protein, where it participates in a small hydrophobic core located at the inner end of the proteins cavity (between helices 1 and 3 and the -sheet). residue ile30 is also located in the protein interior with its aliphatic side chain forming part of the internal cavity and does not contribute to the protein surface. of note, because the loop between strands 6 and 7 is shorter by one residue in mal d 1 than in bet v 1, ser111 and thr112 of 7 in mal d 1 occupy the 7 positions of ser112 and ile113 in bet v 1. the surface patch formed by thr10, ser111, and thr112 in mal d 1 thus appears to be less hydrophobic than the corresponding surface patch in bet v 1 (thr10, ser112, and ile113). as a matter of fact, also the protein surface surrounding these three residues displays a considerably lower level of similarity between mal d 1.0101 and bet v 1.0101 than other parts of the protein surface, as can be seen in figure 4b. this might in part be responsible for the different ige binding properties of these allergens. it has been noted, on the other hand, that epitope coincidence between bet v 1 and mal d 1 may be limited, as exemplified by a recent study describing the isolation of human ige binding to bet v 1 but not to mal d 1. moreover, different mal d 1 isoforms contain amino acid substitutions within potential ige interaction surfaces, suggesting that they may influence the immunologic reaction. it is clear that high-resolution structural data provide the basis to determine and compare structural details of (cross-reactive) binding epitopes in allergenic proteins. in addition, grafting of conformational epitopes by transferring stretches of residues between homologous allergens has become a valuable experimental tool. epitope grafting was used to characterize the role of the bv16 epitope in mal d 1 by recreating this epitope on the mal d 1 surface, confirming its importance for ige binding and cross-reactivity with bet v 1. in an orthogonal approach, several mal d 1 stretches encompassing residues that are crucial for ige binding were transferred to bet v 1 to investigate the role of these structural segments for cross-reactivity, and chimeras of bet v 1 and mal d 1 were created to map the epitope of a human monoclonal ige, which was isolated from a phage library, to the c-terminus of bet v 1. in addition, epitope grafting provides access to chimeric allergens with fine-tuned antigenic properties, such as reduced ige binding capacitites, for molecule-based allergy diagnosis and specific immunotherapy. knowledge of the structural details of these allergens elements is required to generate correctly folded chimeras, because transfer of (partly) mismatching stretches of secondary structure between different allergens may well be the reason for a loss of protein fold and, consequently, reduction of ige-binding capacities. the three-dimensional structure of mal d 1.0101 presented here provides the biophysical basis for elucidating the molecular details of immunological cross-reactivity in great detail.

more than 70% of birch pollen-allergic patients develop allergic cross-reactions to the major allergen found in apple fruits (malus domestica), the 17.5 kda protein mal d 1. allergic reactions against this protein result from initial sensitization to the major allergen from birch pollen, bet v 1. immunologic cross-reactivity of bet v 1-specific ige antibodies with mal d 1 after apple consumption can subsequently provoke severe oral allergic syndromes. this study presents the three-dimensional nmr solution structure of mal d 1 (isoform mal d 1.0101, initially cloned from granny smith apples). this protein is composed of a seven-stranded antiparallel -sheet and three -helices that form a large internal cavity, similar to bet v 1 and other cross-reactive food allergens. the mal d 1 structure provides the basis for elucidating the details of allergic cross-reactivity between birch pollen and apple allergens on a molecular level.

PMC5334782

pubmed-456

the presence of high levels of dental anxiety amongst dental care seekers yielded patients with negative attitudes towards dental treatment and rendered dental treatment more difficult to accomplish successfully4,16. assessment of anxiety in such patients is a crucial factor for the success of their management. avoidance of dental care could be attributed to dental fear and anxiety in many patients7,9,23. dental anxiety might also affect patient-dentist relationship and obscure proper diagnosis of the actual dental problem8,25. taani15 (2002) showed that the levels of dental anxiety were higher among jordanian public school children than those from the private schools. yet, dental fear and anxiety were found among the most reasons that underlie the irregular attendance in two thirds of the public school children and half of those from the private schools. it is a simple, easy to score, short, valid and reliable test for dental visit-associated anxiety5,13,19,22. humphris, morrison and lindsay21 (1995) provided a modified scale from the original corah dental anxiety scale. the modified dental anxiety scale was shown to be more comprehensive, highly valid and reliable, with a simpler and more consistent answering system. the modified dental anxiety scale will be, therefore, used to measure dental anxiety in the current study. lack of educational courses specialized in increasing dental awareness amongst non-dental university students in jordan, in addition to shortage of information about correlation between field of study and dental anxiety levels in the dental literature have raised the idea of investigating the level of dental anxiety among the different student populations. this study was therefore designed to investigate the subjective ratings of dental anxiety levels among dental, medical and engineering jordanian university students. in addition, the present study aimed to explore the sources of dental anxiety and the impact of gender on the perceived dental anxiety. anxiety related to dental treatment was assessed by means of corah's dental anxiety scale (das). however, the modified version of das was used21 where an extra item has been included referring to the respondent's feelings toward local anesthetic injection with especial reference to the site of the injection, because the pain experienced with local anesthetic injections varies according to its location in the mouth14. moreover, a simplified 5-point scale-answering scheme was devised ranging from not anxious to extremely anxious. the modified dental anxiety scale (mdas) contains 5 multiple-choice items including the followings:=if you went to your dentist for treatment tomorrow, how would you feel? if you were about to have your teeth scaled and polished, how would you feel? if you were about to have a local anesthetic injection in your gum, how would you feel? the scores for each of the 5 item responses were summed up to give an estimated value of dental anxiety. the questionnaire was distributed to the third to fifth year undergraduate dental, medical and engineering students at the jordan university of science and technology. students were personally approached in the classrooms by the authors at the end of their class. the students were informed about the study and all the points in the questionnaire were explained and clarified. descriptive statistics were obtained and the means, standard deviation and frequency distribution were calculated. group comparisons were analyzed using twotailed student's t tests as well as one-way analysis of variance (anova) test. statistical significance was based on the probability values of p= 0.05. furthermore, as a total score of 15 or more almost indicates highly anxious patient19, the frequencies of subjects with a score of 15 or more in the 3 student groups were also reported. the total number of the participants in the current study was therefore 535, which accounts for a response rate of 89.2 percent. the distribution of the participants according to gender and field of study is presented in table 1. table 2 presents the means and standard deviations of individual items and total scores of the modified corah dental anxiety scale with the results of one-way anova analysis comparing the various groups based on the field of study. out of the several anxiety scale items, the highest anxiety score (3.4) was given for tooth drilling (item 3) and scored by the engineering students. the next most anxiety-producing item was the local anesthetic injection (3.32) which was scored by the medical students. however, for the items 1, 2, and 5, the only statistically significant differences were found between the dental and the medical students (f=7.92, p=0.00; f=4.69, p= 0.01, f=3.39, p=0.03, respectively). dental students were significantly less anxious about scaling and polishing of teeth (f=8.99, p=0.00) and about tooth drilling (f=19.58, p=0.00) than other groups. however, there were no statistically significant differences between medical and engineering students in relation to these items (table 2). as expected, dental students scored the lowest total dental anxiety scores (table 2) which were significantly lower than those scored by either medical or engineering students (f=14.13, p=0.00). generally, in terms of total anxiety scores, women were relatively more anxious than men (t=-2.21, p=0.03) (table 3). women were particularly more anxious than men concerning items related to waiting in the dentist's sitting room (t=-2.56, p=0.01) and local anesthetic injection (t=-2.62, p=0.01). however, such gender variation was not significantly different when items related to going for dental visit tomorrow, having teeth drilled or having teeth scaled or polished were considered. sd=standard deviation the numbers and percentages of subjects who had a total score of 15 or more are shown in table 4. surprisingly, medical students were found to be the most frequent among those who scored 15 or above. this study revealed that dental students do have lower levels of dental anxiety than their engineering and medical counterparts (p<0.01). the mean total scores for the mdas showed that severe dental anxiety was mostly associated with drilling and intraoral local anesthetic injection. surprisingly, the medical students showed the highest total anxiety scores and the greatest percentage of subjects scoring 15 or more. the engineering students were already expected to score the highest anxiety scores as they do not receive health or dental awareness education, whilst the medical students are supposed to be more familiar with stress management related to health measures. however, some of the highly anxious patients may avoid showing their anxiety in order not to interfere with the dentist's procedure. thus, the dentist should be aware of the patient's possible adverse reaction or distress19,21. however, when the data are critically appraised, statistically significant difference between groups related to total anxiety scores or the scores of individual items of the anxiety scale could be seriously misleading and possibly do not always reflect an actual clinical significance. this could be attributed to the fact that the sample size is large, which added to the power of the study and revealed even the minor statistical differences between the groups. thus conclusions made on actual differences in numbers and on absolute statistical significance should be drawn with caution. lack of dental health education might result in patients ' fear and anxiety which in turn might end with poor patient compliance and attitudes. this will make it more difficult to manipulate patients and yield difficult patients and thus increase the levels of dental profession-related stress1,12. assessing the level of patient anxiety before commencing the dental treatment may offer invaluable insight into the probable patient attitudes and behavior towards the dental treatment. this information will be further utilized in developing the best strategies to manage patient anxiety. health-related behavior depends on oral health knowledge14. in jordan, only students related to the dental field receive adequate dental health education as it is an integral part of their curriculum. jordanian schools and universities pay little attention to the dental health education of their students. students not related to the dental field receive little, if any, dental health education and their curricula lack such courses. since the dental health education is generally ignored in the pre-university stage, medical and engineering students still possess the same ideas about dentistry and dental care at the university. on the other hand, dental field-related students do have the chance to formulate new and better ideas and understanding of the dental health care and for the above mentioned reasons, the dental treatment will still be considered mysterious and stressful for the non-dental students while the dental students will feel better during the dental treatment. this could partly explain the relatively higher scores of dental anxiety among the non-dental students compared to their dental peers. irregular dental attendance may play a major role in increasing the levels of dental anxiety15,23. if this is added to the lack of dental health education in jordan, the high levels of dental anxiety could be explained and clearly demonstrated among the non-dental students. this might form the bases for explaining the presence of higher levels of dental anxiety among women as well as medical students in this study. female students were found to have higher levels of dental anxiety in all groups and they were more anxious about waiting in the sitting room and taking anesthetic injections. this corroborated the results from previous studies that showed higher levels of dental anxiety among women,11,21,24. this finding might be explained on the basis that women have higher levels of neuroticism than men and that anxiety is positively associated with neuroticism9,10,20. however, in the current study, the statistically significant difference between men and women was marginal and the difference in anxiety scores for both genders was minimal (men 12.29%, women 13.17%). therefore, it could be inferred that statistical significance might not be necessarily interpreted as a clinical one. medical students might be prone to have higher levels of neuroticism, due to their courses and stressful field of study2 and thus demonstrate higher levels of dental anxiety than engineering students. on the other hand, although dental students are leading stressful courses and profession they are yet still exposed to better dental health education and knowledge and thus develop favorable dental behavior. presence of suitable dental health education and knowledge seems to be capable, to some extent, of overruling the effect of stress and personality factors on dental anxiety among dental students. the control of dental anxiety might be aided via good dental health education, regular dental visits, good patient-dentist relationship and suitable communication with the patients6. for a successful dental treatment, a gentle, supportive, professional, sympathetic, quiet and more considerate approach should be followed when managing patients with dental anxiety. on their first visit, patients should be dealt with more sensitively in order to avoid increasing their anxiety and thus avoid their repulsion to the dental care9. in view of the current available data, it appears that further dental health education measures are required to be applied among the jordanian non-dental university students and the population in general in order to control the levels of dental anxiety and thus improve patient dental attitudes and compliance. suitable standards of dental health knowledge and education could overcome the negative effects of personality and reduce dental anxiety. interception of dental anxiety at early stages will reduce the chance of resistant dental anxiety and fear which are difficult to deal with in the dental clinic. the importance of dental health education can not be overemphasized in the reduction and control of dental anxiety. pre-university as well as non-dental university curricula should include dental health education in order to help reduction of dental anxiety among the population. although the current study utilized the modified anxiety scale and investigated the levels of dental anxiety among university students from different fields of study and the sample size was representative and large, further studies are required to investigate the effect of various correlates on dental anxiety.

objective: this study was designed to investigate the subjective ratings of dental anxiety levels among university students enrolled at jordan university of science and technology. in addition, the present study aimed to explore the sources of dental anxiety and the impact of gender on the perceived dental anxiety and the correlation between field of study and dental anxiety level. material and methods: the modified corah dental anxiety scale was used to measure dental anxiety among the study population. six hundred subjects were recruited into the study from jordanian undergraduate students from the faculties of medicine, engineering, and dentistry. results:five hundred and thirty five complete questionnaires were returned, which accounts for a response rate of 89.2%. the totals of the mean anxiety scores were the following: medical students, 13.58%; engineering students, 13.27% and dental students, 11.22%. about 32% of the study population has scored 15 or more. dental students had the lowest percentage of those who scored 15 or more. surprisingly, the medical students were responsible for the highest percentage of those who scored 15 or above. although women demonstrated statistically higher total dental anxiety scores than men (p= 0.03), the difference between both genders was small and could be clinically insignificant. the students were anxious mostly about tooth drilling and local anesthetic injection. conclusion:lack of adequate dental health education may result in a high level of dental anxiety among non-dental university students in jordan. further studies are required to identify the correlates of dental anxiety among university students.

PMC4399532

pubmed-457

after hyping deep-etch electron microscopy (em) for my whole career (heuser, 2011), i'll take this invitation to write an ascb award essay to talk it up some more! replicas are not only impervious to beam damage in the electron microscope, forever the big problem, because the electron beam heats up the sample so terribly during viewing, but their electron-scattering power is also excellent, so they are simple to image and give super high-contrast. and the key thing to remember is that replicas are utterly faithful to whatever they are replicating they're just surface renderings, copying exactly the contours of the sample and displaying these contours in the electron microscope image. so the whole approach boils down to worrying about how to prepare your biological samples for replication. it takes the right equipment and some practice to make a proper replica, but, once mastered, it's utterly routine and simple to learn. when mark kirschner first watched me do it while helping me to put it on the map by providing gorgeous cytoskeletons [heuser and kirschner, 1980]he got bored right away and asked me, ca n't you teach a monkey to do that ?) anyway, replicas have a glorious history, because in the early days of em, way before thin-sectioning techniques had been developed, they were the only way to go the only way to get any sort of biological sample into the electron microscope. thus the em pioneers in the 1940s used metal replicas to discover viruses and phages and to make the first halting characterizations of macromolecular assemblies like collagen and neurofilaments. what they lacked back then was a way to see inside cells, which keith porter achieved for the first time in 1945 by simply growing cells flat enough to see through in the electron microscope really, really flat and then fixing and staining them properly for em (his other huge contribution). people not familiar with em should be reminded that porter's 1945 images opened the door to cell biology, and his development of thin-sectioning techniques for cells in the following 10 years really put cell biology on the map. but back to replicas. the whole field of scanning electron microscopy (sem) was totally dependent on them because everything had to be coated with metal in order to be seen in the scanning electron microscope. likewise, the exciting field of freeze-fracture em took off after hans moor teamed up with a swiss company that made replicating machines (balzers of lichtenstein) and mounted a microtome inside one, so that frozen cells could be fractured open (not quite thin-sectioned, the microtome was n't that good). this made it possible for people to make metal replicas of frozen cells without melting them even a little bit some sort of miracle! deep-etch em is a variant of what moor introduced (heuser and salpeter, 1979) and deserves special attention only because its purpose has been to avoid all of the fixation and staining and dehydrating procedures that had accompanied previous approaches to em and essentially to get living cells replicated after they were frozen (figure 1). we found that freeze fracture works just as well or better on unfixed cells and molecules, and therefore made a huge effort to devise a really good way to freeze living cells, tissues, and cell extracts without introducing such artifacts as ice-crystal damage. a platinum replica of the inside surface of a hela cell prepared by unroofing it in culture before quick-freezing and freeze-drying it in the usual way (heuser, 2000). three-dimensional view was used for the publicity and table cards for our department's centennial celebration three years ago. clathrin lattices found on all cell membranes and illustrates the various stages in their evolution, from totally flat to fully curved and ready to pinch off during endocytosis. such three-dimensional deep-etch images were the first to illustrate that f-actin filaments (highlighted in purple) often become involved in the later stages of such clathrin coated pit formation and stay behind as circular scars after coated vesicles have left the surface (above the wash in washington university). as explained in this essay, the swell opportunity to view such expanses of the plasma membrane at such a high resolution was a lucky outcome of our being able to freeze samples fast enough to avoid ice-crystal formation and then, miraculously, to platinum-replicate such frozen membranes without melting them. the best way to freeze everything turned out to be a spruced-up version of an approach anthonie van harreveld had used in the 1960s at caltech to freeze brains in preparation for classical thin-section em. van harreveld wanted to maintain the proper distribution of electrolytes in the brain and had reason to believe that the classical fixation techniques being used on brain were distorting this distribution. he reasoned that the freeze-substitution technique that ned feder and richard sidman had put on the map in the late 1950s would give him more realistic views. with this technique, a frozen sample is fixed and prepared for embedding in plastic by dissolving the ice out of it at subzero temperatures, using acetone or the like. van reasoned, quite correctly, that this should prevent artifacts from occurring during fixation, because nothing ever melted; but how he came up with the idea to freeze the brain by slamming it onto an ultracold block of copper remains a mystery to this day. (it's fun to mention here that van harreveld did n't start developing this technique until he was already 60 years old !) anyway, it sure worked for van, and it also worked for tom reese and me when we copied his slammer, even though we had to spend years ironing out the bugs and making a freezing machine that was mechanically sound and gave reproducible results (heuser et al., 1979). the result was our so-called liquid helium cooled cryopress (renamed to avoid the distressing idea of a delicate piece of tissue being slammed against anything albeit, it's the abruptness of contact and the superfast extraction of heat from the sample by the copper block that gives such good freezing in the first place). fast-forward to today, and we find that freeze substitution is still the backbone of modern efforts to image cells in the electron microscope, and indeed preserves the structure of cells far better than the techniques of fixation and plastic embedding developed by the pioneers of thin-section em. when combined with thicker sections, higher em voltages, and modern tomographic reconstruction techniques, it yields really outstanding images. so why are n't there more than 10 labs in the world using our (or van harreveld's) cryopress to get the quality of freezing our lab has depended on for decades? the answer lies in part with another advance that hans moor spearheaded in switzerland, again with the same enlightened balzers company producing vacuum evaporators, namely, high-pressure freezing. at the time, phase diagrams of water indicated that water could be frozen into an amorphous glass without the induction of any damaging ice-crystal formation by putting it under extreme pressure (> 2000 atm). today, theories about how water turns into vitreous (noncrystalline) ice are much more complex, but moor went ahead and developed ways to put a biological sample under huge pressures and only then freeze it by spraying liquid nitrogen at it rather than slamming it against a liquid nitrogen cooled copper block. (the rapidity of freezing, he reasoned, should no longer be important if the pressure trick works as apparently it does.) today, most em labs have a high-pressure freezer, and most of the em papers that are published on freeze-substituted cells have availed themselves of these devices. so why not use our slammer (or cryopress) for freezing before freeze substitution, since it's cheaper, faster, more reliable, and handles larger samples? frankly, we do n't get it! not only that, but high-pressure frozen samples can not be freeze-fractured at all at least no one has yet devised a way to do so because the samples end up encased in various sorts of metal pressure chambers, whereas our quick-frozen or cryopressed samples are spread out and open to the world (mandatory for freeze fracture, but also good for freeze substitution). and for that matter, why are n't more labs making good old replicas of quick-frozen, deep-etched molecules (heuser, 1983; goodenough and heuser, 1984; hanson et al., 1997)? that is, of course, the ultimate mystery to us. probably it's just because people do n't realize that there are still good replicating machines available for purchase, and people do n't realize that these machines are n't so expensive and are easy to operate. well, as i said at the outset, i've been hyping our technique for decades and ca n't stop now. i believe that an opportunity is being missed and that simplifying techniques so that even a monkey could do it will attract not monkeys to the field, but serious young investigators who want to get their hands on electron microscopes and want to get the most true to life i'm a photographer at heart and love sharing images, all sorts of images, with people who appreciate them and can learn from them i love that more than anything. what fun it was, to be able to interact on a daily basis with the mark kirschners, tom pollards, ron vales, bernie gilulas, and ira mellmans of cell biology (and sorry to all those whom i did n't mention you know who you are!). plus, a handful of people really fired me up: tom reese, my boss as a postdoc at the national institutes of health, with whom i became so intertwined for so many years that he and i will never know who did what or who deserves what credit in the original development of quick-freeze, deep-etch em (heuser and reese, 1973; heuser et al., 1979); and then nobutaka hirokawa, who came to my lab as a postdoc, and immediately orchestrated a host of collaborations with leading cell biologists around the world that put deep etching on the map (before leaving for the university of tokyo to become chairman of the department of cell biology, and then dean of the medical school, and now head of the whole human frontier science program); and finally, my ex ursula goodenough, who absorbed my images and simply took off, making huge advances in several fields, thanks to her deep grasp of all aspects of cell biology. finally, i'd like to simply add this: biological em was terribly interesting for me in the early days, back when it first allowed people to zoom in on the structures that light microscopists had been studying for so long and show what they actually were i used to wait with eager anticipation for each new issue of the journal of cell biology to arrive in the mail and then would devote a whole evening (maybe with a glass of wine) to carefully examining every new electron micrograph published that month. but em became even more captivating for me as people began more and more to systematically manipulate cells by physical and pharmacological (and eventually genetic) methods and then to look in the microscope to see how this altered the fine structures of their cells. this opened the door to true structure/function correlations at least when the effects of these experimental manipulations of cell physiology and biochemistry were properly determined, along with the microscopy. this era of em was the most fun for me, personally, but as it happened, this heyday was cut short by an overwhelming urge in some quarters to improve the methods of em, in an attempt to make the imaging of cells more this trend particularly captivated the equipment manufacturers and led to an arms race of microscope development that ended up making electron microscopes so very costly that only a few centers could support them anymore. the result was actually a curtailment of general, everyday em as it had been practiced by individual investigators in command of their own microscopes and published every month in the journal of cell biology. and as a consequence, over the past 15 years or so, em has gradually been relegated to a service status, carried out largely by em cores in most major institutions. electron microscopists, and gone also is the use of em for all sorts of fun structure/function correlations. and helping to eclipse the routine em that i enjoyed so much have been all the tremendous advances in light microscopy, coupled with all the advances in digital camera recording of live-cell dynamics (not to mention the burgeoning field of superresolution light microscopy, crowned this year with the nobel awards). these huge advances have captivated nearly everyone still interested in functional correlations of cell structure and have left traditional em sort of out in the cold, an outcome i find most unfortunate. i feel strongly that seeing cell structures at the em level still is the only way to fully grasp their molecular architecture, and that seeing changes in their molecular architecture at this level is the only way to truly understand their function. i'm permanently stuck with the founding fathers ' view that cell ultrastructure will ultimately display and explain all of cell function! george palade was my greatest hero, and his fun explanation in his nobel lecture of why he chose to study the pancreatic acinar cell is my favorite quote: perhaps the most important factor in this choice was the appeal of the amazing organization of the pancreatic acinar cell, whose cytoplasm is packed with stacked er cisternae studded with ribosomes. its pictures had for me the effect of the song of a mermaid: irresistible and half transparent. its meaning seemed to be buried under only a few years of work, and reasonable working hypotheses were already suggested by the structural organization itself. irresistible and half transparent, indeed! thanks, george. and thanks to all of you who cared to look at my images and all the institutions and funding agencies that made it possible for me to generate them! every picture i take, i already have an audience for it right as i take it. (of course, they're not actually there, they may be continents away, but i'm imagining them being there and already planning how i will get that picture to them and what i'll tell them about it as soon as it's in the computer.) it's for showing to someone who immediately comes to mind as soon as that field pops into view in the electron microscope. oh, pietro will love that huge neuromuscular junction; fulvio will be amazed by that quality of membrane preservation in freeze-substituted yeast; ursula will be psyched by that run of axonemal dynein; tom will be impressed with such a clear view of actin branchpoints. only rarely am i lucky enough to have someone actually sitting next to me and to be able to talk to him or her right then, person to person maybe a new postdoc or a close collaborator who really needs to look over my shoulder to see how his or her prep came out. anyway, i want each of my real or imaginary viewers to like that picture, to think it's a good picture attractive, clear, understandable, useful, illuminating, that is, illuminating something about the subject (be it a personal portrait or a picture of a cell interior or a molecule). i want my audience's appreciation! my whole drive of focusing all my work on improving techniques of preparation for em has come from wanting to take better pictures and get more of that appreciation. besides that, there's just that darn old curiosity: what does it actually look like, what does it look like exactly? how good a picture of it can i take? how good-looking can i make it (or him or her, with my personal portraits)? (nic spitzer once irritably dubbed the latter my thin sections of life as i was clicking away while canoeing with him down a rapids on the allagash river, but not paddling.) always on my mind is what's the most expressive or most characteristic or attractive attire or decoration i can outfit it (them) with? osmium or platinum or gold or furs and silks? capturing that best picture will help me to get to know my subject better, to really see it for what it is. even artifacts can be extremely beautiful and informative, if one knows how one got them and what they say about what the structure was, before it got altered. all these aspects of photography i can appreciate by myself, all alone, but never as much as when there is just one other person with me, with the same inclination and proclivity. sharing, mutual appreciation, communion that has been the whole name of the game for me in my research career. my advisor don fawcett, one of the great masters of em of all times, told me when i graduated from medical school, do n't become an electron microscopist, you'll become everybody's slave. actually, i think i can say that it turned out just the opposite: everyone else turned out to be my audience, my source of appreciation and self-worth, my foils, my mentors, and, most important of all, my best source for interesting things to look at in the electron microscope !

this brief essay talks up the advantages of metal replicas for electron microscopy and explains why they are still the best way to image frozen cells in the electron microscope. then it explains our approach to freezing, namely the van harreveld trick of slamming living cells onto a supercold block of metal sprayed with liquid helium at 269c, and further talks up this slamming over the alternative of high-pressure freezing, which is much trickier but enjoys greater favor at the moment. this leads me to bemoan the fact that there are not more young investigators today who want to get their hands on electron microscopes and use our approach to get the most true to life views of cells out of them with a minimum of hassle. finally, it ends with a few perspectives on my own career and concludes that, personally, i'm permanently stuck with the view of the founding fathers that cell ultrastructure will ultimately display and explain all of cell function, or as palade said in his nobel lecture, electron micrographs are irresistible and half transparent their meaning buried under only a few years of work, and reasonable working hypotheses are already suggested by the ultrastructural organization itself.

PMC4214773

pubmed-458

pycnodysostosis was first reported in 1923 by montanari, and he called it as atypical achondroplasia. the main characteristics are short stature, cranial dysplasia, increased bone density and fragility. other clinical features include open cranial sutures, hypoplastic paranasal sinuses, dysplastic lateral clavicle, shortened terminal phalanges, proptosis, blue sclera and frontal or occipital bossing. oral manifestations include obtuse gonial angle, grooved palate, anterior cross-bite, malpositioned teeth associated with increased incidence of dental caries and periodontitis, hypoplastic maxilla, receded chin, delayed eruption of permanent teeth, delayed exfoliation of deciduous teeth and hypoplasia of root-obliterated pulp spaces. a 47-year-old man reported to the department with a complaint of deformed lower jaw for the past 10 years. history revealed that the patient had undergone extraction of his teeth that was uneventful, following which there was fracture of jaw at the extraction site. subsequently, there was frequent exfoliation of teeth with fracture at different sites in the lower jaw. his medical history revealed that he had multiple fractures of the upper and lower limbs and a history of diabetes mellitus and hypertension. general examination revealed that the patient's height was 127 cm and weight was around 49 kg, with proportionate dwarfism [figure 1]. the hand and feet had short digits with overlying cutaneous wrinkles that tapered off with large overriding nails [figure 2]. a 47-year-old man, 127 cm in height shortened digits and cutaneous wrinkles on extraoral examination, there was facial dysmorphia with prominent forehead (frontal bossing), proptosis, beaked nose, deep nasolabial skin folds, micrognathia and obtuse mandibular angle on the right side [figure 3]. mild proptosis and facial dysmorphia intraoral examination revealed multiple clinically missing teeth, chronic periodontitis, narrow and grooved palate, no features of enamel hypoplasia, malposed teeth [figure 4] and evidence of sequestrum in relation to the tooth 46 [figure 5]. based on the history and clinical presentation, a provisional diagnosis of a bone dysplasia, probably pycnodysostosis, was made and differential diagnosis of cleidocranial dysplasia and osteopetrosis was included. grooved palate and clinically missing maxillary anteriors sequestrum in relation to tooth 46 laboratory findings were within normal limits, including hemoglobin conc., differential count calcium, phosphate, alkaline and acid phophatase level. computed tomography of the bone window of the skull showed open sutures and fontenalles with nonaerated paranasal sinuses, flattening of the mandibular angle on the right side with evidence of fracture and loss of bone architecture on the left side involving the ramus of the body of the mandible and hypoplastic maxilla [figures 68]. orthopantomograph revealed generalized bone loss, multiple missing teeth and obtuse gonial angle with loss of bone structure on the left side of the mandible involving the body and ramus [figure 9]. 3d-reconstructed computed tomography showing open sutures and fontanelles 3d-reconstructed computed tomography showing fractured body of the mandible and obtuse gonial angle 3d-reconstructed computed tomography showing fractured body of the mandible opg showing generalized bone loss, multiple missing teeth, and obtuse gonial angle lateral skull showing open fontanelles with nonaerated paranasal sinus and fractured body of the mandible the patient was surgically managed for osteomyelitis by removal of the sequestrum and curettage, and further mandibular reconstruction was performed. the patient presents with characteristic facies, dwarfism, beaked nose, prominent head and generalized increase in the density of bones not sufficient to obliterate medullary canals or cranial orifices. frequent fractures due to trauma can aid in diagnosing this condition. in our case, the patient was negligent about the condition and reported to us with a fractured jaw. intraoral clinical presentation included altered pattern of exfoliation of deciduous teeth and eruption of the permanent dentition. the disease is diagnosed at an early age, wherein the main reasons for consultation are generally short stature and open anterior fontanelles. in later stages, consultation is usually for fracture resulting from slight or moderate trauma, given the severe bone fragility. symptoms include dental abnormalities, with hypoplasia of the enamel, obliterated pulp chambers and hypercementosis. protrusion of the incisors with anterior open bite may be found, and dental crowding associated with extensive caries and periodontitis is frequent. in our case, multiple clinically missing teeth, chronic periodontitis, narrow and grooved palate, features of enamel hypoplasia and malposed teeth these conditions cause the premature loss of dentition that may already be complete by the fourth decade of life, similar to our patients. our patient showed evidence of sequestrum in relation to the tooth 46 as a result of osteomyelitis. orofacial infections are commonly encountered by the dentist and there are wide ranges of modalities that can be implemented in managing them. this is caused by the increased bone volume of the sella turcica that, on compressing the pituitary gland, causes its hypoplasia and a deficient production of the growth hormone. our patient's height was 127 cm and weight was around 49 kg, with proportionate dwarfism. the hand and feet had short digits with overlying cutaneous wrinkles that tapered off with large overriding nails. diagnosis of pycnodysostosis is based on the clinical presentation, and medical treatment for the condition is symptomatic. the differential diagnosis of pycnodysostosis includes osteopetrosis, acroosteolysis, mandibular acral dysplasia and cleidocranial dysplasia. unlike osteoporosis, hepatosplenomegaly and difference between pycnodysostosis and cleidocranial dysplasia is that dense and brittle bones are found in pycnodysostosis but not in cleidocranial dysplasia. new treatment modalities like gene therapy and bone marrow transplant can be expected to be the mainstay in the future, now that the abnormal expression of cathepsin k and the gene defect has been located. now-a-days, symptomatic treatment is provided for patients with pycnodysostosis, with the main intention of prevention of fractures. as pycnodysostosis is associated with inappropriate bone remodeling, it can pose a challenge for a dental health care professional to provide treatment as there can be serious complications, such as osteomyelitis arising as a result of dental infections. tooth extractions in these patients demand certain special care, such as carrying out the surgery atraumatically with proper asepsis. oral hygiene practices and frequent visits to

pycnodysostosis, a sclerosing bone dysplasia, is a rare autosomal recessive disorder with an estimated prevalence rate of one in one million. patients with pycnodyostosis usually have normal intelligence, sexual development and life span. this condition is characterized by increased bone density and fragility along with oral manifestations like malposition teeth, hypoplastic maxilla, receded chin and delayed eruption of permanent teeth with discharging sinuses in the jaws because of poor blood supply. this is one such rare case report of a 47-year-old patient presenting with a complaint of fractured jaw and reviewing the clinical and radiographic characteristics of pycnodysostosis.

PMC4260389

pubmed-459

the accumulation of metals in the environment, stemming from their origination in the earth's crust, as well as from anthropogenic sources, creates the potential for significant human exposures and subsequent health hazards. deleterious metal-induced health effects, including carcinogenesis and neurodegeneration, have been reported in all body systems, with exposure stemming from multiple sources, including contact with contaminated food, water, air, or soil. the particular metals considered in this review, antimony, arsenic, cadmium, manganese, mercury, silver, and uranium, are among the classes of essential nutrients, as is the case with copper and manganese, as well as the nonessential, naturally occurring metals, such as arsenic, cadmium, and mercury, all of which can induce toxicity depending on the concentration level and exposure duration. over time, organisms have developed protective mechanisms to deal with metal exposure, most of which function in one of three ways: (1) decreasing the uptake of the metal, (2) stimulating the expulsion of the metal, or (3) activating the organism's general stress response mechanisms. metals can disrupt homeostasis by generating oxidative stress, inhibiting enzyme activity, impairing dna repair, and disrupting protein binding and normal cellular function, including proliferation, cell cycle progression, and apoptosis [14]. elucidating the mechanism(s) of metal detoxification has been difficult due to the complexity of mammalian systems and the reductionist approach inherent to cell culture systems. the model organism, caenorhabditis elegans (c. elegans), offers the advantage of an in vivo system that is less complex than the mammalian system while still sharing high homology. c. elegans possess ~60%80% of human genes and contain conserved regulatory proteins [68]. this soil nematode has been used in a number of toxicity studies due to its well-characterized genetic, physiological, molecular, and developmental stages. some of the advantages afforded by the c. elegans model system are small size (~1.5 mm adult), short lifespan (~3 weeks), and rapid lifecycle (~3 days) [810]. at adulthood, a single c. elegans hermaphrodite is capable of producing ~300 progeny. c. elegans are hermaphrodites, but approximately 1% of c. elegans are male, allowing for genetic experimentation. the nematode's small genome and relative anatomical simplicity (less than 1000 cells) contribute to the appeal of this model system for genetic manipulation. in addition, the use of rna interference (rnai) and chromosomal deletion in worms has provided valuable information regarding the increased sensitivity of mutant strains to metal toxicity [1214]. maintenance of nematode strains is relatively simple; they grow on bacteria-seeded plates and can be maintained at 20c. strains can also be frozen indefinitely, easily allowing for the accumulation of large stocks of worms. c. elegans provide the researcher with a uniquely powerful model, as the worm's translucent body allows for the in vivo visualization of fluorescently labeled individual cells and proteins. the in vivo c. elegans model system is especially valuable for the investigation of metal detoxification and is particularly amenable for examining gene-environment interactions, albeit with a few considerations to take into account (table 1). several toxicity endpoints are readily detected and well documented in the nematode, including mortality, lifespan, reproduction, and feeding [1719]. acute toxicity can also be assessed in the nematode through behavioral endpoints, such as locomotive behavior, head thrashing, body bending, and other basic movements [2024]. recently, the role of c. elegans as a biomonitor in environmental risk assessment has also been explored [2528]. several cellular systems such as the glutathione (gsh), metallothioneins (mts), heat shock proteins (hsps), as well as various pumps and transporters work in concert to detoxify and excrete metals. it remains to be established whether the knockdown or overload of one detoxifying system upregulates other compensatory mechanisms. in this review, we offer a brief summary of the ways in which the c. elegans model has shed novel insights on the various mechanisms of metal detoxification. metals considered herein are antimony (sb), arsenic (as), cadmium (cd), lead (pb), mercury (hg), silver (ag), and uranium (u). glutathione (gsh) is a cysteine-containing tripeptide, consisting of glutamic acid, cysteine, and glycine and is found in most life forms. gsh possesses antioxidant properties, since the thiol group of cysteine is a reducing agent and can be reversibly oxidized (gssg) and reduced (gsh). gsh is maintained in the reduced form by the enzyme, glutathione reductase (gr), and functions by reducing other metabolites and enzyme systems. glutathione peroxidase (gpx) catalyzes the oxidation of gsh to gssg in the presence of ros (figure 1). gssg can be converted back into gsh via gr and the conversion of nadph to nadp+. proton-translocating mitochondrial nicotinamide nucleotide transhydrogenase (nnt) catalyzes the reduction of nadp+ by nadh and is an important source of nadph, as has been demonstrated using nnt-1 deletion mutants. glutathione s-transferases (gsts) can catalyze the conversion of gsh to gs, which can then form complexes with various xenobiotics to facilitate excretion. these enzymes are found at particularly high levels in the liver, and gsh is typically the most abundant sulfhydryl-containing compound in cells. additionally, gsh is known to offer protection against metal-generated ros by binding free radicals. gshs have been reported to increase, decrease, or remain constant after exposure to metals [5557]. the gsh system is found in animals, plants, and microorganisms. c. elegans express approximately 50 gsts. additionally, phytochelatins (pcs), a family of metal-inducible peptides synthesized enzymatically from gsh by pc synthase (pcs) in the presence of heavy metal ions, have been identified in c. elegans. although pcs are synthesized from gsh, they are broadly classified as class iii metallothioneins and have been shown to be important in the detoxification of heavy metals. liao and yu investigated the involvement of the gsh system in response to inorganic arsenic exposure. results confirmed that oxidative stress plays a role in arsenic-induced toxicity by mutating glutamylcysteine synthetase (gcs) (gcs-1), the rate-limiting enzyme in gsh synthesis, in worms. the gcs-1 loss-of-function strain demonstrated hypersensitivity to arsenic exposure in lethality testing, as compared to wild-type animals, an effect that was rescued by the addition of gsh to the medium, indicating that these enzymes are crucial for mediating arsenic-induced toxicity. furthermore, helmcke and aschner reported a significant increase in fluorescence in a gst-4::gfp strain following both acute and chronic exposure to mehg. it was also demonstrated that knockout gst-4 worms did not display greater sensitivity to mehg than did n2 wild-type worms. gsh levels were found to be increased in worms subjected to acute exposure, whereas worms subjected to chronic exposure exhibited depleted levels of gsh. in their hormetic model, helmcke and aschner demonstrated an increase in gst-4::gfp expression after low concentration, acute exposure, a finding which indicates that gst-4 may be involved in the hormetic response to mehg. taken together, the data from these studies suggest that gst-4 contributes to the response to mehg exposure, but that knockdown of this gene does not affect with overall lethality. in a study examining the role of pcs in the elimination of cadmium, vatamaniuk and colleagues reported that the c. elegans pcs-1 gene encodes a functional pc synthase critical for heavy metal tolerance. using double stranded rnai against pcs-1, this group showed that, although the progeny of worms injected with the dsrnai and exposed to cadmium (525 m) managed to reach adulthood, these worms were small, necrotic, sterile, and had a much shorter lifespan than did the wild-type controls. after being exposed to 50 and 100 m concentrations of cadmium, the pcs-1 worms arrested at the l2l4 stage were necrotic and died by day 6. the results were definitively dependent on the presence of cadmium, because pcs-1-deficient worms, in the absence of cadmium exposure, responded identically to the wild-type controls. hughes and colleagues examined metabolic profiles following cadmium exposure in phytochelation synthase-1 (pcs-1) mutants. results from these studies showed that the primary response to low levels of cadmium is the regulation of the transsulfuration pathway, due to the fact that cadmium exposure caused a decrease in cystathionine concentrations and an increase in phytochelation-2 and -3. these results were corroborated by additional studies which demonstrated that pcs-1 mutants were an order of magnitude more sensitive to cadmium than were the metallothionein mutants. furthermore, the mt-pcs-1 triple mutant was found to display an additive sensitivity toward cadmium. metallothioneins (mts) belong to a family of cysteine-rich low-molecular-weight metal-binding proteins (mw 3,50014,000 da) involved in metal detoxification and homeostasis. mts bind both metals of physiological importance such as copper and zinc, as well as xenobiotics including arsenic, cadmium, mercury, and silver. the binding of these metals occurs via the interaction of the cysteine residues with thiol groups. binding of metals by mts may be transient, as mts are capable of rapidly releasing metal ions. the protective roles of mts can be ascribed to their three primary functions: (1) metal homeostasis, (2) heavy metal detoxification, and (3) protection from oxidative stress. additionally, these proteins have been identified as contributors to the hormetic response [39, 61]. mammals express four known metallothionein isoforms (mt-i, mt-ii, mt-iii, mt-iv). mt-i and mt-ii are expressed in almost all tissues and have been best characterized with regard to their protection of the brain. mt-iii is especially enriched in the central nervous system, although its role has not yet been clearly defined. mt-iv is most abundantly expressed in the stratified squamous epithelia [6466]. mt expression has been shown to be induced under stressful cellular conditions such as exposure to cytokines, glucocorticoids, reactive oxygen species (ros), and metal ions. mts can bind directly and sequester the toxicant; they also can provide protection by acting as antioxidants (figure 1). further, mts can limit apoptosis and promote the survival of mitochondrial dysfunctional cells by serving as highly efficient reducing elements against reactive oxygen species (ros). c. elegans contain two distinct isoforms of mts, known as mtl-1 and mtl-2, which can be induced in response to exposure to various metals. jiang and colleagues examined the effects of mts on depleted uranium (du) in c. elegans. results from their study showed that mtl-1 was an important factor in uranium accumulation in c. elegans as knockouts displayed increased cellular accumulation. in a study investigating lead and methylmercury toxicity, ye and colleagues demonstrated the involvement of mts in affording a protective cross-adaptation response to neurobehavioral toxicity. this endpoint was assessed by observing behavioral alterations (head thrashing and body bending) in worms that were exposed during the l2 phase to either pb or mehg. the study was conducted in conjunction with mild heat shock, wherein pretreatment of the larva with heat shock prevented the neurobehavioral deficits and the stress response at lower concentrations (50100 m) but not at higher concentrations (200 m). additionally, mild heat shock coupled with exposure to a low concentration of either metal was found to induce mtl-1 and mtl-2 promoter activity and gfp gene expression, results that were not observed in either the metal-exposed or heat-shocked cohort alone. finally, the overexpression of mtl-1 or mtl-2 at the l2 stage was shown to significantly repress neurobehavioral toxicity, suggesting that the accumulation of mt protein is necessary to confer the protective response to the toxicant. similarly, helmcke and aschner reported that mtl knockouts displayed increased lethality upon exposure to mehg. this group also demonstrated increases in mtl-1::gfp fluorescence in response to acute mehg exposure at the l1 stage; however, chronic mehg exposure produced no change in florescence. their results indicate that mtl-1 is important in mediating mehg toxicity and that the effects occur in a concentration- and time-dependent manner. meyer and colleagues examined the aggregation of silver nanoparticles (agnps) in wild type and mtl-2 c. elegans. results from these studies showed that the mtl-2 strain displayed greater agnp sensitivity than did the wild-type controls. these data indicate that there may be a differential preference for mtl-1 over mtl-2 depending on the particular metal to which an organism is exposed. in a 2004 study by swain and colleagues, using gfp-expressing transgenes, mt-null alleles, and the rnai knockdown of mts, this group demonstrated that cadmium but not copper or zinc was able to influence a concentration-dependent, temporal transcription response. cadmium exposure caused a reduction in body size, generation time, brood size, and lifespan, effects that were magnified in the mt knockdown worms. hughes and colleagues studied metabolic profiles using proton nmr spectroscopy and uplc-ms following cadmium exposure in single and double mtl knockouts. results showed that the metallothionein status did not influence the metabolic profile in cadmium-exposed or unexposed worms. the primary response to low levels of cadmium was the regulation of the transsulfuration pathway, due to the fact that cadmium exposure resulted in a decrease in cystathionine concentrations and an increase in phytochelation-2 and -3. these results were corroborated by data showing that pcs-1 mutants (phytochelation synthase-1) were an order of magnitude more sensitive to cadmium than were mt mutants. further, an additive sensitivity toward cadmium was observed in the mt-pcs-1 triple mutant. a study by bofill and colleagues examined zinc and cadmium toxicity; results indicated differential metal binding behavior for mt-1 as compared to mt-2. specifically, the mt-1 isoform showed optimal behavior when binding zn, and mt-2 showed optimal behavior when binding cd. accordingly, it was hypothesized that, due to its induction following cd exposure, mt2 is primarily responsible for detoxification, whereas mt1 possesses some degree of constitutive expression and is, therefore, primarily involved in physiological metal metabolism (e.g. zinc). these findings were corroborated by additional studies which showed that mt-knockout worms exhibited significantly decreased levels of overall fitness after the knockout of mt1 than after mt2 knockout. further, both mt isoforms displayed a clear preference for divalent metal ion binding as opposed to copper coordination, likely due to the presence of histidines in the mts. using both in vitro and in vivo models, zeitoun-ghandour and colleagues examined zinc and cadmium exposures and showed different roles for mtl-1 and mtl-2. both isoforms their affinities and stoichiometries were measured, and both isoforms displayed equal zinc- binding ability; however, mtl-2 had a higher affinity for cd than did mtl-1. these experiments were repeated in vivo in mtl-1, mtl-2, and double knockouts following exposure to 340 m zn or 25 m cd. zinc levels were found to be significantly increased in all knockout strains, but mtl-1 knockout worms demonstrated the most acute level of sensitivity. however, cadmium accumulation was found to be the highest in the mtl-2 knockout and double mutant strains. additional studies assessed metal speciation, and results indicated that o-donating ligands play an important role in maintaining zinc levels, independent of metallothioneins status. further, cadmium was shown to interact with thiol groups, and cd speciation was significantly different in the mtl-1 strain when compared with both the mtl-2 strain and the double knockout strain, suggesting that the two mt isoforms have distinct in vivo roles. the authors suggested that mts are not functioning as metal storage proteins but, rather, are mediating the accumulation and excretion of metals. a follow-up study, showed in vitro evidence for the partitioning of zinc and cadmium with different metallothionein isoforms. employing electrospray ionization mass spectrometry (esi-ms) to directly observe zinc and cadmium binding preferences, more cadmium ions were found to be preferentially bound to mt-2 than to mt-1; however, cd was shown to be capable of inducing both isoforms. finally, partitioning was also demonstrated to be more effective at lower cd: zn ratios. using daf-2 (insulin receptor-like protein) and age-1 (phosphatidylinositol-3-oh kinase catalytic subunit) mutants, barsyte et al. examined the expression of mt genes under noninducing conditions and after exposure to cadmium and copper. they reported that mt-1 mrna levels were significantly higher in daf-2 mutants compared to both age-1 mutants and wild-type worms under basal conditions. exposure to cadmium treatment resulted in a three-fold induction of mt-1 and a two-fold induction of mt-2 mrna in daf-2 mutants as compared to wild-type controls. copper did not induce mt-1 or mt-2 mrna expression in any of the strains tested. collectively, these studies show differential metal preferences for one mt isoform over another depending on the metal to which an organism is exposed. most significantly, these studies indicate that the mts play crucial roles in metal detoxification (table 2). indeed, mts have been associated with a protective effect in cells under numerous states of disease and stress. interestingly, serum mt levels of cancer patients are three times higher than those of control patients. a study conducted in denmark revealed the increased expression of mt-1 and mt-2 mrna and protein in many human cancers such as breast, kidney, lung, nasopharynx, ovary, prostate, salivary gland, testes, urinary bladder, cervical endometrial skin carcinoma, melanoma, acute lymphoblastic leukemia, and pancreatic cancers. this information is of particular import given the use of metals for the treatment of certain cancers, for example, arsenic as treatment for promyelocytic leukemia. it is interesting to postulate that higher levels of mts may enhance the efficacy of metal therapeutic agents or, conversely, may lead to resistance to such therapies. understanding the factors that modulate mt expression will allow for the improved understanding of metalloid toxicity and will provide more effective therapeutic approaches to metalloid-based chemotherapy. there are a number of pumps and transporters that have been implicated in metal detoxification. these include atp-binding cassette (abc) transporters, such as the multidrug resistance-associated protein (mrp) as well as two members of the p-glycoprotein subfamily (pgp-1 and pgp-3), which have been shown to contribute to heavy metal tolerance through the use of c. elegans deletion mutants. in c. elegans, there are approximately 60 genes encoding abc transporters, and these genes make up the largest family of transporters. c. elegans have four mrp homologues and fifteen pgp homologues. the pgps are ubiquitously expressed and are most abundantly found in the apical membranes of the gut and in the excretory organs of the worm. pgp-2 is expressed in the intestine and is required for the acidification of lysosomes and lipid storage; pgp-1 and -3 contribute to heavy metal and drug resistance. tseng and colleagues investigated the arsa protein-mediated detoxification of the metalloids, as(iii) and sb(iii). bacterial arsa atpase is the catalytic component of an oxyanion pump that is responsible for resistance to arsenite and antimonite. in this study, wild-type and asna-1-mutant nematodes were evaluated for as and sb response and toxicity. the asna-1 gene of c. elegans was found to be stimulated by as(iii); further, sb(iii) was determined to be crucial for establishing tolerance. although these results occurred in response to as and sb exposure, the ubiquity of the arsa atpase-dependent pathway has not been observed in other species or in response to other metals. the role of multidrug resistance-associated protein (mrp) in arsenite and cadmium toxicity was explored in a study by broeks and colleagues. the targeted inactivation of mrp-1 rendered the arsenite and cd exposed worms incapable of recovering from temporary exposure to high arsenic and cadmium, whereas the wild-type controls were able to recover. additionally, worms were also shown to be hypersensitive to arsenite and cd exposures when both mrp-1 and pgp-1 (p-glycoprotein-1) were deleted. lastly, no increased sensitivity in response to exposure to antimony was observed in mrp-1-deletion mutants as compared to wild-type controls. vatamaniuk and colleagues characterized the half-molecule abc transporter of the heavy metal tolerance family-1 (hmt-1) subfamily in response to cadmium exposure. the suppression of hmt-1 expression by rnai was shown to produce punctuate refractive inclusions within the vicinity of the nucleus of the intestinal epithelial cells upon exposure to toxic levels of cadmium. similarly, schwartz and colleagues described the c. elegans hmt-1 following exposure to arsenic, copper, and cadmium. hmt-1 conferred tolerance in response to exposure to all three metals as shown by lethality testing following the knockdown of hmt-1. kurz and colleagues demonstrated the three-fold induction of pgp-5 following cadmium exposure. results of this study showed that strong fluorescence was induced in the intestinal cells of pgp-5::gfp worms, where the gfp-encoding gene is under the control of the upstream pgp-5 promoter. copper and zinc were also found to be capable of inducing pgp-5 expression in these worms. accordingly, it was concluded that pgp-5 is required for establishing full resistance to cadmium and copper. in addition, the rnai knockdown of tir-1, an upstream component of the p38 mapk pathway in the pgp-5 transgenic reporter strain, was shown to significantly reduce pgp-5 induction following exposure to cadmium. however, the double-stranded rna knockdown of erk (mpk-1) and jnk (med-1 and kgb-1) did not affect the induction of pgp-5 in response to cadmium exposure. au and colleagues studied the divalent-metal transporter (dmt1) following exposure to manganese. the dmt1-like family of proteins has been shown to regulate manganese and iron in the cell. the deletion of the three worm dmt1-like genes resulted in differential effects on manganese toxicity. the deletion of smf-1 and smf-3 increased mn tolerance, whereas the deletion of smf-2 increased mn sensitivity. heat shock proteins (hsps) are cytosolic molecular chaperones. hsps promote the refolding and repair of denatured proteins and facilitate protein synthesis upon activation by cellular stress [75, 76]. hsps, particularly those in the hsp70 family, have also been shown to participate in the hormetic response. hsp70s are atp-binding proteins that convert atp to adp and bind to peptides, thereby, inactivating them and preventing aggregation (figure 1). oxidative stress can cause a reduction in cellular atp levels. decreased levels of atp result in the continued prevention of the aggregation of damaged proteins. the functions of the hsp70 products are mediated by the conserved n-terminal atpase and the c-terminal peptide-binding region. the human and c. elegans hsp70 genes have a high degree of homology and share a conserved core the hsp16 family of stress proteins is produced in c. elegans only under stress conditions [8082]. in a study examining the effects of mehg exposure, hsp-4::gfp was measured immediately following the treatment of l1 worms for 30 minutes and l4 worms for 15 hours with this toxicant. after 30 minutes of acute exposure to mehg, however, in l4 worms chronically exposed to mehg for 15 hours, hsp-4::gfp was induced. at the same time point in the chronic treatment paradigm, a four-fold increase in gst-4 fluorescence was detected, but there were no changes in either mtl-1 or mtl-2::gfp expression. jones and candido exposed nematodes to cadmium or mercury and measured feeding behavior. for these studies, transgenic lines containing the promoter sequence for hsp16 genes which regulate the production of e. coli-galactosidase were used. accordingly, to measure stress, levels of this protein were assessed. results showed that cadmium inhibited feeding behavior significantly but not completely, as a minimal rate of feeding continued at high cadmium concentrations. further, exposure to cadmium (1 ppm) induced a detectable production of -galactosidase without inhibiting feeding behavior. the stress response was induced at a concentration of cadmium that was ten times lower than the lc50. mercury also was shown to inhibit feeding at concentrations similar to those necessary for the induction of a stress response; however, the difference in this instance was less than two fold. the use of c. elegans as an experimental model has produced considerable insight and valuable information regarding the multiple and varied processes of metal detoxification. conclusive biochemical evidence has indicated that different metals are not handled in the same capacity. the selectivity and sensitivity of each of these proteins is highlighted in the large body of accumulated research on different metal toxicities as well as various systems of metal detoxification. however, the overall mechanisms, temporal activation, and interplay between different cell detoxification systems remain elusive. future studies are necessary in order to enhance our understanding of the complex interplay of multiple-cell detoxification systems in response to exposure to different metals. the c. elegans model system will be critical for these investigations, as knockouts are easily generated and provide a wealth of information about metal detoxification in a genetically retractable, inexpensive, and in vivo model.

metals have been definitively linked to a number of disease states. due to the widespread existence of metals in our environment from both natural and anthropogenic sources, understanding the mechanisms of their cellular detoxification is of upmost importance. organisms have evolved cellular detoxification systems including glutathione, metallothioneins, pumps and transporters, and heat shock proteins to regulate intracellular metal levels. the model organism, caenorhabditis elegans (c. elegans), contains these systems and provides several advantages for deciphering the mechanisms of metal detoxification. this review provides a brief summary of contemporary literature on the various mechanisms involved in the cellular detoxification of metals, specifically, antimony, arsenic, cadmium, copper, manganese, mercury, and depleted uranium using the c. elegans model system for investigation and analysis.

PMC3157827

pubmed-460

obesity is a complex heterogeneous disease that is caused by genes, environmental factors, and the interaction between the two. obesity is also a multifactorial condition, and many endocrine and inflammatory pathways are involved in its development and in obesity-related diseases. excess weight in obesity may come from muscles, bone, fat, and/or body water, but obesity specifically refers to having an abnormally high proportion of total body fat. the world health organization defines overweight as a body mass index (bmi) of 25 or more and obesity as a bmi of 30 or more. the prevalence of obesity has been stated as being near epidemic size [13, 57], and obesity has been associated with type ii diabetes, hypertension, coronary artery disease, stroke, and many forms of cancer [8, 9]. therefore, it is important that the underlying pathophysiology of obesity-related diseases is understood. obesity results from the combined effects of genes, lifestyle, and the interactions of these factors, and both familial and nonfamilial factors play an important role in its development. a genetic predisposition to obesity has been reported as a major risk factor for individuals. with the increasing prevalence of obesity, studies on candidate genes for obesity most obesity-predisposing genes encode the molecular components of physiological systems related to energy balance. leptin is a protein product of the ob gene and is expressed and secreted by adipose tissue in amounts proportional to the body weight content; studies on its receptor have greatly advanced the comprehension of the mechanism for regulating body weight and energy homeostasis. the lipostat system, mediated by leptin and its hypothalamic receptor, reduces food intake and increases thermogenesis [10, 12]. the leptin (lep) and leptin receptor (lepr) genes have been evaluated for polymorphisms that could potentially be related to the pathophysiology of obesity and its complications. although the polymorphisms in these genes have been evaluated [1315], the association of these polymorphisms with obesity is still controversial. therefore, we investigated whether the lep gene g2548a polymorphism and lepr gene 668a/g (q223r) polymorphism might be involved in the pathogenesis of obesity. this study included 127 obesity patients (93 women, 34 men) and 105 controls (62 women, 43 men) provided from the department of internal medicine, gazi osmanpaa university in tokat, turkey. informed consent was in accordance with the study protocol, and all patients and controls signed a written consent form. all patients received a complete clinical evaluation, and all individuals in the control group were healthy and were selected by excluding the diagnosis of obesity. genomic dna was isolated from white blood cells by a kit procedure (invitrogen life technologies, carlsbad, ca, usa) and stored at 20c. lep g2548a and lepr 668a/g polymorphisms were analyzed by polymerase chain reaction based restriction fragment length polymorphism (pcr-based rflp) methods. the pcr protocol consisted of an initial melting step of 2 min at 94c, followed by 35 cycles of 30 s at 94c, 30 s at 55c (for lep), 30 s at 60c (for lepr), and 30 s at 72c, and a final elongation step of 5 min at 72c. amplification was carried out using primers forward 5-ttt cct gta att ttc ccg tga g-3 and reverse 5-aaa gca aag aca ggc ata aaa a-3 for the lep gene and forward 5-tcc tct tta aag cct atc cag tat tt-3 and reverse 5-agc tag caa ata ttt ttg taa gca at-3 for the lepr gene. pcr was performed with a 25 l reaction mixture containing 2550 ng/l dna, 1 l of 10 pmol/l of each primer, 1 l of dntp mixture (5 mm dntp, 1 l 2.5 mm mgcl2, 1 u taq dna polymerase), 2.5 l 10x pcr buffer (mg free, invitrogen life technologies, carlsbad, ca, usa), and dh2o. amplified products were digested with hhai at 37c for lep and mspi at 37c for lepr, and the resulting fragments were separated by 2% agarose gel electrophoresis. the fragments were stained with ethidium bromide and visualized through a vilber-lourmat gel quantification and documentation system (quantum-st4; vilber lourmat bp 66, torcy, france). analysis of the data was performed using spss 16.0 (spss, chicago, il, usa) and openepi info (http://www.openepi.com). the frequencies of the alleles and genotypes (hardy-weinberg equilibrium) in patients and controls were compared with analysis, and 95% confidence intervals were calculated. a p value less than 0.05 (two-tailed) was regarded as statistically significant. the mean age and bmi were 44.86 1.51, 35.45 4.56 and 34.25 15.43, 21.57 1.89 in patients and control groups, respectively. the mean age of obesity patients with bmi>35 kg/m and bmi<35 kg/m was 45.01 1.38 and 44.7 1.65, respectively, while the mean bmi of these patients was 38.68 3.11 (bmi>35 kg/m) and 32.06 1.56 (bmi<35 kg/m). patients and controls were genotyped for both the g2548a polymorphism in the lep gene promoter and the 668a/g polymorphism in the lepr gene. the distribution of the lep g2548a and lepr 668a/g polymorphisms of the patients and control groups are presented in tables 2 and 3. we found no statistically significant difference in the genotype frequencies of the lep gene polymorphism in patients and control groups (p>0.05). the lepr genotypes differed between the obesity patients and controls, but this was not statistically significant after the bonferroni correction (p=0.05). allele frequencies in the lepr gene showed no statistically significant association (p>0.05) (table 3). the lepr gene a allele was 61.4% in patients and 69.5% in the control group, while the g allele frequency was 38.5% in patients and 30.4% in the control group. in the combined analysis of the lep and lepr genes, the lep/lepr gg/gg combined genotype was found to increase the risk of obesity compared to the controls (p<0.05) (table 4). in the combined genotype analysis based on the mean bmi of obesity patients, there was no association of the lep/lepr combined genotype and obesity between patients with a bmi 35 kg/m and patients with a bmi near 30 kg/m (p>0.05) (table 4). human obesity is a complex trait determined by the interaction of multiple genes and environmental factors. obesity may arise as a result of increased energy intake, decreased energy expenditure, or increased partitioning of nutrients into fat, either alone or in combination. the prevalence of obesity and being overweight continues to increase worldwide, not only causing serious personal health problems but also imposing a substantial economic burden on societies. genetic influences are difficult to elucidate, and identification of the involved genes is not easily achieved. in the present study, we analyzed the frequencies of lep g2548a and lepr 668a/g polymorphisms in obesity patients in a turkish population. there was no statistically significant difference between the groups with respect to the lep genotype distribution (p>0.05) and allele frequencies (p>0.05). suggested that the lep g2548a variant may influence gene expression of leptin and leptin secretion by adipose tissue. noted that the lep g2548a polymorphism may influence a bmi increase by means of its effects on leptin secretion; however, they identified a significant and independent association between the lep 2548gg carrier status and higher leptin levels. an association of the lep g2548a polymorphism and increased bmi was reported in overweight europeans and in taiwanese subjects with obesity and the combined lep 759c/t and lep g2548a genotype may be a determinant of obesity. the results of our study do not support the results of these studies but do support those of other studies that showed no association between the lep g2548a polymorphism and obesity-related phenotypes [11, 14, 15, 21]. we found that lepr genotypes show a difference, but not statistically significant, between obesity patients and controls. we attribute this lack of significance to the low number of patients included in our study, but finding obese patients that have no other disease is difficult. some researchers have proposed that the polymorphisms of the leptin receptor gene (especially lepr 668a/g polymorphism) may contribute to common forms of human obesity [11, 14, 2224]. our results with respect to the lepr polymorphism are in agreement with the results of these studies. our results showed a statistically significant difference between groups with respect to the distribution of the lep/lepr gg/gg combined genotype. obesity results from both gene-gene and gene-environment interactions, and in our study we examined the gene-gene interactions of the lep/lepr genes and their link to obesity. demonstrated that the haplotype association of the lep g2548a and lepr q223r variants was related to a 58% increase in obesity risk, and they considered the interactions between lep and lepr gene polymorphisms to intensively influence modulation of energy homeostasis. in agreement with the findings of our study, boumaiza et al. reported that the lep g2548a and lepr q223r polymorphisms and haplotype combination were associated with a metabolic syndrome and obesity risk in tunisian subjects. the g2548a and 3hvr variants of the lep gene have been noted as being in linkage disequilibrium, and i/g combined genotypes are associated with obesity. in addition, the interactions between the polymorphisms of the lep and lepr genes have been shown to increase the risk of non-hodgkin's lymphoma and influence insulin plasma concentrations and blood pressure levels. our findings indicate that the lep g2548a polymorphism is not a relevant obesity marker and that the lepr 668a/g polymorphism may be related to obesity in a turkish population. additionally, the lep/lepr gg/gg combined genotype was found to increase the risk of obesity in patients compared to controls. however, the association of these polymorphisms with obesity is still controversial, and further research with larger patient populations is necessary.

objective. obesity is a complex heterogeneous disease that is caused by genes, environmental factors, and the interaction between the two. the leptin (lep) and leptin receptor (lepr) genes have been evaluated for polymorphisms that could potentially be related to the pathophysiology of obesity and its complications. the aim of this study was to investigate the role of lep g2548a and lepr 668a/g polymorphisms in the pathogenesis of obesity. subjects. the study included 127 patients with obesity and 105 healthy controls. polymerase chain reaction and restriction fragment length analysis for lep g2548a and lepr 668a/g polymorphisms were applied. results. there was no statistically significant difference in the genotype frequencies of the lep gene polymorphism between patients and control groups (p>0.05). we found a difference in the lepr genotypes between patients and controls, but this was not statistically significant (p=0.05). additionally, we found an increased risk of obesity in the lep/lepr gg/gg combined genotype (p<0.05). conclusion. our findings indicate that the lep g2548a polymorphism is not a relevant obesity marker and that the lepr 668a/g polymorphism may be related to obesity in a turkish population. further researches with larger patient population are necessary to ascertain the implications of lep and lepr polymorphisms in obesity.

PMC3836355

pubmed-461

multiple sclerosis (ms) is a chronic idiopathic disorder of the central nervous system (cns) sustained by a multifocal inflammatory process predominantly affecting myelin-sheathed axons. although traditionally viewed as a white matter (wm) demyelinating disorder, ms is characterized by acute and chronic axonal and neuronal loss, as shown for long by pathological and neuroimaging studies [1, 2]. acute inflammation causes the development of plaques, characterized by blood-brain barrier (bbb) breakdown, perivascular cellular infiltration, demyelination, and axonal degeneration. notably, axonal damage occurs not only in the acute phase but also in inactive ms lesions [3, 4]. plaques represent the underlying pathological substrate of clinical events, with occurrence of focal/multifocal neurological symptoms and signs that eventually subside in many cases as inflammation ceases. lesions may also involve the cortical gray matter (gm) in which case they are characterized by myelin/axonal injury and microglial activation but not bbb disruption and less cellular infiltration compared to wm lesions [6, 7]. it is increasingly perceived that the severity of ms clinical outcome does not simply result from the extent of wm damage, but it rather represents a complex balance among wm and gm tissue damage, tissue repair, and cortical reorganisation [810]. the evidence that axonal loss highly correlates with neurological disability and disease progression has spurred the search for reliable markers of axonal degeneration. although ms aetiology still remains undetermined, genetic and environmental risk factors have been identified or are suspected (i.e., female gender, hla-drb1 allele, genome-wide association studies candidate genes, epstein-barr virus infection, low vitamin d levels, cigarette smoking, etc.) mainly influencing immune system modulation and although much less evidently myelin and axonal repair mechanisms [1115]. the complex and unique interplay between genetic background and environmental exposure in each case likely determines the clinical heterogeneity of ms both between and within subjects varying from benign or even subclinical types to highly disabling forms and making it a challenge to predict the clinical course at the individual level. given that ms is mostly diagnosed in subjects in the third and fourth decade of life, the availability of reliable predictors of long-term prognosis is extremely important. the objective of this paper is to review the current literature and to discuss evidence on clinical, paraclinical, magnetic resonance imaging (mri), and cerebrospinal fluid (csf) markers as predictors of disability progression in ms. the typical clinical course of ms is relapsing-remitting (rr), characterized by an initial event of acute or subacute neurological disturbance, generally indicated as clinically isolated syndrome (cis) followed by recurrence of symptoms over time. cis is the type of onset in around 85% of ms cases, while the remaining 15% of patients have a progressive disease from onset (primary progressive (pp) ms). progressive onset is an unfavourable prognostic predictor per se, since motor, sphincter control, and cognitive impairment are prominent features of the clinical picture, neurological disability continues to worsen over time, and no effective treatment exists. conversely, ciss generally recover well and may remain monophasic for a long time interval before conversion to clinically definite ms occurs. since cis represents the earlier clinical manifestation of rr ms, this patient population is of great value for identification of predictive and prognostic disease markers compared to definite ms cases who are necessarily in a more advanced stage. typical cis presentation includes acute partial myelitis (3050% of cases), brainstem/cerebellum syndromes (2530%) unilateral optic neuritis (2025%), and cerebral hemisphere syndromes (5%); more than 20% of ciss present with symptoms and/or signs of more than one anatomical location (multifocal presentation) [1820]. the percentage of cis patients who develop clinically definite ms in prospective observational studies ranges from 16% at 1 year to 80% at 25 years [21, 22]. however, these figures date back to studies conducted before the introduction of the most recent revision of ms diagnostic criteria according to which patients previously classified as cis already have ms at the time of initial symptoms if mri demonstrates space and time dissemination of demyelinating lesions. after ms develops, irreversible disability may be the result of accumulation of fixed sequelae after each attack or may be due to transition to a secondary progressive (sp) phase, in which insidious neurological deterioration substitutes the preceding rr stage of the disease (3258% of cases in major prospective studies). clinical predictors of long-term disability in ms include male gender, older age, multifocal symptoms, efferent systems involvement, incomplete remission of the initial event, a short interval to the second event, and high relapse rate in the first 25 years after onset, although not all studies replicated the same findings (table 1) [2427, 30, 31, 48, 73]. one single study reported a shorter time to secondary progressive ms in patients with family history of ms. the relevance of age as a prognostic factor is subject to interpretation depending on the temporal frame in which disability levels are captured. indeed, while older age at onset is associated with a more rapid disability progression likely due to prevalence of the primary progressive disease course, age-dependent degenerative processes, and dysfunction of repair mechanisms in older subjects early onset ms patients reach disability milestones at a younger age compared to late onset ms cases, even though in a longer time interval [26, 75]. in addition, it has been shown that the progressive phase of ms is an age- rather than a disease duration-dependent process, since age at pp and sp ms onset overlaps significantly in large observational studies and subsequent disability progresses along a common age-driven trajectory independent of onset epoch and previous clinical course. in this perspective, older age at onset may be viewed as a favourable prognostic factor, meaning a longer disease-free interval before ms symptoms occurrence in life and an older age at which significant disability milestones are reached, compared to early onset. also the influence of relapses on later disability progression is debated. according to the authors who found a positive correlation between relapses and long-term disability, the association is stronger in younger patients (< 25 years old at ms onset), it diminishes significantly after the first 25 years of disease, and it is minimal after the progressive phase has begun [32, 78]. a sizable proportion of ms patients does not accumulate clinically relevant disability during the entire natural history of the disease. this type of course is known as benign ms, although there is no general agreement on its definition and consequently, on its prevalence in the ms population. while initial definitions predominantly stressed the absence of significant ambulatory disability (expanded disability status scale (edss) score<3.5 or 2.5) after a reasonable time interval from initial symptoms (10 or 15 years) [81, 82], more recent studies highlighted the importance of carefully considering cognitive status and quality of life when defining benign ms. indeed, edss, which is the most largely used disability scale in ms clinical practice, is clearly unbalanced towards ambulation impairment. scores range from 0 meaning no disability to 10 meaning death due to ms: from score 1 to 3.5, there can be a wide range of neurological deficit but ambulation is unrestricted; from 4 to 5.5, independent ambulation is below 500 meters; from 6 to 7.5, ambulation is only possible with support; and from 8 on, the patient is wheelchair-bound or bedridden. whatever the definition, it has been shown that benign status at 10 years after ms onset persist at 20 or more years in 5269% of patients, leading to the conclusion that benign ms is a transient condition for a considerable proportion of cases [82, 8486]. however, studies addressing this topic are generally limited by the clinic-based design in which ms patients with mild disease who are not seen on a regular basis in the neurology practices are not included in the analysis falsely reducing the proportion of benign cases. there are no diagnostic tools or validated markers to identify ms patients who will have a favourable clinical course; however, female gender, younger age, and absence of motor symptoms at onset have been associated with a benign disease form. neurophysiological assessment with visual, somatosensory, motor, and brainstem auditory evoked potentials is traditionally used as a paraclinical tool for ms evaluation, although its diagnostic relevance has progressively decreased after mri became largely available as a more sensitive technique. however, evoked potentials still maintain a prognostic significance likely because they reflect the functional integrity of specific anatomical pathways and consequently tend to better correlate with neurological disability than conventional mri, which provide purely morphological information. several cross-sectional and longitudinal studies established that the degree of evoked potentials abnormalities is significantly associated with the edss score at the time of neurophysiological evaluation and up to 14 years later in patients with ms [8893]. a recent study found that cis patients with at least three abnormal evoked potentials at baseline have an increased risk of reaching moderate disability over a mean follow-up period of six years, independent of initial mri features. in recent years optical coherence tomography (oct) has emerged as a powerful tool to detect retinal nerve fiber layer (rnfl) thinning in ms patients with and without optic neuritis history. rnfl thickness decrease results from axonal loss in optic nerve, possibly reflecting diffuse neuroaxonal injury in the cns, and correlates with markers of ms activity such as relapses, new/gadolinium-enhancing lesions, and parenchymal atrophy on brain mri [96, 97]. the extent of rnfl thinning in optic neuritis patients predicts visual recovery and exhibits a modest correlation with overall neurological disability in ms patients. it has been recently suggested that thinning of inner and outer nuclear layers of the retina identifies a subset of ms patients with primary retinal neuronal pathology and more aggressive disease course [98, 99]. given its increased availability and its sensitivity in detecting ms lesions, conventional mri has become the main imaging tool in the ms diagnostic work up as well as in monitoring treatment response to disease-modifying drugs. its diagnostic sensitivity reflects the ability to identify clinically silent lesions, thus, favouring the early demonstration of dissemination in space and time of the lesions (figure 1) according to the recent revision of diagnostic criteria. however, while it may seem obvious that patients who develop new wm lesions are worse off than those without new lesions, conventional mri has been shown to have a prognostic value only in patients at disease onset: high t2-weighted lesion load in patients with a cis has been associated with an increased risk of subsequent conversion to clinically definite ms and long-term disability accumulation [101, 102]. by contrast, in a more advanced phase of the disease, the strength of relationship between conventional mri measures and subsequent disability progression is rather weak [33, 34]. in a recent study including 548 placebo-treated rr ms patients, the multivariate analysis indicated just edss score and t2 lesion load as factors independently predicting the clinical progression. nevertheless, these two variables taken together were able to account for only 3% of the probability to have an edss increase over follow-up time, thus, confirming the limited value of these metrics in predicting short-term disability changes in rr ms. such result is in line with those of several previous cross-sectional and longitudinal studies conducted on smaller groups of patients with different clinical characteristics, which have shown only modest correlation between t2- and t1-weighted brain mri activity and subsequent changes in disability [3638]. although edss is not without limitations in terms of reliability and responsiveness to disease changes, the lack of a strong correlation between wm lesion load and clinical disability had prompted investigations of the so-called normal-appearing brain tissue. for this purpose, unconventional and quantitative mri techniques, having increased sensitivity and specificity to irreversible tissue damage, have been consistently applied to monitor and predict ms evolution. given these premises, several studies have been focused on brain atrophy showing its relevant clinical impact not only in the diagnostic phase but also in predicting subsequent disability progression both in rr ms and in pp ms (figure 2). a recent study published by the magnims group, included 261 ms patients who had mr imaging at baseline and after 1-2 years and edss scoring at baseline and after 10 years; in the whole patient group, after correction for imaging protocol, whole brain and central atrophy were good predictors of edss at 10 years (r=0.74). despite the good sensitivity of brain atrophy, even better results in predicting disability progression have been further achieved by the regional analysis of brain atrophy. jasperse and colleagues, for example, suggested that atrophy of central brain regions was related to decline in ambulatory function, whereas atrophy of both central and peripheral brain regions was associated to decline in neurologically more complex tasks for coordinated hand function. the best results, however, have been obtained by the study of gm and wm atrophy separately. indeed several voxel-based and surface-based studies, both in rrms and in ppms, revealed strong relationship between gm, but not wm, atrophy and disability progression [4345]. even when a very long followup, a very large sample size, or more sophisticated disability scales (i.e., ms functional composite) were considered, gm atrophy reflected disease subtype and disability progression to a greater extent than wm atrophy or lesions [4547]. a further step forward in the comprehension of the pathological mechanisms underlying the accumulation of irreversible disability in ms was obtained by the regional analysis of gm atrophy; since the first studies, indeed, it was clear that some cortical and deep gm structures were more prone to inflammatory and degenerative damage [49, 50] than others and that, when damaged, some cortical areas had a greater impact on the accumulation of physical [8, 51] and cognitive disability [52, 53] than others. in particular thalamus and cerebellum were consistently related to clinical disability and its progression over time. thalamus was found to be one of the earliest structures involved by the neuropathological process taking place in the gm and the rate of thalamic atrophy in ms subjects was correlated with changes in edss. moreover, in a longitudinal study, baseline thalamic fraction (odds ratio=0.62) was identified as independent predictor of worsening disability at 8 years. cerebellum has been indicated as a preferred site of demyelination, especially in patients with progressive ms, whose cerebellar cortex was found to be affected by ms-related pathology in up to 92% of its extension [105, 106]. in a recent 5-year longitudinal study cerebellar cortical atrophy, together with age and cortical lesion load, was indicated among the predictive parameters of progression in those rr ms patients who convert to the sp phase. beyond diffuse gm damage, the relevance of cortical damage in determining disability has been pointed out by the strong correlation observed between focal gm damage as visible by double inversion recovery (dir) sequence (i.e., cortical lesions; figure 3) and clinical progression. indeed, high number of cortical lesions has been demonstrated to characterize patients with the poorest prognosis and having early and severe cortical atrophy and cognitive impairment. in a 5-year longitudinal study on more than 300 ms patients with different clinical phenotypes, cortical lesion volume and gm atrophy were found to be associated to each other and to physical and cognitive disability progressions. patients having high cortical lesion load at baseline showed the worse clinical evolution and a significant progression of cortical atrophy after 5 years. of course a complete and accurate evaluation of the risk of clinical progression should not disregard the evaluation of spinal cord damage that has suggested as a major determinant of disability in patients with ms. in line with what has been happened for the brain damage, the application of quantitative mri techniques to the spinal cord damage has convincingly demonstrated that cord area, rather than t2 lesion load, might have a role in predicting the accumulation of disability [56, 57, 108]. in the last 10 years, finally, other non-conventional sequences have received considerable attention since their high sensitivity for the most disabling pathological features of ms (i.e., irreversible demyelination and neuroaxonal injury) and their ability to detect occult changes occurring in the normal-appearing brain tissue. among these unconventional techniques magnetization transfer and diffusion tensor imaging gave the most interesting results. in 73 patients, who were followed prospectively with clinical visits for a median period of 8 years, a multivariable model identified baseline gm magnetization transfer ratio histogram peak height and average lesion magnetization transfer ratio percentage change after 12 months as independent predictors of disability worsening at 8 years (r=0.28). in a longitudinal study on 54 primary progressive ms patients, lower level of disability and gm damage evaluated at study entry on the base of average gm mean diffusivity identified patients with high risk of disease progression over the following 5 years. in a more recent prospective study fractional anisotropy of normal appearing gm and t2 lesion load were independent predictors of edss score, while change in fractional anisotropy of normal appearing gm (b=0.523) and disease duration (b=0.342) were independent predictors of edss change. finally, the application of diffusion tensor imaging to the spinal cord damage revealed that baseline cord cross-sectional area and its fractional anisotropy correlated with increase in disability at follow-up. all together these studies confirmed that neurological and neuropsychological disability in ms are likely the consequence of both visible and invisible wm and gm damage. the strength of correlation between gm tissue loss and progression of disability exceeds that related to wm lesions or atrophy (table 2). unfortunately, gm damage is poorly evaluated by conventional mri and to achieve more accurate estimates of such a damage it requires multiparametric mri approach including unconventional and quantitative mri techniques, many of which are not yet available or practicable in routine diagnostics. the examination of csf represents a valuable procedure in investigating a number of inflammatory and degenerative neurological disorders. in addition to the classical biochemical and electrophoretic approaches, the proteome complexity of csf can be tackled today by a number of methods, hence, indicating that scientists involved in this frontier are fishing in the right pond. however, in a disorder with a complex pathogenesis, such as ms, individual biomarkers, taken singly, are likely to reflect only isolated components of ongoing neuroinflammation and neurodegeneration, hence, lacking prognostic significance. moreover, most of the investigated ms biomarkers, while of invaluable diagnostic help, are currently unsuitable for predicting disease progression. according to their biological role, molecules of potential prognostic significance for ms may be classified as follows: (i) markers of immune activation (e.g., cytokines, chemokines, antibodies, complement factors, adhesion molecules, etc.); (ii) markers of blood-brain barrier disruption (e.g., matrix metalloproteinases); (iii) markers of demyelination (e.g., myelin basic protein, myelin oligodendrocyte glycoprotein, proteolytic enzymes, proteases inhibitors, etc.); (iv) markers of oxidative stress and cytotoxicity (e.g., advanced oxidation protein products, total thiol, hydroxyl radicals, divalent iron, etc.); (v) markers of axonal/neuronal damage and gliosis (e.g., neurofilaments, tau, 14-3-3 protein, glial fibrillary acidic protein, etc.); and (vi) markers of remyelination/neural repair (e.g., nerve growth factor, brain-derived growth factor, nogo-a, etc.). a correlation with ms disability progression over time has been suggested for several csf markers, including but not limited to 14-3-3 protein; tau; neurofilament heavy chain [66, 67]; chitinase 3-like 1; and cystatin c. csf igg oligoclonal bands, which have a recognized relevance for the diagnosis of ms and predict conversion from cis to ms, do not influence the long-term risk of disability, although a contrasting observation has been described. conversely, csf oligoclonal igm, particularly if directed against myelin lipids, have been associated with a poor ms outcome in terms of frequency of relapses and disability progression [68, 69]. several csf markers of inflammation have been investigated for potential prognostic value in cis and early ms patients. some studies have identified novel biological predictors of conversion from cis to ms, for instance measles-rubella-varicella zoster virus igg antibody reaction (mrzr) and high levels of c-x-c motif ligand 13 (cxcl13) and in the csf. however, no predictive value for progression of disability has been shown for such molecular candidates [111, 112]. since neurodegeneration is regarded as the biological determinant of irreversible neurological disability in demyelinating disorders, csf markers of neuroaxonal injury (e.g., tau, 14-3-3 protein, and neurofilaments) are the most promising candidates for predicting disease progression [113, 114]. csf tau concentration in ms patients with both relapsing and progressive forms of the disease has been reported to be higher compared to controls in several studies [110, 115119], although other researchers did not replicate this finding [120122]. a correlation between csf tau and progression of disability in ms patients has been shown only in one 3-year follow-up study. in a small group of patients with cis and clinically definite ms, investigated either at the acute attack (i.e., within 30 days) or several weeks or months later, our group found values of tau within normal limits, a finding that we later confirmed in a larger mostly independent cohort of cis patients. interestingly, it has been shown that csf tau levels decrease during the course of ms, as a likely effect of progressive parenchymal brain loss, hence, showing a negative correlation with clinical severity. the latter findings are consistent with studies showing progressive brain atrophy in ms patients, regardless of disease subtype. taken together, while the determination of csf tau concentration in ms deserves further scrutiny, it is possible that in a subset of ms patients, this protein represent a reliable marker of axonal injury. conversely, available evidence shows that determination of p-tau has no value as a biomarker. 14-3-3 protein has also been detected in the csf of subjects with cis/ms by several [61, 62, 116, 126, 127] but not all research groups.. showed that a positive csf 14-3-3 assay at the first neurological event suggestive of ms predicted the development of significant neurological disability over a median follow-up period of 32 months, while colucci et al. found that 14-3-3 positive ms patients had a higher rate of edss progression over 10 months compared to 14-3-3 negative cases. fiorini et al. found variable upregulation of csf 14-3-3/in cis/ms patients investigated at active or inactive disease stages (as observed in other inflammatory/demyelinating conditions) but not overexpression of 14-3-3 and, the isoforms typical of disorders characterized by ongoing axonal and neuronal degeneration, such as sporadic creutzfeldt-jakob disease (scjd) and motor neuron disease. these findings encourage an in-dept-analysis in larger cohorts of patients, before ruling out the usefulness of this biomarker. among csf biomarkers of neurodegeneration that have been tested in ms, concentrations are increased in the csf of ms patients compared to age-matched normal controls. furthermore, csf nfl levels seem to better correlate with ms acute inflammatory activity (higher levels in cis patients who convert to ms and during relapse compared to remission phase), while csf nfh concentrations appear to be related to irreversible neuroaxonal injury as indicated by the correlation with confirmed edss score progression and brain atrophy both in cross-sectional and longitudinal studies [64, 65, 122, 128130]. table 3 shows csf markers for which a correlation with ms disability progression has been reported in longitudinal studies. which is not straightforward in all cases, clinicians have to be prepared for the challenge of prognostic predictions in order to give adequate responses to patients concern about their future life with ms. among the determinants of ms burden, development of irreversible neurological disability, particularly when affecting motor and cognitive functions, has the highest impact on patients quality of life and health system costs. therefore, prognostic markers of long-term disability progression are strongly needed in ms. a prognostic marker is a specific parameter or a combination of parameters that can be measured in a subject with a given condition and that is significantly correlated with a relevant clinical outcome of that condition. ideally, reliable prognostic marker studies should fulfil the following methodological requirements: (1) prospective or longitudinal design; (2) long-term followup; (3) adequate marker and outcome measurement; (4) clinical significance of the marker (i.e., good correlation and consistency with relevant clinical outcomes); and (5) reproducibility. clinical prognostic markers that are associated with an increased risk of disability progression in the longterm (e.g., male gender, older age, progressive onset, etc.) have been identified in several ms natural history studies. however, besides identifying subjects who are more likely to experience a severe disease course, such markers do not offer real advantages in terms of prediction potential, since they are not modifiable risk factors, do not directly reflect biological processes, and do not generally distinguish between responders and nonresponders to available ms treatments. conversely, mri and csf parameters, which can be classified as biomarkers as they express more closely biological mechanisms underlying the disease pathophysiology, have a good potential of quantitative assessment as well as variation according to disease stage. considering the complex pathogenesis of ms however, families of biomarkers representative of specific pathogenetic pathways particularly those related to axonal/neuronal damage may correlate with irreversible neurological dysfunction and be used as prognostic indicators to identify patients at risk of a more aggressive disease course. furthermore, such a biomarker might be helpful for identifying patients who could benefit from therapy in case it showed a reliable correlation with the response to a given treatment. unfortunately, no conventional mri measure has shown strong correlation with long-term disability progression in ms, while unconventional mri techniques particularly those assessing gm damage are currently being investigated with promising results, although they are still difficult to apply in clinical settings. on the other hand, research on csf biomarkers has gathered convincing preliminary evidence only for nfh and nfl as predictors of disability progression. so far, biomarkers studies have mainly focused on selected candidates and have generally recruited relatively small sample of cases with a cross-sectional design, often showing conflicting results. it is likely that discrepancies across studies are at least in part explained by differences in selection of patients, marker measurement, and outcome assessment. although a considerable level of international agreement has been reached on methodological requirements of mri studies in ms, an effort is being made by the csf markers research community in order to standardize collection and biobanking of samples from well clinically characterized ms patients to develop reproducible laboratory assays for csf analysis and to find common definitions of healthy and diseased controls [132, 133]. to identify reliable prognostic markers, future ms research will need to focus on large longitudinal observational studies and clinical trials exploring the correlation of unconventional mri measures and selected csf proteins with the development of irreversible neurological disability.

multiple sclerosis (ms) is a chronic disorder of the central nervous system (cns) in which the complex interplay between inflammation and neurodegeneration determines varying degrees of neurological disability. for this reason, it is very difficult to express an accurate prognosis based on purely clinical information in the individual patient at an early disease stage. magnetic resonance imaging (mri) and cerebrospinal fluid (csf) biomarkers are promising sources of prognostic information with a good potential of quantitative measure, sensitivity, and reliability. however, a comprehensive ms outcome prediction model combining multiple parameters is still lacking. current relevant literature addressing the topic of clinical, mri, and csf markers as predictors of ms disability progression is reviewed here.

PMC3842089

pubmed-462

peptide- and protein- hydration is the dominant factor in the stabilization of spatial molecular structure, in the process of protein folding by gating hydrophobic residues, and in the mechanisms of peptide and protein mediated reactions [14]. water molecules, therefore, can be considered as an integral component of biomolecular systems with dynamic, functional, and structural roles [47]. investigation of the structural and functional role of water molecules, bound to proteins and peptides, requires a sufficient understanding of the hydration process of their building blocks [1, 2]. the hydration of amino acids and their derivatives at a molecular level, therefore, is of great importance and has been extensively studied with x-ray crystallography [1, 3] and a variety of spectroscopic techniques including multinuclear magnetic resonance spectroscopy [2, 813], ir and raman spectroscopy [1416], icr mass spectrometry, and laser ablation in combination with microwave spectroscopy. we present here, for the first time in the international literature, a comparative investigation of literature d, c, n, and o nmr and crystallographic data in order to provide a coherent hydration model of amino acids and selected derivatives at different ionization states in aqueous solution and in the crystal state. o nmr has received little attention in amino acid and peptide research [2, 12, 13, 19, 20]. this neglect is due to the fact that of the three naturally occurring oxygen isotopes, only o possesses a nuclear spin (i=5/2). owing to its electric quadrupole moment (qe=2.6 10 em) and, thus, broad line widths, and its low absolute sensitivity compared with that of h (~1.1 10), the o- isotope is one of the more difficult to observe by nmr spectroscopy [12, 13, 21, 22]. o nmr studies, therefore, of compounds at natural abundance require high concentrations (> 0.1 m) and extensive signal averaging. recording of spectra can be greatly facilitated by the use of o enriched samples [2327]. figure 1(a) illustrates the natural abundance o nmr spectrum of glutamic acid, 0.1 m in o-depleted water at 40c. despite the extensive signal averaging (number of scans (ns) =3 10) and the total experimental time of 4.2 hours, the achievable signal-to-noise (s/n) ratio is very poor and practically prohibitive for the accurate determination of chemical shifts and line widths. figure 1(b) illustrates the clear advantages of working with o-labelled glutamic acid (o enrichment 1 at.%). o shieldings of various chemical functional groups are very sensitive for studying hydrogen bonding interactions because of the large chemical shift range of the o nucleus [12, 13]. the effect of solvent-induced hydrogen bonding interactions on (o) of the carboxyl groups is, however, rather small compared with the substantial sensitivity of over 80 ppm to hydrogen bonding interaction of (o) of amide and carbonyl oxygens [12, 13]. only a single o resonance absorption is observed for the carboxylic group since the shifts of the individual resonance absorptions (c=o) and (oh) are averaged out by rapid intermolecular proton transfer with protic solvents, traces of h2o, and/or through hydrogen bonding aggregates of the cooh groups in organic solvents [12, 13, 23, 24, 26, 28]. reuben from dilution studies of acetic acid in 1,2-dichloroethane estimated a deshielding effect of ~12 ppm due to breaking of a hydrogen bond involving the carbonyl oxygen of the acid and a shielding effect of 6 ppm due to breaking of a oho hydrogen bond. therefore, a total shift of only+6 ppm is expected for the monomeric acetic acid in apolar media (dichloroethane) compared with the dimeric form. despite the relatively low sensitivity of the o shieldings of the carboxyl group to hydrogen bond interactions, spisni and collaborators attempted to estimate the solvation state of the -carboxyl group of amino acids in the different ionization states. figures 2(a) and 2(b) show the dependence of (o) of l-alanine and l-proline as a function of molar fraction of dmso in the ph range 7-8 and 12-13. since dmso can not form a hydrogen bond interaction with the carboxylate group, contrary to the case of h2o, the shielding difference of 1017 ppm between the two solvents was interpreted with the hypothesis that the carboxylate group of these amino acids is hydrated by two water molecules in aqueous solution with one hydrogen bond per carboxylate oxygen. in the acidic ph range (figures 2(c), 2(d)), a nonlinear behaviour of the chemical shift at high dmso molar fractions was observed. for dmso molar fractions up to 0.6, a linear dependence of the chemical shift was observed which, on extrapolation to 100% dmso, results in a shielding of 1517 ppm, the same as in the neutral ph. this was interpreted with the hypothesis that two hydrogen bonds (one to each oxygen) are being ruptured. when the dmso molar fraction is between 0.6 and 0.8, it was suggested that a third molecule of water, which is hydrogen bonded to the hydroxyl hydrogen, is dissociated due to the interaction with dmso. this might explain the deflection from linearity and the plateau-like dependence of the o shielding. the protonated form, therefore, of the carboxyl group of the amino acids is more hydrated with an access of a bound molecule of h2o than the deprotonated form. this conclusion is in qualitative agreement with multinuclear nmr relaxation data (see below). for quadrupolar nuclei, such as d, n, and o, the longitudinal (t1) and transverse (t2) relaxation times are essentially due to quadrupolar interaction (1)1t1=1t2(1+23)2f(,d), where is the nuclear quadrupole coupling constant. the asymmetry parameter varies from 0 to 1 and describes the deviation of the electric field gradient from axial symmetry, and f(, d) is the correlation function, which depends on the rotational diffusion constant d and its relative orientation with respect to the principal axes of the field gradient tensor [12, 13]. when isotropic reorientation is assumed, f(, d) reduces to a single overall correlation time mol which is given by the stokes-debye formula (2a)mol=vmkbt, where vm is the molecular volume, the viscosity of the solution, kb the boltzman constant, and t the absolute temperature. vm can be estimated as (2b)vm=0.74mwn0, where n0 is the avogadro's number and mw and are the molecular weight and the density of the solute (amino acid), respectively. the removal, therefore, of these impurities is necessary in studies of t2 and t1 relaxation times. figure 3 illustrates the ph dependence of the o line widths of 0.1 m glycine in h2o. this o line width minimum has been previously explained by a decrease of the molecular tumbling time attributable to a reduction in hydration and, thus, intermolecular association of glycine in the zwitterionic form. in the high ph region, a broad maximum at ph 11 was observed. addition of 2 mm ethylenediamine-n, n, n,n-tetraacetate (edta) to the original solution resulted in no line width variation in the neutral and high ph region. it can, therefore, be concluded that this broad minimum at ph 11 should be attributed to the effect of paramagnetic impurities and not to a hydration change of glycine in the neutral and high ph region. d t1 relaxation times of cd2 of glycine at acidic ph were shown to be shorter relative to those at neutral ph. this shortening in t1 implies an increase in mol and, thus, in the effective molecular weight mw (( 1), (2a), and (2b)), which was interpreted with an increase in the hydration state in the cationic form. tritt goc and fiat investigated in detail the viscosity and temperature dependence of the o nmr line width of glycine, alanine, proline, leucine, histidine, and phenylalanine at ph 2, 7, and 12.5. the experimentally observed viscosity/temperature (/t) dependence of the reorientation correlation time was compared with various hydrodynamic models. a model of the hydration state in the primary solvation sphere of the carboxylic group of amino acids in their cationic state was suggested in which two water molecules are hydrogen bonded to the oxygens and one to the hydrogen of the oh group. in the zwitterionic and anionic states, the hydration model of the carboxylate group can be presented by a structure in which one water molecule is hydrogen bonded to each of the oxygens. the o [10, 11] and n nmr line widths of several protein amino acids were measured in aqueous solution to investigate the effect of molecular weight on the line widths (table 1). the n and o line widths, under composite proton decoupling, increase with the bulk of the amino acid, and increase at low ph. assuming an isotropic molecular reorientation of a rigid sphere and, thus, a single correlation time from overall molecular reorientation (mol), then, the line width 1/2 can be expressed in the following form: (3)1/2=1t2=0+1mw, where mw is the molecular weight, 1 is the contribution to the line width of the quadrupolar coupling constant, density and temperature, (1), (2a), and (2b), and 0 is the solvent viscosity-independent contributions to the line width due to the primary hydration sphere of the amino acids. the linear correlation between 1/2 and mw at ph 6 for both n and o nuclei (figure 4) is in agreement with the hydrodynamic model of (3). furthermore, the (o) of the amino acid is independent of both the ionization and the degree of hydration of the carboxyl group. the increase in the o line widths at acidic ph (~100 31 hz), relative to those at neutral ph, was interpreted by a change in the rotational correlation time and, thus, effective mw of the amino acids, (3). this implies that the cationic form of the amino acids is more hydrated by an access of 1.3 to 2.5 molecules of water relative to that in the zwitterionic form with lifetimes that are longer than the overall molecular rotational correlation time, presumably 210 ps. in the case of a stochastic diffusion of the amino and carboxyl groups comprising contributions from internal (int) and overall (mol) motions, the correlation time c for n or o is given by (4a)c=mol[a+(b+c)(12/r)intmol+(12/r)int] with (4b)a=34(3cos21)2, b=3sin2cos2, c=34sin4,where is the angle between the rotation axis and the main field gradient (r denotes an r-fold jump mechanism). since the sum of a, b, and c is equal to 1, (4a) can be rewritten as(5a)c=molamol+imol+i, where (5b)i=(12r)int.equations (5a) and (5b) can be rewritten as (6)c=(1a)i+amol(1a)i2mol+i. since a and i can be assumed to be constant for all the amino acids, (4a) and (4b) can be written as (7)1/2=0+1mw+2mw+3, where 03 are constants. the minimization of (7) on the basis of the o experimental data gave the mean difference of 35.8 17.3 in mw between ph 0.5 and 6.0 for three different 1/2 values: 250, 350 (figure 4(b)), and 500 hz. the difference in the n line widths at the two ionization states (figure 4(a)) should be attributed to differences in the correlation times and to a decrease in the (n) on deprotonation of the carboxyl group. in the case of the linear model, the influence of variations of values of the (n) to the line width, 1/2, is less for small molecular weights. therefore, for 1/2=70 hz (figure 4(a)), the difference in mw will be a reasonable approximation of the difference in hydration in the two states. the calculated value was found to be 45.2 7.4, which corresponds to an excess of 2-3 water molecules in the cationic form compared to that in the zwitterionic form, in reasonable agreement with the o nmr data. more recently, takis et al. investigated the cc longitudinal relaxation times (t1) and n line widths (1/2) of amino acids and acetyl-amino acids in aqueous solutions at acid and neutral ph. both c and n values indicate that amino acids and acetyl-amino acids at acid ph interact with an access of one water molecule with respect to their deprotonated form at neutral ph. on the contrary, c and n values of betaines (r3nch(r)coo) crystal structure databases provide a rich source of information to extract details on the architectures and interactions of molecules. this kind of search provides the opportunity to examine the formation of intramolecular and intermolecular hydrogen bond in small molecule crystal structures [33, 34]. propensities for the hydration of the -carboxylate group of amino acids and their derivatives were derived on the basis of exhaustive searches in the cambridge crystallographic database (csd). since intermolecular hydrogen bonds are preferred when five- or six-membered conjugated rings are formed, particular attention has been given to the hydrogen bond patterns in the vicinity of the carboxylate group that involves two simultaneous hydrogen acceptors. the concept of five- and six-membered conjugated rings, along with three-center (bifurcated) and 4-center (trifurcated) hydrogen bonds, has been acknowledged and accepted widely as an important factor in determining the structure and function of molecules ranging from inorganic to organic and biological molecules [1, 3539]. furthermore, port and pullman studied theoretically the formate ion-water interaction as a prototype of the carboxylate group. three energetically favourable hydration sites were obtained, two equivalent sites on the carboxylate oxygens at the exterior of the ion and one water bridging the two oxygen atoms. the conquest 1.13 program was used for all the statistical analysis described in this paper. specifically, the csd version 5.32 (november 2010) for small molecules was searched, with the following general search flags: r>0.5,, and only organic. in order to extract the number of entries present in the current database that form six-membered conjugated rings between the two oxygens of the -carboxylate and the carboxylic group with a molecule of water in the vicinity, the following geometric cut-offs were used: upper limits d=3 for (ow)ho=c and (ow)ho c, and d=3.5 for owo=c and owo c 44 hits were obtained for the carboxylate state (figure 5), whereas only one was derived for the protonated form. figure 5 demonstrates that the oxygen of water, ow, is reasonably close to the carboxylate oxygens and displays a significant preference for the o1c there is a general correlation between hydrogen bond lengths and hydrogen bond angles (figure 6) similar to that observed by jeffrey and maluszynska in the case of water molecules in the hydrates of small biological molecules. furthermore, crystallographic database searches were performed to identify the propensity for the formation of intramolecular hydrogen bond interaction in the carboxylate (nho) and the carboxylic acid (nho=c) state. interestingly, 946 and 118 hits were retrieved for the carboxylate and 621 and 6 hits for the carboxylic form in the absence and presence of two molecules of water, respectively. it is evident from figure 7 that in the presence of two bound water molecules there is a significant reduction in the number of structures with intramolecular hydrogen bond interaction for the carboxylate group and, concurrently, a significant increase in the distance (nho). it is important to note that no intramolecular nh3ooc hydrogen bonds were observed for 82 amino acid carboxylates with sp-hybridized c-atoms in agreement with an early survey of amino acid structures determined by neutron diffraction. o shielding changes of amino acids as a function of molar fraction of dmso/h2o, the decrease in the longitudinal relaxation times (t1) of c d and c, and the increase in line widths of n and o at acidic ph relative to those at neutral ph may be interpreted with the hypothesis that the cationic form of amino acids is more hydrated by 1 to 3 molecules of water than the zwitterionic form. similar behaviour was also observed for acetylated derivatives of amino acids, but not for betaines, between the protonated and deprotonated carboxyl group. although the precise hydration differences observed for various nuclei deviate somehow, it may be concluded that these hydrated complexes have lifetimes that are shorter than the nmr chemical shift time scale, but presumably longer than the overall molecular rotational correlation time of 210 ps. an exhaustive search in the cambridge crystallographic database (csd) demonstrates a strong tendency of the two oxygens of the deprotonated carboxylate group to form hydrogen bonds with a single molecule of water. even though statistical analysis of structural parameters in crystals can not be used in a straightforward way to derive quantitative structural models in solution, it is of interest to note that this mode of six-membered conjugated ring, which is absent in the case of the carboxylic group, might result in a more compact and, thus, less hydrated structure in aqueous solution, in accordance with the nmr data (figure 8). furthermore, it may be concluded that the bound molecules of water alleviate the nho interaction and very probably this effect is even more pronounced in aqueous solution. from the above, it is evident that the reduced hydration of the carboxylate group, relative to the carboxylic group, should be attributed mainly to the strong tendency of the carboxylate group to form a six-membered conjugated ring with a single molecule of water. constructively, the tentative models illustrated in figure 8 should be further validated by in silico and experimental approaches. computational methods complement the experimental results by providing information on the microscope and physicochemical details on the interplay between water and the biomolecule of interest [4447]. for example, introduction of solvent effects into molecular dynamics can provide an atomic description of the folding and unfolding of a protein. furthermore, there is an array of theoretical approaches that have been utilized for treating nmr shieldings in solution, that can be classified as continuum models [49, 50] and molecular dynamics simulations. experimental approaches could involve o nmr both in powders and in the crystal state with varying degrees of hydration.

2d, 13c, 14n, and 17o nmr and crystallographic data from the literature were critically evaluated in order to provide a coherent hydration model of amino acids and selected derivatives at different ionization states. 17o shielding variations, longitudinal relaxation times (t1) of 2d and 13c and line widths (1/2) of 14n and 17o, may be interpreted with the hypothesis that the cationic form of amino acids is more hydrated by 1 to 3 molecules of water than the zwitterionic form. similar behaviour was also observed for n-acetylated derivatives of amino acids. an exhaustive search in crystal structure databases demonstrates the importance of six-membered hydrogen-bonded conjugated rings of both oxygens of the -carboxylate group with a molecule of water in the vicinity. this type of hydrogen bond mode is absent in the case of the carboxylic groups. moreover, a considerable number of structures was identified with the propensity to form intramolecular hydrogen bond both in the carboxylic acid (nho=c) and in the carboxylate (nh o) ionization state. in the presence of bound molecules of water this interaction is significantly reduced in the case of the carboxylate group whereas it is statistically negligible in the carboxylic group.

PMC3361190

pubmed-463

aspergillus species are ascomycetes that are classified in the form subdivision deuteromycotina, as many of them do not show a sexual reproductive phase. generally, they are common ubiquitous saprophytes in soil and on dead organic substrates. being classic opportunistic pathogens, invasive infections by aspergillus species almost exclusively develop in immunocompromised patients, while localized infections and allergic bronchopulmonary aspergillosis occur in individuals without immunosuppression. generally, the species aspergillus fumigatus represents the most common inducer of invasive and allergic manifestations, followed by a. terreus, a. flavus, and a. niger [1, 2]. invasive aspergillosis (ia) considerably contributes to the morbidity and mortality among immunocompromised individuals, including patients with haematological malignancies, recipients of haematological stem cell and solid organ transplants, aids patients, and patients treated with immunosuppressive regimens due to autoimmune diseases. the most important single risk factor is prolonged and profound neutropenia (< 500 neutrophils/l for more than 10 days) [1, 46]. over the last decades, invasive fungal infections, particularly aspergillosis, have become more frequent due to a higher number of immunocompromised patients (new chemotherapy regimens, increasing number of solid organ transplant recipients, and immunosuppressive regimens) and extended survival time in hiv patients (haart therapy) [1, 79]. on the side of the pathogen, several characteristics and various putative virulence factors that may facilitate the infection have been described for a. fumigatus. it differs from nonpathogenic species by its growth at 37c; furthermore, it is rapidly growing and has very small conidiospores (35 m). these include melanin and a hydrophobic protein-coat layer on the surface of conidia that may help to protect them against recognition, ingestion and/or elimination by complement and phagocytes [1014]. various proteases that may help to pass tissue barriers and to degrade proteins of the immune response are secreted by the fungus [1517], and mycotoxins like gliotoxin might also contribute to undermine the host defence [1820]. the most important path of aspergillus infections is via inhalation of the conidia into the respiratory tract. as conidia of pathogenic aspergillus species are very small, they can be inhaled deeply into the lung and even into the pulmonary alveoli. in immunocompetent individuals, conidia are effectively phagocytosed and eliminated by alveolar macrophages and infiltrating neutrophils [12, 21, 22], but in the case of immunologic deficits, they are able to germinate and to penetrate the lung tissue, thus causing an invasive pulmonary aspergillosis. infections of the lung are the by far most frequent type of ia. by penetration of blood vessels, aspergillus can disseminate and invade other organs, including the heart, the liver, and the central nervous system (cns). cerebral aspergillosis occurs in 10%20% of all cases of ia and thus is the most common extrapulmonary form. neuropathologic features include hemorrhagic infarcts and/or necrosis, vascular thrombosis, meningitis, granuloma, and formation of solitary as well as multiple abscesses [6, 2325]. according to the division of bacterial and mycotic diseases (dbmd), the incidence of aspergillosis is 1-2 per 100,000 per year. incidence rates of ia in high-risk populations depend on the respective group and rise up to 24% in patients with prolonged and profound neutropenia. furthermore, ia is the most expensive opportunistic infection in immunosuppressed patients, with annual treating costs in europe of approximately 200 million. in-hospital stays complicated by ia cause additional costs of 75,000 per patient. despite antimycotic therapy and surgical interventions, the fatality of ia is high and depends on the degree of immunosuppression and on the affected organs. without treatment, the mortality is nearly 100%, while under treatment the overall case-fatality rate is nearby 60% and rises to more than 90% in cases of cns aspergillosis [1, 6, 26]. complement consists of approximately 30 fluid-phase and membrane-bound proteins that cooperate to form the cascade. regulatory factors control and modulate its activity, and cellular receptors mediate the interaction between complement factors and immune cells. representing a potent component of the innate host defence and an interface to adaptive immunity the most outstanding roles are the direct and indirect defence against infections, the stimulation and regulation of b- and t-cell response, and the disposal of debris [2731]. hepatocytes are the main producers of complement factors; however, several other cell types participate in the synthesis. activation of the complement system is triggered by a multiplicity of danger signals, such as pathogen-associated molecular patterns (pamps), antigen/antibody complexes, and the presence of transformed cells, apoptotic cells, or cell debris. three different activation pathways start the complement cascade, all of them resulting in the cleavage of the central complement factor c3 by proteolytic enzyme complexes (c3 convertases), and subsequently leading to the common terminal pathway (figure 1). in the classical pathway, binding of complement factor c1q to immunoglobulin class g or m (igg, igm) of antigen-antibody complexes represents the initial step. alternatively, the globular heads of c1q can interact with microbial surfaces that had been covered by pentraxins, a class of soluble pattern recognition molecules. the thereby induced conformational changes of c1q subsequently activate the associated proteases c1r and c1s, which cleave the factors c4 and c2. the resulting fragments form the c3 convertase c4b2a. in the lectin pathway, foreign carbohydrate molecules on the surface of pathogens are recognized by mannose-binding lectin (mbl) or the related ficolins. mbl-associated serine proteases (masps) cleave c4 and c2, and the fragments build up the c3 convertase c4b2a, which is identically equal to the one of the classical pathway. ficolin-2 can also interact with pentraxin-covered microbes, thus starting the lectin pathway in an alternative manner. interestingly, mbl was recently described to support c3 cleavage by a c2 bypass mechanism, which results in activation of the alternative pathway. the alternative pathway is triggered via activating foreign surfaces and creates an amplification loop by spontaneous reaction of c3 with h2o (c3(h2o)); alternatively, c3b generated by the other pathways represents the starting trigger. surface-bound c3b associates with factor b, which is then cleaved by the plasma serine protease factor d. these steps result in the formation of the c3 convertase c3bbb [27, 36]. proteolytic cleavage of c3 by one of the c3 convertases is the common and central step of all three activation pathways. this split generates the fragments c3a and c3b, which are two important components that mediate a multitude of complement functions (see below). the product c3b associates with the c3 convertases, thus forming the c5 convertases, which cleave factor c5 into c5a and c5b. this step initiates a chain of assembly processes of the proteins c6, c7, c8, and c9. the bound and polymerized c9 units create the terminal complement complex (tcc) that can form a pore in the target lipid bilayer, called membrane attack complex (mac). targeted cells, bacteria and viruses die or are inactivated by efficient disruption of the membrane integrity [31, 37]. beneath the mac formation and direct pathogen destruction, complement displays several additional antimicrobial mechanisms aiming to neutralize invading microbes and to restore body homeostasis. surface-bound c3b undergoes internal cleavage steps; the derived products ic3b, c3d, and further, coat and label the pathogens for phagocytosis (opsonization). effector cells with specific membrane-bound complement receptors (crs) recognize the opsonizing complement fragments, ingest the labeled pathogens, and eliminate them. furthermore, the interaction of the opsonized particles with cr-bearing immune cells results in their activation and their increased proliferation. the receptor cr3, a heterodimer of cd11b and cd18, is regarded to be the most important mediator for complement-driven phagocytosis. being expressed on phagocytes like dendritic cells, neutrophils, macrophages, and microglia, it interacts with ic3b on the pathogen [27, 38]. the complement receptors cr3 and cr4 allow adhesion of cells to cells of the same and other cell types, respectively, (homotypic and heterotypic adhesion). immune cells can bind via these receptors to their ligands on endothelium of the blood vessels, a prerequisite for penetration through the vessel wall into the tissue and migration to the site of infection and inflammation. surface-bound ic3b can be further cleaved proteolytically to generate the opsonizing fragment c3d. binding of c3d-opsonised pathogens to the corresponding complement receptor cr2 (cd35) on b cells induces cross-linkage with the b cell receptor complex, a process that lowers the threshold for b cell activation by the specific antigen by several orders of magnitude. cleavage of c3 and c5 generate the potent anaphylatoxins c3a and c5a, respectively, which exert several biological functions by binding to their corresponding cellular receptors c3ar, c5ar (cd88), and c5l2. they provoke chemotactic attraction of immune cells to the site of infection and an increase of vascular permeability [40, 41]. furthermore, c3a and c5a trigger an efficient proinflammatory response by stimulating cytokine synthesis and secretion [40, 41]. various cell types harboring the corresponding anaphylatoxin receptors on their surface react on ligand binding with cell activation, stimulation of cell specific signaling pathways, or of oxidative burst [27, 42]. the complement cascade needs a tight control to prevent host damage by cell/tissue lysis and excessive inflammation. a variety of both soluble and membrane-bound regulators can influence all steps of the complement cascade, with the c3/c5 convertases as main control targets [27, 37, 4345]. under normal conditions, these regulators should protect all body cells against auto-attack by the complement system. the serine protease factor i cleaves both c4b and c3b and is thereby supported by various cofactor molecules. c4 binding protein (c4 bp) and factor h (fh) are fluid-phase proteins that enable the cleavage of c4b and c3b, respectively, moreover, the membrane-anchored molecules complement receptor 1 (cr1, cd35) and membrane cofactor protein (mcp, cd46) support the degradation of both c3b and c4b. in addition, fh and c4 bp accelerate the decay of assembled c3 convertase; cr1 affects both c3 and c5 convertases. decay accelerating factor (daf, cd55) is another notable membrane-bound regulator that efficiently prevents the assembly and promotes the disintegration of both c3 and c5 convertases [27, 4345]. in the terminal pathway, the membrane-anchored cd59 (protectin) binds to c8 in the c5b-8 complex and thus inhibits further incorporation and polymerization of c9 units to form the mac [27, 4345]. the potency of complement represents a valuable tool to attack invading pathogens and to defend the host against penetration and dissemination. one particular advantage of complement lies in the fact that activation can start within seconds after contact with the microbe and ends with a multifaceted spectrum of antimicrobial reactions. however, the fact that microbial infections occur in a considerable proportion, already implicates that the pathogens have developed appropriate counterstrategies to avoid elimination, thus starting a vicious circle of reaction and counterreaction. furthermore, the antimicrobial effector mechanisms of the complement system might also harbour harmful consequences for the affected host. as known from several infectious and noninfectious diseases, chronic or exceeding complement-mediated inflammation putative mechanisms for such complement-induced tissue damage may include a fulminant inflammatory reaction and opsonization of surrounding bystander cells with subsequent lysis. aspergillus conidia and hyphae activate the complement system via all three pathways [4648] (figure 1). initiation of the complement cascade by resting conidia is mediated predominantly by the alternative pathway. however, when the conidia begin to swell and transform into hyphae, there is a progressive involvement of the classical pathway. these differences in the activation pathways are reflected by different kinetics; the slowest initiation is seen with resting conidia. furthermore, mbl as a pattern recognition molecule of the lectin pathway is able to bind to carbohydrate structures on the surface of aspergillus and promotes complement activation via the lectin pathway, which results in the deposition of c4. as mentioned above, mbl can support c3 cleavage by a c2 bypass mechanism after contact with a. fumigatus conidia, resulting in activation of the alternative pathway and avoiding formation of the classical pathway c3 convertase. this mechanism is not restricted to a. fumigatus, but can also take place in the presence of a. terreus, a. niger, and a. flavus. mbl generally seems to be a molecule of high significance for innate defence against a range of pathogens. in several studies, it was shown to bind to various sugars on the surfaces of viruses, bacteria, yeasts, fungi, and protozoa [4851]. further evidence for the crucial role of mbl arises from findings in patients with chronic necrotizing pulmonary aspergillosis and mouse models of pulmonary aspergillosis [52, 53]; these facts suggest mbl as a promising molecule for prophylaxis and therapeutical treatment (see below). a further mechanism for complement activation driven by aspergillus involves the interaction with the pattern recognition molecule pentraxin-3 (ptx-3). when a. fumigatus is opsonized with ptx-3, the complement cascade can be activated either by interaction between ptx-3 and c1q via the classical pathway, or by interaction between ptx-3 and ficolin-2 via the lectin pathway. after seroconversion, anti-aspergillus antibodies in the serum can trigger the start of the classical complement pathway. the thesis that complement represents a central tool in antifungal host defence is supported by several findings. furthermore, recognition by the complement system and activation of the cascade seems to interfere with efficient dissemination in the host. this conclusion is strongly indicated by the fact that the level of complement deposition on different aspergillus species correlates inversely with their pathogenicity: highly virulent species like a. fumigatus and a. flavus bind less c3 on their surface than nonpathogenic species like a. glaucus or a. nidulans. the antimicrobial potency of the complement cascade appears to be independent from direct killing via formation of a mac; presumably, the thick fungal cell wall block the formation of a pore by the c9 polymers and the subsequent lysis of the cells. opsonization of the fungal surface with c3-derived fragments are presumably the most relevant complement-associated weapon, stimulating efficient phagocytosis or release of damaging compounds, oxidative burst and killing by monocytes, bronchoalveolar macrophages, and polymorphonuclear cells [46, 47, 57]. the capacity to opsonize pathogens and to exert antifungal effects strictly depends on the available complement levels in the respective compartment of the body. the complement concentrations in the central nervous system (cns) are low and thus only allow a rather weak deposition. consequently, the complement amounts in the cerebrospinal fluid are unable to induce a significant oxidative burst in immune cells and to result in reduced fungal viability, thus making the cns a highly vulnerable organ. however, the brain cells react to the fungal presence with an upregulation of complement synthesis to enable better opsonization and therefore a more efficient clearance of the fungus. mice deficient in complement factor c5 can exert the early opsonization processes with c3 fragments, but are unable to fulfil the complete cascade. when infected with a. fumigatus, these mice show decreased resistance and lower 50% lethal conidia dosage for a disseminated infection [59, 60]. since this enhanced susceptibility is unlikely to be due to absent mac formation in the fungal cell membrane, it might be supposed that the inability to form the anaphylatoxin c5a could be the relevant deficit in these mice [59, 60]. c5a exerts a wide range of proinflammatory effects; by binding to its receptor c5ar, c5a recruits inflammatory cells to the site of infection, enhances cellular adhesion, and stimulates oxidative metabolism. in addition, c5a can trigger the release of lysosomal enzymes and of inflammatory mediators such as tumor necrosis factor-alpha (tnf-) and interleukin-6 (il-6) [61, 62]. furthermore, a higher susceptibility to aspergillosis in c5-deficient mice might be attributed to missing tcc. low doses of this soluble complex were shown to bind to the membrane of a range of cell types, thereby triggering various effects like activation, rescue from apoptosis, and secretion of prostaglandins, which are important regulators of the immune response [6367]. the potency of aspergillus to cope with the complement system and to undermine its mechanisms for elimination determines how successful the fungus can establish an infection. aspergillus has developed a complex repertoire of effector mechanisms for this purpose (table 1). single or multiple abscess formation is a characteristic feature of aspergillosis, particularly in the central nervous system (cns). the fungal hyphae are found in brain blood, vessels with invasion through vascular walls into adjacent parenchymal tissue. fungal brain abscesses may arise from these sites of localized parenchymal infection. in this case, white blood cells collect in the affected part of the brain, and fibrous tissue forms around this area, creating a mass. cns abscesses typically present with headache, focal neurological abnormalities, and/or seizure, which is the consequence of local destruction or compression of adjacent brain tissue. mature fungal abscesses exhibit a central necrotic area with fungal hyphae, surrounded by a capsule of newly formed fibrous tissue. the formation of abscesses represents a host mechanism to inhibit further spreading of invading pathogens. however, this sealing off not only inhibits fungal dissemination, but also forms some kind of protection shields against the complement attack. immunohistochemical staining revealed that effect: whereas the fibrous surrounding tissue was intensely stained for complement proteins, the central necrotic area contained only minor complement concentrations. no deposition of complement factors on the fungal surface in the abscess was visible, implying that the encapsulation protects the fungus within the abscess from any efficient complement attack. putative complement recognition sites on the conidial surface of a. fumigatus are optimally masked to minimize the stimulus for complement activation. experiments aiming to identify the relevant fungal structure indicated that melanin could play a substantial role for masking; for this purpose, knock out mutants lacking enzymes of the melanin biosynthesis pathway were used [10, 11, 76]. disruption of the gene alb1, which encodes a polyketide synthase in the synthesis of melanin, results in increased opsonization of the conidia with c3 and in a better ingestion by human neutrophils. deposition of pigments on the conidial surface might mask the c3 binding sites, and disruption of the alb1 gene might expose these sites and thus allow enhanced c3 binding. a mouse model confirms this function of alb1 in fungal pathogenesis, since the alb1-deficient mutant of a. fumigatus turned out to be less virulent than the wild-type fungus [11, 76]. inactivation of the gene for the pigmentation protein arp1 similarly increased the deposition of c3 on conidia. this pigment seems to be a central element of aspergillus against the host defence, as it is also involved in scavenging reactive oxygen species (ros) and inhibits the acidification of phagolysosomes of alveolar macrophages, monocyte-derived macrophages, and human neutrophil granulocytes after ingestion of conidia [12, 13, 77]. as mentioned above, the activity of the complement cascade is strictly limited by several fluid-phase inhibitors. immunofluorescence analysis, adsorption assays and flow cytometry studies showed that aspergillus acquires fh, factor h-like protein 1 (fhl-1), factor h-related protein 1 (fhr-1), and c4 bp from the host [70, 71]. fhl-1 is a splicing product of the fh gene, and fhr-1 is a related protein belonging to the fh family. bound to the conidial surface, fh maintained its regulatory activity and could act as a cofactor for the factor i-mediated cleavage of c3b. as a consequence of covering the fungal surface with these complement inhibitors, all three pathways might be downmodulated. the attachment molecules on aspergillus are not yet known, whereas the corresponding binding regions within fh were recently described. one of them was identified within n-terminal short consensus repeats (scrs) 1 to 7 and a second one within c-terminal scr 20. a. fumigatus not only acquires complement inhibitors from the host, but also produces and releases its own soluble factor that inhibits complement activation and opsonization of the fungus [72, 73]. this complement inhibitor (ci), which is also synthesized by a. flavus, selectively abolishes activation of the alternative pathway and interferes with c3b-dependent phagocytosis and killing. the exact chemical composition of ci is as yet unknown; it contains 15% protein and 5% polysaccharide. recent own results raise the possibility that this described ci or a closely related activity also contributes to the pathogenesis in cerebral aspergillosis, since immunohistochemical studies show deposition of c1q and c4, but not of c3, on the fungal hyphae in the cns. studies by sturtevant revealed the synthesis of a proteolytic enzyme that is able to degrade c3 (, reviewed in:). this is confirmed by own experiments showing complement-degrading proteolysis in the supernatant of aspergillus when grown in cerebrospinal fluid (csf). the fungus-induced degradation of complement in csf evoked a drastic reduction of the opsonization of the fungal hyphae. in parallel, the fungal serine protease alp1 might participate in complement degradation and thus be partly responsible for the complement evasion [15, 17]. to date, degradation of c1q, c3, c4, c5, mbl, and factor d were shown [15, 17]. the high morbidity and lethality of invasive aspergillosis strongly demands for an expansion of the current treatment options. the antimycotic therapy might be completed by new approaches aiming to strengthen the host immune response against the fungus. putative approaches could be to increase the available complement concentrations or to improve the efficiency of complement attack by undermining the fungal evasion strategies. appropriate strategies that target the complement system may aim to the following. our own studies about cerebral aspergillosis showed a clear correlation between the complement levels in the csf and the capacity of csf to opsonize fungal hyphae and designate them for phagocytic killing. a therapeutic increase of mbl concentrations in invasive aspergillosis might be an appropriate approach, since patients with chronic necrotizing pulmonary aspergillosis show more frequently mbl haplotypes that encode for low levels of the protein than healthy control persons. further support comes from a murine model of invasive pulmonary aspergillosis: those mice with externally administered recombinant mbl reveal better survival, compared to untreated animals. detailed studies confirmed that mbl-treated mice show a significant increase in the levels of the proinflammatory cytokines tnf- and il-1, together with a marked decrease of anti-inflammatory il-10 and of fungal hyphae in the lung. blocking of the fungal surface pigments by specific antibodies or peptides might be a hypothetical approach that could help to expose the c3 binding sites and thus improve complement deposition and ingestion of conidia by phagocytes. another approach might target the acquisition of the negative complement regulators fh, fhl-1, and c4 bp to the fungal surface. for candida albicans, some molecules that bind c4b and fh have recently been identified [78, 79], while the attachment sites on aspergillus are still unknown but might include related molecules. blocking antibodies, designed peptides or other inhibitors against these complement regulator binding molecules might help to make the fungus more vulnerable towards complement attack. a similar approach might be developed for the aspergillus-derived complement inhibitor described by washburn [72, 73]. our own results open the possibility to neutralize the fungal protease(s) that is/are secreted by aspergillus to degrade complement proteins. two different strategies were tested by first in vitro experiments: the neutralization of the protease by specific inhibitors or interference with the production of this proteolytic enzyme. in our studies, we could prevent the complement degrading activity by serine protease inhibitors. however, a therapeutically used protease inhibitor must be highly specific, since a general block of serine proteases might be fatal for the host. alternatively, our experiments exhibited that the secretion of complement-degrading enzymes strictly depends on the availability of nitrogen sources. thus, the supply of amino acids in the infected host might downmodulate the secretion of the relevant fungal protease that cleaves the complement factors of the host. despite new antifungal drugs and improved medical treatment, invasive aspergillosis remains a dangerous threat for immunocompromised patients, as the innate immune defence is the most crucial weapon against this infection. the complement system is of particular importance, as it harbours multiple effects against infectious diseases, bridges the elements of the human defence network by a multitude of factors, and helps to preserve the homeostasis of the body. the presence of fungal pathogens is detected by different pattern recognition molecules; three pathways guarantee the activation of the complement cascade by resting, swollen, and germinating conidia as well as by hyphae of aspergillus. direct lysis of fungal cells by the membrane attach complex (mac) appears to be of minor importance for the antifungal defence. presumably, attraction and activation of immune cells (monocytes, pulmonary macrophages, and polymorphonuclear neutrophils) are the most essential mechanisms. anaphylatoxins (c3a, c5a) chemotactically recruit immune cells to the site of the infection and induce further inflammatory reactions. opsonization of conidia and hyphae with complement fragments like c3b and ic3b mediate phagocytosis, oxidative burst, and release of damaging compounds by binding to corresponding receptors on immune cells. however, highly virulent aspergillus species have evolved mechanisms to evade the attack by complement. they hide from recognition, acquire complement regulatory molecules from the host, and secrete proteases to degrade complement factors. the multifaceted interactions between complement and aspergillus represent promising approaches for future therapeutic strategies that may help to improve the outcome of invasive aspergillosis.

invasive aspergillosis shows a high mortality rate particularly in immunocompromised patients. perpetually increasing numbers of affected patients highlight the importance of a clearer understanding of interactions between innate immunity and fungi. innate immunity is considered to be the most significant host defence against invasive fungal infections. complement represents a crucial part of this first line defence and comprises direct effects against invading pathogens as well as bridging functions to other parts of the immune network. however, despite the potency of complement to attack foreign pathogens, the prevalence of invasive fungal infections is increasing. two possible reasons may explain that phenomenon: first, complement activation might be insufficient for an effective antifungal defence in risk patients (due to, e.g., low complement levels, poor recognition of fungal surface, or missing interplay with other immune elements in immunocompromised patients). on the other hand, fungi may have developed evasion strategies to avoid recognition and/or eradication by complement. in this review, we summarize the most important interactions between aspergillus and the complement system. we describe the various ways of complement activation by aspergillus and the antifungal effects of the system, and also show proven and probable mechanisms of aspergillus for complement evasion.

PMC3423931

pubmed-464

obesity has become a heavy public health problem in the united states, with a prevalence among adults increasing to 32% from 13% between the 1960s and 2004. currently, 66% of adults and 16% of children and adolescents are overweight or obese. although obesity has long been recognized as an independent risk factor for cardiovascular diseases and diabetes mellitus, newer research points to obesity as an important risk factor for chronic kidney diseases (ckds) [24]. in 1974, weisinger et al. subsequent studies confirmed that obesity could induce renal injury, namely, obesity-related glomerulopathy (org) [68]. a large-scale clinicopathologic study including 6818 renal biopsies from 1986 to 2000 revealed a progressive increase in biopsy incidence of org from 0.2% in 19861990 to 2.0% in 19962000. the tenfold increase in incidence of org over 15 years suggests a newly emerging epidemic. the clinical characteristics of subjects with org typically manifest with nephrotic or subnephrotic proteinuria, accompanied by renal insufficiency [810]. histologically, org presents as focal segmental glomerulosclerosis (fsgs) and glomerular hypertrophy or glomerular hypertrophy alone and relatively decreased podocyte density and number and mild foot process fusion [8, 11, 12]. clinically, it is distinguished from idiopathic fsgs (i-fsgs) by its lower incidence of nephrotic syndrome, more benign course, and slower progression of proteinuria and renal failure [8, 11]. potential mechanisms by which obesity affects renal physiology include altered renal hemodynamics, insulin resistance, hyperlipidemia, activation of renin-angiotensin-aldosterone system (raas), inflammation, and oxidative stress. increases in both glomerular filtration rate (gfr) and renal plasma flow (rpf) were observed in obese subjects and animals [13, 14]. this likely occurs because of afferent arteriolar dilation as a result of proximal salt reabsorption, coupled with efferent renal arteriolar vasoconstriction as a result of elevated angiotensin ii (angii). these effects may contribute to hyperfiltration, glomerulomegaly, and later focal glomerulosclerosis [8, 9]. insulin resistance can raise the transcapillary pressure gradient and cause hydrostatic pressure and hyperfiltration by reducing norepinephrine-induced efferent arteriolar constriction, leading to glomerular hypertrophy and sclerosis. hyperinsulinemia also has been shown to stimulate the synthesis of growth factors such as insulin-like growth factor- (igf-) 1 and igf-2 and transforming growth factor-1 (tgf-1), which accelerate production of extracellular matrix and promote glomerular hypertrophy and sclerosis [17, 18]. hyperlipidemia may promote glomerulosclerosis through mechanisms that involve engagement of low-density lipoprotein receptors on mesangial cells, direct podocyte toxicity, oxidative cellular injury, macrophage chemotaxis, and increase renal expression of sterol regulatory element-binding proteins (srebp-1 and srebp-2), resulting in the renal accumulation of cholesterol and triglycerides and together with significant renal increase of fibrogenic cytokines [19, 20]. obese subjects usually have increases in plasma renin activity, angiotensinogen, angiotensin-converting enzyme activity, and circulating angii, which trigger or promote renal damage by renal hemodynamic changes and nonhemodynamic pathways such as hyperinsulinemia, oxidative stress, and inflammation [2123]. inflammatory abnormalities and oxidative stress are characteristic findings of obesity and play important roles in the renal damage associated with obesity, which will be discussed in detail in the following. recent studies have demonstrated that obesity causes chronic low-grade systemic inflammation and thus contributes to the development of systemic metabolic dysfunction that is associated with obesity-related disorders and renal disease [2427]. levels of some inflammatory markers and cytokines such as c-reactive protein (crp), tumor necrosis factor- (tnf-), interleukin-6 (il-6), and macrophage migration inhibitory factor (mif) are elevated, whereas concentrations of adiponectin, a protein hormone that exerts anti-inflammatory activities, are reduced in obesity [2834]. leptin is a 16-kda-peptide hormone encoded by obese (ob) gene that is mainly produced by adipose tissue. leptin serves as a regulator of energy balance by binding to the full-length leptin receptors obese receptor b (ob-rb) in the hypothalamus, leading to reduction in food intake and elevation in temperature and energy expenditure. leptin receptors can be classified as secreted-forms (ob-re), short-forms (ob-ra, c, d, and f) mainly expressed in peripheral tissue, and long-forms (ob-rb) predominantly expressed in hypothalamus. the kidney expresses abundant concentrations of the truncated isoform of the leptin receptor ob-ra, but only a small amount of the full-length receptor ob-rb. leptin production is associated with increased size of adipocytes and is positively correlated with the body mass index (bmi). increased circulating leptin, a marker of leptin resistance, is common in obesity. obesity-induced leptin resistance injures numerous peripheral tissues including kidney, liver, myocardium, and vasculature [36, 38]. leptin results in the development of renal disease by binding to its specific receptors in renal endothelial cells and mesangial cells. in glomerular endothelial cells, leptin stimulates cellular proliferation, tgf-1 synthesis, and type iv collagen production [36, 39]. in mesangial cells, leptin upregulates synthesis of the tgf- type ii receptor, but not tgf-1, and stimulates glucose transport and type i collagen production through signal transduction pathways involving phosphatidylinositol-3-kinase. however, both those cell types increase their expression of extracellular matrix in response to leptin. transgenic mice with leptin overexpression demonstrated an increase in collagen type iv and fibronectin mrna in the kidney. leptin is involved in the development of glomerulosclerosis through a paracrine tgf- pathway (between glomerular endothelial and mesangial cells) that promotes the deposition of extracellular matrix, proteinuria, and, eventually, glomerulosclerosis. infusion of leptin into normal rats for 3 weeks fosters the development of focal glomerulosclerosis and proteinuria. leptin also has proinflammatory actions through its interaction with mediators of innate and adaptive immunity and crp. leptin regulates components of innate and adaptive immunity, including t lymphocytes and monocytes/macrophages [42, 43]. central leptin administration in ob/ob mice accelerates renal macrophage infiltration through the melanocortin system. leptin stimulates central t-cell production and a peripheral shift in favor of t helper (th) 1 adaptive immune responses (proinflammatory) as opposed to th2 responses (anti-inflammatory). leptin has been shown to modulate adaptive immunity by enhancing t-cell survival and stimulating production of proinflammatory cytokines such as ifn- and il-2. leptin also has structural and functional resemblance to proinflammatory cytokines, such as il-6, and may modulate crp, a leptin-interacting protein. therefore, these direct and indirect effects of leptin on the kidney, including stimulating cellular proliferation and hypertrophy, increasing extracellular matrix expression, and exhibiting proinflammatory activities, may partially explain obesity-related kidney disease. adiponectin is a 30 kda adipocyte-derived protein hormone encoded by the adipose most abundant gene transcript 1 (apm1), which plays a role in the suppression of inflammation-associated metabolic disorders. adiponectin is highly abundant in human serum, but its level is decreased in most obese animal and human subjects, particularly in those with visceral obesity [4951]. recent clinical studies show a negative association of adiponectin in obese patients, [52, 53] suggesting that adiponectin may play a key role in the development of obesity-related albuminuria and alteration of renal function. studies with the adiponectin knockout mouse provide evidence that adiponectin can regulate podocyte function and thus contribute to the initial development of albuminuria [37, 53]. sharma et al. showed that knockout of adiponectin in mice increased albuminuria and caused fusion of podocyte foot processes. in cell culture studies with podocytes, (zo-1) to the plasma membrane, and reduced the renal predominant nadph oxidase nox 4, largely via a 5-amp-activated-protein kinase- (ampk-) dependent pathway. treatment of the adiponectin knockout mice with exogenous adiponectin was able to decrease albuminuria and improve podocyte morphology. chronic hyperadiponectinemia significantly alleviated the progression of proteinuria in early-stage diabetic nephropathy by several mechanisms. it led to an increase in nephrin expression, improvement of the endothelial dysfunction due to decreases in endothelin 1 (et-1) and plasminogen activator inhibitor 1 (pai-1), and an increase in endothelial nitric oxide synthase (enos) expression in the renal cortex. recent studies suggest that adiponectin exerts anti-inflammatory effects by suppressing tnf--induced activation of nuclear factor-b (nf-b) in human aortic endothelial cells and aortic smooth muscle cells through inhibition of ib phosphorylation [55, 56] and inhibition of vascular cell adhesion molecule 1 (vcam-1) and intercellular adhesion molecule 1 (icam-1) expression, thereby reducing monocyte adhesion and macrophage-induced cytokine production and crp expression in human adipose tissue. human adipose tissue expressed crp, which was negatively correlated with adiponectin expression in adipose tissue. low levels of adiponectin are associated with higher levels of highly-sensitive c-reactive protein (hs-crp) and il-6, two inflammatory mediators that are involved in the initiation and progression of atherosclerosis and renal disease. therefore, hypoadiponectinemia contributes to development of a low-grade systemic chronic inflammation state, suggesting that hypoadiponectinemia may play a causative role in the systemic and vascular inflammation commonly found in obesity and obesity-related disorders, including renal injury, through its proinflammatory effects. resistin, also known as adipocyte-specific secretory factor (adsf) or as found in inflammatory zone (fizz), is a cysteine-rich 12.5-kda polypeptide that belongs to a small family called resistin-like molecules (relms) [60, 61]. in rodents, resistin is secreted from white adipocytes [62, 63]. in human, it is produced largely by macrophages and expressed in adipose tissue predominantly by nonadipocyte resident inflammatory cells [6466]. current evidence suggests that resistin has been variably associated with obesity, insulin resistance, inflammation, and renal dysfunction. resistin levels are elevated in both genetic and diet-induced animal models of obesity [62, 67]. studies of obese subjects have frequently noted higher serum levels of resistin as well as direct correlations between resistin level and adiposity as measured by bmi [68, 69]. there has been a link between circulating resistin and low-grade inflammation that accompany obesity. resistin is associated with elevated crp and white blood cells, suggesting that the role of resistin may be a component of obesity-related inflammation. it has recently been found that resistin involves in the regulation of proinflammatory cytokine expression. resistin strongly upregulates il-6 and tnf- in human peripheral blood mononuclear cells (pbmcs) via nf-b pathway. human resistin enhanced secretion of proinflammatory cytokines, tnf- and il-12 in macrophages by nf-b-dependent pathway. studies also show that increased levels of resistin in patients with ckd are associated with declined renal function and inflammation [73, 74]. this suggests that resistin may play an important role in obesity and obesity-associated disease by triggering the release of other proinflammatory cytokines. a growing body of evidence indicates that obesity-related glomerulopathy is associated with upregulation of inflammatory mediators. obesity leads to adipose tissue macrophage infiltration in white adipose tissue and increased levels in proinflammatory cytokines. several inflammatory mediators released from adipocytes and macrophages, such as tnf-, il-6, il-1, crp, monocyte chemoattractant protein 1 (mcp-1), pai-1, and mif, contribute to a low level of chronic inflammatory state in obesity and may be responsible for renal injury in obesity-associated glomerulopathy. an emerging pattern of gene expression was observed in adipose tissue in mice fed high fat-fed diets, indicating a shift toward global upregulation of inflammatory genes, including tnf-, il-6, and mcp-1. tnf-, a proinflammatory cytokine, is predominantly produced by macrophages infiltrating adipose tissue [77, 78] and can also be produced by the kidney. this cytokine is involved in the genesis of inflammation and contributes to obesity-associated insulin resistance [8082]. within the kidney, angii, advanced glycation end-products (ages), and oxidized low-density lipoprotein (ldl) a recent study demonstrates that tnf- reduces the expression of klotho, a protein expressed by renal cells, through an nf-b-dependent mechanism, which contribute to renal injury. tnf- enhances the expression of pai-1 in human adipose tissue and plasma pai-1 levels in obesity subjects and is responsible for reduced fibrinolysis and also a component of extracellular matrix, leading to renal fibrosis and terminal renal failure [8890]. tnf- also has been shown to induce the expression of mcp-1 via p38 mitogen-activated protein kinase (mapk) signaling pathway in renal mesangial cells. mcp-1, a key regulator in recruiting monocytes to the glomeruli, may also contribute to renal damage at a later stage of kidney disease in obesity. il-6 is another important proinflammatory mediator systemically secreted from adipose tissue and locally produced in the kidney. studies have demonstrated the positive relationship between bmi and plasma il-6 concentrations [9294]. studies also suggest that il-6 plays a key role in the development of renal disease. endogenous il-6 enhances the degree of renal injury, dysfunction, and inflammation caused by ischemia/reperfusion by promoting the expression of adhesion molecules and subsequent oxidative stress. transgenic knockout of il-6 ameliorates renal injury as measured by serum creatinine and histology. blocking the il-6 receptor prevents progression of proteinuria and renal lipid deposit as well as the mesangial cell proliferation associated with severe hyperlipoproteinemia. il-6 also stimulates the synthesis of crp, which is well known as both a marker and important risk factor of atherosclerosis in the general population and ckd patients. mif was initially described as an immunomodulatory factor isolated from the supernatants of t lymphocytes and was found to inhibit the random migration of macrophages. subsequent studies have indicated that mif acts as a proinflammatory cytokine and pituitary-derived hormone that potentiates endotoxemia. immunologically induced crescentic antiglomerular basement membrane glomerulonephritis, treatment with anti-mif antibody reduced proteinuria, prevented the loss of renal function, attenuated histological damage including glomerular crescent formation, inhibited renal leukocytic infiltration and activation, and reduced il-1 expression by both intrinsic kidney cells and macrophages. thus, mif is a key mediator of the inflammatory and immune response and plays a pathological role in immune-mediated renal injury. oxidative stress is caused by an imbalance between increased production of reactive oxygen species (ros) and/or reduced antioxidant activity, leading to oxidative damage to cells or tissue including lipids, proteins and dna. it is known that oxidative stress is involved in pathological processes of various diseases, such as cancer, diabetes mellitus, hypertension, and cardiovascular disease. studies have suggested that obesity is associated with increased oxidative stress [103, 104]. analysis of oxidative markers in obesity subjects indicates that oxidative damage is associated with increased bmi and percentage of body fat [105, 106]. conversely, parameters of antioxidant capacity are inversely related to the amount of body fat and central obesity [107, 108]. the possible mechanisms of obesity-related oxidative stress include increased oxygen consumption and subsequent production of free radicals derived from the increase in mitochondrial respiration, diminished antioxidant capacity, fatty acid oxidation, lipid oxidizability, and cell injury causing increased rates of free radical formation [104, 109, 110]. it is also reported that the increase in obesity-associated oxidative stress is due to the presence of excessive adipose tissue accumulation. accumulated adipose tissue generates an immune response leading to the secretion of proinflammatory cytokines, including tnf-, il-1, and il-6, which lead to increased generation of ros. excessive fat accumulation also stimulates nicotinamide adenine dinucleotide phosphate (nadph) oxidase activity, which contributes to ros production. ros, in return, augmented the expressions of nadph oxidase (nox) subunits, including nox4 and pu.1 in adipocytes, establishing a vicious cycle that augments oxidative stress in white adipose tissue and blood. oxidative stress in adipocyte seems to be responsible for the low-grade proinflammatory state commonly observed in obesity [113, 114]. oxidative stress is increasingly viewed as a major upstream component in cell-signaling cascades involved in inflammatory responses, stimulating the expression of proinflammatory cytokines. ros activate redox-sensitive transcription factors, particularly nf-b, inducing the release of proinflammatory cytokines and the expression of adhesion molecules and growth factors, including tnf-, il-6, il-1, tgf-1, connective tissue growth factor, igf-1, platelet-derived growth factor, and vcam-1 [115, 116]. h2o2 stimulates il-4 and il-6 gene expression and cytokine secretion by an apurinic/apyrimidinic-endonuclease/redox-factor-1- (ape/ref-1-) dependent pathway. ros increased the expression levels of pai-1, il-6, and mcp-1 through nadph oxidase pathway. oxidized high-density lipoprotein (hdl) enhances proinflammatory properties such as tnf- and mcp-1 in renal mesangial cells partly via cd36 and ldl receptor-1 and via mapk and nf-b pathways. oxidized ldl can stimulate tnf- synthesis from renal cells and initiate local effects of renal damage. increased ros production and mcp-1 secretion from accumulated fat may cause infiltration of macrophages and inflammation in adipose tissue of obesity. moreover, enhanced macrophage migration induces the release of proinflammatory cytokines, which further stimulates the generation of ros [119, 120]. therefore, oxidative stress-induced cytokine production is likely to further increase oxidative stress levels, setting a vicious cycle that may promote the progression of kidney damage in obesity. oxidative stress has been commonly identified in obesity-related renal diseases and may be the mechanism underlying the initiation or progression of renal injury in obesity [122, 123]. previous studies suggest that oxidative stress triggers, at an early age, the onset of kidney lesions and functional impairment in zucker obese (zo) fa/fa rats, a good model of obesity-related renal disease, in absence of hyperglycaemia, hypertension, and inflammation. ros are highly reactive molecules that oxidize lipids and proteins, cause cellular injury, and promote glomerular and renal tubule injury and associated proteinuria. ros are produced by various cells, such as vascular cells, inflammatory cells, and renal cells, and have distinct function on different types of cells, such as endothelial dysfunction, inflammatory gene expression, and renal tubule ion transport. a major source for vascular and renal ros is a nox family of nonphagocytic nad(p)h oxidases, including the prototypic nox2 homolog-based nad(p)h oxidase, as well as other nad(p)h oxidases, such as nox1 and nox4. numerous reports indicate that within the kidney, nad(p)h oxidase, an enzyme that produces superoxide (o2) by transferring electrons from nadh/nadph to molecular oxygen and thereby forming o2, h, and nad/nadp, is capable of modulating renal epithelial ion transport [130, 131]. nap(d)h oxidase-derived ros can alter renal pressure natriuresis and blood pressure regulation through its effects on renal hemodynamics and renal tubular sodium transport. recent data suggest that nadph oxidase-mediated oxidative injury to the proximal tubule, like that seen in the glomerulus, contributes to proteinuria in insulin-resistant states. oxidative stress also plays an important role in the pathogenesis of renal damage through its effects on vascular biology. ros are generated by all types of vascular cells, including endothelial, smooth muscle, and adventitial cells. ros influence vascular cell growth, migration, proliferation, and activation [132, 133]. physiologically, ros can mediate cellular function, receptor signals, and immune responses on vascular cells. in pathophysiological condition, ros contribute to progressive vascular dysfunction and remodeling through oxidative damage caused by decreased nitric oxide (no) bioavailability, impaired endothelium-dependent vasodilatation and endothelial cell growth, apoptosis or anoikis, endothelial cell migration, and activation of adhesion molecules and inflammatory reactions [134, 135]. obesity accelerates the progression of renal injury, associated with augmented inflammation in adipose and kidney tissues. studies in il-6 transgenic mice suggested that high concentrations of il-6 contribute to development of renal injury. treatment with anti-il-6 receptor antibody mr16-1 prevented progression of proteinuria, renal lipid deposit, and the mesangial cell proliferation in hypercholesterolemia-induced renal injury. inhibition of tnf- by etanercept, a tnf- antagonist, also decreased blood pressure and protected the kidney through reduction of renal nf-b, oxidative stress, and inflammation. tnf- blockade increases renal cyp2c23 expression and slows the progression of renal damage in salt-sensitive hypertension. tnf- inhibition also reduces renal injury in deoxycorticosterone-acetate- (doca-) salt hypertensive rats via suppression of renal cortical nf-b activity. treatment of adiponectin-knockout mice with adenovirus-mediated adiponectin results in amelioration of albuminuria, glomerular hypertrophy, and tubulointerstitial fibrosis and reduces the elevated levels of vcam-1, mcp-1, tnf-, tgf-1, collagen type i/iii, and nadph oxidase components. excessive fat accumulation contributes to macrophage infiltration in adipose tissue and increased production of proinflammatory cytokines, such as tnf-, il-8, and il-6 [142145]. consequently, it is possible that weight loss may be a potential method to reduce inflammation. evidence indicates that weight loss induced by nutritional intervention or gastric surgery markedly improves the systemic and adipose tissue inflammatory states linked to obesity [143, 146, 147]. studies by gene profiling analysis have shown that caloric restriction-induced weight reduction leads to the regulation of a wide variety of inflammation-related molecules in human adipose tissue. weight loss globally improves the inflammatory profile of obese subjects through a decrease of proinflammatory factors and an increase of anti-inflammatory molecules in white adipose tissue. roux-en-y-gastric-bypass- (rygb-) induced weight loss has been shown to reduce mcp-1, il-18, il-6, and tnf- concentrations [149, 150]. a longer-term weight reduction induced by rygb in corpulence also prevails in regulating circulating cytokine concentrations. weight loss also ameliorates the low-grade inflammation state that leads to glomerular dysfunction in obesity. in morbidly obese individuals with glomerular hyperfiltration, weight loss by surgical interventions normalizes glomerular filtration rate (gfr) and reduces blood pressure and microalbuminuria. weight loss improves renal function as shown by reduced levels of serum creatinine and improved creatinine clearance. since ros play a key role in the pathogenesis of renal injury such as glomerulosclerosis and tubulointerstitial fibrosis, approaches to reduce oxidative stress by antioxidants supplementation, nutritional and surgical interventions may have renoprotective effects. garcinia protects against obesity-induced nephropathy by attenuating oxidative stress through reduced lipid peroxidation and levels of oxidized ldl. the obese zucker rat is a good model for studying obesity-related kidney disease because it develops proteinuria, glomerular hypertrophy, and focal segmental glomerulosclerosis [155157]. using these rats, it has been demonstrated that nephropathy is associated with oxidative stress, and supplementation with an antioxidant ebselen improved kidney damage by ameliorating proteinuria and renal focal and segmental sclerosis. chronic ebselen therapy also improved vasculopathy with lipid deposits, tubulointerstitial scarring, and inflammation. for example, administration of grape seed proanthocyanidin extract (gspe), an efficient phytochemical antioxidant, can protect against the nephrotoxicity effects induced by cisplatin and gentamicin [159, 160] and reverse experimental myoglobinuric acute renal failure. quercetin, a flavonoid that exhibits antioxidant properties in many diseases, could also protect the rat kidney against lead-induced injury and improve renal function. thus, antioxidants may be a potential therapeutic to prevent the renal damage in org. nutritional and surgical interventions are additional approaches to reduce oxidative stress and prevent kidney injury in obesity. caloric restriction and protein restriction reduce free radicals and ros formation and inhibit accumulation of oxidative biomarkers in animal models. in genetically obese animals, diet restriction can prevent or greatly delay the onset of specific degenerative lesions, in particular glomerulonephritis associated with obesity. since adipose tissue mass in obesity contributes to oxidative stress, bariatric surgery-induced weight loss also results in decreasing systemic oxidative stress in adiposity. weight loss induced by diet restriction or bariatric surgery not only improves inflammation state but also reduces oxidative stress state in obesity, which may protect renal function in obesity-related glomerulopathy. obesity causes chronic low-grade inflammation and systemic and local oxidative stress, which may play a pivotal role in the initiation or progression of obesity-associated glomerulopathy. elevated inflammation in obesity is the result of the production of adipokines and increased inflammatory cytokines and decreased anti-inflammatory factors. oxidative stress is triggered by an imbalance between increased production of ros and/or reduced antioxidant activity. both inflammation and oxidative stress induce damage to renal tubule and glomerulus and result in endothelial dysfunction in the kidney. therefore, anti-inflammation and antioxidant interventions may be the potential therapies to prevent and treat obesity-related renal diseases.

obesity-related glomerulopathy is an increasing cause of end-stage renal disease. obesity has been considered a state of chronic low-grade systemic inflammation and chronic oxidative stress. augmented inflammation in adipose and kidney tissues promotes the progression of kidney damage in obesity. adipose tissue, which is accumulated in obesity, is a key endocrine organ that produces multiple biologically active molecules, including leptin, adiponectin, resistin, that affect inflammation, and subsequent deregulation of cell function in renal glomeruli that leads to pathological changes. oxidative stress is also associated with obesity-related renal diseases and may trigger the initiation or progression of renal damage in obesity. in this paper, we focus on inflammation and oxidative stress in the progression of obesity-related glomerulopathy and possible interventions to prevent kidney injury in obesity.

PMC3332212

pubmed-465

in recent years, nanoparticles have been used increasingly for biomedical applications, including drug or gene delivery, imaging, sensing, or photothermal therapy. in particular, gold nanoparticles (nps) have been suggested as highly useful sensitizing agents in phototherapy due to their unique size and shape-dependent optical properties, high absorption coefficients, ease of synthesis, biocompatibility, and their ability to hold a variety of functional ligands. it is well-known that citrate-stabilized gold nps are endocytosed by cells and remain in intracellular vesicles. moreover, targeting of specific sites inside the cell by functionalization of the surface with cell penetrating peptides or peptides containing nuclear localization sequences has been reported. alternatively, to selectively target specific types of cells, gold nps can be modified with suitable antibodies. this approach has been studied for potential use in photothermal cancer therapy, where cancer cells overexpressing human epidermal growth factor receptor 2 (her2) or epithelial growth factor receptor (egfr) were incubated with gold nps conjugated to anti-her2 or anti-egfr antibodies, respectively. the nps were then irradiated with light within their plasmon resonance absorbance band to heat the cells to temperatures leading to cell death. most interestingly, it has also been demonstrated that endocytosis of gold nps by cancer cells and subsequent irradiation of such intracellular nps can lead to cell death even at irradiation levels that are not high enough to cause significant heating. this nonthermal route to laser-induced cell death has been ascribed to an as yet not fully characterized photochemical reaction, although irradiation of endocytosed nps was reported to be accompanied by increased levels of reactive oxygen species. the use of such a photochemical mechanism could be of great advantage in situations where different types of cells coexist in close vicinity, since it would allow for more selective targeting of particular cells, whereas due to the fast diffusion of heat over the relevant length scales photothermally induced cell death will affect all cells within the irradiated volume more or less indiscriminately, as long as some of them contain nps. irradiation of gold nps with continuous wave (cw) lamp or laser light has been shown to lead to photogeneration of singlet oxygen (o2) in vitro, suggesting that this highly reactive species, which is widely used in photodynamic therapy, may be involved in the photochemical pathway of cell killing by gold nps. in vitroo2 photogeneration by irradiation of spherical gold nps with short laser pulses or cw laser light at comparable powers and intensities has also been reported. in this study, it was suggested that the mechanism of singlet oxygen photogeneration may involve hot electrons, i.e., the highly excited conduction band electrons which upon absorption of a short laser pulse by a np can reach quasi-equilibrated energy distributions corresponding to temperatures of several thousand degrees. this seems somewhat surprising, since cw light at the intensities used does not yield hot here we present new experimental results on the photogeneration of singlet oxygen by irradiation of gold nanoparticles with continuous or pulsed laser light, as well as theoretical work pertaining to the underlying mechanism(s), which so far had not been addressed. we show that electron temperatures in excess of 2000 c are easily achieved in pulsed laser irradiation experiments, whereas cw light under similar conditions yields electronic temperatures of at most 10 c above room temperature. thus, the photogeneration of o2 by gold nps proceeds by different mechanisms under different irradiation conditions; the implications for the further development of medical applications of the effect are discussed in detail. furthermore, we also found that even a moderately thick, but dense, ligand layer significantly reduces the efficiency of o2 photogeneration at the np surface, which also has important consequences for practical applications. citrate-stabilized spherical gold nps with 15 and 46 nm diameter were prepared according to the turkevich frens and a seeded growth method, respectively. gold nanorods (nrods) were synthesized using the seeded-growth method reported by dickerson et al. with slight modifications. more details of the np preparation are provided in the supporting information. nps were characterized using uv vis spectroscopy (genesys 10 uv), differential centrifugal sedimentation (cps instruments dc24000), and transmission electron microscopy (tem, fei tecnai spirit microscope at 120 kv) (see figures s1 and s2 in the supporting information). the uv vis absorbance spectrum of nrods showed a transverse plasmon resonance band at 522 nm, and a longitudinal plasmon resonance band at 798 nm and tem revealed nrods to have a length of 40 nm and a diameter of 12 nm. functionalization with thiolated peg ligands or peptides was achieved by overnight incubation with excess ligand, followed by repeated centrifugation for excess ligand removal. the capping ligands used here were peg-oh (hs-(ch2)11-(eg)4-oh), mpeg5000 (hs-(ch2)2-(eg)n-o-ch3, average mw 5000 g mol) and peptide c-tat (primary sequence calnnagrkkrrqrrr); see figure s3 for the structures of the peg ligands. o2 was detected via the bleaching of 1,3-diphenylisobenzofuran (dpbf), which is widely used for this purpose. all experiments involving dpbf were carried out in the dark. because dpbf is not soluble in neat water, all experiments were conducted in 50/50 (v/v) mixtures of water and ethanol. a fresh solution of dpbf (3.1 mg, 0.115 mm, a412 nm=2) in etoh (100 ml) was kept stirring in the dark. a 10 mm quartz cuvette and a 3 mm stirrer bar were left in aqua regia (1:3 hno3:hcl) for 15 min and thoroughly rinsed multiple times with milli-q water (mq h2o) and etoh. in the clean cuvette with stirrer bar, either mq h2o (600 l) or np solution (600 l) was mixed with the ethanolic dye solution (600 l). where appropriate, the np concentration was adjusted prior to mixing to yield an absorbance of 0.4 at 532 nm in the final solution. the cuvette was sealed with an airtight lid and parafilm, and the uv vis absorbance spectrum was recorded (genesys 10 uv). the sample was then placed on a stirring plate (in the dark), and its absorbance spectrum measured every 10 min for 30 min to ensure the solution was stable. for the irradiation experiments, the cuvette containing 1200 l of sample solution was placed on a stirring plate in front of the laser. for experiments with nps, the cuvette was fitted with heat fins using non-silicone heat transfer paste and cooled using a fan. no significant increase of the cuvette temperature beyond a slight warming was observed. for most experiments, the sample was irradiated at 1000 mw (unless stated otherwise) using a 532 nm continuous-wave diode pumped solid state laser (laser quantum opus 532) with a 1/e beam diameter of 1.85 mm; for some experiments the beam was expanded to 8 mm diameter using a lens, as stated explicitly. the sample was irradiated for 10 min and then removed from the laser setup to record the absorbance spectrum; this was repeated until the sample had been irradiated for 60 min in total. irradiation of gold nrods in the longitudinal plasmon resonance band at 800 nm was performed in the same setup, but using a titanium: sapphire laser (coherent mira 900), aligned on the auxiliary cavity, which prevents mode-locking and thus provides continuous-wave laser operation at 800 nm with 1000 mw power; the beam was expanded to 3.4 mm using two lenses. pulsed laser irradiation was performed in the same setup, but using the second harmonic of a q-switched nd: yag laser (quantel brilliant) (532 nm, 5 ns pulse length, 10 hz repetition rate, 3.5 mm beam size, 15 mj pulse energy). for experiments that included nps, the absorbance of dpbf at 412 nm was calculated by subtracting the np absorbance at 412 nm (obtained from neat np samples) from the measured sample absorbance. the concentration of dpbf in the samples always yielded an initial absorbance very close to 1 at 412 nm; for comparative data analysis, the irradiation-time-dependent dpbf absorbance at 412 nm was therefore normalized to 1 at zero irradiation time. 1,3-diphenylisobenzofuran (dpbf) readily undergoes a 1,4-cycloaddition on reaction with o2 to form endoperoxides which irreversibly yield 1,2-dibenzoylbenzene. dpbf strongly absorbs light at 412 nm (figure 1a), but due to the loss of the -system of isobenzofuran, the product does not absorb light at this wavelength; it is this loss of absorbance that is used to detect the presence of singlet oxygen. here, dpbf was chosen as singlet oxygen sensor as it has no absorbance at 532 or 800 nm, the wavelengths used for laser irradiation of gold nanoparticles in their plasmon resonance bands, and no photobleaching of dpbf was expected to occur upon irradiation in the absence of nps. contrary to this expectation, some bleaching was observed under our cw irradiation conditions, with the dpbf absorbance decreasing by ca. 10% upon irradiation at 1 w (37 w cm) for 60 min (figure 1a). as shown in figure 2a, and described in more detail in the supporting information, this photobleaching of dpbf has two phases: (i) a rapid phase, extending over the first 1020 min of irradiation under the conditions used here, and (ii) a slower phase, which on the time scale investigated appears linear with time. we found that phase i depends on laser power, beam size, and the presence of oxygen, whereas the slope of the time dependent bleaching after 20 min, phase ii, is essentially independent of the presence of oxygen and of the beam power/intensity in the range used here (see figure s4 for details). it should be noted that similar effects have been reported previously, with cw irradiation at 514 nm for 60 min at significantly lower powers (40 mw) than employed here, leading to a ca. 5% decrease of the absorbance of dpbf in benzene, although no explanation was suggested in that report. the oxygen dependence of phase i is in agreement with direct photogeneration of singlet oxygen by visible light, which has been suggested to be the reason for dpbf photobleaching upon irradiation with light at wavelengths above 470 nm. however, the slower phase ii is not affected by removing oxygen from the solution (see figure s4b and figure 3) and hence can not be ascribed to singlet oxygen formation. at the present moment, the mechanism of both phases of dpbf photobleaching remains unclear, although both effects are highly reproducible and bleaching does not occur when the sample is not exposed to light (see figure s4a). photobleaching of the dpbf absorbance upon cw irradiation at 532 nm, 1 w (37 w cm), in a 50/50 (v/v) mixture of water and ethanol: (a) in the absence of nps and (b) in the presence of 15 nm citrate-stabilized spherical gold nps. shown are absorbance spectra taken at intervals of 10 min from before the irradiation up to a maximum irradiation time of 60 min; the arrows indicate the direction of change. (a) time dependence of the photobleaching of the dpbf absorbance at 412 nm, a412(dpbf), upon cw irradiation at 532 nm, 1 w (37 w cm), in the absence and presence of citrate-stabilized spherical nps with 15 and 46 nm diameter; shown here are the results from several individual experiments (dashed lines) and the average (solid lines), after subtraction of the np absorbance and normalization to 1 at time zero; see the experimental section for details of data treatment and analysis. (b) effect of the nps alone, calculated by subtracting the photobleaching effect of dpbf in the absence of nps from the results obtained in the presence of nps; the solid lines in (b) are linear fits of the data in the range 2060 min. gradient of the time-dependent dpbf absorbance photobleaching in the irradiation time window 2060 min for different samples under cw irradiation at 532 nm, 1 w (37 w cm). for experiments in the presence of nps, the np concentration was adjusted to yield an absorbance of 0.4 at 532 nm. experiments for dpbf in the absence of nps and in the presence of 15 nm spherical nps were also undertaken after bubbling the sample with nitrogen for 10 min, as indicated (+ n2). the error bars correspond to the standard deviation of several repeat experiments, and indicates statistically significant differences with respect to the experiment on dpbf only, as determined by the anova f-test at p<0.001; it should be noted that the results for 15 or 46 nm spherical nps without nitrogen bubbling or a peg capping layer were found to be different to all other results at this statistical significance level. no repeat experiment was undertaken for irradiation of nanorods at 532 nm, but the same result (no additional bleaching in the presence of nanorods) was obtained for irradiation at 800 nm (figure s5). in the presence of citrate-stabilized spherical gold nps, the photobleaching of dpbf upon irradiation at 532 nm, i.e., within the nanoparticle plasmon resonance band, is significantly increased (figure 1b). the time dependence of the dpbf photobleaching in the absence of nps and in the presence of nps with 15 and 46 nm diameter is shown in figure 2a. it can be clearly seen that the presence of nps leads to a significant increase of the dpbf photobleaching effect and that larger nps yield a larger effect although the np absorbance was adjusted to be the same for all samples, so that the same amount of light was absorbed. the additional photobleaching of dpbf caused by the presence of nps can be clearly ascribed to the generation of reactive oxygen species (ros), since purging of the samples with nitrogen removes this effect (see below). moreover, previous experiments had shown that light irradiation of citrate-stabilized spherical gold nps leads to the characteristic luminescence of o2 at 1280 nm and does not result in the generation of superoxide, o2, or hydroxyl, oh, radicals, which leads us to conclude that the predominant ros species produced here is singlet oxygen, o2. the fact that significant dpbf photobleaching occurs even in the absence of nps requires careful consideration for the analysis of these data. like dpbf photobleaching in the absence of nps, the effect in the presence of nps shows two phases. however, subtraction of the effect observed when only the dye is present yields an essentially linear time dependence for the additional effect ascribed to the nps on the time scale of the experiment (figure 2b), indicating that the np-induced photogeneration of o2 is essentially constant over our experimental time interval. moreover, the second phase of direct dpbf photobleaching is largely independent of the laser power, at least down to 0.1 w, i.e., significantly lower powers than those used for most of the experiments reported here, as shown in detail in the supporting information (figure s4a), and therefore should be independent of the presence of nps whose absorbance leads to a decrease of the laser power along the beam path, allowing a direct comparison of the results obtained in the time frame of 2060 min. moreover, this slower phase of direct dpbf photobleaching is also independent of the presence of oxygen. for these reasons, only data starting at 20 min irradiation will be used for quantitative comparisons. figure 3 summarizes the main results obtained here after np photoexcitation at 532 nm. it is very obvious that citrate-stabilized spherical gold nps induce significantly faster dpbf photobleaching than is observed in their absence (black); this is found for 15 nm gold nanoparticles (red) but is even more pronounced for larger nps with 46 nm diameter (blue). increasing the size of the spherical nps from 15 to 46 nm increases the np-induced dpbf photobleaching (i.e., the additional effect, after subtracting the direct dpbf photobleaching effect) by ca. it is interesting to note that unlike the direct dpbf photobleaching, which is not affected by nitrogen purging, the np-induced additional bleaching is significantly reduced by nitrogen purging, almost down to the level of the direct dpbf photobleaching effect. this strongly supports the conclusion that the np-induced effect is caused by the formation of o2 which then leads to dpbf photobleaching. furthermore, capping the 15 nm gold nps with peg-oh, a moderately large ligand (figure s3), essentially removes the np-induced bleaching and reduces the observed effect to the level of the direct dpbf photobleaching. gold nanorods (nrods) with a length of 40 nm and a diameter of 12 nm, which have two plasmon resonance bands, namely the transverse band at 522 nm and the longitudinal one at 798 nm (figure s1a), stabilized with a capping layer consisting of a mixture of a peg polymer (mpeg5000) and a peptide (c-tat) (see the experimental section), were also investigated. irradiation at 532 nm, i.e., in the transverse plasmon resonance band, showed no additional dpbf bleaching above the direct dpbf effect (figure 3). irradiation with 1 w (11 w cm) cw laser power at 800 nm, i.e., in the longitudinal plasmon resonance band (figure s5), showed reduced direct dpbf photobleaching compared to irradiation at 532 nm, but again no additional photobleaching was observed in the presence of nrods. these results show that the nrods used here, which have a ligand layer consisting of a mixture of a peg polymer and a peptide, do not induce the formation of o2 upon laser irradiation, independent of the plasmon resonance which is photoexcited. irradiation at 532 nm in the presence and absence of citrate-stabilized spherical gold nps with 15 nm diameter was also investigated using laser pulses with a pulse duration of 5 ns (figure 4). in this case, the uv vis spectra show a slight broadening of the np plasmon resonance band at 520 nm during the first 5 min of irradiation, suggesting that some aggregation occurs; after the initial 5 min, however, the nps remain stable. analysis of the spectra shows that photobleaching of dpbf in the absence of gold nps is similar to the results obtained using cw laser irradiation at comparable average powers (figure s4a). in the presence of gold nps with 15 nm diameter, on the other hand, the np-induced bleaching effect (i.e., the effect remaining after subtracting the direct dpbf photobleaching effect) which is induced by pulsed laser irradiation is larger than that caused by cw irradiation by almost 1 order of magnitude, in spite of the significantly lower laser power employed during the pulsed irradiation experiments (0.15 w vs 1 w), which leads to a correspondingly lower number of absorbed photons; compare figure 4b (pulsed irradiation) with figure 3 (cw irradiation). photobleaching of the dpbf absorbance upon laser irradiation with 5 ns laser pulses at 532 nm, 0.15 w, 10 hz repetition rate (corresponding to a power density of 1.5 w cm and a pulse energy density of 0.15 j cm). (a) absorbance spectra in the presence of citrate-stabilized spherical gold nps with 15 nm diameter, taken at intervals of 5 min from before the irradiation (gray) up to a maximum irradiation time of 30 min; the arrow indicates the direction of change. (b) gradient of the time-dependent dpbf photobleaching (measured at 412 nm) in the irradiation time window of 2030 min in the absence and presence of nps. it is straightforward to estimate the quantum yield of np-induced dpbf photobleaching from the absorbed laser power and the observed rate of absorbance bleach. for the citrate-stabilized spherical nps with 15 and 46 nm diameter under cw irradiation at 532 nm, this yields values of 5 10 and 8 10, respectively. thus, less than one of each 1 million photons absorbed by a np leads to the photobleaching of a dpbf molecule under cw irradiation. for irradiation of 15 nm nps with 5 ns laser pulses, on the other hand, the quantum yield of np-induced dpbf photobleaching is 3.5 10, i.e., almost 2 orders of magnitude larger than for cw irradiation, but still very small. diffusion of oxygen over the lifetime of o2 (approximately 6 s in 50/50 water/ethanol) covers a distance larger than the average distance between dpbf molecules at the concentrations used here, and hence it can be concluded that a significant fraction of the photogenerated o2 which escapes from the np surface should be detected. 10 m, which is close to the concentration used here, is sufficient to detect 50% of photogenerated o2. thus, the observed low quantum yield of dpbf photobleaching indicates a very low quantum yield of o2 photogeneration by gold nps (i.e., number of o2 generated for each photon absorbed), having values of the order of ca. 10 for cw irradiation and ca. 10 for irradiation with nanosecond laser pulses. our results show that irradiation of citrate-stabilized spherical gold nps at 532 nm, i.e., in their surface plasmon band, with pulsed or cw laser light leads to the production of o2, detected here by monitoring the bleaching of dpbf absorbance. however, the rate of o2 production is much larger when using short laser pulses than when using cw light of comparable intensity. whereas ca. 24% of the dye is bleached after only 10 min of irradiation of 15 nm nps with 5 ns laser pulses at an average power of 150 mw (figure 4), only 12% of the dye is bleached over this time by irradiation of the same nps with cw light at significantly higher power (1 w) (figure 2). this difference allows one to draw important conclusions on the mechanism of o2 production by irradiation of gold nanoparticles. irradiation of spherical gold nps at 532 nm leads to the excitation of their plasmon resonance, which can be described as a coherent oscillatory motion of the conduction band electrons; this oscillation dephases and decays on the sub-100 fs time scale, with only a very small radiative contribution, so that most of the excitation energy is retained as electronic excitation in the form of electron hole pairs. since the photon energy is close to the minimum energy required for direct excitation of d-band electrons into the conduction band of gold, a minor contribution of this excitation mechanism can not be ruled out; however, d-band holes are filled by conduction band electrons on the 10 fs time scale, yielding essentially the same outcome as excitation of the plasmon resonance band. the excited electrons initially have a nonthermal energy distribution, and often are referred to as primary hot electrons, although the concept of temperature does not strictly apply to such a distribution. they rapidly (within less than 500 fs) equilibrate by electron electron scattering to yield a fermi distribution corresponding to an elevated temperature and can then be referred to as hot electrons. thiol bond dissociation at the surface of gold nps and have been suggested to be responsible for the creation of o2 by irradiation of gold nps with nanosecond laser pulses. they lose their energy on the time scale of a few picoseconds by interaction with the lattice (electron phonon scattering) with coupling times that are essentially size-independent for nps above 10 nm in diameter, although they strongly depend on the amount of energy deposited due to the temperature-dependent electronic heat capacity. because the lattice heat capacity is much larger than the electronic heat capacity, this leaves the electrons and the lattice in equilibrium at a temperature which is significantly lower than the initial electronic temperature; finally, cooling occurs by heat transfer to the solvent and heat diffusion on the time scale of 10 to a few 100 ps, strongly depending on np size. since the dissipation of the absorbed energy proceeds on the picosecond time scale, excitation with a nanosecond laser pulse yields a highly nonequilibrium situation during the duration of the laser pulse where energy is continuously deposited into the electronic system and at the same time flows through the lattice into the surrounding solvent. it is therefore not straightforward to predict the temperature of the hot electrons achieved in such experiments. we used the two-temperature model for the electron and phonon heat baths coupled to finite-element heat transfer and diffusion simulations in the surrounding solvent to estimate this temperature; details of these simulations are given in the supporting information. as shown in figure 5, under our conditions the electrons are expected to reach a temperature of 2100 c, whereas the lattice reaches temperatures of the order of 1400 c and the solvent in the immediate vicinity of the np a temperature of about 900 c. time-dependent temperatures of the conduction band electrons (red), lattice (black), and first solvent layer (blue), calculated for our experiments using nanosecond-laser pulse excitation (15 nm spherical nps in 50/50 etoh/water, 5 ns laser pulses with 0.15 j cm intensity, solid lines) and for the experiments described in ref (24) (40 nm spherical nps in 80/20 etoh/water, 7 ns laser pulses with 0.03 j cm intensity, dashed lines) using the two-temperature model for the electron and phonon heat baths, coupled to finite-element heat transfer and diffusion simulations in the surrounding solvent (see supporting information for details); time zero corresponds to the center of the laser pulse. a lattice temperature of 1400 c, which is above the melting temperature for solid gold, albeit present for only a few nanoseconds, might be sufficient to cause temporary melting of the nanoparticle, although the pulse intensities used here are still below the reported threshold for size reduction of gold nps of 15 nm diameter by nanosecond laser pulses. nevertheless, a minor effect on the shape and/or size of the nps, especially at the upper end of the size distribution, can not be ruled out and may be related to the minor np spectral changes observed upon irradiation which suggest some aggregation to occur (see above). because of the slower heat dissipation around larger nps, these are heated to higher temperatures and hence are more likely to fragment, in agreement with experimental results on nanosecond-laser pulse induced fragmentation, which also show that fragmentation is finished after 5 min under conditions similar to the ones used here. since our nps were prepared without excess citrate, the resulting increase in the ratio of surface area to volume may indeed cause some aggregation. it also can not be ruled out that the solvent near the np surface temporarily forms bubbles, although the pulse intensities used here are still below the reported threshold for bubble formation by nanosecond pulses for 15 nm nps. such bubbles could lead to better thermal insulation, thus potentially increasing the maximum temperatures, but on the other hand, they might prevent oxygen from reaching the np surface, thus reducing the chance of o2 formation. the formation of o2 from the triplet ground state by interaction with a photoexcited sensitizer requires a change of the electron spin and hence can not result from dipole dipole (frster) interaction, but only from dexter-type electron exchange coupling; the latter can be described as simultaneous transfers of an electron from one of the 2*molecular orbitals on oxygen to a photogenerated hole on the sensitizer and of an electron with opposite spin from a high-energy excited sensitizer level to the same or the other 2*orbital, resulting in the formation of the or singlet oxygen state, respectively. this mechanism requires significant overlap of the relevant electronic wave functions and hence only occurs at short distances of at most 10. since the photoexcited hot electrons have such a short lifetime, this reaction can only occur if an oxygen molecule happens to be in the vicinity of the np or is temporarily adsorbed to its surface at the moment of excitation. a similar reaction occurs on photoexcited si nanocrystals, although in this case the reaction is more efficient than for au nps due to the much longer lifetime of the photoexcited excitons in si. thus, the short lifetime of hot electron excitation in gold nanoparticles easily explains the low quantum yield of o2 photogeneration which is observed here. the excitation of electrons to temperatures exceeding 2000 c means that a significant number have sufficient energy to excite an oxygen molecule to the state, which has an energy of 0.98 ev above the ground state (see figure 6). the number of hot electrons available at the higher energy and the number of holes available at the lower energy involved in this two-electron exchange reaction can be estimated from the density of states of gold and the fermi distribution, as described in more detail in the supporting information. for example, for a spherical np with 15 nm diameter at an electron temperature of 2100 c, there are 260 electrons within an energy interval of 0.1 ev around the state. (it should be noted that the relevant energy interval is the width of this state for an oxygen in the vicinity or temporarily adsorbed onto a np, which is not known, so only relative numbers will be used here.) thus, the electron temperatures achieved in our experiment are sufficient for a significant population of hot electrons and holes at the relevant levels. schematic diagram showing the population probability f(e) for a np electron state at energy e near the fermi level, ef, under different conditions: (a) in equilibrium at room temperature, (b) at an electron temperature of te=2100 c after electron electron equilibration (hot electrons), and (c) immediately after the absorption of photons by single electrons (primary hot electrons, with population changes highly exaggerated to make them visible). also shown are the energies of the ground-state triplet () and lowest-excited singlet state () of oxygen as well as the next singlet state () under the assumption that ef is equidistant from the and energies. excitation of an oxygen molecule to o2 requires the simultaneous transfer of an electron from the oxygen to a hole at the energy of the state and of a hot electron with the opposite spin and an energy at the (or) level to the oxygen molecule. the large number of equilibrated hot electrons available during a nanosecond-laser pulse also rules out the primary hot electrons as the main source of o2 photogeneration. the number of primary hot electrons available during the pulse duration can be estimated from the number of photons which are absorbed per nanosecond, multiplied by their lifetime, which is less than 500 fs. however, these primary hot electrons populate np states at energies from the fermi level ef to ef+2.34 ev (the energy of a photon at 532 nm) (see figure 6c). for 15 nm nps and excitation with 5 ns laser pulses with 0.15 j cm intensity, this predicts that not more than three primary hot electrons are available within an energy interval of 0.1 ev around the level at any time during the laser pulse, assuming that all levels are equally populated and taking into account that the density of states for gold has an essentially constant value in the relevant energy range around ef. this is significantly less than the number of hot (equilibrated) electrons available at the maximum electron temperature, which was estimated to be on the order of 260 for the same energy interval (see above). thus, we can conclude that it is indeed the hot electrons which are responsible for the observed o2 photogeneration when using short laser pulses for excitation. similar results to those obtained here using pulsed irradiation have recently been reported for spherical gold nps with 40 nm diameter. although a significantly lower laser pulse energy density (0.03 j cm, compared to 0.15 j cm here) was used under otherwise similar experimental conditions, slightly faster o2 photogeneration was observed in this study, with the dpbf absorbance decreasing by ca. 24% decrease observed here in the first 10 min (figure 4a). in this context, it is interesting to note that in spite of the lower laser pulse energy density the nps are heated to almost the same electron temperature as in our experiments (see figure 5). this is largely due to (i) heat dissipation from larger nps being slower and (ii) the use of 80/20 etoh/water as solvent in ref (24), which has slower heat transport than a 50/50 etoh/water mixture. an explicit calculation of the number of hot electrons available at the energy of the oxygen state for the two experiments is given in the supporting information; together with a detailed consideration of all other experimental differences, these numbers yield very good agreement between the expected and the observed relative o2 photogeneration rates (see the supporting information for details). this provides further support for the conclusion that o2 photogeneration is mediated by the equilibrated hot electrons of gold nanoparticles under nanosecond-pulsed laser irradiation. compared to the effect of pulsed irradiation, the rate of o2 photogeneration is much smaller when using cw light of comparable intensity. whereas ca. 24% of the dye is bleached after only 10 min of irradiation of spherical nps with 15 nm diameter with 5 ns laser pulses at an average power of 150 mw (figure 4), only 12% of the dye is bleached over this time by irradiation of the same nps with cw light at significantly higher power (1 w) (figure 2). in a previous publication, significantly higher rates of dpbf photobleaching had been reported under conditions which appear to be similar to the ones used here. in this context, we note that we also observed such significantly higher rates of bleaching, but only when the sample preparation protocol described in the experimental section was not followed accurately; for example, the use of dye solution that had not been freshly prepared or of a cuvette that had not been cleaned thoroughly and rinsed multiple times with mq water or the use of a cell that was not sealed during irradiation, leading to some loss of ethanol from the solution, all resulted in larger and highly irreproducible bleaching of dpbf under cw irradiation, up to levels comparable to those reported in ref (24), even in the absence of nps. in the following, we will show that photogeneration of o2 under cw irradiation, unlike pulsed irradiation, is mediated by the initially created primary hot electrons; i.e., it occurs during the short time during which the excited electrons have not yet relaxed to a thermal distribution (see figure 6c). under the cw irradiation conditions used here (1 w, 1.85 mm beam diameter), a spherical np with 15 nm diameter absorbs photons at an average rate of 1.3 10 s, as estimated from the absorption cross section and the beam intensity. this means that after absorption of a photon there is enough time for full relaxation and transfer of the photon energy into the solvent, which occurs in less than 100 ps, before absorption of the next photon. absorption of one photon by a 15 nm np yields hot electrons at a temperature of 10 k above the surrounding after electron electron equilibration; these hot electrons lose their energy by electron phonon scattering within a few picoseconds to yield a np whose temperature is only 80 mk above the surrounding. neither of these effects is expected to yield any significant photochemical effects; estimates analogous to those described above predict that for a spherical 15 nm np with an electron temperature of 35 c there are ca. 2.6 10 electrons within an energy interval of 0.1 ev around the oxygen state and the same number of holes around the state energy. this means that there are on the order of 10 times less hot electrons and 10 times less holes available for the photoreaction than under our pulsed laser irradiation conditions, which rules out any significant reaction; this is also confirmed by the fact that these population numbers are less than a factor 2 larger than those for room temperature, where no o2 is generated in the absence of light. this leaves only the primary hot electrons, i.e., those electrons that are excited upon absorption of a photon but have not yet equilibrated by electron electron scattering, as potential cause for photogeneration of o2. absorption of a single photon can potentially excite electrons to energies of up to 2.34 ev above the fermi level. if one assumes excitation of only one electron by each photon and equal excitation probability for all available electrons, as shown in figure 6c, on average there will be 0.043 electrons within an energy interval of 0.1 ev around the oxygen state, which is approximately 5000 times less than during a single laser pulse in the pulsed experiments, see above. taking into account that the same factor also applies to the holes required for the dexter mechanism, but correcting for the lifetime of the excitation (500 fs for primary hot electrons created during cw irradiation, ca. 3 ns for the hot electron distribution induced by a single laser pulse; see figure s6) and the repetition rates (1.3 10 s for single photon absorption during cw irradiation, 10 s for the pulsed laser irradiation), one would predict a rate of o2 photogeneration under our cw irradiation conditions which is smaller than that expected for our pulsed irradiation conditions by a factor on the order of 10. this is significantly closer to the experimental results (ratio of o2 photogeneration rates under pulsed vs cw irradiation of 1020) than any estimate based on the equilibrated hot electrons after the absorption of a single photon. the main discrepancy between the predicted and observed cw results arises from the assumption of direct excitation of single electrons in the above estimate, which is not valid for irradiation at 532 nm, i.e., in the gold np plasmon resonance band, since this leads to the coherent excitation of many electrons which rapidly dephases without the electrons exchanging energy. thus, absorption of a single photon yields more than one primary hot electron, with the photon s energy distributed over all of them. consequently, the energy distribution even of the primary hot electrons will not extend up to 2.34 ev above ef but will be shifted toward the states nearer the fermi level, thus increasing the population of states around the oxygen energy and hence the yield of o2. a more quantitative estimate of this effect is beyond the scope of this paper. an alternative possibility for the mechanism of o2 photogeneration by gold nps could be envisaged, which is based on increased direct photoexcitation of oxygen due to the well-known local electric field enhancement in the vicinity of metal nps by the plasmon electrons. however, significant field enhancement extends to distances comparable to the dimensions of the nanoparticle, so that this mechanism is in disagreement with our observation that a peg-oh capping layer, which has a thickness of only 2 nm, completely inhibits o2 photogeneration (figure 3). dexter-type electron exchange coupling, on the other hand, is known to be of significance only over distances of less than 1 nm, as discussed above, and thus is further supported by this observation. in conclusion, cw irradiation is less efficient than pulsed laser irradiation in photogenerating o2 since the (equilibrated) hot electrons, which are the main mediator of the photochemistry in the case of pulsed irradiation, do not have sufficient energy/temperature to drive o2 photogeneration. nevertheless, our results confirm that cw irradiation of spherical gold nps produces detectable amounts of o2. the absence of significant amounts of (equilibrated) hot electrons means that o2 photogeneration proceeds via a different mechanism under cw irradiation compared to pulsed laser generation; the above estimates indicate that it is the primary hot electrons, i.e., the directly photoexcited electrons, which are responsible for the photochemistry here and that the photochemical reaction must occur before these equilibrate by electron electron thermalization. photodynamic therapy holds great promises for medical applications, such as the treatment of cancer, because of the ability to selectively affect diseased tissue only. however, wider use of photodynamic cancer therapy is currently prevented by several limitations imposed by the available photosensitizers. these limitations include toxicity, poor stability and photostability, poor selectivity for cancer tissue, and the need of using visible light with poor tissue penetration. all of these limitations, in principle, can be overcome by the use of gold nanoparticles, which are nontoxic, have excellent stability even under irradiation, can target cancer tissue either passively by the enhanced penetration and retention (epr) effect or by active targeting, have extinction coefficients that are larger than those of dye molecules by several orders of magnitude, and can be tuned to absorb in the near-infrared spectral region for maximum tissue penetration. gold nanoparticles have been reported to have three potential modes of operation for inducing cell death by irradiation, namely (i) hyperthermia, which is based on the rapid conversion of the absorbed light energy into heat, (ii) np-assisted photodynamic therapy, in which the efficiency of a standard sensitizer is amplified by the np plasmon field enhancement effect, or (iii) a direct photochemical mechanism without involvement of a photosensitizer. the feasibility of photothermal therapy has been clearly established by careful experiments in vitro(1113,15,54) and has been shown to work in vivo. however, it should be noted that most studies reporting successful photothermal therapy made no attempt to either confirm significant heating or rule out photochemical effects, which means that some of these reported results could in fact arise from photochemical rather than photothermal effects or from a synergistic combination of photochemical and photothermal effects. as has been pointed out recently, another problem with many reports on photoinduced hyperthermia using nps is the relatively high light intensity required to reach sufficient temperatures, which often were well above the generally accepted skin tolerance threshold. the presence of gold nps can also lead to increased photogeneration of o2 by traditional photodynamic sensitizers, which can be ascribed to the local electric field enhancement in the vicinity of metal nps by the plasmon electrons, similar to the well-studied sers (surface-enhanced raman spectroscopy) effect. this approach might help to alleviate some of the drawbacks of traditional photodynamic therapy but does not directly overcome them, since it still requires the presence of a sensitizer. in fact, the requirement for two active components, gold np and sensitizer, which must be colocalized, introduces an additional complication, and care must be taken to avoid a reduction of the photosensitizer effect due to quenching of its excited state by the metal np. in an alternative approach, cell death has been demonstrated to occur following irradiation of intracellular (endocytosed) gold nps even at irradiation levels that are not high enough to cause significant heating. this photochemical effect has been related to the observation that irradiation of nps in vitro results in the formation of singlet oxygen, which is the active species in traditional photodynamic therapy. because of the short lifetime of o2 (3.4 s in water), its action is highly localized an oxygen molecule only diffuses over the length scale of 100 nm in this time. for this reason, only intracellular nps are expected to trigger cell death by the photochemical route, although they may initially be located inside endosomes which are known to be broken up by nps under cw irradiation. on the other hand, the localization of the photochemical effects within individual cells also means that in situations where different types of cells coexist in close vicinity, selective targeting of particular cells and minimization of collateral damage should be achievable. this is different than photothermally induced cell death, which affects all cells within the irradiated volume more or less indiscriminately, as long as some of them contain nps, because of the fast diffusion of heat over the relevant length scales. the results of the experiments described here provide more insight into the direct photochemical mechanism and allow some important conclusions to be made for the further development of practical applications. they show that detectable amounts of o2 are generated by irradiation of nps with short laser pulses or cw light even in the absence of a photodynamic sensitizer, albeit with low quantum yield. short laser pulses are significantly more efficient at this process, since they can heat a significant fraction of the np conduction band electrons to high enough temperatures to excite oxygen to the singlet state. however, this requires pulse energy densities that are well above generally accepted safe levels for the irradiation of skin with pulsed laser light. since the effect requires the absorption of many photons by a np during one laser pulse, it is highly nonlinear with respect to irradiation intensity, and thus it will not be possible to compensate lower irradiation levels by longer irradiation times. furthermore, pulsed irradiation at the required intensities also can cause other effects, such as np fragmentation or bubble formation, and it is not clear what consequences these effects may have when occurring in tissue. although the use of femtosecond laser pulses, as compared to the nanosecond pulses used here, might alleviate some of these problems to some extent, they would require even more sophisticated equipment which may not be suitable for a clinical environment. the use of cw light, on the other hand, is straightforward and does not even require a laser but can be achieved with simple lamps. in spite of the lower quantum yield of o2 photogeneration by nps under cw irradiation which is reported here, cell death induced by cw light in the presence of nps has been reported in vitro and in vivo. although the experiments described here, which did not involve any biological material, were undertaken at light intensities that are above safe irradiation levels, the mechanism of o2 photogeneration by nps under cw illumination is shown to be based on the absorption of single photons. this means that lower light intensities can be compensated for by longer irradiation times, allowing one to reduce the intensity to safe levels for biological or medical applications without affecting the amount of o2 generated. o2 photogeneration and cancer cell destruction in vitro and in vivo have indeed been observed upon irradiation on the minute time scale at intensities below safe levels and using nonlaser light sources. the results presented here indicate another design criterion which needs to be fulfilled for successful implementation of the photochemical route of inducing cell death by irradiation of nps, namely that the nps must not possess a dense capping layer. even the thin capping layer formed by peg-oh, with a thickness of only 2 nm, is sufficient to completely inhibit o2 photogeneration (figure 3). this is in full agreement with the suggestion that energy exchange between the np and the oxygen molecule occurs via the dexter (two electron exchange) mechanism, which is limited to distances of less than 1 nm. similar effects have been reported for o2 photogeneration by si nanocrystals, which is significantly affected by a thin oxide layer. it is important to point out that the assay used here for o2 detection (bleaching of dpbf) only reports on singlet oxygen found outside the np capping layer; thus, it can not be ruled out that even on nps with a peg-oh capping layer some o2 is photogenerated at the np surface but reacts with the np ligands and hence is quenched. on the other hand, such singlet oxygen would not be of any direct use for practical applications, such as the induction of cell death, so that the assay results in fact report the effects relevant for such applications. uncapped (citrate) nps are rapidly covered by a protein corona after they have been taken up into live cells, but it appears that this corona is permeable enough for oxygen to not completely prevent singlet oxygen formation, as evidenced by fact that cell death by the photochemical route has been observed with such nps; it may be that because of their size proteins are not able to form a capping layer (corona) of similar density as the smaller ligands used here. similarly, a lipid bilayer appears to allow oxygen access to the surface of gold nanorods, whereas a dense pentapeptide (calnn) capping layer on spherical gold nanoparticles is sufficient to suppress the photochemical mechanism of cell death. it also seems likely that the thick peg capping layer present on the nanorods used here is the main reason for the absence of o2 photogeneration by nanorods upon irradiation in either the transversal or the longitudinal plasmon resonance band that was observed here. because of their longitudinal plasmon resonance band, which is in the near-ir spectral region with high tissue penetration, nanorods are more suitable for practical photodynamic applications in tissue. however, standard synthesis protocols yield nanorods within a bilayer of cytotoxic ctab, so that ligand exchange is required before any biological or medical application, and care will need to be taken to choose suitable ligands to allow access of oxygen to the nanorod surface. potential examples for these are poly(vinylpyrrolidone), lipid bilayers, or mesoporous silica, all of which have been used successfully in experiments showing photochemically induced cell death using gold nanorods. dpbf has been used successfully to detect singlet oxygen that is formed when spherical nps are irradiated at 532 nm, either with cw or pulsed laser irradiation. singlet oxygen generation by pulsed laser irradiation has been shown to act via the equilibrated hot electrons that can reach temperatures of several thousand degrees during the laser pulse; cw irradiation, on the other hand, can act only via the directly excited primary hot electrons, which rapidly lose their energy by electron electron equilibration, and hence is significantly less efficient for the formation of singlet oxygen. nevertheless, even cw irradiation can produce enough singlet oxygen for photodynamic therapy applications and will allow practical applications of the effect at safe irradiation levels. photodynamic therapy using gold nanoparticles will also require careful design of the nanoparticles with respect to size, shape, and capping layer and will require internalization of the nps, not just attachment to the cell surface, which is sufficient for photothermal therapy.

the formation of singlet oxygen by irradiation of gold nanoparticles in their plasmon resonance band with continuous or pulsed laser light has been investigated. citrate-stabilized nanoparticles were found to facilitate the photogeneration of singlet oxygen, albeit with low quantum yield. the reaction caused by pulsed laser irradiation makes use of the equilibrated hot electrons that can reach temperatures of several thousand degrees during the laser pulse. although less efficient, continuous irradiation, which acts via the short-lived directly excited primary hot electrons only, can produce enough singlet oxygen for photodynamic cancer therapy and has significant advantages for practical applications. however, careful design of the nanoparticles is needed, since even a moderately thick capping layer can completely inhibit singlet oxygen formation. moreover, the efficiency of the process also depends on the nanoparticle size.

PMC4878812

pubmed-466

chronic kidney disease (ckd) is defined as the slow and steady damage of kidney function in an irreversible manner, which ultimately results in end-stage renal disease (esrd) (1, 2). this chronic disorder is a serious health problem with a high prevalence rate in adults and children. it causes mortality and other health complications, and high costs are incurred due to the frequent medical diagnosis and poor prognosis of patients (1, 2). the causes of ckd are very different in children than in adults. in a recent north american pediatric renal transplant cooperative study (naprtcs), congenital causes, including congenital anomalies of the kidney and urinary tract (cakut) (48%) and hereditary nephropathies (10%), were the most common causes of ckd in the children (3). based on previous studies, such a chronic disorder impairs the quality of life of children due to the development of various clinical symptoms, especially developmental disorders and psychiatric disorders (4-6). ckd clinical manifestations in children are presented as edema, hypertension, hematuria, and proteinuria, and specifically during the neonatal period as weight gain, polyuric dehydration, and urinary tract infection (7). in addition to the above mentioned symptoms, ckd may lead to a type of hyperactivity and central nervous system (cns) dysfunction (especially affecting sympathetic function) (8). based on the literature, the prevalence of cognitive disorders such as memory disorder, and different psychiatric disorders including anxiety disorders (ads), depression, and adjustment disorders in children with different levels of ckd was significantly higher compared to the group of healthy children and children with very early stages of ckd (9-12). based on the available literature, patients with ckd who develop psychiatric and cognitive disorders face longer hospitalization times, more health complications, and greater mortality compared to other patients at the same stage without these disorders (11, 12). therefore, proper diagnosis of psychiatric and cognitive disorders in these patients is paramount. more specifically, one disorder whose relationship with ckd in children has not been adequately examined is obsessive-compulsive disorder (ocd). obsessive-compulsive disorder (ocd) is a chronic disabling illness characterized by repetitive ritualistic behaviors over which the patients have little or no control (13, 14). a review of the literature revealed no study that assessed the association between ocd (based on the obsessive compulsive inventory-child version (oci-cv)) and ckd. the aim of this study was to investigate ocd in children with early stages of ckd and to compare it with healthy children. this case-control study was performed on 160 children in the age bracket of 7 to 17 years old who were referred to the pediatric clinic of amir kabir hospital in arak (iran) in 2015. the ethics committee approved the study (approval code: 93-162-1, registration code: 1087). eighty children with early stages of ckd (stages 1, 2 and 3) comprised the case group, and the control group consisted of 80 healthy children without ckd; children were included in the study based on the inclusion criteria. the sample number was calculated with regards to the prevalence of cognitive disorders due to ckd (= 0.05%, =0.2%). ckd was defined as the presence of kidney damage (for example, any structural or functional abnormality involving pathological, laboratory, or imaging findings) for 3 months or a glomerular filtration rate (gfr)<60 ml/minute/1.73 m for 3 months (2). in this study, as per the ckd definition (2), children with early stages of ckd (stage 1, 2 and 3 ckd) who were diagnosed with ckd due to renal and urinary-genital tract anomalies, such as obstructive uropathy, renal dysplasia, or reflux nephropathy, were included in the case group. those children who were diagnosed with ckd due to reasons other than renal and urinary-genital tract anomalies, or those with stages 4 and 5 stage 1, 2, 3 and 4 ckd were defined as kidney damage with normal or increased gfr (gfr 90 cc/minute/1.73 m), kidney damage with mild decreased gfr (gfr=60-89 cc/minute/1.73 m), kidney damage with moderately decreased gfr (gfr=30-59 cc/minute/1.73 m) and kidney damage with severely decreased gfr (gfr=15-29 cc/minute/1.73 m), respectively (15). esrd or stage 5 ckd was defined by the amounts of gfr<15 cc/minute/1.73 m which are indicative of the start of dialysis (15-17). we included children of both sexes in the age range of 7 to 17 years old, and children with stage 1 to 3 ckd (for at least 6 months). we excluded patients with the following conditions or circumstances: a history of considerable psychiatric disorders; intellectual disabilities, or nervous system disorders; a history of any type of anxiety disorder before developing ckd; congenital and chromosomal abnormalities; a chronic medical condition; a family history of major psychiatric disorders in first-degree relatives; parents not consenting to participating in the study; and not completing the questionnaire. intellectual disability was defined in terms of the intelligence quotient (iq) of 70 (18). healthy children were selected from children who had been referred to the hospital as outpatients for minor conditions such as the common cold or abdominal pain. the matching method was used for selecting the healthy children, and children were matched in groups based on age, sex, and socioeconomic status. in this 3-month study (april 2015 to july 2015), a total of 40 children were excluded based on the inclusion and exclusion criteria. among 22 (100%) patients who were excluded in the case group, 17 (77.27%) and 5 (22.72%) patients were excluded due to parental unwillingness to complete the oci-cv and a history of considerable psychiatric disorders (anxiety disorders before the diagnosis of ckd), respectively. for the control group, the remaining children were excluded due to lack of parental consent. after obtaining informed consent from the children s parents, demographic, clinical, and perinatal data (age, sex, residence, birth weight, current weight, height, body mass index (bmi), mother s age at birth, gestational age, maternal education, household incomes, marital status, type of delivery, age at diagnosis of ckd, and duration of ckd) were recorded. ocd in children was evaluated using the obsessive compulsive inventory-child version (oci-cv) by a psychologist (consultant). this self-reporting questionnaire has been designed for people aged 7 to 17 years, containing 21 items and 6 subscales, including doubting/checking (5 phrase), obsessing (4 phrase), hoarding (3 phrase), washing (3 phrase), ordering (3 phrase), and neutralizing (3 phrase) (19-21). the subjects are supposed to indicate their degree of agreement or disagreement with each item through a 3-point likert scale ranging from never to always. the scoring options on this test were as follows: never=0, sometimes=1, and always=2. based on multiple sources of evidence, the oci-cv is considered to be a reliable and valid method for identifying children with ocd. the oci-cv was modestly correlated with obsessive compulsive symptom severity on the children s yale-brown obsessive compulsive scale (cy-bocs), as well as with clinician-reported ocd severity (19, 20). the persian version of the oci-cv questionnaire was tested for reliability in a pilot study by the researchers with 30 patients in each of the case and control groups; the cronbach s alpha was 0.89. the collected data was analyzed with spss software (statistical package for the social sciences, version 18.0, spss inc. categorical data are expressed as numbers (percentage) and compared with a chi-square test. the mean weight, height, and bmi of the children were 38.15 6.8 kg, 127.61 8.5 cm, and 17.31 3.52, respectively. of the 160 subjects, 86 (53.75%) were boys and 74 (46.25%) were girls. in the group comprised of children with ckd, the duration of the disease and the age of diagnosis were 1.48 2.12 and 6.9 5.4 years, respectively. abbreviations: bmi, body mass index; ckd, chronic kidney disease; cs, caesarean section; nvd, normal vaginal delivery. household incomes: low mean monthly incomes<5000000 rials; moderate mean incomes between 5,000,000 and 10,000,000 rials; high means incomes>10,000,000 rials. the mean age (p=0.211), weight (p=0.51), height (p=0.113), gender distribution (p=0.112), average bmi (0.32), mother s age at childbirth (p=0.66), and the child s birth weight (p=0.08) were not significantly different between the children in the case and control groups. furthermore, the children in both groups were identically distributed in terms of residency status (p=0.3), family s monthly income (p=0.376), gestational age (p=0.12), marital status (p=0.311) and type of delivery (p=456). however, the maternal educational level was significantly different between the two groups (p=0.001), so that 30 (37.5%) and 37 (46.25%) of mothers in the group of the children with ckd had college and high school education, respectively, whereas 16 (20%) and 50 (62.5%) of the mothers in the control group had college and high school education, respectively. the mean scores of doubting/checking (case: 3.52 2.54, control: 2.5 2.32, p=0.007) and ordering (case: 2.59 1.81, control: 1.5 2.56, p=0.005) of the children with ckd was significantly higher than the scores of the healthy ones. moreover, the mean total scores for the oci-cv of the children with ckd at 15.32 7.69 was significantly higher than those of the healthy ones at 11.12 2.54 (p=0.021). nevertheless, the mean scores of obsessing (p=0.11), hoarding (p=0.117), washing (p=0.211), and neutralizing (p=0.41) were not significantly different between the case and control groups (table 2 and figure 1). according to the results of spearman s test, there was significant correlation between the duration of ckd and doubting/checking (p=0.004, correlation coefficient (cc): 0.4), obsessing (p=0.06, cc: 0.02), washing (p=0.031, cc: 0.8), ordering (p=0.001, cc: 0.2), and the total scores of the oci-cv questionnaire (p=0.04, cc: 0.4) (table 3). according to the results of the current study, ocd and certain subscales of this disorder are more likely to occur in children with early stages of ckd than in healthy children. in this respect, doubting/checking and ordering had higher rates among children with ckd than among healthy children. furthermore, the results indicated that the duration of ckd was significantly correlated with the mean total scores of the oci-cv and certain subscales, such as doubting/checking, obsessing, washing, and ordering. on the basis of previous evidence, chronic disorders or diseases in adults and children can be associated with reduced quality of life and social, occupational and educational poor performance (22). in fact, it has been revealed that such an association can in particular cases have a negative effect on the clinical course of the underlying disease (22). according to relevant studies, the risk of mood and anxiety disorders occurring among people with chronic medical illnesses tends to be greater as compared to the corresponding occurrence rates among healthy people in the general population (22). although there have been a number of studies assessing the psychiatric disorders in children with ckd, no study has been carried out on the relationship between ocd and ckd based on the oci-cv. (9) studied 19 children with ckd who did not need dialysis and 19 others with esrd in order to determine if any of them had a psychiatric disorder. the evidence indicated that 18.4%, 10.3%, 7.7%, 5.1%, and 2.6% of the subjects had adjustment disorder, depression, cognitive disorder, anxiety, and elimination disorder. the total prevalence of the above disorders were calculated at 68.4% and 36.8% in dialysis and non-dialysis groups, respectively. in another study on 30 children with both ckd and continuous peritoneal dialysis (cpd), 30 children who had undergone renal implantation, and 33 healthy children, fukunishi and honda (10) revealed that there was a significant difference in the prevalence of adjustment disorder between the children in the three groups; the dialysis and control groups had the highest and lowest prevalence of this disorder, respectively. with the objective of investigating cognitive function in children with ckd, slickers et al. studied 29 children aged 7 to 19 simulated through creatinine clearance (crcl). the results showed that the severity of ckd was significantly correlated with decreased iq and impaired memory function. furthermore, it was revealed that a longer duration of ckd and its onset at a younger age were considerable risk factors for the development of cognitive disorders (11). in another study on children with esrd and healthy children, fukunishi and kudo (23) concluded that the prevalence of anxiety and depression was significantly higher in children with esrd and peritoneal dialysis (pd) than in healthy children. (24) examined 15 children aged 8 to 16 with renal disease (8 children with esrd and 7 children with mild kidney disease as the control group) and showed that the depression rate in the case group was significantly higher than that in the control group. in 2015, yousefichaijan et al. (25) compared the occurrence of attention deficit/hyperactivity disorder (adhd) among 75 children aged 5-16 with ckd (stages 1 to 3) and 75 healthy children. the results of this study indicated that the prevalence of adhd (case group: 12%, control group: 16%, p=0.664) was not significantly different between the two groups. in another study on adhd, yousefichaijan et al. (26) examined 100 children with esrd undergoing peritoneal dialysis and 100 healthy children. they found that the rates of attention deficit (p=0.01) and hyperactivity (p=0.002) among children with esrd were significantly higher than the corresponding rates among healthy children. according to previous studies, it can be argued that the risk of various psychiatric disorders in children with ckd at early stages and children with esrd tend to be significantly higher as compared to healthy children. moreover, such disorders can be found more frequently in children with esrd, particularly in those undergoing dialysis, than in patients experiencing the early stages of ckd. similarly, the results of this study were indicative of the higher prevalence of ocd as an anxiety disorder in children with ckd as compared to healthy children. although it can be concluded that psychiatric interventions, particularly in the case of anxiety disorders, can be beneficial for children with varying degrees of ckd, there is an insufficient number of studies exclusively focused on each of the psychiatric disorders (such as ocd in our study) and a lack of evidence concerning the clinical impact of these disorders on the clinical course of ckd. therefore, it is highly recommended that future studies be conducted so as to yield more definitive conclusions. one limitation of our study involved the lack of cooperation of some parents in completing the oci-cv questionnaire. although this criterion led to the exclusion of some otherwise eligible children, we tried to encourage the parents by describing the possible usefulness of the study and by offering to help them fill in the questionnaire. according to our findings, the risk of ocd among children with ckd is significantly higher than the risk among healthy children. although the results suggest that psychiatric interventions could be helpful in treating children with ckd, the limited number of studies on this particular issue suggests that further investigation into this medical condition is required so as to obtain more conclusive results.

background: chronic kidney disease (ckd) is a common medical condition among children and obsessive-compulsive disorder (ocd) is a frequent, chronic, costly, and disabling disorder among them. objectives:the aim of this study was to investigate obsessive-compulsive disorder (ocd) in children with early stages of ckd, and to compare it with the occurrence of ocd in healthy children. patients and methods: in this case-control study, we evaluated 160 children aged 7 to 17 years old who were visited in the pediatric clinics of amir-kabir hospital, arak, iran. the control group consisted of 80 healthy children and the case group included 80 children with stage 1 to 3 ckd. the ages and sex of the children in the two groups were matched. ocd in children was evaluated using the obsessive compulsive inventory-child version (oci-cv). results: the mean scores of doubting/checking (case: 3.52 2.54, control: 2.5 2.32, p=0.007) and ordering (case: 2.59 1.81, control: 1.5 2.56, p=0.005) in the children with ckd was significantly higher than in the healthy ones. moreover, the mean total scores for the oci-cv of the children with ckd at 15.32 7.69 was significantly higher than the scores of the healthy ones at 11.12 2.54 (p=0.021). there was a significant correlation between the ckd duration and doubting/checking (p=0.004, correlation coefficient (cc): 0.4), obsessing (p=0.06, cc: 0.02), washing (p=0.031, cc: 0.8), ordering (p=0.001, cc: 0.2), and the total scores of the oci-cv questionnaire (p=0.04, cc: 0.4). conclusions: the risk of ocd in children with ckd is significantly higher than that in healthy children. although the results seem to suggest that psychiatric intervention can be helpful in treating ocd in children with ckd, further investigation into the medical condition is required so as to obtain more definitive conclusions.

PMC4779309

pubmed-467

problems with shoe wear have long been recognized as an endemic issue among the geriatric population with a prevalence rate of nearly 80%. women in particular are more susceptible to these problems than men. individuals having foot pathology are often severely physically impaired, making it increasingly difficult to perform activities of daily living. consequentially, this leads to physical inactivity, which is cited as one of the first signs of deterioration and the overall decrease in quality of life. moreover, some studies have linked physical inactivity to suicide, depression, and increased risk of cardiovascular-related problems. individuals with diabetes, chronic disease, nondiabetic neuropathy, and inflammatory conditions are at a severe disadvantage. further complications of foot pathology, which include cellulitis, ulcerations, and difficulty in maintaining balance, have increased the risk of serious injuries and fractures from falls. studies have shown that adults older than 65 years fall at least once per year on average, some of which are attributed to generalized thinning of skin and fat pad atrophy. the increased risk of falls may highlight the need to promote preventative measures to combat this issue. unfortunately, little current data or research focuses on the role of footwear in the prevention of inactivity in the elderly patients. common foot pathologies like corns, hallux valgus (bunions), and hammertoes have been known to increase plantar pressure, cause discomfort, pain, and swelling. multiple etiologies have been noted to cause foot problems, and studies have indicated ill-fitting shoes as one of the major underlying cause. a prevailing hypothesis is that wearing the appropriate footwear will improve factors such as plantar pressure, thereby mitigating pathology while facilitating balance. this review aims to form a general basis of understanding footwear-associated foot pathologies pertaining to the elderly patients. elderly individuals with preexisting clinical conditions such as diabetes, neuropathies, and musculoskeletal disorders are at a higher risk of developing foot problems when compared to normal, healthy individuals. furthermore, factors such as exercise and living conditions contribute to the development of foot problems. finally, the uneven pressure provoked by ill-fitting footwear has been documented as a key factor that can cause, accelerate, or exacerbate foot-related conditions. some common foot pathologies include hallux valgus (bunions), corns (callus), and hammertoes. callus formations, or corns, result from continuous pressure from tight footwear, leading to hyperkeratosis of the skin. if left unattended, callus formations can lead to ulcerations, which are particularly troublesome for diabetic patients. the hallux valgus deformity, or bunion, is also frequently linked to ill-fitting (ie, tight, narrow) footwear. it is caused by the lateral deviation of the great toe, which causes a valgus deformity in the first metatarsophalangeal (mtp) joint. lesser toe deformities, such as hammertoes, are also associated with ill-fitting footwear. these deformities of the second, third, fourth, and fifth toe are caused by contracture of the phalangeal joints. clinical presentations include the deformity accompanied by pain over the dorsal surface of the foot, which tends to worsen with ill-fitting footwear. one of the unifying elements to these 3 common forms of foot pathology is their link with ill-fitting footwear and their tendency to go unnoticed in the elderly population. nonoperative approaches have been employed as the first line of treatment for the most common foot pathologies. measures are taken to adjust or alleviate pressure from the affected area. as a result, foot inserts (ie, dr scholls) are also used to facilitate balance by improving arch support, which can decrease the width of the foot during weight bearing maneuvers. furthermore, met pads and splits operative management is considered only when conservative measures fail to relieve progressed, advanced-stage pathology. usually, symptomatic relief can be achieved with more accommodating shoe wear after the soft tissue envelope around the foot is stabilized. this process of relieving the pressure areas and encouraging natural healing of ulcerations or soft tissue problems to heal before using modified shoe wear and orthotics often involves close follow-up in the office or wound care center. special consideration is given to diabetic patients, who have a higher risk of developing ulcerations due to peripheral neuropathy. therefore, routine physical examination is encouraged to detect and address these issues early before they lead to progressive deterioration. studies have shown that women are more susceptible than men to problems associated with inappropriate footwear in terms of the choice of footwear in the elderly patients, lord et al concluded that individuals had better balance when wearing shoes with higher collars than with lower ones. wide shoes are effective against bunions, and extra-depth shoes are more appropriate if the individual has hammertoes or mid-foot arthrosis. low-heeled footwear is known to reduce the risk of falls, likely due to the associated lowering of the center of gravity. while assessing different footwear, athletic and canvas shoes (sneakers) are associated with the lowest risk of a fall when compared to other types of shoes. moreover, some suggested that sole hardness have a little effect on overall balance in the absence of a specific condition, such as hallux rigidus, which may benefit from application of hard sole. as an extra preventative measure, better fitting footwear with slip-resistant soles is recommended both inside and outside the household to reduce the risk of falls. taken together, these studies seem to support the use of footwear with low heels, slip-resistant soles and wider frames to ensure comfort, better balance, and reduced risk of injury in the elderly population. in the event of multiple deformities, an individual may benefit from the combination of both extra-depth, wide, and low-heeled footwear to increase foot stability and facilitate balance. the use of appropriate shoe wear in the elderly patients is related to the socioeconomic factors. millions of elderly individuals live at or below the poverty level. in fact, women older than 75 years are 3 times more likely to become poor. without established financial programs such as social security, nearly half of all elderly individuals would be considered poor today. changes in living conditions (ie, community homes, nursing homes, etc) can often worsen the socioeconomic status of elderly patients. deteriorating health conditions followed by reduced physical activity can lead to a shift in clinical care, one that emphasizes the management of illness. in the face of such socioeconomic constraints, furthermore, retail stores often do not stock the customized shoes (ie, extra wide) needed for elderly customers, making availability an issue for elderly patients. the neglect of appropriate footwear in the elderly has been linked to early amputation, and in rare cases, early demise. to help alleviate the financial burden, elderly patients enrolled in medicare part b may benefit from the medical coverage of certain medical supplies and preventative services. eligible patients typically have diabetes or other severe foot diseases. under the medicare part b provision, such patients are entitled to a pair of custom-molded shoes, 3 pairs of inserts, or a pair of extra-depth shoes per calendar year. the patient will be held responsible for only 20% of the cost while medicare covers the rest.. some of these hazards such as wet pavements and bad weather conditions are capable of causing serious injury. these pathologies, in return, have contributed to the growing risk of even more serious injuries such as falls and fractures. ultimately, the noticeable decrease in physical activity in the elderly patients has been linked to depression and an overall decline health and quality of life. clinically, preventative measures such as appropriate footwear should be continually emphasized to stop or delay the progression of some foot problems. while recommending appropriate footwear is a cost-effective approach when compared to other options, it is often hindered by socioeconomic obstacles. nevertheless, studies have shown that it can be a vital step to ensure prolonged musculoskeletal and overall health among the elderly patients.

foot pathologies are common in nearly 80% of all elderly patients, and studies have indicated inappropriate footwear as one of the major underlying cause. it has been postulated that ill-fitting shoe wear affects plantar pressure, thus exacerbating weak balance. complications arising from foot pathologies, which include difficulty in maintaining balance, have increased the risk of falls that can result in fractures and other serious injuries. the link between footwear and the onset or progression of certain foot pathologies has emphasized the need to explore and promote preventative measures to combat the issue. wider and higher toe boxed shoes, along with sneakers, are examples of footwear documented to evenly distribute plantar pressure, increase comfort, and facilitate appropriate balance and gait. ultimately, the use of appropriate footwear can help to better stabilize the foot, thus reducing the risk of sustaining debilitating physical injuries known to drastically decrease the quality of life among the geriatric population.

PMC4647201

pubmed-468

how does the pattern of retinal activity produced by looking at the image of halle berry ultimately lead to the perception of the actress rather than a violin? how do the myriad different associations with halle berry (actress, specific roles) or violin (inanimate object, music) form through learning and memory? they have been approached by a variety of techniques that fall into two broad categories, watching brain activity and disrupting brain function. in this review we will discuss the development of genetic techniques that bridge the divide between these two approaches and allow the targeting of molecular changes specifically to anatomically dispersed neural representations that are activated by discrete environmental stimuli. these new tools allow the establishment of causal relationships between the activation of sparsely distributed neural ensembles and changes at the behavioral level. the use of single unit recordings in awake behaving animals provides an exquisitely precise measure of the temporal activity of neurons. this has been used to extract information about how the brain encodes information by studying the correlation between neuronal activity and the presentation of specific sensory stimuli. the best-studied example is probably in the primate visual system where a hierarchical pathway has been defined (van essen et al., 1992). neurons in the primary visual cortex (v1) fire in response to very general visual features such as orientation whereas following processing through the ventral visual pathway, neurons in inferior temporal cortex respond to complex object features. this visual information is then relayed to the medial temporal lobes, which integrate multimodal sensory information and play a critical role in memory. single unit recording studies in the medial temporal lobe in humans have detected neurons with responses to highly defined categories. in the limit, units were found that responded to the presentation of a single individual in a variety of contexts (quiroga et al., 2005). one neuron in the right anterior hippocampus responded to the actress halle berry, presented in a photograph, as a masked character (catwoman), as a drawing, or as the letter string this level of response specificity shows that the cells are not tuned to general visual features common to images of halle berry but to the concept of the specific actress. the striking degree of responsive specificity of these units strongly suggests that they participate in the neural representation of specific individuals. however, these studies are still correlative in the sense that they allow us to watch neurons that fire in a manner suggesting a role in encoding specific information, but they do not allow us to disrupt these neurons specifically to test this hypothesis. historically much of what we know about the functional parceling of the brain has been obtained from lesion studies in experimental animals and in patients with damage to specific brain regions (squire, 2004). in learning and memory, studies of patient hm, who underwent a bilateral resection of the medial temporal lobe, have helped define this area, and in particular the hippocampal formation, as critical in the formation of long-lasting declarative memories. at a more molecular level both pharmacological and genetic manipulations have been used to test ideas about the cellular signaling mechanisms that underlie behavioral plasticity. however, each of these approaches is limited in that they act as sledgehammers, altering every neuron in a given brain region, when the electrophysiological studies suggest that it is really a very sparse group of neurons that is truly of interest in any given experimental context. one approach to circuit analysis is to describe the precise pattern of wiring within specific processing units like the hippocampus or a cortical column. for example, in the hippocampus there is the classic tri-synaptic circuit where information from the entorhinal cortex enters through the dentate gyrus, is relayed to ca3 neurons via the mossy fiber pathway and then to ca1 neurons via the schaffer collateral pathway and finally back out to the entorhinal cortex (squire, 2004). this connectivity diagram can be obtained to finer and finer levels of resolution and in principle an entire wiring diagram of a single brain at a single time point could be produced to the level of individual synaptic connections, similar to that obtained in c. elegans (white et al., 1986). however, even if this were precisely defined down to the level of single synapses it is not likely that the mechanisms that give rise to a neural representation or memory trace would become apparent. as the example of the halle berry neuron indicates, these representations are likely to be quite sparse and embedded within a matrix of apparently identical neurons. the particular response patterns of an individual neuron are likely determined by the strength of specific synaptic connections that have been altered through experience. moreover, even if one could explain how these specific firing patterns arise through circuit plasticity, it would be difficult to experimentally establish the contribution of a particular ensemble of neural activity to an actual representation of the environment. an alternate view is that what defines a circuit is the environmental contingencies that lead to its activation (figure 1). in the mammalian brain this is generally referred to as an ensemble code or neural representation of the particular environmental stimulus. in the case of simple systems or reflex pathways, the wiring diagram often predicts the location of the neural ensembles that encode specific environmental information. for example, in the aplysia gill withdrawal reflex a group of sensory neurons are activated by tactile stimulation and synapse directly onto motor neurons to control withdrawal behavior (kandel, 2001). here the primary sensory neurons are defined by their enervation of the gill and their activation by tactile stimulation of the gill. the behavioral plasticity of the withdrawal reflex is controlled by synaptic plasticity within these sensory neurons. because of the uniformity and anatomical isolation of this group of cells, it has been possible to apply techniques for both watching and manipulating neurons within the context of a defined circuit (representation) in a behaving animal. the application of these convergent approaches has proven quite powerful in defining the cellular and molecular mechanisms that underlie behavioral plasticity in this system. the goal of this review is to discuss recent attempts to develop approaches that allow similar convergent molecular and physiological access to the more dispersed neural representations of the mammalian brain. the top panel shows a simplified version of the gill and siphon withdrawal circuit in aplysia. the sensory neuron cell bodies are located adjacent to each other in a cluster and possess similar biochemistry and response properties. the bottom panel represents the hippocampal circuit of the mammalian brain. the green circles represent neurons that are activated by a specific pattern of sensory stimulation and that when activated contribute to a specific behavioral response. the hippocampal circuit, like many other circuits in the brain, responds to sensory stimulation with activation patterns that can not be predicted from their wiring diagram. each of these neural ensembles involves a sparse subset of neurons that have an unpredictable spatial distribution. the aplysia neurons are primary sensory neurons and their response properties can be predicted by their physical location in the ganglion. one technique that has been used for many years to watch brain activity has taken advantage of a class of immediate early genes or iegs that are expressed in response to high-level neural firing (sagar et al., 1988). the three most commonly used iegs for this purpose are cfos, arc, and zif268. the expression of these genes is induced by action potential firing, and the -life of the gene products are relatively short. thus the expression pattern of iegs in brain sections from an animal provides a record of the neural activity from several hours prior to sacrifice and has been used extensively to map brain activation from a wide variety of environmental stimulation and in learning and memory relevant paradigms (guzowski et al., 2005). one of the limitations of this approach is that it provides only a single time point record of activity patterns, making it difficult to determine how plasticity modulates activity or even how stable this pattern of gene expression is in relation to an identical stimulus. this problem was addressed using the expression of the ieg arc (guzowski et al., 1999). by using fluorescent in situ hybridization to examine expression of arc mrna they were able to detect the pattern of arc expression at two separate time points in the same animal. they took advantage of the fact that they could detect the expression of the arc precursor rna while it was still in the nucleus as well as the mature mrna which was present in the cytoplasm and dendrites. the nuclear arc signal represented very recent and ongoing expression reflecting neural activity several minutes prior to sacrifice of the animal, while the cytoplasmic signal reflected activity that had occurred 30 min or more prior. they used this approach to examine the consistency of activation of the hippocampus when an animal was repeatedly exposed to the same environment. they found that when animals were allowed to explore the same environment, they re-expressed arc in many of the same neurons that had also expressed it on the first exposure. this is a critical result in that it demonstrated for the first time that ieg expression could be used to consistently reflect patterns of activity associated with a discrete representation and provided results that were qualitatively and quantitatively similar to results obtained with electrophysiological recordings of the hippocampus. the temporal information regarding neural activity that can be obtained using ieg expression is clearly limited relative to electrophysiological recordings. for example, while it is clear that high-level firing induces expression, it is not clear what the precise threshold is and how this might vary among different neuronal cell types. one advantage is that large brain regions can be surveyed and precise anatomical information can be obtained. a second advantage is that the promoter regulatory elements that confer neural activity dependence can, in theory, be used to drive expression of any linked heterologous transgene. 1992) using the cfos promoter to drive activity dependent expression of e. coli -galactosidase. more recently, an axonally targeted -galactosidase was expressed from the cfos promoter, providing the potential to trace the projections of specific active neuronal populations (wilson et al., 2002). the use of these promoter elements is general and provides the potential to introduce functional effector molecules directly into activated neural ensembles to allow their molecular manipulation. the use of ieg promoters as tools for both watching and potentially manipulating functional neural circuits is limited in a number of ways. for example, the direct introduction of toxins or other molecular regulators via the cfos promoter could be complicated by developmental effects of their expression. in addition, it would be useful for many studies to allow the molecular change introduced into the activated neurons to be maintained for more prolonged periods than the short (minutes to hours) times afforded by the promoters themselves. we therefore set out in a recent study to develop a genetic system with the following features. (1) the expression of any transgene of interest should be linked to neural activity only during a specific experimenter controlled time window. (2) the transgene expressed in those active neurons should be maintained for a prolonged period, but no further labeling of active cells should occur following closure of the permissive time window. we achieved activity dependent regulation of transgene expression with these two features by combining elements of the tetracycline system for gene regulation with the cfos promoter as shown in figure 2 (reijmers et al. the first uses the cfos promoter to drive expression of the tetracycline transactivator (tta or tet-off). in mice carrying only this transgene high-level neural activity will result in the induction tta, which is a transcription factor that can be blocked by the antibiotic doxycycline (dox). in the absence of dox tta drives expression of genes linked to a teto-promoter sequence. the second transgene incorporates both a teto-linked reporter (in this case the somato-axonal marker taulacz) as well as a transcriptional feedback loop to maintain teto-linked gene expression indefinitely once it is activated. the tta (tta *) in this construct was made dox insensitive by introduction of a point mutation in the tet binding domain. in the presence of dox the teto-linked reporter is not activated even in those neurons in which the cfos-linked tta is expressed. however, if dox is withdrawn then both taulacz and tta*are expressed, but only in those neurons that were active to a high enough level to induce the cfos-linked tta. once activated, the tta*sets up a transcriptional feedback loop that can be maintained even in the presence of added dox. in this manner discrete time windows for genetic tagging of active neurons can be opened and closed through the use of dox. in the absence of dox, any neuron that has sufficient induction of cfos-linked tta to activate the feedback loop will persistently activate the taulacz reporter, as well as any other teto-linked transgene that is introduced into the mouse (aiba and nakao, 2007). this expression will be maintained even when the time window for sampling active neurons is closed by the readministration of dox. in this way a persistent record of neurons that were active during the off-dox period can be maintained. we called this the tettag mouse, which stands for tetracycline transactivator controlled genetic tagging of active neural circuits. the tettag mouse. mice carrying two transgenes were used. the first transgene uses the cfos promoter to drive expression of the tetracycline transactivator (tta). tta activates the teto promoter in the absence but not presence of doxycycline (dox). the second transgene uses the teto promoter to drive expression of a dox insensitive tta (tta *), which, once expressed, sets up a positive feedback loop that continuously drives expression of a -galactosidase reporter coupled to the tau protein (taulacz). neurons activated during fear conditioning (while off dox) were tagged with long-lasting expression of taulacz (lac; red circle). mice were put back on food with doxycycline and a retrieval test was done 3 days later, followed by analysis of the brains 1 h after retrieval for expression of lacz and zif268. neurons activated during learning expressed lacz and those active during retrieval expressed zif268 (zif; green circle). the number of neurons in the amygdala that expressed both lac and zif, indicating that they were activate during both learning and retrieval, was positively correlated with the strength of the fear memory that the animal displayed. we used the tettag mouse to examine the neural circuit that mediates fear memory (reijmers et al., 2007). we asked whether neurons that are stimulated during learning in a pavlovian fear-conditioning paradigm were reactivated during retrieval of the memory as shown in figure 2. we subjected tettag mice to a learning trial consisting of paired presentations of a tone (cs) and a foot-shock (us). this results in a long-lasting fear memory for both the tone (cued conditioning) and the conditioning box in which the animals were shocked (context conditioning). the learning trials took place during a time window in which the animals were free from dox, allowing activated neurons to be tagged with long-lasting expression of taulacz. the animals were then returned to dox to prevent further tagging of activate neurons, tested for retention of the memory in a retrieval trial, and sacrificed after 1 h for analysis using the endogenous ieg zif268 as a measure of recent neural activity. by comparing the expression of lacz (activity during learning) and zif268 (activity during memory retrieval) we could determine the degree of circuit reactivation. we found that the number of reactivated neurons in the amygdala, a region critical in fear conditioning (ledoux, 2007), correlated with the retrieval of the fear memory. we hypothesized that these reactivated neurons represent a component of the memory trace for conditioned fear. to test this idea we weakened the strength of the memory by extinction training; giving repeated cs presentations without the shock us. animals were first fear conditioned while free from dox to tag the learning activated neurons. the extinction training then took place following re-exposure to dox (to prevent further labeling) and a memory retrieval trial was conducted 1 h prior to analysis. we found that there was a significant correlation between the strength of the remaining fear response and the degree of circuit reactivation; animals with a high fear response during retrieval showed strong reactivation of the learning circuit while those with low fear responses showed a low degree of reactivation. in addition, we found some specificity in the anatomy of the responses so that reactivation in the basal amygdala was correlated with context fear while reactivation in the lateral amygdala was associated with the strength of the cued (tone) fear memory. these results are consistent with the known role of these subdivisions of the amygdala with the two different forms of fear memory (quirk et al., 1995; these results demonstrate that memory retrieval results in a reactivation of some of the same neurons that were active during the initial learning. we suggested that neurons activated by the us (shock) but also receiving weak cs inputs were altered during learning such that the cs alone could now activate them. in this way presentation of the cs alone after learning would recapitulate a portion of the aversive us leading to downstream fear responses. this approach represents a somewhat elaborate way of simply watching neural activity and the results are still purely correlative. however, by using a regulatable and binary genetic system, it should be relatively easy to introduce additional effector transgenes into the mice to control the activity or biochemistry of these neurons and directly test their role in memory. one recent study by josselyn and coworkers has achieved this direct manipulation of neural ensembles associated with a specific memory trace using a somewhat different approach. the use of neural activity to introduce genetic alterations into neurons offers the possibility of obtaining direct molecular control over the neurons that participate in a specific neural representation or memory trace. an alternate approach that realized this goal took advantage of the finding that certain molecular manipulations could recruit neurons to participate in control of a specific memory. in one recent study, (han et al., 2007), it was found that over expression of the transcription factor creb in neurons resulted in their preferential recruitment into a fear memory trace. in this study, neurons in the amygdala were randomly infected with a viral vector that over expressed creb and the animals were then trained in fear conditioning. they then performed a retrieval trial and examined the expression of the ieg arc in the amygdala. they found that the creb over expressing neurons were more likely to be activated during the memory retrieval. this result suggests that these creb over expressing neurons were predisposed to participate in the memory trace. the mechanism by which these neurons are preferentially recruited is unclear but it does demonstrate that, at least in the amygdala, there is a good deal of flexibility in which neurons can be used to encode a specific memory. the system is not hard wired at the level of individual neurons but there is a sort of competition, with creb over expression favoring a neuron's recruitment into the memory trace. josselyn and coworkers went on to take advantage of this creb priming trick to directly manipulate a specific fear memory trace (han et al., study the viral vector that delivered creb to amygdala neurons also carried a gene that allowed for the expression of the diphtheria toxin receptor (dtr) (figure 3). the creb over expression recruited the neurons to the memory trace and expression of the dtr allowed for the selective ablation of these specific neurons with diphtheria toxin (dt). ablation of the creb over expressing neurons disrupted the fear memory while ablation of a similar number of random neurons in the amygdala did not. the memory effect was long lasting and specific (the same animals could learn a second fear association) demonstrating that this limited group of neurons played a critical role in the specific memory encoded during the creb expression time window. this is the first example of the disruption of a specific memory within a distributed network. disrupting a specific memory in the mouse. a viral vector expressing both creb and cre recombinase was injected into the amygdala of idtr mice leading to the expression of both cre and creb in a random subset of neurons (circles with creb/cre). the cre recombinase removed a transcriptional stop sequence and allowed for expression of dtr in these creb expressing neurons. an earlier study from the same authors (han et al., 2007) demonstrated that the creb expressing neurons participate in the storage of the fear memory (green circles symbolize neurons that participate in the storage of the memory). after fear conditioning, mice were injected with diphtheria toxin (dt), which killed the creb expressing neurons which participated in the encoding of that memory (red circles). this caused a significant reduction in the strength of the fear memory measured during retrieval. while the ability of creb over expression to recruit neurons to participate in a specific memory is interesting in its own right, it would be useful to have an approach to manipulate neurons that were naturally activated by any general environmental stimulus. this has recently been accomplished using a technique in which neurons expressing -galactosidase can be specifically disrupted with a pharmacological agent (koya et al., 2009). the study used rats that carry a cfos-promoter driven -galactosidase to label activated neurons. to manipulate the neurons they use a drug that is inactive in the absence of -galactosidase (daun02) but can be hydrolyzed to a compound that can reduce ca dependent action potentials (santone et al., 1986). they examined context specific sensitization to cocaine, which is an associative paradigm where the response to a drug of abuse is potentiated when it is administered in the same environment in which it has been repeatedly taken. animals were given repeated injections of cocaine over 1 week in context a to produce the context specific sensitization (measured as increased locomotor response to the drug). following the training, a final sensitization trial to induce -galactosidase was given and 90 min later daun02 was injected into the nucleus accumbens to disrupt the -galactosidase expressing neurons. previous studies had suggested that the nucleus accumbens was a critical site of plasticity mediating this behavior (mattson et al., 2008). the injected animals showed a reduction in the context specific component of the sensitization but retained normal responses to cocaine in a novel context b when tested several days later. like the results with the creb over expression, this study suggests that a specific associative representation (context a+ cocaine) is being interfered with selectively. while the behavioral results in this study are intriguing and the linkage to cfos based expression provides a potentially general approach for manipulating discrete neural representations, there are a number of important questions that remain to be addressed with this technique. first, the electrophysiological effect of the daun02 treatment was not examined directly in the neurons but inferred from studies in cell lines. whether the effects are mediated by suppression ca dependent action potentials or some other effect of the time course of any neural excitability or ca channel changes is also a critical parameter. the effect of the daun02 treatment was examined 3 days after the initial injection of the compound and it is unclear whether the observed effect was due to ongoing suppression of activity or to a persistent effect manifest during the initial treatment. nevertheless, it demonstrates the general principles of this approach, which could be combined with the host of recently developed genetic regulators of neural activity. while neural representations are encoded in the specific ensemble of neurons that are activated in response to a stimulus, the plasticity that molds these patterns of activation is thought to occur at the synapse. it has been known for some time that long-term memory lasting 24 h requires new gene expression initiated at the time of learning, while short-term memory lasting a few hours lacks this requirement (davis and squire, 1984). since the short and long-term memories for the same event presumably involve the same pattern of synaptic changes, it raises the question of how the required gene products exert their effects selectively on the appropriate synapses. a potential answer to this question was suggested by frey and morris in studies of long-term potentiation (ltp), a form of synaptic plasticity thought by many to underlie memory (frey and morris, 1997, 1998). they found that synaptic activity could produce a sort of molecular tag at a synapse that would allow it to utilize newly expressed gene products to maintain ltp for long periods. we recently used the cfos based genetic approach to demonstrate a similar mechanism in behavioral learning and memory. a number of studies have implicated the regulated trafficking of the glutamate receptor glur1 to synapses as an important mechanism in both ltp and fear learning (kessels and malinow, 2009). in addition to allowing the genetic tagging of activated neurons, the ieg promoters like cfos show a very rapid onset and offset of expression, making them useful for cellular trafficking and turnover studies. we took advantage of this property to examine the trafficking of glur1 following learning in the fear-conditioning paradigm (matsuo et al., 2008). mice carrying both a cfos-tta and teto-gfpglur1 fusion transgene were used in this study (figure 4). in the absence of dox neural activity will induce a pulse of expression of the gfp tagged glur1 and the distribution to synapses of this newly synthesized receptor can be followed histologically. animals were fear conditioned in the absence of dox to both induce a contextual fear memory and to induce synthesis of the gfp tagged receptor in activated neurons. the distribution of the receptor to dendritic spines (the site of most excitatory synapses) in the hippocampus was examined 24 h after the conditioning. we found that the receptor was not evenly distributed but present in only about 50% of spines, even in controls. in the fear conditioned animals we found a similar distribution except that there was an increase in trafficking to one morphological type of spine, the mushroom spines. this preferential trafficking only happened when the conditioned stimulus (cs: novel box) and the unconditioned stimulus (us: foot shock) were paired, but not when cs or us were presented separately. the newly synthesized glur1 requires 2 h to begin to reach the dendritic spines, yet is somehow preferentially recruited to a specialized class of spine based on the associative conditioning that occurred 2 h prior to its arrival. this is indicative of a synaptic tagging event acting in behavioral memory similar to that described for ltp (frey and morris, 1997). learning regulated targeting of glutamate receptors. the first transgene is identical to the one described in figure 2 and uses the cfos promoter to drive expression of a tetracycline transactivator (tta). the second transgene was a teto-promoter gfp tagged glutamate receptor subunit (glur1-gfp). animals were fear conditioned in the absence of dox to produce both a fear memory and a pulse of gfp-glur1 expression in active neural ensembles in the hippocampus. the distribution of gfp-glur1 in dendritic spines was analyzed 24 h following the conditioning using dii to label all spines on a given neuron. fear conditioning led to an increase in trafficking of the receptor specifically to mushroom type spines. this experiment demonstrates how genetic tools can be used to image a specific molecular event selectively within an activated neural circuit. the ability to genetically manipulate activated neuronal ensembles or neurons participating in a sparsely encoded memory trace offers a number of advantages that are only beginning to be realized. a parallel line of technological development has focused on generating genetic tools for manipulating neuronal activity. the light regulated channelrhodopsin chr, developed by deisseroth and colleagues (zhang et al., 2006), allows for the very precise light regulated control of action potential firing in neurons expressing the channel. there is an expanding tool box of genetic effectors like chr that are light or ligand controlled and can be used to either stimulate or suppress neural activity (luo et al., 2008). in addition, there are a number of similar effectors that can be used to regulate second messenger signaling pathways when expressed in heterologous cells (isiegas et al., 2008; the combination of these new tools with activity based genetic delivery and multi time point brain activity mapping at cellular resolution will open up a variety of new questions to experimental analysis. to return to the initial discussion of neural representations, the fusion of these approaches should allow one to address the question of what neural firing patterns mean to the animal. to take the example of the halle berry neurons (actually the equivalent in genetically accessible animal models) one could ask what would be the consequences of silencing this specific group of neurons. what fraction of neurons in the representation need to be silenced in order to impair recognition? how does the silencing of a specific group of neurons in one brain region affect the activation patterns in downstream areas? an alternate approach is to ask whether a representation can be built by experimenter driven stimulation of the appropriate neurons. in one recent study, svoboda and colleagues delivered the chr2 molecule to random populations of neurons in the somatosensory cortex. they found that stimulating as few as 300 neurons could be detected by the animals and used to alter behavior in a conditioning task (huber et al., 2008). however, the coordinated activation of those neurons presumably does not form any natural representation. suppose that instead of a random group of neurons one could activate a subset of neurons that responded to a natural stimulus, say the tone cs in a fear-conditioning task. the artificial stimulation of those neurons paired with a foot shock would presumably lead to a conditioned fear of the extrinsic stimulation cs. if so would all the features of this sensory representation be maintained, for example frequency selectivity? with what fidelity does artificial stimulation of the tone representation in one brain region recapitulate the brain activity patterns produced by the natural tone itself? this type of approach should now be achievable and allow direct functional investigation of the structure of neural representations. finally, the ability to genetically alter activated neural ensembles provides an entre into a more specific biochemistry of the brain. in the glur1 trafficking studies discussed above, the gfp tagged receptor provides not only a signal to watch molecular movements, but also a tag for specific biochemical analysis. for example, one could ask how the synapses from activated neurons that received new receptor differ biochemically from those that did not by using the antibodies to the gfp tag to affinity purify positive from negative material. a similar approach could be used to tag specific cellular compartments or molecular complexes so that biochemical studies can be limited to just the activated neurons, sparsely embedded in a matrix of inactive neurons and glia. it should also be possible to improve the specificity of the genetic modifications using a variety of genetic tricks such that only neurons active in one brain region or active at time point 1 but not time point 2 would be tagged. the increased specificity along with the new genetic tools and biochemical tagging should provide a new level of circuit analysis in the brain and break down the barrier between watching neural firing and manipulating neural function. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

the use of molecular tools to study the neurobiology of complex behaviors has been hampered by an inability to target the desired changes to relevant groups of neurons. specific memories and specific sensory representations are sparsely encoded by a small fraction of neurons embedded in a sea of morphologically and functionally similar cells. in this review we discuss genetics techniques that are being developed to address this difficulty. in several studies the use of promoter elements that are responsive to neural activity have been used to drive long-lasting genetic alterations into neural ensembles that are activated by natural environmental stimuli. this approach has been used to examine neural activity patterns during learning and retrieval of a memory, to examine the regulation of receptor trafficking following learning and to functionally manipulate a specific memory trace. we suggest that these techniques will provide a general approach to experimentally investigate the link between patterns of environmentally activated neural firing and cognitive processes such as perception and memory.

PMC2802553

pubmed-469

angiogenesis, the formation of new blood vessels by sprouting of pre-existing ones, is a main mechanism of vascularization during embryonic development, growth, formation of the corpus luteum and endometrium, regeneration and wound healing. however, deregulated, abnormal angiogenesis is involved in many pathological processes [1, 2]. the complex sequence of events involved in angiogenesis is related to changes in endothelial cell biosignalling. the relationships of angiogenesis with cancer have special relevance, since angiogenesis has been described as one of the hallmarks of cancer, playing an essential role in tumour growth, invasion, and metastasis. since tumour blood vessels show many differences from normal vessels and are not genetically unstable, they are potential targets for therapy of all types of cancer. due to the pivotal role played by endothelial cells in tumour angiogenesis, most previous efforts were devoted to the development of agents that could block their activation by an angiogenic signal (mainly vegf), or to inhibit one or several specific functions of activated endothelial cells (proliferation, adhesion to extracellular matrix, proteolytic activities, migration, invasion or differentiation). national cancer institute database showed that in august 1999 a total of 20 angiogenesis inhibitors were being tested in clinical trials. remarkably, most of them were monotherapies with the antiangiogenic agent, and those compounds that had then reached the phase iii, including several inhibitors of matrix metalloproteinases, were discontinued due to their lack of activity or the appearance of undesirable toxicities. in spite of the great number of angiogenesis inhibitors described so far (estimated to be>300 drug candidates in 2001), and the interesting results obtained in experimental models, even showing complete tumour regressions in pre-clinical studies, modest or even negative results emerged from the first generation of compounds entered in clinical trials. nevertheless, there is no reason for premature pessimism, as revealed by current ongoing trials and the clinical developmental status of anti-angiogenic drugs. a critical analysis of the disappointing results obtained in previous clinical trials points to different reasons for this failure. these include flaws in the methods used to select these inhibitors and in the design of the clinical trials to test their effects, as well as an oversimplified view of tumour vasculature pathophysiology. angiogenesis inhibitors are initially selected by means of in vitro assays that make use of endothelial cells from different sources. the results obtained in this primary screening can be dependent on the type of endothelial cell. afterwards, the antiangiogenic activity of the selected compounds is usually tested with several in vivo assays (for a review, see). although useful, these have limitations. some of them do not take into account the tumour microenvi ronment (this is the case of vascularization assays in the chicken chorioallantoic membrane and in the mouse cornea). other assays make use of rapidly growing tumours and/or animals that do not fit into the clinical reality. most of the tumour systems used with laboratory animals show angiogenic responses much higher than those induced by human tumours. furthermore, the effectiveness of an angiogenesis inhibitor can be hampered by the intrinsic heterogeneity of human tumour angiogenesis. animal and preliminary clinical trials have revealed that different tumours respond very differently to antiangiogenic therapy. in fact, the responses have been extremely heterogeneous, most probably due to the randomized design of the trials. a clinical challenge in antiangiogenesis is the finding of biological markers that help to identify subsets of patients more likely to respond to a given antiangiogenic therapy, as well as to determine optimal dosing of therapy, to detect early clinical benefit or emerging resistances and to decide whether to change therapy in second-line treatments., microvessel density has been proven to be a useful prognostic indicator but, at the same time, does not seem to be a good direct indicator of antiangiogenic treatment efficacy. surrogate biomarkers could include those related to the various steps of the angiogenic process, including variations in endothelial-cell survival, alterations in the endothelial-cell signaling, and variations in the number of circulating endothelial progenitor cells [3, 13, 15, 16]. another possibilityis the fractal analysis of the vascular network in tumour biopsies. however, these approaches are far from an ideal biomarker for clinical practice. although some authors consider taking biopsies repeatedly to be feasible, most physicians consider this to be very cumbersome for patients. since tumour angiogenesis produces interstitial hypertension in tumours, the determination of interstitial pressure of tumours can be considered an alternative surrogate biomarker. easier to determine and less invasive approaches include the measurements of circulating levels of several angiogenic factors, but so far no growth factor has been validated for predicting response to antiangiogenic therapy, as well as high resolution image analysis that requires expensive instrumentation that could not be available in all institutions. on the other hand, the clinical end-points for dose-defining trials (phase i) and efficacy trials (phase ii) should be reconsidered. the expected good tolerability and low toxicity of well selected antiangiogenic compounds give little relevance to the determination of maximum tolerated doses (mtd) and dose-limiting toxicity (dlt), which could be replaced by the determination of optimal biological dose (obd) in phase i trials. more and better-designed pharmacokinetic studies are required not only to determine the obd, but also to determine the optimal schedule of drug administration. some years ago, trials with this antiangiogenic compound were discontinued due to low responses and toxicity. however, an improvement in the treatment regimen yielded enhanced response with decreased toxicity in phase i and ii trials. probably, a readjustment of doses and/or schedule could contribute to diminish or even abolish some of the side effects produced by other previously tested antiangiogenic compounds. in phase ii trials, objective responses (i.e. the degree of tumour regression) alternative parameters such as disease stabilization, progression-free survival and time to progression should be used. however, these parameters are more difficult to be evaluated properly and they require larger patient samples and more prolonged treatments. validation and standardization of monitoring techniques for antiangiogenic therapy are urgently required. the fact that tumour vasculature has been understood in an oversimplified fashion is another explanation for the poor results obtained in the first generation clinical trials. it is now known that different tumour types may acquire their blood supply by different mechanisms. tumour vasculature is not necessarily derived from endothelial cell sprouting;instead cancer tissue can acquire its vasculature by a number of alternative mechanisms that could be the basis for developing effective clinical modalities using antivascular therapy of cancer. the recruitment of circulating endothelial progenitor cells, mainly from bone marrow origin, can contribute to the tumoural neovascularization by vasculogenesis, a process that was initially thought to be limited to embryonic development. in fact, as recently reviewed, there is accumulating evidence that progenitor cells as well as other stromal cells are actively recruited into tumours and that this recruitment is essential for the proangiogenic environment of tumours. another possibility is the co-option of vessels during early growth of tumours in the absence of angiogenesis [11, 28]. on the other hand, vascular mimicry, the generation of microvascular channels by genetically deregulated and aggressive tumour cells, is an alternative way to provide blood supply to tumours and is independent of angiogenesis. the potential of mono-cytes/macrophages to contribute to neovascularization has recently come into focus. some experimental evidences indicate that infiltrating mononuclear cells may incorporate in the lumen microvessels and that peritoneal macrophages may form capillary-like lumens and branching patterns in vitro[31, 32]. pathological angiogenesis is characterized by structurally and functionally abnormal vessels and lymphatic vessels [1, 2]. these abnormalities result from an imbalance between levels of pro- and antian-giogenic molecules. as a result, this, in turn, compromises the delivery and effectiveness of conventional therapies, as well as molecular targeted therapies. finally, in this brief analysis of the rationale behind the failure of the first generation of antiangiogenic agents, concern should be given to the way in which scientists communicate their findings to society. angiogenesis research is an especially competitive area in which the promising preclinical results have been very often prematurely amplified by mass-media releases. the high prevalence of cancer and the extremely high sensitivity of society towards this primary medical problem facilitate that great expectations could lead to deep disappointment. a second generation of antiangiogenic trials is beginning to show highly significant potential [8, 35]. the first study showing phase iii data validating an antiangiogenesis strategy for treating human cancers was obtained with bevacizumab, a humanized recombinant monoclonal antibody that neutralizes the biologically active forms of vegf that interact with vegf receptors 1 and 2. in a communication that received much attention in the 2003 asco meeting, the authors reported that the beva-cizumab/ifl (irinotecan/fluorouracil/leucovorin) combination led to significantly prolonged survival and had a better ability to shrink tumours than standard ifl alone, without statistically significant increases in adverse events in patients with metastatic colorectal cancer. the results were based on 412 patients in the ifl/placebo arm and 403 patients in the ifl/beva-cizumab arm. the presence of bevacizumab in the treatment produced remarkable and statistically very significant increases in all the four determined survival and response parameters: median survival, progression-free survival, objective response, and duration of response. these impressive results led the fda to approve the use of bevacizumab in patients with metastatic colorectal cancer and they have been finally published in the form of a research article in the new england journal of medicine. however, as stated in the accompanying editorial, although it is tempting to attribute the effect of bevacizumab to a direct antiangiogenic mechanism, the validity of this assumption is presently uncertain. once more, mass-media releases have led to unrealistically high expectations. as commented in the aforementioned editorial, patients need to be informed that beva-cizumab does not cure metastatic colorectal cancer and that there is no evidence as yet that the antibody has antitumour activity when administered as a single agent for this disease. although hurwitz et al. did not measure surrogate markers of antiangiogenesis, they mention that bevacizumab may have altered tumour vasculature and decreased elevated interstitial pressures in tumours, thereby enhancing the intracellular delivery of chemotherapy agents. recently, bevacizumab plus folfox4 (oxaliplatin/5-fu/leucovorin) treatment was approved for second-line metastatic colorectal cancer. in october 2006, bevacizumab in combination with paclitaxel and carboplatin treatment was approved for the first-line treatment of patients with non-small cell lung cancer. currently, more than 100 clinical trials with bevacizumab are ongoing, including phase iii trials in kidney, breast, prostate and ovarian cancer, among others. in fact, successful clinical trials of multitargeted compounds have yielded two significant fda approvals. in december 2005, sorafenib (bat 43-9006), an inhibitor of the faf/mek/erk and the vegfr and pdgfr signaling pathways, received fda approval for the treatment of renal cell carcinoma. sunitinib (su11248), an oral inhibitor of vegfr2, pdgfr, flt-3 and c-kit, received fda approval in january 2006 for patients with gastrointestinal stromal tumours (gist) and advanced kidney cancer, being the first time the agency had approved a new oncology product for two indications simultaneously [42, 43]. it seems that, after a period of flawing interest, antiangiogenic compounds have regained their place in the centre of anticancer treatment trials, as shown by the eastern cooperative oncology group portfolio of clinical trials. from the results obtained so far in clinical trials, it can be concluded that the future clinical success of angiogenesis inhibitors could be related to their use in combination with chemotherapy or radiotherapy. since abnormal angiogenic vessels compromise the delivery of drugs targeting tumour cells, the normalization of tumour vasculature with antiangiogenic therapy has emerged as a new paradigm for combination therapy [19, 45]. synergic effects can be expected, since judiciously applied antiangiogenic therapy can increase the penetrability of chemotherapeutic agents, as well as the radiosensibility of tumour cells. a detailed analysis of how antiangiogenic compounds reduce vessel density shows that these drugs reduce vascular permeability, destroy immature vessels and increase the recruitment of pericytes to stabilize other vessels. this transient stabilization has been termed the normalization window, defined as a period of time where tumour blood flow and oxygenation increases, thus providing an opportunity to better deliver chemotherapeutic drugs and radiation therapy. as previously mentioned, the heterogeneity of blood vessel growth, the fact that angiogenesis differs among tumour types is a basis for the observed differences in response to antiangiogenic therapy in both animal and clinical trials. therefore, a multidrug approach might be more successful than monotherapy. the combined used of several antiangiogenic compounds targeting different steps of angiogenesis should be explored. as stated before, reliable biomarkers are strongly needed to validate the efficacy of antiangiogenic therapy, to identify responsive patients and optimal doses, to predict efficacy of regimens that include anti-angiogenic agents, and to detect and prevent tumour escape. the lack of reliability of measurements of circulating levels of angiogenic factors has made the search for new biomarkers to shift away from measuring their levels to measuring their effects, such as the recruitment of endothelial progenitor cells from the bone marrow to the tumour where they contribute to neovascularization. preclinical studies have shown that circulating endothelial cells, which are probably derived from blood vessel wall turnover, and circulating endothelial progenitors kinetics correlate well with several standard laboratory assays, that can not be used in humans. the initial suggestion that variation in the levels of circulating endothelial progenitor cells could be a useful surrogate marker to monitor angiogenesis has been confirmed and extended in an outstanding report published in cancer cell. this report provides evidence that the levels of circulating endothelial progenitor cells are genetically predetermined and regulated by regulators of angiogenesis, including vegf, tie-2 and thrombospondin-1. moreover, antiangiogenic therapy can be optimized by monitoring the levels of both circulating endothelial cells and circulating endothelial progenitor cells [13, 25]. therefore, the kinetics of these cells in peripheral blood is suggested to be useful surrogate markers of pathological angiogenesis with potential application for the monitoring of antian-giogenic therapy response. there are clear signs that during the last year antian-giogenesis research has entered a new age. table 1 tries to summarize the trials and errors in past failures and possible solutions to them. antiangiogenic cancer therapy: past and future; clinical trial lessons the development of new and better models for the in vivo assay of potential inhibitors of human angio-genesis should be considered a priority in this field of research. there is increasing concern that by using approaches based on traditional end-points, potentially interesting angiogenic modulators might be rejected prematurely. consequently, the extensive use of correlative studies in the early phases of drug development to establish surrogate biomarkers for use in efficacy trials is strongly recommended. methods of imaging will be helpful to assess the efficacy of treatment [12, 13, 20]. a careful selection of the clinical setting for the investigation (for example, tumour type and stage of disease) and innovative statistical designs to optimize the selection of patients must be carried out before expensive, definitive phase iii clinical trials. an example is the randomized discontinuation trial design (rdtd), aimed to select a subset of enrolled patients who are more homogeneous with respect to important prognostic factors than the group of patients that would otherwise be randomized in the trial. frequent administration of chemotherapy at low doses, ranging from one-tenth to one-third of the mtd, significantly increases the antiangiogenic effect. metronomic scheduling has shown impressive antitumour activity in animal models and is now being tested either alone or in combination with other antiangiogenic agents in clinical trials [35, 47, 48]. furthermore, this metronomic approach is also used with radiation therapy, when administered at lower than normal doses, known as hyper-fractionated radiation. the concept of vascular targeting is related to antiangiogenesis but involves a different approach. juliana denekamp outlined the concepts behind vascular targeting for cancer treatment in the early 1980s, showing that physical occlusion of the blood supply to tumours in rodents led to tumour regressions. vascular targeting agents exert their primary action on the pre-existing blood vessels of solid tumours. vascular targeting therapies would share the advantages of antiangiogenic therapies and could offer some additional advantages. first, blood flow is a defined surrogate marker of biological activity that can be measured in the clinic. second, temporary effects on vascular functioning may be sufficient. and third, unlike angiogenesis inhibitors, vascular targeting agents should require only intermittent administration to synergize with conventional treatments rather than chronic administration. the clinical studies completed to date with vascular targeting agents are encouraging. progression into combination studies has begun. recently, concerns have been raised on the possibility of resistance to antiangiogenic therapy [13, 21]. in fact, an effective antiangiogenic therapy could select for resistant and aggressive cancer cells during therapy-induced tumour regression. ideally, the most effective therapy would suppress all cancer cells, avoiding relapse. on the other hand, hypoxia is common in tumours, despite the increase in their vascularization, because of a poor perfusion caused by aberrant vessels. strategies that target hypoxic cells may therefore synergize with antiangiogenic treatments. the contribution of inflammatory cells to tumour angiogenesis the inhibitors of this pathway exhibit high tolerability and they can be administered chronically. their performance in clinical studies is currently being tested. however, the recent problems with cycloxygenase inhibitors in cancer prevention treatment raise serious concerns for their use [55, 56]. inhibitors of angiogenesis could slow the progression of premalignant lesions and reduce the risk of developing invasive tumours. they have the potential to be used in primary, secondary or tertiary cancer prevention settings. in fact, monotherapies with antiangiogenic compounds could be useful as adjuvant treatments in situations of minimal residual disease following either cytore-ductive surgery or cytotoxic treatment. however, in spite of the fact that past clinical studies have shown that many angiogenesis inhibitors can be given safely to patients, more long-term toxicity studies are needed. how much antiangiogenic therapy will be incorporated in the future to the treatment of cancer patients depends on further advances in the understanding of the molecular mechanisms involved in tumour angio-genesis, the development of standardized methods to assess surrogate predictive markers of response, and the capability of performing a new generation of appropriately designed clinical studies. a convergence of the efforts carried out in basic, applied and clinical research would contribute to achieve these goals. this knowledge will be applied not only to cancer treatment but also to other diseases characterized by abnormal vasculature such as hemangiomas, diabetic retinopathy, macular degeneration and psoriasis, among others for which antiangiogenic approaches have already shown benefits [1, 2].

abstractangiogenesis inhibition has been proposed as a general strategy to fight cancer. however, in spite of the promising preclinical results, a first generation of antiangiogenic compounds yielded poor results in clinical trials. conceptual errors and mistakes in the design of trials and in the definition of clinical end-points could account for these negative results. in this context of discouraging results, a second generation of antiangiogenic therapies is showing positive results in phases ii and iii trials at the beginning of the twenty-first century. in fact, several combined treatments with conventional chemotherapy and antiangiogenic compounds have been recently approved. the discovery and pharmacological development of future generations of angiogenesis inhibitors will benefit from further advances in the understanding of the mechanisms involved in human angiogenesis. new styles of trials are necessary, to avoid missing potential therapeutic effects. different clinical end-points, new surrogate biomarkers and methods of imaging will be helpful in this process. real efficacy in clinical trials may come with the combined use of antiangiogenic agents with conventional chemotherapy or radiotherapy, and combinations of several antiangiogenic compounds with different mechanisms of action. finally, the existing antiangiogenic strategies should include other approaches such as vascular targeting or angioprevention.

PMC3922346

pubmed-470

regulation of gene expression is one of the most enigmatic facets of molecular genetics that results in intricate appearance of a biological entity. scientists have been attempting to elucidate the regulatory mechanisms of gene expression for long and the radical discovery of regulatory function of endogenous small noncoding rnas is overwhelming the scientific community with their ever increasing potentials. small noncoding rnas of 1840 nucleotides (nt) in size have been proved to play a vital role in a remarkably wide range of biological processes, including cell proliferation, developmental timing and patterning, chromatin modification, genome rearrangement, and stress response in plants and animals. small rnas regulate a variety of biological processes in plants by interfering with messenger rna (mrna) translation, directing mrna cleavage or promoting the formation of compact, transcriptionally inactive chromatin. several distinct classes of small rnas have been reported so far including micrornas (mirnas), small interfering rnas (sirnas), repeat-associated small interfering rnas (ra-sirnas), piwi interacting rnas (pirnas), natural antisense transcript derived small interfering rnas (nat-sirnas), transacting small interfering rnas (ta-sirnas), heterochromatic small interfering rnas (hc-sirnas), secondary transitive small interfering rnas, primary small interfering rnas, competing endogenous rnas (cernas), and long small interfering rnas [1113]. it has only been a few years since it was appreciated that micrornas provide an unanticipated level of gene regulation in both plants and metazoans. mirnas are well differentiated due to some of their particular characteristics; they are derived from distinct genomic loci and processed from transcripts that can form local rna hairpin structures, and usually mirna sequences are nearly always conserved in related organisms [13, 14]. most mirnas are transcribed by rna polymerase ii which folds into a stable, usually imperfect, hairpin structure; pri-mirna transcript is cleaved to pre-mirna by rnaseiii-type dicer-like 1 (dcl1) protein to produce a distinctive ~21 nt, double-stranded rna. this duplex is exported into the cytoplasm by hasty and methylated at the 3 end by hen1. a cytoplasmic helicase unwinds the translocated duplex into a single-stranded mature mirna, which is finally incorporated into rna-induced silencing complex (risc) [5, 17, 18]. a mature mirna sequence can range from 19 to 24 nucleotides (nt) in length and act as a regulatory molecule in posttranscriptional gene silencing by base pairing with target mrnas. within the risc complex, mirnas function in the direct cleavage of 3 untranslated region of protein-coding genes or translational repression depending on its perfect or imperfect match with the targets. the same mature mirna can also be present as several length variants; these populations of mirna variants are called isomirnas, which are isoforms of micrornas caused by an imprecise or alternative cleavage of dicer during pre-mirna processing. several mirnas have been identified in plants, and they have been characterized in a wide variety of metabolic and biological processes with important functions. the first plant mirnas were described in arabidopsis thaliana; currently the latest mirbase release (v20, june 2013) contains 24,521 microrna loci from 206 species, processed to produce 30,424 mature microrna products. earlier, mirnas have been identified through either bioinformatics analysis or sequencing; various methods have been used to identify mirnas in rice, wheat, tomato, and maize. besides the mirnas that are highly conserved in different species, there are species-specific mirnas originating from recently evolved mirna genes [28, 29]. the expression of these species-specific mirnas is often low and can therefore be difficult to detect by traditional methods. in recent times, high-throughput sequencing platforms are showing significant promise for small rna discovery and genome-wide transcriptome analysis at single-base pair resolution [23, 31]. sequencing techniques such as the solexa platform, solid, and 454 technology as well as other massively parallel sequencing strategies have been successfully applied in order to identify mirnas in many plant species, such as rice, alfalfa, grape, tomato, orange, soybean, peanut, poplar, and black gram. in comparison with microarray, deep sequencing has several advantages, the major one being its application in comprehensive identification and profiling of small previously unknown rna populations. nevertheless, analyses of these data are not perfect, especially in the absence of native genome sequence. jute (corchorus olitorius and corchorus capsularis) is a bast fibre, like flax and hemp. cultivation of this environmentally friendly as well as the most affordable fibre producing plant is concentrated around the ganges delta region of bangladesh and india where the warm, wet climate during the monsoon season provides ideal growing conditions. in terms of usage, production, and global requirement, jute is second only to cotton. jute plants are easy to grow, have a high yield per acre, and, unlike cotton, have little need for pesticides and fertilizers. the leaves and roots left after harvest enrich the soil with micronutrients, maintaining soil fertility. when used as a geotextile, it puts nutrients back in the soil when it decomposes. this rain-fed crop during its growth helps to clean the air by assimilating three times more co2 than an average tree, converting the co2 into oxygen. despite its great agronomic importance, research on jute at the molecular level so far only 1,210 sequences are found in the genbank with no deposits of any mirna sequences in mirbase database. within this context, the current study has employed the deep sequencing strategy in an attempt to effectively identify conserved and novel jute mirnas. quantitative real-time pcr (qrt-pcr) has been performed to determine the expression of these mirnas. for mapping the identified mirnas, genome of vitis vinifera seeds of farmer popular o-9897 variety of tossa jute (corchorus olitorius) were collected from bangladesh jute research institute (bjri). they were surface sterilized with 70% ethanol, subsequently washed in distilled water, and allowed to germinate on sterile petri dishes containing 3 mm moist filter paper (whatman) at 30 1c and 65% relative humidity. seeds were allowed to grow for 4 days under the specified conditions. on the fourth day of germination, seedlings were collected and immediately snap-frozen in liquid nitrogen and stored at 80c for subsequent use. rna was isolated from collected seedlings using trizol reagent (invitrogen, usa) by following the manufacturer's instructions. later, rna samples were sent to beijing genome institute (bgi, shenzhen, china) for deep sequencing of small rna by illumina hiseq high-throughput sequencing platform. in short, after ligation with 5 and 3 adaptors, the short rnas so obtained were reverse-transcribed to cdna according to the illumina protocol. the resulting small rna library was then sequenced following sbs method (sequencing by synthesis) by illumina hiseq high-throughput sequencing. raw data obtained from illumina hiseq high-throughput sequencing was at first filtered by removing contaminants which include low quality reads, reads with 5 primer contaminants, reads without 3 primer, reads without the insert tag, reads with poly a, and reads shorter than 18 nt. after cleaning, clean reads fully matching other rnas, including mrna, rrna, trna, snrna, snorna, and repeat rna, were excluded by using blastn-short alignment (blast2.2.26 +, ftp://ftp.ncbi.nih.gov/blast/executables/blast+/2.2.26/) and aligning against sanger rna family database (rfam 11.0, ftp://ftp.sanger.ac.uk/pub/databases/rfam). the remaining unique sequences were further aligned against mirbase-v20 allowing up to 3 mismatches to identify known mirnas present in c. olitorius. mature mirnas present within a genome encoding identical or nearly identical sequences were then grouped together into a family. prediction of novel mirna was done using prediction software, mireap (http://sourceforge.net/projects/mireap/), developed by bgi by taking into consideration secondary structure, cleavage position of dicer protein, and minimum free energy of the unannotated small rna tags. strategic conditions for selecting unique mirna are as follows: (i) the tags which are to be used to predict novel mirna should be from unannotated tags which can match reference genome (vitis vinifera), from the tags which align to introns as well as antisense exons; (ii) genes, whose sequences satisfy the above standards and their secondary structures which allow hairpin mirnas to fold within them and the presence of mature mirnas in one arm of the hairpin precursors, are considered as candidate genes for mirna; (iii) the possible candidate mature mirna strand contains 2-nucleotide 3 overhang; (iv) hairpin precursors of the candidate mirnas are devoid of large internal loops or bulges; (v) secondary structures of the hairpins are stable, with the minimum folding energy (mfe) lower than or equal to 20 kcal/mol. the rules used for target prediction in plants are based on those suggested by allen et al. and by schwab et al. these are (i) presence of maximum 4 mismatches between small rna and target (g-u bases count as 0.5 mismatches), (ii) not more than 2 contiguous mismatches in the mirna/target duplex, (iii) no end-to-end mismatches at the 5 of mirna from 2 to 12 positions of the mirna/target duplex, (iv) no mismatches in positions 10-11 of mirna/target duplex, (v) a maximum of 2.5 mismatches in positions 112 of the mirna/target duplex from 5 region of mirna, and (vi) minimum free energy (mfe) of the mirna/target duplex which should be 74% of the mfe of the mirna bound to its perfect complement. the targets of mirnas were further validated by a well-recognized mirna-target prediction tool: psrna target. in addition to potential target prediction for known and novel mirnas, pathways which include the corresponding target genes as well as the biological function of such genes are taken into consideration by using the grape genome as a reference. gene ontology (go) is an international standard classification system for gene function, which provides a set of controlled vocabulary to comprehensively describe the property of genes and gene products. there are 3 ontologies in go: biological process, cellular component, and molecular function, containing lists of biological functions that illustrate each gene and its product (http://www.geneontology.org/). each category defines precise participation of a given gene within an organism. as for pathway identification, kegg (http://www.genome.jp/kegg/) kegg analyses reveal the main pathways with which the target gene candidates are involved. to verify the identified known and potential novel mirna candidates in jute, stem-loop reverse transcription-pcr was performed. stem-loop primers were designed according to the method described by chen et al.. this primer binds to specific mirna at the 3 region owing to the precision conferred by the primer with the exact reverse complement of six nucleotides from the 3 end of each particular mirna sequence, which is reverse-transcribed by the rt enzyme. two thousand nanograms of total rna were used to perform the rt reaction with superscript iii first strand synthesis system (invitrogen, usa) according to the protocol of varkonyi-gasic et al., which has been further standardized for jute. for a reaction volume of 20 l, 0.5 l of 10 mm dntps was at first taken together with an appropriate amount of rna and an adjusted amount of depc-treated h2o followed by heating the mixture for five minutes at 65c and then immediately transferring the same on ice. while keeping on ice for approximately 2 minutes, 4 l of 5x fs buffer, 2 l of 0.1 m dtt, 1.2 l of 1 m stem-loop primer, 0.25 l of m-mlv superscript iii rt (200 u/l), and 0.1 l rnaseout (40 u/l) were added to the reaction mix. the rt reaction was carried out in a thermal cycler (mastercycler, eppendorf, germany), followed by a pulse rt cycle starting from incubation at 16c for 30 minutes, then 60 cycles of 30 sec. at 30, 30 sec. at 42c, and 1 sec at 50c. this step was followed by another incubation step of 5 mins at 85c to inactivate the rt enzyme. end point pcr was then conducted with mirna specific forward primer and a universal reverse primer to check the presence of the specific mirnas. pcr products were electrophoresed on 3% agarose gel in 1x tae and stained with ethidium bromide before visualization under a transilluminator. we further conducted quantitative real-time pcr for confirming the expression of some selected known and novel mirnas using a 32-well plate roche lightcycler nano system and the roche sybr green master i (roche diagnostics, germany). briefly, equal amount of cdna was taken in a reaction volume of 7.5 l in triplicate with 0.1875 l of each primer (forward and universal reverse) and 3.75 l of sybr green master i. thermo cycling conditions were set at an initial polymerase activation step for 600 seconds at 95c, followed by 45 cycles of 5 sec at 95c for template denaturation, 10 sec at 60c for annealing, and 1 sec at 72c for extension and fluorescence measurement. later, a dissociation protocol with a gradient from 50c to 95c was used for each primer pair to verify the specificity of the rt-qpcr reaction and the absence of primer dimers. in order to identify micrornas in jute, rna was isolated from the total tissue of four-day seedlings and subjected to illumina hiseq high-throughput sequencing by synthesis (sbs) technology. among a total of 16912862 raw reads, the details of tag cleaning are summarized in table 1, which shows that a total of 16644324 clean reads were obtained by removing 3 adapter null, insert null, 5 adapter contaminants, sequences smaller than 18 nt, and poly a. this comprises about 99% of the high quality reads. distribution of these clean reads that contain a pool of small rnas ranging from 18 to 30 nucleotides is shown in figure 1. however, the sizes of small rnas were not found to be uniform; majority (92.69%) of the srnas are 2024 nt in size, with 21 nt being the most abundant (42.19%), followed by 24 nt (22.95%) and 20 nt (14.25%), respectively. these sequences were then aligned to rfam 11.0 and genbank database (blastn) to identify common srnas other than mirnas, as well as to remove mrnas (see supplementary file-1 in supplementary material available online at http://dx.doi.org/10.1155/2015/125048). the remaining sequences were matched against mirbase-20 database for the prediction of mirnas, revealing 33433 unique and 8994892 redundant reads, which were finally used to identify known mirnas. the novel mirnas in jute were identified from unannotated tags by using mireap, software developed by bgi (described in section 2). in the absence of the complete genome sequence and with practically no information on mirna of jute in mirbase, clean reads were aligned to the mirna precursor/mature mirna of all plants in mirbase allowing up to three mismatches or free gaps to identify known mirnas. the expression of mirna is generated by summing the count of tags which can align to the temporary mirna database, generated by choosing the most expressive mirna of each mature mirna family. a total of 227 known mirnas were identified in this study, of which 164 belong to 23 conserved and 63 to 58 nonconserved families. conservancy of mirna families found in jute showed high homology with their respective homologs in other model plants (figure 2). however, the number of family members of conserved mirnas was highly variable, with mir156 being the largest family, consisting of 26 members, whereas mir403, mir394, mir827, mir477, and mir2111 were the smallest among the families, comprising only one member. mir166 and mir169 were the second largest with each having 18 members and mir171 was the third largest family with 14 members (figure 3). most of the conserved families contain both 5p and 3p mature mirna sequences, attaching a high confidence to the data set. highly variable reads numbers were also found among the families, even in members of the same family, indicating different expression levels of these mirnas. among them, col-mir157a had the highest level of expression, having 5531609 counts, and the other mirnas like col-mir156a, col-mir166a, and col-mir167h also had relatively high reads numbers, counting more than 150000. several conserved mirnas (like mir171, mir398, and mir159) and, as expected, most of the nonconserved mirnas had relatively low copy numbers. interestingly, a mirna named col-mir3954 from an undefined family had very high level of expression, having 868222 reads, third highest of all mirnas found in jute. high expression frequency of mirnas derived from the 3 arm of some pre-mirnas, like col-mir166h-3p, col-mir166g-3p, col-mir166j-3p, col-mir165a-3p, col-mir396b-3p, col-mir396e-3p, and so forth, compared to their corresponding 5 arm-mirnas, supports the observations of functional activity of both arms of pre-mirna hairpins [55, 56]. the mirna hairpins are mostly located in intergenic regions, introns, or reverse repeat sequence of coding sequences. thus tags belonging to these regions characteristic hairpin structure of mirna precursor was used to predict novel mirna with prediction software mireap (http://sourceforge.net/projects/mireap/) by exploring the secondary structure, the dicer cleavage site, and the minimum free energy of the unannotated small rna tags which could be mapped to the reference vitis vinifera genome. predicted secondary structures were further validated by mfold (supplementary file-3) and novel mirnas were identified based on the selection criteria described in section 2. 17 potential novel mirnas have been identified in this study of which 9 mirnas were derived from 3 arm of the pre-mirna sequences and 8 from the 5 arm (table 3). average length of the pre-mirnas sequences ranged from 78 to 349 nt, similar to those found in maize and rice; minimum folding energy (mfe) for jute mirnas was observed to be within a range from 21 to 105.3 kcal/mol, similar to the range observed in cucumber (supplementary file-4). expression of novel mirna was determined by summing the count of such mirnas which have no more than 3 mismatches on either the 5 or 3 ends and with no mismatch in the middle. novel mirnas usually have lower levels of expression than the conserved mirnas, as evident from findings of several plant species like soybean, brassica napus, maize, arabidopsis, and wheat [59, 6164]. for a precise elucidation of the role of mirnas, target identification and determination of their biological functions it is now evident that cleavage or translational repression site of most known plant mirnas is located in the cds (coding sequence) region of their target mrna with perfect or nearly perfect sequence complementarity, making it feasible to identify plant mirna targets [4, 21, 66]. in this study, target genes of mirnas were identified by blastn against the genome sequence of vitis vinifera, following methods described by allen et al. and schwab et al. [9, 45]. among a total of 79 identified mirna (both conserved and nonconserved) families, 116 potential target genes were predicted for 39 families (supplementary file-5). a total of 46 genes from this prediction overlapped with targets identified by psrna target which found 99 target genes for 19 mirna families (supplementary file-9). highest number (16) of targets was identified for mir397 family, all of which are laccase, an enzyme, involved in plant cell wall lignification. most of the other families targeted only a single gene. for the novel jute mirnas, a total of 11 targets were predicted for 4 among the 17 identified mirnas (table 4, details in supplementary file-6), with a maximum number of target genes (6) recognized for col-mirn7. most of col-mirn7 targets are nb-arc domain containing protein, which is a resistance (r) protein, involved in pathogen recognition and subsequent activation of innate immune responses. to better understand the functions of mirnas, target genes were analyzed by gene ontology (go) level 3 to divulge the regulatory network of mirnas and target genes. such analysis demonstrates that for jute 133 predicted target genes (both for known and novel mirnas) can be classified into 20 having biological, 5 cellular, and 5 molecular functions. same gene was found to be involved in multiple processes with the reverse being also true (figure 4 and supplementary file-7). as illustrated by kegg pathway analysis (supplementary file-8), the predicted target genes of jute mirnas were found to be involved in 42 different pathways. some of the mirnas identified through deep sequencing were verified by the standard stem-loop rt-pcr method followed by end point pcr and qrt-pcr. the stem-loop primers were designed with a 3 specificity for a particular mirna which hybridizes to the same and is reverse-transcribed by the rt enzyme. these primers increase the sensitivity of the reactions such that this method can significantly distinguish two mirnas with only one single nucleotide change. the rt product is then subjected to end point and qrt-pcr. forward primers were precisely designed from the first 15 bases of each mirna with 5 extension of random gc rich sequence to increase the melting temperature as mentioned by varkonyi-gasic et al. in 2007 while the reverse primer is a universal sequence that is designed from the 5 region of the stem-loop rt primer. a set of 11 randomly selected conserved mirnas as well as 9 novel mirnas were used for verification. in this study, the stem-loop primer used was 50 bp long together with 5 (forward primer) and 3 extensions, and the end point pcr product size ranged from 60 to 70 bp. amplification of the product gave a sharp band for each of the selected known and novel mirnas (shown in figure 5). cdnas were further amplified by qrt-pcr in technical triplicates from which log 2 values of cq were calculated for each of the mirnas and average of these values was compared with the log 2 value of read counts obtained from deep sequencing. most of the qrt-pcr results acceded with the sequencing data; however, in some cases, discrepancy was observed (figure 6). widespread discovery of mirnas and their critical role in gene regulation has made it ever important to recognize them in different species. while a large amount of mirnas are reported and deposited in databases from different plants, mirna associated research in jute is still to be instigated. without the genome sequence of jute at hand, identification of mirna and their targets in jute by deep sequencing of small rnas use of closely related species ' genomes as proxy references can facilitate mirna identification in nonmodel species like jute for which genome sequence is not available. we have used the grape genome as the background because of sequence similarity between these two species.. analyses of size distribution patterns of the reads show that the most abundant srnas in jute are 21 nt in size which is about 42.19%, consistent with recent identification of srnas in different plant [34, 62, 64]. sequencing frequencies for mirnas in a library can be used as an index for estimating the relative abundance of mirnas. numerous small rna sequences, engendered from illumina hiseq high-throughput sequencing platform, show the presence of different mirna families and are even able to differentiate between distinct members of a given family. mir156 family which is highly conserved across the species was found to be the largest family in jute seedlings with the highest expression of col-mir157a followed by col-mir156a. two other members of the same family, namely, col-mir156c and col-mir156k, also show significant levels of expression. during shoot development, mir156 regulates the transition of plants from juvenile to adult phase by targeting spl genes. in arabidopsis, mir156 is strongly expressed during seedling development and shows weak expression in mature tissues. this could explain the relative abundance of the members of mir156 family since rna used in sequencing was extracted from jute seedlings. deep sequencing technology allows distinguishing and measuring mirna sequences with only a few nucleotide changes. for example, the abundance of mir156 family varied from 1 read (col-mir156p) to 5531609 reads (col-mir157a). this was also the case for some other mirna families, such as col-mir166 (from 3 to 215636 reads) and col-mir167 (from 12 to 154973 reads). presence of a prevailing member in a mirna family may indicate the dominant role of this member during the growth phase at which the samples were collected. it is also to be noted that most of the conserved mirna families consist of more than one member, whereas nonconserved mirnas identified in this study are mostly represented by a single mir (mirna) gene. it has been hypothesized that mir genes originate by gene duplication events followed by random mutation processes to evolve in multiples of imperfectly paired hairpins [77, 78]. consequently, ancient evolutionarily conserved mirnas are represented by multiple mir genes whereas nonconserved mirnas (believed to be evolutionarily recent) generally originate from a single locus. it is plausible that the conserved mirnas are responsible for control of basic cellular and developmental pathways common to most eukaryotes whereas nonconserved mirnas are involved in regulation of species-specific pathways and functions. species-specific mirnas are believed to have recently evolved and, in general, expressed at levels lower than those of strictly conserved mirnas [34, 77]. data acquired from sequencing frequencies of conserved and nonconserved mirnas fits well with this extrapolation, where the nonconserved and species-specific mirnas show residual accumulation in the tested tissue. however, one mir-3954, a single member of an undefined family, appears to be expressed in significantly high levels. its only homolog deposited in mirbase v20 is in c. sinensis, showing high frequency of reads. 17 new jute specific mirnas identified in this study show a size anticipated for srnas derived from dcl1 processing, although sequence variants that possess shortened or extended 5 or 3 ends were also found. ten among the seventeen new col-mirnas are 21 nt in size, consistent with canonical dcl1 products. however length variation was also found. two, col-mir2 and col-mir9, are 20 nt in size; three, col-mir5, col-mir6, and col-mir14, are 22 nt long. col-mir13 was found to be 23 nt in size which can probably be explained by the fact that diverse mirna families are also independently processed by dcl3 to generate a new class of bona fide (2325 nt) mirnas with no canonical size, called long mirnas. a total of 20 mirnas of both conserved and species-specific origin were corroborated by stem-loop rt-pcr and their expression pattern was assessed by qpcr to validate the data obtained from deep sequencing. discrepancies in the expression pattern of some mirnas found by deep sequencing and qpcr can be attributed to practical differences between the sensitivity and specificity of these two techniques. the sensitivity and large dynamic range of next generation sequencing (ngs), along with its consistent prediction of fold changes when compared with gold-standard qpcr, support its use for discovery-oriented and exploratory mirna profiling experiments [84, 85]. to evaluate and outline a putative function for a mirna in plants, we have predicted target genes for known and potential new mirnas identified in this study using the genome of vitis vinifera as a reference. most of the target genes for conserved mirna families predicted in jute have already been confirmed in model plants, as target genes are commonly conserved [78, 80]. mir156/157-squamosa promoter-binding protein, mir166-homeodomain leucine zipper protein iii (hd-zip iii), mir167-auxin response factor (arf), mir164-nac domain protein, mir172-transcription factor apetala2, mir159-myb transcription factor, mir171-gras family transcription factor, mir394-f-box family protein, and mir395-atp sulfurylase well characterized mirna-target pairs in other plants have been found in jute. however, a number of widely studied mirna-target pairs, such as mir398-copper superoxide dismutase, mir399-e2 ubiquitin conjugating protein, and mir162-dicer-like 1(dcl1), were not found in this study. this could possibly be due to the fact that the jute genome sequence is not available to be used as a reference. however, conserved mirnas with their nonconserved targets, including mir167-peroxidase29, mir396-eukaryotic translation initiation factor 2c, mir168-nac domain containing protein, mir164-growth regulating factor 1, mir390-ap domain containing transcription factor, mir160-myb transcription factor, and mir393-gtp-binding protein alpha subunit, were also found to be present in jute, allowing presumption of nonconserved targets for conserved mirnas. highest number of target genes were identified for mir397, which is laccase, a well-studied enzyme, encoded by multigene families in poplar, arabidopsis, rice, and liriodendron tulipifera, reported to be involved in lignin biosynthesis of plants [99101]. toughness of this biopolymer also poses a major obstacle to pulping, forage digestibility, and biofuel production. it has been reported that transgenic p. trichocarpa plants overexpressing ptr-mir397a result in a reduction of klason lignin content, supporting the idea that use of mir397 would be an attractive means for reducing lignin-related problems. future experiments including in-depth studies of mir397-laccase pair may help in producing quality products from jute. this set of experimentations for identification of mirnas and their potential targets can initiate further study on understanding the mechanisms of regulation of jute mirna.

micrornas play a pivotal role in regulating a broad range of biological processes, acting by cleaving mrnas or by translational repression. a group of plant micrornas are evolutionarily conserved; however, others are expressed in a species-specific manner. jute is an agroeconomically important fibre crop; nonetheless, no practical information is available for micrornas in jute to date. in this study, illumina sequencing revealed a total of 227 known micrornas and 17 potential novel microrna candidates in jute, of which 164 belong to 23 conserved families and the remaining 63 belong to 58 nonconserved families. among a total of 81 identified microrna families, 116 potential target genes were predicted for 39 families and 11 targets were predicted for 4 among the 17 identified novel micrornas. for understanding better the functions of micrornas, target genes were analyzed by gene ontology and their pathways illustrated by kegg pathway analyses. the presence of micrornas identified in jute was validated by stem-loop rt-pcr followed by end point pcr and qpcr for randomly selected 20 known and novel micrornas. this study exhaustively identifies micrornas and their target genes in jute which will ultimately pave the way for understanding their role in this crop and other crops.

PMC4378336

pubmed-471

the use of laparoscopy in gynecology has developed into a vital tool for the evaluation and treatment of pelvic pathology. the predominant method of entry in gynecologic surgery worldwide remains a closed technique, with or without pneumoperitoneum. this approach unfortunately has been demonstrated in multiple studies to have the potential for visceral and vascular injury due to the blind insertion of veress needles or trocars. complications reported in the literature from the closed entry technique range from 0.05% to 0.67%, and include vascular injury, enterotomy, urinary tract injury, subcutaneous emphysema, and gas embolism. these complications arise due to the normal anatomic relationship of the periumbilical site to the underlying great vessels and viscera and are particularly problematic in patients with adhesions or at extremes of body weight. additionally, delay in diagnosis of bowel injuries frequently sited with this closed technique accounts for significant morbidity and mortality. this technique consists of creating a small umbilical incision under direct visualization to enter the abdominal cavity followed by the introduction of a blunt trocar. hasson proposed its potential benefits to be the avoidance of blind insertion of the veress needle and bladed trocar, prevention of preperitoneal insufflation and gas embolism, guaranteed pneumoperitoneum, and a more anatomical repair of the abdominal wall. since that time, hasson and others have corroborated these proposed benefits with data obtained from large case series. this large chart review was undertaken to examine our experience with open laparoscopy and to determine whether preoperative characteristics can predict open laparoscopic entry complication. we completed a retrospective chart review of all patients who underwent laparoscopy via an open technique at the mayo clinic arizona in the department of gynecology (8 surgeons) from january 1, 1998 through december 31, 2006. data were extracted from a computer-generated search, and each electronic medical record was individually reviewed to ensure inclusion criteria and obtain end points. all charts were reviewed from the point of surgery through the remainder of their care at our institution. intraoperative complications (bowel and vascular injury, failure to enter), postoperative complications (hernia, hematoma, cellulitis, abscess, cosmetic issues), body mass index (bmi), number of previous abdominal surgeries, and length of follow-up were extracted from charts. statistical analysis was performed using jmp 6.0 for windows (sas institute, cary, nc). the open laparoscopic technique used in this series is similar to that originally described by hasson and is as follows: the umbilicus is held and everted with 2 allis forceps. a vertical skin incision, 10-mm to 15-mm long, is created at the deepest portion of the umbilicus. the underlying fascia is grasped with 2 kocher forceps, elevated, and incised in a vertical midline fashion. if the peritoneal cavity is still intact, it is grasped with kocher or allis forceps and entered with metzenbaum scissors. finally, the laparoscope is introduced and the organs below the entry site are inspected as is the remainder of the abdominal cavity. closure of the umbilical entry site is performed with direct visualization and identification of the fascial layer. a 0-polyglactin suture on a #2 urologic needle is used for the fascial layer, and a 40 polyglactin suture is used for skin approximation. an occlusive dressing is removed from the site in the ensuing 24 hours to 36 hours. mean patient bmi was 26.5 (range, 14 to 57), mean previous abdominal surgery was 1.3 (range, 0 to 18), and mean follow-up was 340 days (range, 0 to 3028). at the time of entry with the open technique, we experienced 2 (0.1%) instances of enterotomy and 3 (0.1%) cases of failed entry. of the enterotomies, in one patient dense adhesions from a previous debulking laparotomy (requiring over 3 hours of laparoscopic adhesiolysis) were present with jejunum adherent immediately under the umbilicus. the second patient had the transverse colon adherent to the umbilicus; she had had 4 previous laparotomies. of the failed entries, the first patient had dense umbilical adhesions from 2 prior laparotomies, and the procedure was aborted. in the second patient, surgeons were unable to reach the peritoneal cavity due to the patient's pannus (bmi 43), and a left upper quadrant entry was chosen. the third patient had dense periumbilical adhesions from a previous colectomy, so the procedure was converted to a minilaparotomy. patients with umbilical infection were subclassified based on whether the diagnosis was made over the phone due to patient complaints of erythema or drainage, by examination and diagnosis of cellulitis, or an umbilical abscess requiring evacuation. patients with subsequent umbilical hernias were subclassified into symptomatic (noted by a physician or patient) or asymptomatic (discovered serendipitously at a subsequent surgery) (table 1). fisher's exact test was used to determine the association between each of the recorded complications and the number of previous abdominal surgeries and obesity (bmi>30) (table 2). a significant association was noted between umbilical infection and previous abdominal surgery (p=0.049), and between umbilical hernia and obesity (p=0.024). this chart review confirms previous findings of case series, literature reviews, and meta-analyses of the complications associated with open laparoscopic entry. a large dutch review of 12,444 cases of open laparoscopy culled from 6 previously published case series found a 0.048% rate of enterotomy and no cases of vascular injury or gas embolism. these rates contrast with an enterotomy rate of 0.083% and vascular injury rate of 0.075% from 489,335 cases of closed laparoscopic entries in this same review. similarly, an australian meta-analysis examined 22,465 cases of one published open laparoscopy series and 760,890 patients from 22 published closed laparoscopy series. they noted an enterotomy rate of 0.049% (11 patients) in open cases and 0.067% (515 patients) in closed cases (nonsignificant difference). no cases occurred of vascular injury in open cases, while the rate of vascular injury in closed cases was 0.044% (336 patients), which was statistically significant (p=0.003). similar rates of umbilical infection and hernia occurred with both techniques. to date, there are no reports of fatal vascular injuries and only 2 instances of nonfatal major vascular injuries in the literature associated with the use of the open technique. the first case occurred during the skin incision, when the scalpel directly entered the aorta (the hasson trocar had not been used). the second case involved a damaged metal hasson cannula with a protruding spike that caused an aortic laceration. the lack of fatal vascular injuries noted in this and other large series is of utmost significance. data from litigious allegations related to 135 laparoscopic procedures over a 19-year period revealed a disproportionate percentage of the cases involving vascular injuries. additionally, these injuries are associated with a significant mortality risk, with 5 of 9 vascular injuries at closed laparoscopic entry reported to the medical defense union resulting in death. moreover, the morbidity incurred with major vascular injury includes transfusions, prolonged hospitalization, loss of limb, or other long-term sequelae. visceral injuries, in particular small bowel and colon, are also life-threatening, and because they are commonly missed during a closed entry, and injury may later be recognized only when symptoms of peritonitis develop. although intestinal injuries occurred in this case series, a major advantage of the open technique is the immediate recognition and repair of the enterotomy. neither of the 2 patients in this series suffered any long-term complications related to enterotomy. previous abdominal surgery and obesity are therefore not contraindications to an open laparoscopic entry, and in fact the open approach may offer some advantage over the closed technique in these patients. several series have reported lower hernia formation with the open technique, perhaps because of the ability to easily identify the fascial layer during closure. the fact that none of the hernias occurred prior to 6 weeks postoperatively also suggests that a good primary reappoximation was achieved at surgery. although this is a retrospective review of patients presenting to a referral center with somewhat limited follow-up, the large number of procedures performed via a consistent technique enhances the strength of this study. additionally, each case was hand reviewed for complications rather than using icd-9 codes to assess for rate of complications. finally, when available, patients charts for the entire time they received care at our institution, in some cases up to 8 years, were reviewed. the use of an open laparoscopic entry is advocated because it is a safe, simple means of accessing the peritoneal cavity. this case series confirms previous reports of the low risk of enterotomy, absence of fatal vascular injury, and comparable rates of umbilical infection/hernia associated with an open entry technique. the rapid recognition of enterotomy with this entry technique, and the utility of this technique in obese patients or those with previous abdominal procedures are additional advantages.

objective: we assessed safety and efficacy of an open laparoscopic entry technique. methods:a retrospective review of all patients undergoing laparoscopy via open laparoscopic access over an 8-year period from january 1, 1998 to december 31, 2006 is presented. results:during the study period, 2010 consecutive subjects underwent laparoscopy. recorded intraoperative complications include enterotomy (0.1%) and failure to enter (0.1%). there were no instances of vascular injury related to entry. recorded postoperative complications include hernia (0.9%), infection (2.5%), hematoma (0.05%), and noncosmetic healing (0.4%). a statistically significant association existed between obesity and postoperative hernia, and between previous abdominal surgery and postoperative infection. conclusion:though typically straightforward, initial entry is one of the most common causes of injury in laparoscopy. the predominant entry method of entry in gynecologic surgery remains a closed technique. this technique has unfortunately been demonstrated in multiple series to have the potential for visceral and vascular injury due to its blind insertion of veress needles and trocars. the open laparoscopic technique is a safe and effective method of obtaining access to the abdominal cavity with no associated vascular injury.

PMC3015995

pubmed-472

pulmonary hypertension (ph) is predominantly defined by a mean pulmonary artery pressure at rest greater than or equal to 25 mm hg. it is an enigmatic vascular disease and the pathogenesis of ph is multifactorial of origin and, hence, is categorized as idiopathic type [14]. as ph develops in a wide variety of clinical circumstances and is associated with diverse histological manifestations, a classification system is developed [5, 6]. the dana point expert group has published a consensus of ph classification based on pathology, survival, natural history/epidemiology, etiology, and response to the treatment. among them, pah specifies that the disease primarily restricted to the pulmonary arterioles, a typical characteristic which shows an elevated pulmonary arterial pressure [2, 3]. the pathological consequence of pah is the structural remodeling of pulmonary arteries (pa), where increased proliferation of pulmonary artery smooth muscle cells (pasmc) and dysfunction of pulmonary artery endothelial cells (paec) occur in the vascular bed [79]. the morphological changes consist of hypertrophy of the tunica media, multicellular vascular lesions which obstruct and obliterate pulmonary arterioles leading to intimal thickening. the obstructed vessels limit the blood flow via pa and increase right ventricular afterload leading to right ventricular hypertrophy (rvh) and rv dysfunction [1012]. at molecular level, it is believed that the remodeling events in ph demand the participation of all cell-types present in the pulmonary arteries and that influence the pathological manifestation in the pulmonary vessel wall. the contributing factors that influence the remodeling process are hypoxic state, inflammation, vessel injury, and oxidative stress in the pulmonary vessels. as all forms of ph have in common an altered production of various endothelial vasoactive mediators, such as nitric oxide, prostacyclin, or endothelin- (et-) 1, to establish the correct balance between vasoconstriction and vasodilatation [1316]. currently, the management for ph is aimed at optimizing cardiopulmonary interactions by targeting prostacyclin, endothelin, and nitric oxide signaling pathways. the most commonly used treatment regimen of ph is the use of prostacyclin analogues (alprostadil, epoprostenol, treprostinil, and iloprost), endothelin receptor antagonists (bosentan, ambrisentan), and inhaled no. in addition, phosphodiesterases (pdes) inhibitors; pde-3 inhibitors (e.g., milrinone and enoximone), and pde-5 inhibitors (e.g., sildenafil and tadalafil) are used to treat ph. they were used as an alternative therapeutic strategy which targets downstream components of the no signaling pathway by inhibiting pde-5, the enzyme that catalyzes the conversion of cgmp to gmp. despite the advancement of modern surgery or ph-specific therapy, although ph (or pah) is well-studied encompassing both cardiac and vascular boundaries, the precise cellular and molecular mechanism of initiation and progression of ph are not completely understood and are still being explored. there is no cure of this disease and current therapies are limited to reverse the vascular remodeling. evolving evidence indicates that dysregulation of micrornas (mirna or mir) contributes to ph pathogenesis [1924]. indeed, an emerging body of evidence demonstrates that a fine balance in mirna levels seems to be a fundamental to maintaining homeostasis in the pulmonary vasculature and an imbalance with mirna level playing a critical role in the pathogenesis of ph by regulating a set of targeted genes. this review will collate vascular remodeling during ph, mirna biogenesis, recent advances on mirna modulation in ph, therapeutic opportunity, and conclusion. the mirna(s) are composed of a vast family of short, noncoding rnas (~22 nucleotide long). mirnas are found from a single cell organism to plant and higher animals and even viruses [3739]. in humans, the biogenesis (canonical) pathway of mammalian mirnas is a two-step enzymatic process initiated in the nucleus and then transported to cytoplasm (figure 1). the transcription of mirnas generally processed by rna polymerase ii (less frequently by rna polymerase iii) and are typically capped and polyadenylated [41, 42]. as a result, a long primary mirna transcripts (pri-mirna) containing a stem-loop structure is developed which is recognized by a large protein complex, called the microprocessor, the main components of which are the rnase iii drosha and digeorge syndrome critical region 8 (dgcr8) [4348] (figure 1). for most pri-mirna, drosha is contributing the cleavage process called cropping with the assistance of its binding partner protein, dgcr8 [41, 48, 49]. the resultant product is a ~60 nucleotides long, hairpin-structured, called precursor mirna (pre-mirna). dgcr8 directly interacts with the pri-mirna stem and flanking segments, a crucial measurement for one end of the mature mirna. in addition to the canonical pathway of mirna biogenesis described above, an alternative pathway also exists that are independent of drosha. in alternative pathways, the mirna can be released as mitrons (pre-mirna like introns) from pri-mirna and proceed for mirna processing unit without assistance of microprocessor [50, 51]. the pre-mirnas are actively transported from the nucleus into the cytoplasm by exportin-5 (exp-5) (figure 1). in the cytoplasm, another rnase iii enzyme, called dicer, catalyzing the process with the assistance of partner molecules argonaute (ago2), hiv-1 transactivation response rna-binding protein (trbp) and/or protein activator of pkr kinase (pact), producing a short dsrna duplex, mirna/mirna of approximately 22-nucleotide length [44, 5356]. the mirna/mirna duplexes are then incorporated into a ribonucleoprotein (rnp) complex called mirisc (rna-induced silencing complex)that plays a critical role in the mirna-mediated mechanism of gene regulation. during assembly process of the mirisc, the mirna/mirna duplex is loaded into the ago, and the strands (called the passenger strand) are released and degraded. eventually, the bound mirna strand (called the guide strand) dictates mirisc to interact with partially complementary sequences in target transcripts (localized within the 3utr) and primarily triggers mrna deadenylation and degradation. micrornas (mirnas) are small, endogenously expressed noncoding rnas that regulate gene expression at posttranscriptional level, via degradation or translational inhibition of their target mrnas [37, 59]. mirnas are ~22 nucleotides in length which bind to the 3 untranslated region of specific target genes and thereby suppress/inhibit the translation of target genes [60, 61]. mirnas are key regulators of a wide range of cellular processes and play a pivotal role in vascular inflammation and cardiovascular pathologies inclusive of ph. from extensive studies from the past few years, it has become apparent that mirnas are expressed in a cell- and tissue-specific manner and are critically involved in various biological processes. emerging evidence indicates that mirnas contribute an important role in the maintenance of pulmonary vascular homeostasis and in the pathogenesis of ph. in the following part, a discussion of the roles of mirnas in ph-related signaling pathways is provided. mir-21 is a ubiquitously expressed mirna that is traditionally considered to be an oncogenic mirna (oncomir). the two-channel microarray was performed to quantify mirna expression in whole lung extracts during the development of ph or pah caused by chronic hypoxia or monocrotaline in rats. it was suggested that mir-21 expression was downregulated in mct-induced ph model, but not in chronic hypoxia rats. mir-21 showed a similar expression level in both normoxic hypoxic cells, whereas tgf-1, an important regulator of pulmonary vascular remodeling in ph, reduced expression of mir-21. this suggests that although hypoxia- and monocrotaline-induced ph shares some common cellular processes driving the characteristic vascular remodeling, the different pathobiology induced by hypoxia and monocrotaline may lead to the different regulation of mir-21 expression. furthermore, the downregulation of mir-21 was confirmed in human lung tissue and serum from patients with idiopathic ph. since bmps induce smooth muscle cell differentiation through upregulating the expression of mir-21, the downregulation of mir-21 in this setting may relate to the reduced bmp signaling and contribute to the alteration of smooth muscle cell phenotype in ph. however, yang and colleagues reported that mir-21 expression was increased in distal small pulmonary arteries of hypoxia-exposed pah mice and levels of bmpr2, wwp1, satb1, and yod1, the putative mir-21 targets, were decreased in the same tissue. transfection of mir-21 mimics also led to the reduced expression of bmpr2, satb1, and yod1 in pasmcs. the crucial role of mir-21 in vascular pathology has been evidenced by the results that the blockade of mir-21 impeded the development of intimal hyperplasia after acute vascular injury and bleomycin-induced pulmonary fibrosis [28, 64]. yang et al. also found that inhibition of mir-21 alleviated chronic hypoxia-induced ph and attenuated pulmonary vascular remodeling. in human pasmcs, overexpression of mir-21 promoted, whereas sequestration of mir-21 abrogated cell proliferation and the expression of cell proliferation-associated proteins. the mir-21 null mice showed an exaggerated ph response to hypoxia, suggesting a major role of mir-21 in the pathogenesis of chronic hypoxia-induced pulmonary vascular remodeling and ph. although an association between mir-21 and ph (pah) is identified, the function of mir-21 in the development of ph is inconsistent in many experimental models. therefore, further investigations are required to clarify the role of mir-21 in the pathogenesis of ph. the mir-204 appears to be the first mirna that showed a mechanistic link between pulmonary arterial remodeling and cellular function. mir-204 was reported to be decreased in rodent lungs with hypoxia- and mct-induced pah and lung specimens from patients with pah. the reports suggested that stat3 activation contributed a crucial role in regulating mir-204 in pasmc. it was further demonstrated that mir-204 inhibition increased expression of shp2, triggered the activation of src kinase, stat3, and nuclear factor of activated t cells (nfat), and thereby reduced apoptosis and was promoted proliferation of pasmcs. finally, delivery of synthetic mir-204 mimic to the lungs lowered pulmonary artery pressure, reduced medial wall thickness, normalized levels of mir-204, shp2, and stat3, and alleviated the disease severity. this study may indicate the safe and effective use of mimic delivery to the pulmonary vasculature for future therapeutic purpose. interestingly, wei et al. have identified reduced level of mir-204 in the buffy coat of human subjects may correlate with pah severity and might serve as a circulatory biomarker for ph. expression of mir-143/145 was driven by tgf- and bmp4 and induced contractile gene expression through downregulating klf4 and myocardin [31, 68, 69]. the elevated levels of mir-145 were observed in primary pasmcs cultured from patients with bmpr2 mutations and also in the lungs of bmpr2-deficient mice, suggesting a role of bmpr2 signaling in modulating mir-145 expression. caruso and colleagues also found that mir-145 was downregulated in patient samples obtained from idiopathic and congenital ph but upregulated in plexiform lesions. the role of mir-145 in pah was further explored and elevated expression of mir-145 was shown in the wild type mice exposed to hypoxia. mir-145 deficiency and a locked nucleic acid anti-mir-145 resulting in significant protection from hypoxia-induced ph may represent a potential therapeutic target. the mir-17-92 cluster encodes seven related mirnas, which result from the transcription of a single pre-mirna and is further processed and cleaved to the mature mirnas. mir-17-92 cluster was retrieved as potential modulators of bmpr2 signaling by performing a computational algorithm on the bmpr2 gene. overexpression of mir-17-92 resulted in a marked reduction of bmpr2 protein level, and bmpr2 was proved to be directly targeted by mir-17 and mir-20a by using a bmpr2 reporter in hek293 cells. stat3 was found to be involved in il-6 signaling-mediated upregulation of mir-17-92 cluster and the subsequent downregulation of bmpr2, since a highly conserved stat3-binding site exists in the promoter region of mir-17-92 gene. the role of a cholesterol-modified antagomir to mir-20a in hypoxia-induced ph was explored by brock et al. mir-20a restored functional bmpr2 signaling in human pasmcs and intraperitoneal administration of anti-mir-20a increased bmpr2 levels and alleviated vascular remodeling in lung tissue of hypoxic mice. antagomirs to mir-17 and mir-92a reduced muscularization of pulmonary arteries in the hypoxic mouse and monocrotaline rat models of ph, but only anti-mir-17 decreased rvsp and parameters of right heart dysfunction. bertero and colleagues identified mir-130/301 family as a master regulator of cellular proliferation in ph by constructing in silico a network of genes and interactions based on curated seed genes with known importance in ph. mir-130/301 expression was found to be increased in lungs of mice suffering from ph induced by su5416 administration with chronic hypoxia, in lungs of rats with mct-induced ph and in lungs of juvenile sheep with shunt-induced ph. in both human paecs and pasmcs, multiple ph inducers, including hypoxia, il-1, and il-6, increased mir-130/301 expression and hypoxia was found to upregulate mir-130/301 via a dependence on hif-2 and pou5f1/oct4. it was further validated that mir-130/301 modulated apelin-mir-424/503-fgf2 signaling in paecs and mir-130/301-ppar- axis controlled proliferation of pasmcs by increasing stat3 expression and activity and repressing mir-204 expression. in hypoxia-induced ph mouse model, induction of mir-130/301 was promoted while mir-130/301 inhibition prevented ph pathogenesis. ph is broadly considered to be a vascular disease as vasoconstriction in the pulmonary artery is a prime cause for the development of ph. the hyperproliferation of pulmonary vascular cells (mainly vsmc) and subsequent neointima formation in the small pas are hallmark in ph. on the other hand, pulmonary arterial endothelial cells (paecs) further contribute a critical role in vascular homeostatic balance in the pulmonary vascular bed by orchestrating the vessel tone, leucocytes trafficking, and so forth [74, 75]. proliferation of fibroblasts is reported in hypoxia-induced ph that plays a role in adventitial thickening [77, 78]. under pathological situation like ph (pah), the endothelium becomes dysfunctional which allows the penetration of infiltrating molecules, increased leucocytes adhesion, getting resistant to apoptosis that result in severe remodeling in the pulmonary vessels. these infiltrating molecules invade the barrier of neighboring cells like smooth muscle cells and activating the resident of adventitious fibroblasts. the remodeling process is basically an interaction between these cell-types present in the pulmonary arterial layers that resulting in a marked histological change in the pulmonary vasculature. an emerging body of evidences suggests that the mirnas play a pivotal role in the vascular cell integrity and plasticity during the progression of ph. here, although identification of mirnas in vascular diseases like ph is relatively new, but the earliest association connecting vsmc remodeling with mirnas was reported in 2007. described the mirna signature in the vascular wall of balloon-injured carotid artery rat model. they showed that mir-21 was upregulated and the cellular effects were targeted by pten and bcl2 for neointimal lesion formation. the initial studies of mir-21 in vascular remodeling were followed by series of milestone works that include mir-143/145 cluster [24, 31, 7983]. two of the key mirnas involved in regulation of the phenotype of smcs are mir-143 and mir-145. mir-145 is transcribed bicistronically along with mir-143 from human chromosome 5. with relevance to pah, downregulation of mir-145 downregulation of target genes klf4 and klf5 by activation of mir-143/mir-145 upregulates smc-specific genes such as sma, calponin, and sm22-, triggering differentiation and lowering the proliferation rates in vascular smcs [31, 82]. another mirna, mir-221/222 that triggers pdgf signaling in vsmc is thought to contribute in neointimal proliferation [35, 84, 85]. it is demonstrated that inhibition of mir-221 prevented pdgf induced proliferation, while forced expression of mir-221 increased proliferation and reduced the expression of vsmc marker [35, 84]. in addition to the above mirnas, other mirnas are reported to regulate smc gene expression in hypoxia-induced ph. it is demonstrated that mir-210 that acts as a hypoxia-inducible mirna both in vitro and in vivo, inhibits pasmc apoptosis in hypoxia by specifically repressing e2f3. a cell-based high throughput screening of a human mirna library with the nfat luciferase reporter system identified mir-124 as a new candidate in ph. it showed decreased nfat reporter activity and, decreased dephosphorylation and the nuclear translocation of nfat. an elegant study by courboulin et al. demonstrated that mir-204 downregulation correlates with ph severity and responsible for proliferative and antiapoptotic phenotypes of ph-pasmcs targeting stat3 and nfatc. interestingly, a recent study showed that a serine/threonine kinase mst1, a modulator of cell death, appears to be a target of mir-138. the authors suggest that mir-138 may be a negative regulator of pasmc apoptosis in hypoxic mediated pah. a panel of mirnas which are shown to modulate smc fate and modulation are listed in table 1 [2636]. therapeutic target which defines the treatment of a disease by means of a well-defined biological molecule. in this tiny rna molecule, have become an important gene modulator of various biochemical, physiological, and cellular functions. as we observed that either deficiency or abundance of a specific mirna or cluster of mirnas contributed a pathological state of many cardiovascular diseases including ph, it is reasonable to accept those mirnas as therapeutic target for diagnosis and therapeutic intervention. this is because we can utilize ability of a single mirna to control the expression of multiple (hundreds) proteins suggest that changes of a single mirna may influence several signaling pathways associated with pathological disease state. while each target is regulated subtly, the additive effect of coordinated regulation of a large suite of transcripts is believed to result in strong phenotypic outputs. several animal models in diverse disease pathology showed usefulness of mirnas as target molecules for therapeutic benefit; this review will focus on vascular remodeling associated with ph. at therapeutic strategic stand-point, the aberrant mirna expression can be modulated or restored to normal by two main approaches: an anti-mirna (anti-mir) and mirna mimic. the former can be applicable to those mirnas whose expressions were increased in disease pathology and, therefore, silencing or inhibiting will be beneficial. to modulate mirna expression, the current strategy utilized chemically modified, cholesterol-conjugated single-stranded rna analogues complementary to the mature mirnas for antagomir or mimics, respectively. an elegant work demonstrated by liu et al. in a balloon-injury model represents a successful knocking down of mir-221/222 that suppressed vsmc proliferation and neointimal lesion formation. recently, brock et al. have shown that treatment with antagomir-20a restores the levels of bmpr2 in pulmonary arteries and prevents vascular remodeling in hypoxia-induced pah. for downregulated mirnas, a mimic approach is generally undertaken which basically rescued the underexpressed mirna in the tissue under pathological situation. this strategy is sometimes referred to as mirna replacement as it reintroduced the similar depleted mirna those were downregulated during diseases progression. therefore, by introducing the mirna mimic, the cells can restore the function as the mirna mimic is expected to target the same set of mrnas that is also regulated by the endogenous mirna. in a very comprehensive study by courboulin et al., they demonstrated that reestablishing of mir-204 level by delivery of synthetic mir-204 into the lungs of experimentally-induced pah animals significantly reduced the disease pathology. although the mirna-based intervention is attractive and seems to be feasible, however, several challenges are noted. first, the critical mirnas responsible for ph must be confirmed definitively in well-accepted animal models along with explanted human pah samples, biopsies, and so forth, second, the cellular, molecular and physiological function of these mirnas should be performed in diseases progression and prevention; third, the delivery route should be precisely targeted or restricted to lung vascular cells, for example, paec, pasmc, or fibroblasts. finally, the dose of antagomir or mimic delivery to the vascular bed should be carefully monitored further to avoid off-target effects. therefore, our future direction should be focus of vascular cell-specific delivery of mirna mimic or inhibitors. additionally, a regulated release of mirnas by conjugating nanoparticle will be considered for long-term use. finally, mirna-mediated therapeutics can be achieved by inhalation which may reduce the potential off-target issues to the other organs. biomarker generally refers to measurable substance in biological state. at clinical stand-point, it is extremely important as it predicts the medical outcome of a disease and its progression. over the past few years, it has been demonstrated that mirnas are found in the blood, plasma, urine, platelets, and saliva in a surprisingly stable form [9197]. the stability of mirna in the extracellular environment offers a great opportunity to consider as biomarker at clinical settings. using mirna as a biomarker offers many advantages like early prediction of diseases, differential expression during disease progression/pathologies, high degree of specificity, and sensitivity and importantly having longer half-life in the system. accumulating evidence suggested that the stability of mirna in extracellular environment illustrated by packed with lipid vesicles, wrapped in protein or lipoprotein complexes [98100]. in the context of cardiac diseases that include myocardial infarction, hypertrophy and heart failure; several mirnas are identified and predicted to be considered as biomarker [101104]. the mirnas are circulating freely in the mammalian blood and can be predicted as biomarker for early diagnosis of cardiovascular diseases in humans [102, 105108]. several evidences indicate that mirnas are secreted as micro vesicles or exosome and apoptotic bodies that may be responsible for release the mirnas into the circulation [109112] and are extremely stable in the blood or serum. rhodes and colleagues measured plasma levels of mirnas in eight patients newly diagnosed with pah and eight healthy controls by use of microarray analysis. fifty-eight mirnas showed differences in plasma concentration between the two groups and mir-150 was largest downregulated in pah. reduced expression of mir-150 predicted 2-year survival and correlated with disease severity. the mir-150 level was also found to be significantly reduced in circulating microvesicles and lymphocytes from patients with pah. courboulin et al. reported that downregulation of mir-204 in buffy-coat cells correlated with pah severity and might serve as a circulatory biomarker of pah. on the contrary, increasing higher plasma levels of mir-130/301 family members were observed in patients with increasing hemodynamic severity of ph. our previous study detected the pattern of mirnas in buffy-coat samples from mild-to-severe human ph subjects compared with the control subjects by mirna array. our study revealed that moderate to severe ph in human subjects are associated with significant downregulation of plasma levels of circulatory mir-1, mir-26a, and mir-29c. the kinetics of the changes differed from moderate to severe ph human subjects as we observed further downregulation of the above mirnas in severe ph. mir-21, mir-23b, mir-130a, mir-491, and mir-1246 were moderately upregulated in moderate ph subjects and were more pronounced in severe ph. another set of mirnas that include mir-133b, mir-204, and mir-208b were significantly upregulated in all moderate ph subjects and showed further increment in severe ph subjects. our study provided the evidence for the first time that circulating mirnas in the setting of ph may be used as early detection parameters for ph. nevertheless, the data presented were based on limited study population and the results need to be confirmed in a larger cohort study. furthermore, the dysregulation mirna in ph remains to be tested whether these circulatory mirnas have any influence in the target genes. circulatory mirnas are considered to be novel promising biomarkers for detection of ph. however, at current stage, using circulatory mirna as diagnostic tool for detecting ph is still in its infancy. the key advantage of circulating mirnas is a noninvasive testing procedure and relies on the stability of mirnas in the circulation and their storage condition. we give credit to the concept of quick determination of secretory molecules (mirnas) in the blood; but the importance of mirna must be reevaluated at the clinical setting or model at multiple laboratories. another limitation is the small number of cohort study which appears to be heterogeneous clinical sample-type. these points may consider as preliminary-type of observation and need further confirmatory studies for considering mirnas as biomarkers for clinical use. understanding of mirnas role and function in the pulmonary circulation will offer and contribute a great potential to the pathogenesis of ph or pah. the mirna signature can be used as a diagnostic tool for the development of therapeutic strategy. the characterization of these mirnas under various experimental settings (in vitro and in vivo) should be performed along with the human subjects. the outcome will validate both physiological and pathological roles of these mirnas during the development and progression of ph prior to consider them for therapeutic intervention or future clinical trials. furthermore, use of disease specific mouse models, conditional transgenic, and knockouts are necessary to elucidate the functional role in vivo and verify its therapeutic potential, both of which remain largely unexplored. more novel approaches such as combination of argonaute protein immunoprecipitation, deep sequencing, or proteomic profiling approaches are required to identify its potential bona fide targets responsible for its functional impact.

micrornas (mirnas) have emerged as a new class of posttranscriptional regulators of many cardiac and vascular diseases. they are a class of small, noncoding rnas that contributes crucial roles typically through binding of the 3-untranslated region of mrna. a single mirna may influence several signaling pathways associated with cardiac remodeling by targeting multiple genes. pulmonary hypertension (ph) is a rare disorder characterized by progressive obliteration of pulmonary (micro) vasculature that results in elevated vascular resistance, leading to right ventricular hypertrophy (rvh) and rv failure. the pathology of ph involves vascular cell remodeling including pulmonary arterial endothelial cell (paec) dysfunction and pulmonary arterial smooth muscle cell (pasmc) proliferation. there is no cure for this disease. thus, novel intervention pathways that govern ph induced rvh may result in new treatment modalities. current therapies are limited to reverse the vascular remodeling. recent studies have demonstrated the roles of various mirnas in the pathogenesis of ph and pulmonary disorders. this review provides an overview of recent discoveries on the role of mirnas in the pathogenesis of ph and discusses the potential for mirnas as therapeutic targets and biomarkers of ph at clinical setting.

PMC4377470

pubmed-473

coronary artery bypass grafting (cabg) is considered to be the gold standard in patients with multivessel disease and remains the treatment of choice for patients with severe coronary artery disease, including three-vessel or left main coronary artery disease. the use of cabg, as compared with both percutaneous coronary intervention (pci) and medical therapy, is superior with regard to long-term symptom relief, major adverse cardiac or cerebrovascular events and survival benefit [14]. however, because of the use of cardiopulmonary bypass and median sternotomy, cabg is associated with significant surgical trauma leading to a long rehabilitation period and delayed postoperative improvement of quality of life. an alternative hybrid approach to multivessel coronary artery disease combines surgical left internal thoracic artery (lita) to left anterior descending coronary artery (lad) bypass grafting and percutaneous coronary intervention of the remaining lesions [3, 68]. ideally, the lita to lad bypass graft is performed in a minimally invasive fashion through minimally invasive direct coronary artery bypass grafting (midcab). this hybrid approach takes advantage of the survival benefit of the lita to lad bypass, while minimizing invasiveness and lowering morbidity by avoiding median sternotomy, rib retraction, aortic manipulation, and cardiopulmonary bypass [3, 8, 1014]. the purpose of the hybrid approach is to achieve complete coronary revascularization with outcomes equivalent to conventional coronary artery bypass grafting, while ensuring faster patient recovery, shorter hospital stays, and earlier return to work due to lower morbidity and mortality rates. angelini and colleagues reported the first hybrid coronary revascularization (hcr) procedure in 1996, and several patient series using hybrid coronary revascularization have been published since then. these series support the above-mentioned presumptions and indicate that the hybrid approach is a feasible option for the treatment of selected patients with multivessel coronary artery disease involving the left main. moreover, the introduction of drug-eluting stents (dess) with lower rates of restenosis and better clinical outcomes may make hybrid coronary revascularization a more sustainable and feasible option than previously reported [9, 15]. nevertheless, this hybrid approach has not been widely adopted because practical and logistical concerns have been expressed. these concerns implicate the need for close cooperation between surgeon and interventional cardiologist, logistical issues regarding sequencing and timing of the procedures, and the use of aggressive anticoagulant therapy for percutaneous coronary intervention that may worsen bleeding in the surgical patient [7, 14, 16]. this review aims to clarify the place of hybrid coronary revascularization in the current therapeutic armamentarium against multivessel coronary artery disease. second, the results of previous patient series using the hybrid approach are summarized and interpreted. the medline/pubmed database was searched in january 2012 using the medical subject headings (mesh) for coronary artery disease and angioplasty, balloon, coronary combined with the following free-text keywords: multivessel coronary artery disease, minimally invasive coronary artery bypass, percutaneous coronary intervention, and hybrid coronary revascularization. one hundred seventy-seven articles matching these search criteria were found, and the search for additional papers was continued by analysing the reference lists of relevant articles. randomized controlled trials, nonrandomized prospective and retrospective (comparative) studies were selected for inclusion. letters, editorials, (multi)case reports, reviews, and small studies (n<15) were also excluded. studies examining the hcr procedure for multivessel coronary disease were included, while studies investigating the hcr procedure for left main coronary stenosis were excluded. authors and medical centres with two or more published studies were carefully evaluated and were represented by their most recent publication to avoid multiple reporting of the same patients. a total of eighteen included studies remained eligible for analysis after applying these in- and exclusion criteria (figure 1). the primary outcome measures were in-hospital major adverse cardiac and cerebrovascular events (macces), packed red blood cells (prbcs) transfusion rate, lita patency, hospital length of stay (los), 30-day mortality, survival, and target vessel revascularization (tvr). secondary outcome measures were intensive care unit (icu) los and intubation time, as only a limited number of studies reported these outcome measures. in addition, the period of time between pci and lita to lad bypass grafting and the cost effectiveness of hcr were examined. the long-term lita patency was not included as an outcome measure, since only a limited number of studies report this outcome measure in a clear and concise manner. in-hospital major adverse cardiac and cerebrovascular events were defined as postoperative stroke, myocardial infarction (mi), or death during hospital stay. only the fitzgibbon patency class a (widely patent) was considered as a patent lita to lad bypass graft, while the fitzgibbon patency class b (flow limiting) and c (occluded) were defined as a nonpatent lita to lad bypass graft. hospital los was defined as the number of days spent in hospital from operation to discharge. if the need for repeated revascularization involved a coronary artery initially treated with either bypass grafting or pci, this repeated revascularization was considered to be target vessel revascularization. one observer extracted all available outcome measures of each article and a second observer checked and supervised the first observer thoroughly. when an article did not disclose one or more of these outcome measures or reported medians and ranges as central tendency instead of means and standard deviations, the study was excluded from the analysis of that particular variable. the results were analysed using ibm spss statistics 19 software (ibm inc., armonk, continuous data were presented as mean and standard deviation (sd), while categorical data were expressed as numbers and percentages. nine hundred seventy patients undergoing hcr procedures were included for analysis (tables 1 and 2) [6, 7, 1114, 1728]. the most important findings are reported below. the classical indication for an hcr procedure is multivessel coronary artery disease involving lad lesion judged suitable for minimally invasive lita to lad bypass grafting but unsuitable for pci (type c), and (a) non-lad lesion(s) (most of the time right coronary artery (rca) and/or circumflex coronary artery (cx) lesions) amenable to pci (type a or b) [7, 11, 12, 14, 17, 18, 20, 22, 23, 2628]. high-risk patients especially with severe concomitant diseases (e.g., diabetes mellitus, malignancies, significant carotid disease, severely impaired lv function, and neurological diseases), who are more prone to develop complications after cardiopulmonary bypass and sternotomy, might benefit from the circumvention of cpb and sternotomy [11, 18, 20, 2224]. exclusion criteria for hcr consist of contraindications to minimally invasive lita to lad bypass grafting or pci. lita to lad bypass grafting in a minimally invasive fashion requires single-lung ventilation and chest cavity insufflation. therefore, hcr procedures are contraindicated in patients with a compromised pulmonary function (i.e., forced expiratory volume in one second less than 50% of predicted) and a small intrathoracic cavity space [14, 27, 28]. moreover, patients with a nongraftable or a buried intramyocardial lad, history of left subclavian artery and/or lita stenosis, morbid obesity (bmi>40 kg/m), and previous left chest surgery are not well suited for minimally invasive lita tot lad bypass grafting [14, 20, 22, 27, 28]. conditions rendering pci unsuitable include peripheral vascular disease precluding vascular access, coronary vessel diameter smaller than 1.5 mm, tortuous calcified coronary vessels, fresh thrombotic lesions, chronic totally occluded coronary arteries, extensive coronary involvement, chronic renal insufficiency (serum creatinine 200 mol/l), and allergy to radiographic contrast [7, 14, 18, 20, 22, 27, 28]. finally, haemodynamic instability, need for a concomitant operation (e.g., valve repair or replacement), and decompensated congestive heart failure are regarded as exclusion criteria [7, 17, 20, 22, 27, 28]. three hcr strategies can be distinguished: (i) performing pci first, followed by lita to lad bypass grafting or (ii) vice versa; (iii) combining lita to lad bypass grafting and pci in the same setting in a hybrid operative suite. in the included studies, staged hcr procedures (i and ii) were applied much more frequently than simultaneous procedures (iii). in a staged procedure, in which pci and lita to lad bypass grafting are carried out at separate locations and/or different days, both interventions can be performed under ideal circumstances (in a modern catheterization laboratory and modern operating room, resp.) [11, 18, 29]. however, patients have to undergo 2 procedures, while they remain incompletely revascularized and at risk for cardiovascular events for an extended period of time [14, 29]. when pci is performed first, a staged procedure takes place with an unprotected anterior wall, which could pose serious health risks in case the lad lesion is considered the culprit lesion. in addition, lita to lad bypass grafting is performed after aggressive platelet inhibition for prevention of acute (stent) thrombosis, which might lead to unnecessary postponement of following operation or may cause a higher than expected rate of bleeding [12, 13, 21, 29]. moreover, stent thrombosis is risked after reversal of surgical anticoagulation and is related to the inflammatory reaction after cardiac surgery. furthermore, the opportunity for quality control of the lita to lad bypass graft and anastomosis by a coronary angiogram is lost and, therefore, this strategy requires a reangiography [12, 13]. these repeat control angiograms increase overall healthcare costs unnecessarily and decrease cost effectiveness. first, revascularization of non-lad vessels provides an optimized overall coronary flow reserve, thereby minimizing the potential risk of ischemia and myocardial infarction during the lad occlusion for lita to lad bypass grafting [6, 12]. second, it is possible for the interventional cardiologist to fall back on conventional cabg in case of a suboptimal pci result or major pci complications. however, failure of pci leading to emergency conventional cabg has become extremely rare with decreasing incidence since the introduction of coronary artery stenting [12, 20, 2932]. furthermore, this strategy allows hcr in patients with the immediate need for pci in a non-lad target and no immediate possibility for emergency bypass surgery [11, 24]. critical stenosis in the right coronary artery (rca) or the left circumflex coronary artery (lcx) or difficult pci targets are considered as clear indications for a pci first approach because these patients can undergo conventional cabg in case of pci failure. when the lita to lad bypass graft is performed first, antiplatelet therapy is routinely started after surgery to prevent antiplatelet-related bleeding complications during surgery and is present at time of pci [6, 13, 27]. these antiplatelet agents can be administered long term, which is mandatory for preventing stent thrombosis. moreover, the quality control of the lita to lad bypass graft and anastomosis can be performed simultaneously without a further angiogram [6, 12, 13, 18, 20, 23, 25, 26, 29]. in addition, pci is performed in a protective environment with a revascularized anteroseptal wall, which probably reduces the procedural risks and gives the interventional cardiologist the ability to approach lesions that would be quite challenging without a revascularized lad [13, 20, 25, 26, 29]. however, patients undergoing this strategy could require a second, much higher-risk, surgical intervention due to complications of the pci [13, 23, 25]. finally, the cardiac surgeon has to be aware of possible intraoperative ischemia during this hcr strategy because the collateral, non-lad vessels are unprotected. nevertheless, combining the two procedures in one stage under general anaesthesia in a specific hybrid-operating room, which combines the potential of catheterization and cardiac surgery, has advantages compared with staged hcr procedures [7, 14, 25, 28]. this simultaneous approach represents a single procedure that achieves complete revascularization, while minimizing patient discomfort and reducing the need for anaesthetics [12, 14, 18, 20, 28]. this approach eliminates logistic concerns about timing and sequence of two separate procedures and maximizes patient satisfaction [7, 14, 25, 28]. moreover, the quality of the lita to lad bypass graft and anastomosis can be confirmed immediately by an intraoperative angiogram, which enables direct revision of the lita to lad bypass graft [18, 25]. complications and difficulties during pci or midcab can be dealt with immediately in the same setting by conversion to conventional, open-chest cabg. perioperative haemorrhage can become a problem because full antiplatelet therapy and incomplete heparin reversal are necessary instantly after midcab to prevent a transient rebound increase in thrombin formation associated with stent thrombosis and ensure an optimal intraoperative des placement [7, 14, 18]. besides, off-pump surgery may give rise to hypercoagulability and increased platelet activation during the early postoperative period, which is associated with an increased risk of stent thrombosis. therefore, a modified antiplatelet protocol and careful patient selection seem appropriate, especially in one-stop hcr, in order to minimize the risk of stent thrombosis without increasing perioperative bleeding risk. a tried and tested protocol of dual antiplatelet therapy (dapt) includes continuous use of aspirin (100 mg/day) until the operation day and intraoperative administration of a loading dose clopidogrel (300 mg) via a nasogastric tube after confirming lita graft patency, followed by a maintenance dose of 75 mg/day for 12 months. however, caution is required when using dapt, since reversal agents for clopidogrel and aspirin are not available. moreover, newer more potent antiplatelet agents, like prasugrel and ticagrelor, should be reserved exclusively for selected cases (high risk of stent thrombosis) and managed with even more care, since the clinical experience with these newer antiplatelet agents is limited in cardiac surgery and the bleeding risk may be increased. furthermore, intraoperative collaboration and communication among cardiac surgeons, interventional cardiologists, and anaesthesiologists should be outstanding and ongoing to optimize continuity of care [11, 14]. currently, this simultaneous procedure is used in only a few centres, and some authors state that this might be caused by the need to possess catheterization laboratories outfitted to accommodate cardiac surgery or hybrid operating rooms equipped with a mobile coronary angiography c-arm or permanent fluoroscopic equipment [7, 13]. the latter is reflected in the small number of patients undergoing a simultaneous procedure in our sample of included studies [7, 13, 14, 18, 24, 25, 28]. expansion of other percutaneous and hybrid procedures like hybrid af ablation may help to make these hybrid, multipurpose operating rooms more common in the future. however, staged hcr procedures could offer a more realistic alternative for many institutions without a so-called hybrid operating room, and this is supported by the fact that staged hcr procedures are applied much more frequently than simultaneous procedures in the included studies [6, 1113, 1724, 26, 27]. tables 3 and 4 present the period of time between both procedures in a staged hcr strategy, and this period of time varied notably from 0 to 180 days. therefore, some patients remained incompletely revascularized and were in theory at risk for cardiovascular events for a considerable length of time, while complete myocardial revascularization should be the main goal of treatment in patients with multivessel coronary artery disease. moreover, delhaye et al. found that pci with clopidogrel preloading can be performed within 48 hours of lita to lad bypass grafting without increasing the bleeding risk. in addition, zenati et al. performed pci zero to four days after lita to lad bypass grafting without increasing the prbc transfusion requirements, while lowering the hospital length of stay (2.7 1.0 days). the mean hospital length of stay was 5.5 1.8 days (range: from 2.7 to 8.2 days), and hospital length of stay seems not to be influenced by the hcr strategy used (table 2). as shown in table 1, the surgical techniques for lita to lad bypass grafting have evolved continuously since the introduction of the hcr procedure in 1996 by angelini et al. most of the initial patient series performed the lita to lad bypass graft in a minimally invasive fashion carrying out a mini-thoracotomy on the anterolateral chest wall in imitation of angelini et al. [3, 7, 12, 1719]. in this so-called minimally invasive direct coronary artery bypass (midcab) approach the anastomosis to the lad is performed with 8-0 or 4-0 prolene sutures on the beating heart (without cpb) with the help of mechanical stabilizers. in more recent patient series, the lita was identified and harvested thoracoscopically or robotically, which decreased rib retraction, chest wall deformity, and trauma [11, 14, 21, 22, 27]. this approach significantly minimizes the typical thoracotomy-type incisional pain and wound complications of conventional midcab, while optimizing graft length and retaining the reliability of manually sewn lita to lad anastomosis [21, 22]. some teams prefer to place the lita bypass graft to the lad through a ministernotomy (inversed l-shaped or reversed j-shaped), which makes it possible to switch to full sternotomy in case complications may occur during the original operation [20, 23, 28]. nevertheless, this surgical technique increases surgical trauma and, therefore, may raise morbidity and mortality. in addition, some centres even decided to perform the lita to lad bypass graft through a full sternotomy on the beating heart (off-pump cabg), thereby further increasing invasiveness [6, 25, 26]. if the lita bypass graft is placed on the lad through a sternotomy on the arrested heart (on-pump cabg), circumvention of cpb is lost too [6, 25, 26]. thus, both on-pump and off-pump cabg can be seen as suboptimal procedures to carry out the lita to lad bypass graft. this might explain the higher macce rates found by zhao et al. and delhaye et al. and the high 30-day mortality discovered by zhao et al. and gilard et al., who decided to place the lita to lad bypass graft on the arrested heart through full sternotomy in the majority of the patients [6, 25, 26]. lastly, some authors prefer to perform the lita to lad bypass graft in a totally endoscopic, port-only fashion using totally endoscopic coronary artery bypass grafting (tecab) [13, 24]. this most challenging form of lita to lad bypass grafting using robotic telemanipulation techniques was initially performed on the arrested heart with the use of peripherally introduced cardiopulmonary bypass with intraaortic balloon occlusion and cardioplegic arrest [13, 24]. a major disadvantage of this approach is the use of the heart lung machine, which increases the risk of stroke, bleeding, and an inflammatory response to surgery. the latter can be solved by using beating heart tecab (bh-tecab), in which cpb and its considerable drawbacks are avoided. total endoscopic completion of the lita to lad bypass graft on the beating heart requires an additional port subxiphoidally to place a specially designed endoscopic stabilizer, which stabilizes the heart to optimize the quality of the anastomosis. this so-called beating heart totally endoscopic coronary artery bypass (bh tecab) procedure might be the least invasive approach for coronary bypass surgery without making concessions to graft patency [24, 3538]. however, the tecab procedure is an extremely challenging and a potentially expensive procedure with an extensive learning curve, which may raise concerns about widespread adoption and application. the postoperative lita patency seemed to be independent of the surgical technique of lita to lad bypass grafting, since lita patency has shown to be approximately equal for all surgical techniques (table 2). the postoperative lita patency varied between 93.0% and 100.0% (mean: 98.8% 2.3%). the mean in-hospital macce rate was 1.3% 1.9% (range: from 0,0% to 5.6%) with relatively high macce rates shown by katz et al. strikingly, three of these authors (katz et al., zhao et al., and delhaye et al.) performed lita to lad placement on the arrested heart [13, 25, 26]. the percentage of patients requiring prbc transfusion varied considerably between 0.0% and 35.4% (mean: 13.6% 11.7%). the surgical technique or hcr strategy (staged versus simultaneous) used did not appear to affect the percentage of patients requiring prbc transfusion. overall, the 30-day mortality rate was 0.4% 0.8% (range: from 0.0% to 2.6%). interestingly, higher than expected 30-day mortality rates were found in studies (gilard et al. and zhao et al.) using on-pump cabg to perform the lita to lad bypass graft in the majority of patients [6, 25]. finally, the mean overall survival rate in hybrid treated patients was 98.1% 4.7% (range: from 84.8% to 100.0%). besides the technical improvements of lita to lad bypass grafting, innovations occurred in the field of pci. this development was supported by the increased rate of des implantation in later patient series compared to earlier patient series, which used percutaneous transluminal coronary angioplasty (ptca) only or ptca in combination with bms implantation. application of drug-eluting stents should lower the restenosis rate, but their potentially beneficial effect on the target vessel revascularization (tvr) is not supported by data from the included studies (table 2). however, the (early and late) patency rate of new generation drug-eluting stents in non-lad lesions, provided that proper dapt is applied, may already be superior to that of saphenous vein grafts. hard evidence is however lacking, since a head-to-head comparison of (early and late) patency rates between des (in non-lad lesions) and saphenous vein grafts is not available. finally, the introduction of bioresorbable scaffold (brs) technology may improve sustainability, safety and feasibility of future hcr interventions. the application of brs technology can make long-term dapt redundant reducing bleeding complications without increasing the risk of stent thrombosis and may allow future reinterventions or reoperations on the same vessel if necessary due to its bioresorbable features. a relatively small number of studies in our sample (table 5) compared the hcr procedure using minimally invasive lita to lad bypass grafting with conventional cabg or off-pump coronary artery bypass (opcab) [7, 12, 27, 28]. all four of these studies selected matched controls who had undergone elective cabg or opcab with lita and saphenous vein grafts through median sternotomy during the same period using propensity score matching [7, 12, 27, 28]. hu et al. found that patients in the hybrid group had a statistically significant shorter hospital length of stay, icu length of stay, and intubation time compared with opcab, while de cannire et al. reported that hospital and icu length of stay was statistically shorter in hybrid treated patients compared with patients treated with cabg [7, 12, 28]. showed that intubation time, icu, and hospital length of stay were similar between the hybrid and opcab group. moreover, these studies revealed that prbc transfusion requirements were reduced by the hybrid approach [12, 27, 28]. lastly, the in-hospital macce rates were considerably lower in the hybrid groups compared with both the cabg and the opcab groups. currently, only a few studies have explicitly explored the costs associated with hybrid coronary revascularization. de cannire and colleagues were the first to quantify costs associated with hcr and to compare these costs with costs involved in conventional double cabg. costs were calculated using six major expenditure categories: costs of hospital admission (including intensive care unit and postsurgical cardiac ward cost as well as costs associated with delayed repeat procedures), pharmaceutical costs, surgical costs, pci-related costs, costs of blood products, and other miscellaneous fees (including physiotherapy and consultants). the extra cost associated with pci (including stents) in the hybrid group in comparison with the cabg group (2.517 288 versus 0 0), which uses autologous grafts to treat non-lad lesions, counterbalanced the cost savings on all other expenditure categories, which resulted in a nonsignificant cost difference at 2 years between both groups (10.622 1329 versus 9699 2500; not statistically significant). it is worth mentioning that the reduced icu and hospital length of stay due to faster recovery were largely responsible for the cost reduction in the hybrid group compared with the cabg group (3.033 499 versus 4.156 1.413). showed that shorter intubation times, shorter icu and hospital length of stay, and less prbc transfusions resulted in a significant reduction in costs for hybrid treated patients in the postoperative period. conversely, intraoperative costs were statistically significant higher in patients undergoing hcr compared with opcab, largely because of longer operative times and the use of coated stents (des) rather than autologous grafts ($ 14.691 2.967 versus $9.819 2.229; p<0.001). in conclusion, the difference in intraoperative costs was almost completely outweighed by the lower postoperative costs in the hybrid group. this resulted in slightly, but not significantly, higher overall costs in the hybrid group. the nonhealthcare costs after hcr will presumably be lower than after cabg or opcab because both kon et al. and de cannire et al. showed that return to work was significantly faster in the hybrid group, leading to a marked reduction in absenteeism from work in hybrid treated patients [7, 12]. this difference in nonhealthcare costs should be able to compensate the opposite difference in healthcare costs, resulting in a negligible difference in total societal costs. moreover, the emergency of simultaneous hybrid procedures in especially designed multipurpose operating rooms combining the potential of catheter-based procedures and cardiac surgery will reduce the unnecessary costs incurred by staged hcr procedures [12, 25]. lastly, more experience with minimally invasive cardiac surgery will shorten operative times, which might help reduce total healthcare costs. this review is the largest and most comprehensive report to date comparing the clinical outcomes of patients who underwent either hybrid coronary revascularization or conventional on- or off-pump cabg for multivessel coronary artery disease. three principal findings were revealed as follows: (1) hybrid treated patients showed a significantly faster recovery with lower prbc transfusion requirements and less in-hospital major adverse cardiac and cerebrovascular events than patients treated by on- or off-pump cabg; (2) staged procedures were associated with considerable period of times between both procedures, leaving patients incompletely revascularized and in theory at risk for cardiovascular events for a considerable length of time; and (3) the invasiveness of surgical lita to lad bypass grafting appeared to influence the clinical outcome, with higher macce and 30-day mortality rates in patients treated by more invasive surgical techniques using cpb and/or median sternotomy. as with any review, this report shares the limitations of the original studies. first, the initial reports especially included a relatively small number of patients, which may have resulted in biased results due to outliers. furthermore, almost all studies were performed retrospectively with inherent patient selection bias, since the decision to perform the hcr procedure was taken on an individual and highly selective basis according to cardiac surgeon and interventional cardiologist discretion. likewise, the inclusion and exclusion criteria used to select high-risk patients for the hcr procedure differed notably between the included studies, yielding a very heterogenic population. in addition, the used surgical techniques to perform the lita to lad bypass graft varied considerably, with learning curve issues and different levels of expertise and equipment. all these factors potentially contribute to heterogeneity, which may reduce the certainty of the evidence presented in this review. moreover, the mean length of followup was generally short, almost never exceeding two years, which made it difficult to assess long-term clinical outcomes of hybrid treated patients. therefore, this review relies mainly on in-hospital and short-term outcomes to assess the safety and feasibility of the hcr procedure. another limitation was the lack of long-term systematic and routine angiographic followup of graft and stent patency in the majority of studies included in the present review, which precluded any conclusions about the graft and stent longevity of the hcr procedure. furthermore, the comparative studies lacked randomization and nonblinded assessment of outcome, which might have led to selection bias and might have influenced outcome measures by preconceived notions about the superiority of the hcr procedure. finally, postoperative pain, which might be higher in patients treated with conventional midcab, was not included as outcome measure in the present review, because only a limited number of studies assessed this outcome measure. notwithstanding these weaknesses and limitations, this review selected the best evidence currently available to give a broad and comprehensive overview of the preliminary results of the hcr procedure. larger, multicenter, prospective, randomized trials with long-term clinical and angiographic followup and cost analysis comparing hcr with both conventional on-pump and off-pump cabg or multivessel pci will be necessary to further evaluate whether this hybrid approach is associated with similar promising long-term results. in the meantime, the first prospective, randomized pilot trial to compare hcr with conventional cabg in patients with multivessel coronary artery disease has been started. these data are also needed to identify patient populations that would benefit most from this hybrid approach. furthermore, more insights in the different surgical techniques for lita to lad bypass grafting and their clinical outcomes are necessary. therefore, the different surgical techniques for lita to lad bypass grafting in the hcr procedure should be integrated in these large, multicenter hcr studies in order to determine the best way of lita to lad bypass grafting in hcr. moreover, different hcr strategies (staged versus simultaneous) should be compared to decide which strategy will serve which patients best. finally, the advantages and disadvantages of a hybrid operative suite need to be explored further. the large variability in hcr techniques makes it difficult to draw firm conclusions from the currently available evidence, but hcr appears to be a promising and cost-effective alternative for cabg in the treatment of multivessel coronary artery disease in a selected patient population. the hcr procedure was associated with short hospital stays (including icu stay and intubation time), low macce and 30-day mortality rates, low prbc transfusion requirements and tvr, high postoperative lita patency rates, and high survival rates. these promising early outcomes warrant further research with larger sample size, multicenter rcts to determine the definite place of hcr in the current therapeutic armamentarium against coronary artery disease. until then, this review justifies the continued use of the hybrid approach, but careful patient selection and close cooperation between cardiac surgeons and interventional cardiologists will determine the clinical outcomes to a significant extent.

the hybrid approach to multivessel coronary artery disease combines surgical left internal thoracic artery (lita) to left anterior descending coronary artery (lad) bypass grafting and percutaneous coronary intervention of the remaining lesions. ideally, the lita to lad bypass graft is performed in a minimally invasive fashion. this review aims to clarify the place of hybrid coronary revascularization (hcr) in the current therapeutic armamentarium against multivessel coronary artery disease. eighteen studies including 970 patients were included for analysis. the postoperative lita patency varied between 93.0% and 100.0%. the mean overall survival rate in hybrid treated patients was 98.1%. hybrid treated patients showed statistically significant shorter hospital length of stay (los), intensive care unit (icu) los, and intubation time, less packed red blood cell (prbc) transfusion requirements, and lower in-hospital major adverse cardiac and cerebrovascular event (macce) rates compared with patients treated by on-pump and off-pump coronary artery bypass grafting (cabg). this resulted in a significant reduction in costs for hybrid treated patients in the postoperative period. in studies completed to date, hcr appears to be a promising and cost-effective alternative for cabg in the treatment of multivessel coronary artery disease in a selected patient population.

PMC3649801

pubmed-474

globally four million deaths occur every year in the first month of life. almost all (99%) neonatal deaths arise in low-income and middle-income countries [1, 2]. in india alone, around one million babies die each year before they complete their first month of life, contributing to one-fourth of the global burden [1, 3]. the neonatal mortality rate in india was 32 per 1000 live births in the year 2010, a high rate that has not declined much in the last decade [4, 5]. india's neonatal mortality rate dropped significantly, that is, by 25%, from 69 per 1,000 live births in 1980 to 53 per 1,000 live births in 1990 followed by a 15%, decline from 51 to 44 per 1,000 live births between 1991 and 2000. in recent years the nmr has dropped by 15% that is, from 40 per 1000 live births in 2001 to 34 per 1000 live births in 2009. urban-rural differences in neonatal mortality exist with the mortality rates higher by 50% in rural (42.5/1000 live births) compared to urban (28.5/1000 live births) areas, as per the national family health survey (nfhs-3). the common causes of neonatal deaths in india include infections, birth asphyxia, and prematurity which contribute to 32.8%, 22.3%, and 16.8% of the total neonatal deaths, respectively [7, 8]. india is one of the ten countries, along with china, democratic republic of congo, pakistan, nigeria, bangladesh, ethiopia, indonesia, afghanistan, and tanzania, that account for more than 65% of all intrapartum related neonatal deaths. despite the recognition of neonatal survival as a key to child survival, poor progress in neonatal survival in india poses concern regarding attainment of the fourth millennium development goal (mdg) target, that is, to reduce under-5 child mortality by two-thirds by 2015. despite having a comparatively higher neonatal mortality rate, rural india is tackling with the problem of ill equipped public health facilities. the numbers of existing peripheral health facilities fall short of what has been recommended by the government of india. the healthcare in rural areas has been developed as a three-tier structure based on predetermined population norms. the subcenter is the most peripheral institution and the first contact point between the primary healthcare system and the community. primary health centers (phcs) comprise the second tier in rural healthcare structure envisaged to provide integrated curative and preventive healthcare to the rural population. community health centers (chcs) form the uppermost tier and their function is mainly to provide specialized obstetric and child care. a situational analysis done by the neonatal health research initiative (nhri), indiaclen from 20072009, in 24 centers of the country, suggested that less than 20% of the chcs/phcs provide essential newborn care services. also, the availability of a neonatal resuscitation area was relatively low in chcs (46%) and phcs (14%). as per the district level health survey (dlhs-3), newborn care equipment was available in only 27.9% phcs. also, while around 76% of the community health centres had newborn care management facilities, just 35.1% had facilities for managing low birth weight babies. these findings underscore the critical condition of the public health facilities that are meant to cater to the health problems of the newborns in rural india. rural public health facilities across the country are having a difficult time attracting, retaining, and ensuring regular presence of highly trained medical personnel especially the gynecologists and pediatricians that are epochal in ensuring and promoting newborn health. statistics for 2010 suggest a shortfall of 10.3% for doctors at primary health centers (phcs). the condition of 4535 community health centers supposed to provide specialized medical care is even more appalling. as compared to requirements for an existing infrastructure, there was a shortfall of 62.6% of specialists at the chcs, 55.2% of obstetricians and gynecologists and 69.5% of pediatricians. according to the dlhs facility survey (2003), healthcare facilities with newborn care staff and a medical officer trained in newborn care were 59%, 45.0%, and 34% at district hospital, first referral units (frus) and chcs, respectively. as on march 2010, the overall shortfall in the posts of health worker (female)/auxiliary nurse midwife (anm) was 8.8% of the total requirement. similarly, in case of health worker (male), there was a shortfall of 64.1% of the requirement. in case of health assistant (female), the shortfall was 31.9% and that of health assistant (male) was 44%. the lack of qualified child care specialists results in a majority of rural households receiving care for their ill babies from private providers, many of whom are less than fully qualified. the government of india has launched various initiatives envisaging a high priority action with regard to neonatal health. under national rural health mission (nrhm), accredited social health activists (ashas) are being deployed and assigned the responsibility to create awareness in the community regarding maternal and child health issues. they are further expected to mobilize the community and help them in accessing healthcare services. a safe motherhood intervention named janani suraksha yojana (jsy) has been implemented under the nrhm to increase the institutional delivery rates and provide skilled care at birth for the newborn. under the reproductive and child health program (rch-ii), the quality and reach of antenatal care is planned to be expanded and home-based newborn care using integrated management of neonatal and childhood illness (imnci) protocols is envisaged. the imnci strategy encompasses a range of interventions to prevent and manage the commonest major childhood and neonatal illnesses that cause death, that is, acute respiratory infections, diarrhoea, measles, malaria, and malnutrition. the imnci package is planned to be implemented at the level of household and subcentres (through anms) and primary health centres (through medical officers, nurses, and lady health visitors). till october 2011 facility-based care of neonates (f-imnci) is proposed through strengthening of infrastructure, provision of extra nurses, and skills upgradation of physicians and nurses. the government, with the help of unicef, has started setting up special care newborn units (scnus) for managing sick newborns [17, 19, 20]. these units have been established at district hospitals and are expected to have a minimum of 12 to 16 beds manned by 3 physicians, 10 nurses, and 4 support staff. further, newborn stabilization units (nbsus) are being set up in first referral units (frus) and community health centers (chcs) and they aim to provide care to sick newborns referred from peripheral health facilities. as of october 2011, 1134 nbsus have been set up. a total of 8582 new born care corners (nbccs), which are special corners within the labour room where resuscitation, infection control, and early breast feeding can be commenced, have been set up, as of 2011. janani shishu suraksha karyakram (jssk) was launched on 1 june, 2011 with the aim to promote institutional delivery, eliminate out-of-pocket expenses, and facilitate prompt referral through free transport. a program on basic newborn care and resuscitation, named navjaat shishu suraksha karyakram (nssk), is being launched to address important interventions at the time of birth that is, prevention of hypothermia and infections, early initiation of breastfeeding, and basic newborn resuscitation. the objective is to have one person trained in basic newborn care and resuscitation at every delivery. this training is being imparted to medical officers, staff nurses, and anms at chc/frus and 24 7 phcs where deliveries are taking place. provision of comprehensive emergency obstetric and new born care (cemonc) services and basic emergency obstetric and newborn care (bemonc) at various levels has also been given due importance. neonatal health is seemingly one of the priority issues in the agenda of the government which gets reflected in the various programs devised and implemented. the worrisome issue is the fact that improving health systems through facility upgradation and ensuring availability of trained manpower and logistics comprise essential prerequisites for the success of these programs/initiatives. the reluctance of trained manpower, especially doctors, to serve in rural areas has become a major impediment in the government's ability to provide quality health services. the main obstacles to improving newborn survival are that many babies are born at home without being attended by skilled personnel, faulty home-based newborn care practices are widespread, lack of awareness among care givers limits care-seeking for neonatal illness and even if that is taken care of, lack of trained health workforce adds to the problem. this deficiency in skilled manpower undermines the initiatives by the government to improve neonatal health. another set of dilemma exists in bringing the neonates and the health system closer to each other. there are broadly two ways of doing so, either bring the health system closer to the neonate or bring the neonate closer to the health system. both of these are feasible and hold the promise to yield positive results but the real challenge lies in their reproduction and sustainment at the national level. in order to ensure the availability of trained medical personnel in rural areas, we first need to understand the reasons behind the observed shortage. recruiting trained doctors by all means is one of the essential components towards providing quality maternal and neonatal care services. a recent report documents that out of the 264 paediatricians (including both postgraduates and diploma holders) that are produced annually in india, only around half of them (i.e., 158) are available for public sector service, a large chunk either emigrate or get attracted towards private sector jobs in urban setups. the predominant reasons for preference to work in urban areas include adequate infrastructural facilities, high salary, and a decent standard of living [24, 25]. further, in the recent years, there has been substantial emigration of trained doctors to developed countries, much of it coming from lower and middle income countries [2628]. among the developing countries, india is the biggest exporter of trained physicians with india-trained physicians accounting for about 10.9% of british physicians and 4.9% of american physicians. a report of the national commission on macroeconomics and health documented that around 10% of the obstetrician(s) and paediatrician(s) that the country produces eventually emigrate. although the recipient nations and the physicians that emigrate benefit from this migration, the home country loses its important health potentialities. there is no clear-cut solution to the problem of lack of doctors in rural setup. interventions in education and financial incentives along with professional support probably have the potential to ease out the problem, as had been seen in rural australia where the gprip continuing medical education grants and locum grants designed to assist rural general practitioners to maintain and increase their skills in areas relevant to rural practice helped in their retention in rural areas. the provision of better financial incentives oriented specifically to doctors working in the rural areas might be crucial to attract and retain more doctors in these areas. in canada, the distribution of doctors was positively influenced by raising fees in rural and underserved areas and reducing fees in areas, but in the philippines, rural incentives had an unintended negative impact due to the fact that local governments were unable to hire healthcare professionals at the high salary levels specified [3133]. thus, the experience with paying direct financial incentives, such as rural allowances, has been variable and usually depends on the affordability of resources but this should not undermine the potential it might offer to increase the influx of doctors in rural areas. other key initiatives could include establishing rural doctor networks, mentorship programmes, and giving rural practitioners preference in admissions in specialty programs. exposure to rural areas as part of the training of medical graduates, so they can understand the working conditions and acquire rural clinical skills, is essential and has the potential to yield positive results. this has been documented in thailand where a majority of graduates continued in rural practice after completing a compulsory rural residency. to prevent brain drain, international scholar exchange programmes could be thought of as an option besides improving healthcare infrastructure and creating an enabling work environment. certain care practices can be deleterious to the health of the baby like applying ghee/oil on cord, early bathing, avoidance of colostrum feeding (considering it as harmful for the baby) and not practicing exclusive breast feeding. realizing the presence of such traditions in the community and formulating intensive information, education, and communication (iec) campaigns to address these is required. there is a need to develop programs where there is a collective involvement of the communities in order to identify problems and their solutions. several such programs have been implemented in other parts of the globe and have yielded positive results. bolivia's warmi program where the key highlight was participatory planning at the community level, with an emphasis on women's participation to identify obstetric and perinatal health problems and their potential solutions. as a result of the intervention, neonatal mortality decreased from 120 per 1000 live births to 40 per 1000 live births. in rural nepal, a cluster randomized trial suggested that women's groups facilitated by a local female community worker could reduce neonatal mortality rates by about 30%. in eastern india, the ekjut trial (20052008) evaluated the impact of community mobilization on birth outcomes in three districts of jharkhand and orissa. these studies offer evidence to encourage community involvement and leverage the community resources to bring about improvements in neonatal health. further, there is a need to make an effort to integrate community mobilization with health system strengthening. in a review of the evidence-based, cost-effective interventions for reduction of neonatal mortality, darmstadt et al. documented that a combination of outreach and home-based newborn care at 90% coverage could avert 1837% neonatal deaths. home-based newborn care could be explicated as a family as well as community oriented services that involve community mobilization and the empowerment of care givers to demand quality services for their sick newborns. hbnc mainly aims at reducing the neonatal deaths by preventing or treating morbidities such as infections, asphyxia or hypothermia which largely form the preventable causes of mortality. moreover, they are the underlying causes of nearly 55% of the neonatal deaths in india and addressing them could drastically cut down on the mortality rate. have documented that community-based pneumonia case management can lead to a 27% decrease in all-cause neonatal mortality, which indeed is a very high achievement. the most convincing example was set out by bang et al. in rural gadchiroli where female village health workers were selected from the local population and they were also trained to manage neonatal sepsis by providing parenteral antibiotic treatment to sick neonates. in the three years of intervention, there was a 71% reduction in perinatal mortality and a 62% reduction in neonatal mortality compared with the control area. in another example from sirur, a periurban area near pune, maharashtra, india, forty female village health workers were trained to serve a population of 47,000. the village worker identified high-risk cases that required treatment by herself and the nurse, under the supervision of the field medical officer. she also made 3 home visits: on day 1 or soon after delivery and on days 8 and 29. as a result of the intervention, a decline in the neonatal mortality rate of 25% from 51.9 to 38.8 per 1,000 live births was recorded. other successful examples include trials of home-based care in north india, bangladesh, pakistan, and nepal [4649]. in addition to creating awareness among community members and care givers in the family through information, education, and communication (iec) activities, a prerequisite for implementation of home-based care is the development of simple and easily comprehensible standard management guidelines. further, it would be a challenging task to upscale the home care newborn package to the most vulnerable states such as uttar pradesh, bihar, jharkhand, madhya pradesh, orissa, and rajasthan with a high neonatal mortality rate. in rural india, most of the births (53%) occur at home largely unattended by skilled personnel. the lack of a trained personnel predisposes the newborn to a variety of birth related complications mainly birth asphyxia, birth injuries, and infections. moreover, most of the neonatal deaths occur in the first week of life with a majority of them dying on the first day of birth, thus reflecting the poor intrapartum care that the mother receives [1, 50, 51]. with the shortage of trained personnel, nonavailability of adequate healthcare facilities, poor connectivity to a health facility, and lack of transport facilities, providing care at home through training of midwives/traditional birth attendants (tbas) would probably be a better option. they can be a vital link between women and the health system, giving advice, encouraging women to go to the clinic to deliver, and accompanying mothers to provide moral support. one such successful case study is from indonesia [52, 53]. in 2003, nearly half of all newborn deaths in the cirebon district of indonesia were due to birth asphyxia. in order to address this situation in cirebon, program for appropriate technology in health (path) supported by saving newborn lives/save the children began for training community midwives (bidan di desas). these midwives were taught a series of initial steps for assessing and managing a newborn's condition, including the use of a locally produced tube and mask resuscitation device that could be used in home birth settings. one year after the training, it was found that newborn deaths due to birth asphyxia dropped by 47 percent in the district, at a cost of only $42 per asphyxia death averted. in zambia, midwife training programs significantly decreased the seven-day neonatal death rate in community health clinics. the midwives were given training in essential newborn care (enc) and in neonatal resuscitation. after training, the all-cause, 7-day neonatal mortality rate decreased from 11.5 deaths per 1000 live births to 6.8 deaths per 1000 live births. the perinatal mortality rate decreased from 18.3 deaths per 1000 births to 12.9 deaths per 1000 births. similar examples providing evidence for up scaling of trained midwives in order to lower down the neonatal mortality can be drawn from sri lanka, thailand, malaysia, and pakistan [5559]. infant mortality rates (reflecting neonatal mortality as well) are one of the most important indicators of the differentials in health and socioeconomic condition in a community. a substantial progress in lowering down the high burden of neonatal mortality is unlikely unless ways can be found to enhance the economic wellbeing of the lower socioeconomic groups. a pertinent example is that of kerala, a southern state of india, where the state's achievement of stabilizing population growth, attaining high levels of literacy, and life expectancy have led to a significant decline in the infant mortality rates [60, 61]. in a study done in rural haryana to document the determinants of neonatal deaths, it was found that the occurrence of deaths was a multifactorial process with involvement of factors at community level, family level (socioeconomic), and biological level and that the socioeconomic determinants explained a large proportion of neonatal deaths. further, rahman et al. in their study in qatar found that low-cost, community-based interventions, on the background of socioeconomic development, had a stronger impact on neonatal and perinatal survival as compared to high-cost institutional interventions. similar findings documenting the importance of socioeconomic development in reducing the burden of neonatal deaths have been reported from studies done in chile, malaysia, malawi, and arab countries [6468]. neonatal care in rural india is largely provided by a large number of unqualified healthcare providers [6971]. they are the early providers of neonatal care and often attract a large number of ill newborns because of their easier access and comparatively cheaper treatment that they offer. there is a wide range of quality of services provided by these doctors and it would be useful to standardize their services by providing support in the form of training and technical support. though it does not qualify as a paragon solution, but this concept would probably score well, given the limited resources the country has. in alignment with what had been advocated by yadav et al. let best not be the enemy of the good, it would be beneficial to engage these local healthcare providers and equip them with necessary skills to provide acceptable standards of neonatal care until constraints on the supply of qualified and motivated healthcare providers into the system can be alleviated. they could further be involved in promoting key newborn essential care practices as they are popular and acceptable in the community. in china, rural healthcare is provided by village doctors who are trained in preventive and curative medicine of both traditional chinese and allopathic schools. the skills acquired are regularly upgraded by apprenticeship and in-service courses [73, 74]. another example is from usa where the shortage of physicians in the 1960s paved the way for the emergence of physician assistants who were licensed to practice medicine under the supervision of physicians. they made a considerable contribution by working in rural areas which otherwise would not have received any care at all [75, 76]. successful examples of providing quality healthcare through involvement of local healthcare practitioners can also be seen in ghana, mexico, and bangladesh [77, 78]. providing a degree of bachelor of rural medicine and surgery (brms) after three-and-a-half years of training, as opposed to five and-a-half years of training for a usual medical graduate, has recently been discussed as one of the possible options to cater to the need of quality healthcare in rural india. the government of india, in consultation with the medical council of india (mci), is planning to introduce this course in medical schools proposed to be established at district hospitals. the concept of a new degree course of a comparatively shorter duration is to encourage students from rural areas to take up medicine and subsequently provide services in their respective rural areas. the potential impact of selecting medical students of rural origin has been documented by rabinowitz et al. in a longitudinal study that evaluated the impact of the physician shortage area program (psap) in the usa. on multivariate analysis, rural origin was the single variable most strongly associated with rural practice. studies done in south africa, southern australia, and canada have also substantiated that the doctors with rural background have more tendency to work in rural areas [8183]. students enrolled in the proposed brms course will be taught preclinical as well as clinical subjects with more focus on paediatrics and obstetrics/gynaecology. further, it is envisaged to impart special training in care of the newborn and vaccination. chhattisgarh, a state in central india, has come up with the concept of awarding a degree named rural medical assistants (rmas). this three-year course was a response to a major crisis in human resources for health that the state faced. three colleges were inaugurated in 2001 and were situated in rural/tribal districts, but with access to a large government hospital (usually the district hospital) to make it possible for clinical teaching and internship. there has been overwhelming positive response to recruitment of rmas to the most rural and tribal phc postings, where previously no trained physician existed. it will certainly improve health care delivery in rural, remote, and tribal areas by providing qualified practitioners but the training of these rural healthcare practitioners will be a major area of concern. it is doubtful as to how overworked, poorly staffed, ill-equipped district hospitals, which cater to thousands of patients, can become quality training grounds for healthcare practitioners. ensuring that these graduates would practice only in rural areas and not shift to urban setup further, there is a need to document the difference in the quality of care provided by the new cadre of healthcare professional and mbbs graduates. the use of mobile phones to improve the quality of care and enhance efficiency of service delivery within healthcare systems is known as mobile health, or m-health. who defines m-health as the provision of health services and information via mobile technologies such as mobile phones and personal digital assistants (pdas). m-health tools have shown promise in providing greater access to healthcare to populations in developing countries, as well as creating cost efficiencies and improving the capacity of health systems to provide quality healthcare. studies done in kenya, sierra leone and zanzibar unleash the immense potentialities this innovative concept holds in addressing a wide variety of healthcare challenges [8688]. as earlier discussed, in rural setup, access to healthcare professionals and medical facilities is limited. although much work has not been done in context of m-health in india, yet efforts are required to be made to implement this in the indian context based on the initial success in other developing countries. the feasibility does not seem to be highly questionable considering the recent increase in the number of mobile phone users in rural areas. according to the press release by the telecom regularity authority of india (trai), the number of telephone subscribers in india increased to 943.49 million at the end of february 2012. the share of urban subscribers had been 65.59% whereas share of rural subscribers had reached 34.41%. subscription in rural areas had increased from 320.29 million in january 2012 to 324.68 million in february 2012, an increase of 4.39 million in just one month. now with the recent initiative by the government to provide a subsidy of 20 percent on bills of less than rs 300 a month to mobile users in rural india, the increase in the number of mobile users could further be expected. mobile telephone short-message service (sms) can be used for delivering health behaviour change interventions. this service has wide population reach, can be individually tailored, and allows instant delivery, suggesting potential as a delivery channel for health behavior interventions. researchers in korea, croatia, new zealand, and united kingdom have used sms to deliver information pertaining to diabetes and asthma self-management, smoking cessation, and increasing physical activity and this has proved to be beneficial byincreasing awareness and bringing about the desired behaviour change [9194]. mobile technology can also be involved in better training of community health workers in using cellular short messages (sms) to encode and transmit basic health information such as vital signs and health symptoms to a monitoring computer. algorithms on the monitoring computer could recognize emergent conditions and send system-generated notification informing the community health worker of the appropriate management of the baby related to the inputted vital signs and symptoms. given the volume of neonatal care services that are being sought through the private sector in rural areas, one can not hope to reduce neonatal mortality through public sector interventions alone [6971]. because the private sector does not operate within the restrictive confines of a government bureaucracy the advantage with such a partnership could be the wider coverage and increased service utilization. also, using strengths and skills of each partner enhances efficiency. successful examples of improving maternal and neonatal health through public private partnerships have been documented in the literature [9598]. one such example is of pampers/unicef collaboration to eliminate neonatal tetanus. through this collaboration, over 300 million tetanus vaccines, protecting over 100 million mothers and their babies in 25 of the world's poorest countries, have been provided. in even the poorest countries, the private sector is a major provider of goods, services, and information for maternal and child health. there could be different ways to involve the private sector, depending on the resources available and the need of services. one of the strategies could be to use microfinance to allow private sector doctors and other healthcare providers to provide quality practices. one such innovative scheme in india is the chiranjeevi scheme in gujarat [98, 100]. it is an innovative health financing scheme covered through public-private partnership for emergency obstetric care and emergency transport services, for women belonging to below poverty line (bpl) category. have published an evaluation of a pay for performance (p4p) scheme implemented in rwanda. p4p scheme involves for-profit organizations who are provided incentives based on improvements in utilization and quality of care. statistically significant improvements were observed in the maternal and neonatal health (mnh) indicators of institutional delivery and quality of prenatal care which increased by 21%, and 7.6%, respectively over baseline in the p4p districts [101, 102]. introducing public-private partnerships to improve the quality of maternal and child health services is not new in india. key examples include vande mataram scheme in west bengal which involves private sector for provision of safe motherhood and family planning services, janani express yojna in madhya pradesh for transportation in case of obstetric emergencies, and use of vouchers in uttar pradesh where reproductive and child health services for below poverty line (bpl) women and children are provided through private practitioners [103, 104]. under national rural health mission (nrhm), several initiatives based on public-private partnerships have been or/are planned to be implemented. the key issues include sustainability of such initiatives and ensuring that quality services are being provided. to conclude, the neonatal mortality rate in india is still high and skewed towards rural areas. much of the problem lies in the nonavailability of trained manpower and this in turn influences the quality of care the neonates receive. bringing qualified health professionals to rural, remote, and underserved areas is a challenging task which needs to be addressed urgently to avert neonatal deaths. other options such as training of local rural healthcare providers and traditional midwives, promoting home-based newborn care, creating community awareness and community mobilization along with strengthening public-private partnerships should be explored further, as evidence generated from previous studies and large scale projects support these strategies as a way to improve neonatal health. more research should be directed towards upcoming innovations such as m-health in order to exploit the potential they offer in terms of enhancing the quality of care. while the focus should be on devising strategies to recruit and retain trained manpower in rural areas, alternative strategies such as community mobilization, upscaling of home-based newborn care, imparting training and subsequent involvement of local rural healthcare providers and midwives should be attempted as well. more research is required to reveal the potential that innovations such as m-health, telemedicine, and public-private partnership hold in context to improving the quality of care in rural india.

the neonatal mortality rate in india is amongst the highest in the world and skewed towards rural areas. nonavailability of trained manpower along with poor healthcare infrastructure is one of the major hurdles in ensuring quality neonatal care. we reviewed case studies and relevant literature from low and middle income countries and documented alternative strategies that have proved to be favourable in improving neonatal health. the authors reiterate the fact that recruiting and retaining trained manpower in rural areas by all means is essential to improve the quality of neonatal care services. besides this, other strategies such as training of local rural healthcare providers and traditional midwives, promoting home-based newborn care, and creating community awareness and mobilization also hold enough potential to influence the neonatal health positively and efforts should be made to implement them on a larger scale. more research is demanded for innovations such as m-health and public-private partnerships as they have been shown to offer potential in terms of improving the standards of care. the above proposed strategy is likely to reduce morbidity among neonatal survivors as well.

PMC3506889

pubmed-475

the hypersensitivity syndrome, described as drug rash with eosinophilia and systemic symptoms (dress) syndrome is a severe, acute, drug reaction, defined by the presence of fever, cutaneous eruption, and systemic findings including enlarged lymph nodes, hepatitis, or hematologic abnormalities with eosinophilia and atypical lymphocytes1,2). the syndrome can involve several sites, leading to findings such as pneumonitis, renal failure, myocarditis, thyroiditis, or neurologic symptoms, but the liver is the most commonly affected internal organ3). this reaction can be life threatening, with a mortality rate of approximately 10%, most commonly secondary to liver failure1). here we report the first pediatric case of liver transplantation for the treatment of acute liver failure caused by vancomycin-induced dress syndrome in korea. a 14-year-old girl was referred to asan medical center children's hospital on the basis of test results that indicated abnormal liver function. she had been injured by a traffic accident 10 years earlier, and had undergone plastic surgery twice to heal wounds on her forehead. thereafter she had suffered from methicillin-resistant staphylococcus aureus (mrsa) osteomyelitis, which had been treated with intravenous vancomycin over five weeks. following this treatment, she developed fever and whole-body pruritic erythema, and abnormal liver function tests 6 days prior to her transfer. on admission, she presented with fever, nausea, vomiting, and abdominal discomfort. a generalized erythematous rash with variously sized, discrete lesions was noted on the face, trunk, and extremities. the patient reported an itching and heating sensation, which was aggravated after vancomycin injection. laboratory tests showed a total eosinophil count of 3,150/mm (normal,<500 mm), c-reactive protein level of 15.1 mg/dl (normal,<0.6 mg/dl), creatinine level of 2.5 mg/dl (normal, 0.7 to 1.4 mg/dl), aspartate aminotransferase (ast) level of 320 iu/l (normal,<5 to 40 iu/l), alanine aminotransferase (alt) level of 263 iu/l (normal,<5 to 40 iu/l), alkaline phosphatase level of 440 iu/l (normal, 40 to 120 iu/l), gamma glutamyl transpeptidase level of 321 iu/l (normal, 8 to 35 iu/l), lactate dehydrogenase level of 2,437 iu/l (normal, 120 to 250 iu/l), total bilirubin level of 3.3 mg/dl (normal, 0.2 to 1.2 mg/dl), direct bilirubin level of 1.8 mg/dl (normal,<0.5 mg/dl), prothrombin time (pt) international normalized ratio (inr) of 1.68 (normal, 0.8 to 1.3), and activated partial thromboplastin time of 34.5 (normal, 25 to 35). serologic tests for hepatitis a, b, and c, as well as cytomegalovirus, epstein-barr virus, and autoimmune hepatitis were all negative. the vancomycin level was 26.9 mg/l (normal, 20 to 40 mg/l). treatment with vancomycin was stopped, and replaced with ciprofloxacin for the treatment of osteomyelitis. intravenous delivery of a high dose of methylprednisolone was initiated upon an initial suspicion of dress syndrome. however, her liver function worsened progressively on hospital day 7, with an ast level of 1,285 iu/l, alt level of 1,077 iu/l, total bilirubin level of 15.5 mg/dl, direct bilirubin level of 8.0 mg/dl, and pt inr of 4.8, all suggesting acute liver failure. given the presence of aggravated hepatic encephalopathy, azotemia, and that the patient was refractory to medical treatments, she received a living-donor liver transplantation from her aunt on hospital day 9. the detailed care of preliver and postliver transplantation and care of acute liver failure in our program was described elsewhere4,5). after liver transplantation, the skin rash disappeared, with normalization of the eosinophil count and scores in liver and renal function tests. no severe clinical and surgical complications developed postoperatively. serial liver biopsies showed no evidence of acute rejection. over the course of a 25-month follow-up period, there has not been any definite recurrence of dress syndrome, with the exception of persistently elevated levels of liver enzymes and intermittent eosinophilia. the patient is currently suffering from iatrogenic cushing's syndrome owing to the high dose of steroids administered to control her elevated levels of liver enzymes. we here report a pediatric case of acute liver failure resulting from dress syndrome, which was treated by liver transplantation. most reported cases of dress syndrome show that a cure can be achieved by the immediate withdrawal of the causative agent and the administration of methylprednisolone6). liver involvement in dress syndrome is common and may range from a transitory increase in liver enzymes to liver necrosis with acute liver failure. to our knowledge, acute liver failure caused by dress syndrome has been reported at least twice in other european countries8,9). in one of these cases9), the patient died waiting for a liver transplant, whereas in the other case8), fatal recurrence occurred after liver transplantation despite potent immunosuppression and cessation of the precipitating factors. the pathogenesis of dress syndrome is not fully understood, and may be multifactorial10). although it is most commonly associated with antiepileptic drugs, dress syndrome has also been reported after exposure to a range of medications, including sulfasalazine, doxycycline, allopurinol, linezolid, and vancomycin1,8,10). the differential diagnosis includes stevens-johnson syndrome (sjs), life-threatening, cutaneous adverse reaction. the two syndromes overlap clinically, but have different characteristics, treatments and prognoses11). therefore, a high index of suspicion and rapid diagnosis may be necessary to save the patient's life. the only undisputed way to treat the use of systemic corticosteroids is common, although evidence regarding their effectiveness is scant12). in our case, we replaced vancomycin with ciprofloxacin approximately 6 days after symptoms of dress syndrome were evident, and immediately began corticosteroid treatments after the switch to ciprofloxacin therapy. although ciprofloxacin is also known to cause dress13), we finally chose that agent to control infection because other several antibiotics aggravated skin rash as well. these measures were ineffective in preventing liver failure. to our knowledge, this is the first pediatric case report to describe vancomycin-induced acute liver failure occurring as a component of the dress syndrome in korea. we highlight the need for awareness of the association between drugs, dress syndrome and liver failure. given the absence of reports describing the outcomes of liver transplantation in patients with dress syndrome, particular attention should be devoted to identification of its possible recurrence after liver transplantation.

drug rash with eosinophilia and systemic symptoms (dress) syndrome is characterized by a severe idiosyncratic reaction including rash and fever, often with associated hepatitis, arthralgias, lymph node enlargement, or hematologic abnormalities. the mortality rate is approximately 10%, primarily owing to liver failure with massive or multiple disseminated focal necrosis. here, we report a case of a 14-year-old girl treated with vancomycin because of a wound infection by methicillin-resistant staphylococcus aureus, who presented with non-specific symptoms, which progressed to acute liver failure, displaying the hallmarks of dress syndrome. with the presence of aggravated hepatic encephalopathy and azotemia, the patient was refractory to medical treatments, she received a living-donor liver transplantation, and a cure was achieved without any sign of recurrence. vancomycin can be a cause of dress syndrome. a high index of suspicion and rapid diagnosis are necessary not to miss this potentially lethal disease.

PMC3668204

pubmed-476

systemic lupus erythematosus (sle) is an autoimmune disease of connective tissue involving multiple organs. it is currently accepted that there are several genetic, environmental, and hormonal factors responsible for complex immunological disorders contributing to its development. recent studies have shown that abnormal stimulation of innate immunity may have a great influence on the immunopathogenesis of sle. hence, the receptors for pathogen-associated molecular patterns (pamps) have been the source of much recent attention. one of the representatives of this group is toll-like receptors (tlrs). they are associated with innate immunity insofar as they are agents in the pathogenesis of sle and lupus-like syndromes. tlr3, tlr7, and tlr9 seem to be involved in the development of autoimmune diseases. the ligation of a tlr activates a chain of proteins which transmit a signal to the nucleus, which in turn leads to increased production of proinflammatory cytokines, the expression of major histocompatibility complex (mhc) class i and ii antigens, and costimulatory molecules, which effectively activate antigen presentation and acquired immunity [5, 6]. intracellular tlrs, apart from pathogen recognition and initiation of innate immunity, are capable of recognizing endogenous ligands. in sle patients, impaired apoptosis and invalid cell debris clearance lead to increased concentration of serum nucleic acids (ssrna, dsrna, and dna), which are well-known ligands for tlr3, tlr7, and tlr9. nucleic acid-dependent activation of endosomal tlr is mediated by bcr receptor on lymphocytes b and fc, binding immunologic complexes and inducing their endocytosis. the activation of these receptors by specific ligands is thought to initiate autoimmune processes, which has been confirmed by studies on animal sle model. tlr stimulation leads to increased expression of proinflammatory cytokines (il-6, ifn, and tnf), which may reflect the intensity of the disease. on the other hand, synthetic oligodna with tlr receptor inhibitory properties causes the opposite effect, leading to a clinical improvement being observed in animal sle models. the aim of our study was to assess the tlr3, tlr7, and tlr9 expression on peripheral blood mononuclear cells (pbmcs), including cd3 t lymphocytes and their cd4 and cd8 subpopulations, and cd19 b lymphocytes, in patients with sle, compared to healthy controls. the original results of this study serve as the first presentation of a simultaneous analysis of the relationship between the expression of the studied tlrs and disease activity, the degree of organ damage, several clinical and laboratory parameters, and the influence of immunosuppressive treatment. moreover, a correlation between the expression of tlrs and gender as well as pre- and postmenopausal period was evaluated. thirty-five sle patients, diagnosed as having met at least 4 criteria according to the acr, were included in the study. all of the patients had been treated at the department of dermatology and venereology, medical university of lodz and did not present symptoms of active infection or neoplastic disease at the time of the study. the study group comprised 30 women and 5 men aged from 25 to 65 years. the average duration of sle was 7 years, ranging from 3 months to 21 years. disease activity was assessed according to the slam (systemic lupus activity measure) scale. patients who reached 10 and more points were diagnosed as having active sle. during the study, organ damage was then assessed with the slicc/acr (systematic lupus international collaborating clinics/american college of rheumatology) damage index. however, 22 patients received 1 point and 8 of them 2 points, indicating severe organ damage. pbmcs were isolated by gradient centrifugation using ficoll-histopaque-1077 (paa laboratories, pasching, austria). briefly, blood was precisely applied on the surface of the gradient and centrifuged at 1600 rpm for 20 min. the obtained buffy coat at the interphase was collected and dispersed in 5 ml of hank's medium (biomed, lublin, poland) and centrifuged at 1600 rpm for 10 min. the supernatant was collected and cells were washed twice with rpmi 1640 medium (paa laboratories, pasching, austria) at 1100 rpm for 5 min. each time. isolated pbmcs were divided into 1 10 cells per tube (each 100 l of pbs) and incubated with surface monoclonal antibodies against cd3, cd4, cd8, and cd19 conjugated with the fluorochromes allophycocyanin (apc), peridinin chlorophyll protein (per-cp), and phycoerythrin-cy7 (pe-cy7) (all from bd pharmingen, san diego, ca, usa) at a concentration of 20 l/1 10 cells, in darkness at room temperature for 30 min. the cells were then fixed and permeabilized using an intracellular tlr staining kit according to the producer's protocol (imgenex, san diego, ca, usa). the cells were then incubated with monoclonal antibodies against tlr3, tlr7, and tlr9 conjugated with fluorescein isothiocyanate (fitc) and phycoerythrin (pe) and their corresponding isotype controls (invivogen, san diego, ca, usa), at a concentration of 4 l/1 10 cells, in darkness, at room temperature for 30 min. six-color, two laser flow cytometry measurements were performed using the facs canto ii cytometer, equipped with bd facs diva software (all becton dickinson, san jose, ca, usa) as previously reported. the cell fluorescence was estimated using standard fluorescence filters: fl1 (313 nm 10), fl2 (264 nm 10), fl3 (374 nm 10) and fl4 (467 nm 10), fl5 (355 nm 10), and fl6 (653 nm 10). for each sample, the lymphocyte population was discriminated from pbmcs by forward scatter (fsc) versus side scatter (ssc) distribution. then, the percentages of cd3, cd4, cd8, and cd19 expressing tlr3, tlr7 or tlr9 were assessed. finally, the ratios of tlr3, tlr7, and tlr9 in the whole population of pmbcs were calculated. representative dot plots from flow cytometry measurements of tlr3 and tlr9 expression on t- and b-cells in patients and healthy controls (panel b) are presented in figure 1(a). representative dot plots from flow cytometry measurements of tlr7 expression on b-cells in patients and healthy controls are presented in figure 1(b). for measurable characteristics, minimum and maximum values were shown; average values were calculated: the arithmetic mean, median, and mode were calculated as were the parameters describing the internal differentiation (standard deviation). the interquartile range was also calculated as the distance between the third and the first quartiles. for quality characteristics, the percentage of occurrence of the categories was determined. to determine the pattern of distribution of the quantitative variables, the shapiro-wilk test was used. the mann-whitney test was used to assess the significance of any differences in average values between two groups, as the distribution pattern was not normal, and the anova rank test and kruskal-wallis test, followed by a post hoc test of multiple comparisons of average ranks (dunn test), were performed, to evaluate the differences in average values in several groups. the assessment of the relationship between the measurable variables was based on the spearman rank correlation coefficient. in all comparisons, the level of significance was p 0.05. significantly higher percentages of tlr3- and tlr9-positive pbmcs and cd3 t lymphocytes, including those positive for cd4 and cd8 antigens, as well as cd19 b lymphocytes were observed among patients with sle, compared to healthy controls (figures 2 and 3). a higher percentage of cd19 b lymphocytes expressing tlr7 was found in patients with sle than in healthy subjects (p<0.006) (figure 4). with regard to pbmcs and both subpopulations of t lymphocytes, tlr7 expression did not differ between patients and healthy controls (table 2). there were no significant correlations between the proportions of various cell subsets expressing the studied tlrs and disease activity (table 2). no statistically significant correlation was observed between the expression of any of the studied types of tlr among the given cell subpopulations and the degree of organ damage according to the slicc/arc damage index. however, subjects with severe organ dysfunction presented a higher percentage of tlr9-positive pbmcs, cd4 and cd8 t lymphocytes, and cd19 b lymphocytes (table 3). a significant mutual correlation was seen to exist between the expression of tlr3 and tlr9 in pbmcs (p<0.00001). no statistically significant correlation was observed between the expression of studied tlrs and the patient's gender. a significantly higher percentage of cd19 b lymphocytes expressing tlr7 was found in premenopausal women with sle than in postmenopausal women (3.52% 6.46 versus 0.12% 0.17 resp., p<0.03). a significantly lower count of cd4 cells with tlr9 was observed in patients with lymphopenia, compared with patients with a normal lymphocyte count (> 1000/mm) in the peripheral blood (4.59% 5.83 versus 6.86% 6.83 resp., p<0.005). in this subpopulation of cells, there was a significantly higher count of cells among patients with hypogammaglobulinemia, representing less than 12% of all proteins in the proteinogram analysis, compared to subjects with normal concentrations of gammaglobulins (32.12% 13.78 versus 11.46% 12.05, resp., p<0.05). among patients with anaemia, there was a higher percentage of tlr7-positive cd3 (4.19% 5.45), cd4 (4.19% 5.45), and cd19 cells (5.87% 8.71), compared to patients with haemoglobin concentration>12 g/dl (0.55% 1.08, p<0.05; 0.55% 1.08, p<0.03; 0.85% 2.24, p<0.02, resp.). moreover, an erythrocyte sedimentation rate (esr) of more than 25 was significantly more frequent in subjects with lower counts of tlr3-positive, cd19 b lymphocytes compared to esr 25 (2.55% 2.85 versus 5.10% 3.37, resp., p<0.03). a review of the clinical findings reveals that only patients with joint symptoms have lower tlr9-positive cd19 b lymphocyte counts, compared to subjects with no joint symptoms. no statistically significant correlation was observed between the expression of studied tlrs and immunosuppressive treatment. despite intensive research in many centres, the pathogenesis of sle remains poorly understood, and hence, the condition lacks targeted therapy. however, the discovery of tlrs in humans opened a new field in the studies of lupus, and our study of tlr3, tlr7, and tlr9 confirms their potential influence on the disease. tlr expression has been studied on the molecular level (mrna), as well as the protein level, and involves many subsets of peripheral blood cells [1520]. higher expression of tlr9 has been shown in sle patients, compared to healthy individuals, which is consistent with our findings. however, the results of studies concerning tlr3 and tlr7 expression are inconsistent. most of them concentrate on tlr9 expression in b lymphocytes, probably due to the fact that these cells are the main source of pathological antibodies responsible for the propagation of the disease [16, 18, 20]. a higher count of cd19 b and cd3 t lymphocytes expressing tlr9 were seen in our study group, compared to healthy controls. this observation is similar to those obtained by wu et al., who assessed patients with newly diagnosed, untreated sle. on the other hand, papadimitraki et al. observed a higher percentage of tlr9-positive cd19 b lymphocytes in a group of patients with active disease, compared to those with inactive disease. what is more, they observed a decrease in tlr9 expression on b cells of as much as 50% when the patient entered remission. it is plausible that in remission, stimulation of b lymphocytes through tlr9 is less intense, and as a result of this phenomenon, the autoimmune inflammation subsides. a potential confirmation of this hypothesis is a study by wong et al., who demonstrated a positive correlation between the concentrations of proinflammatory cytokines and chemokines produced after tlr9 stimulation and disease activity. however, in our study, no difference was observed between active and inactive sles in terms of the percentage of tlr9-positive cd19 b lymphocytes. this discrepancy between our and other centres may stem from the use of different criteria for patient selection. patients with lupus nephritis dominated in the study by papadimitraki et al., constituting 36% of the whole study group, whereas they only constituted 3% of our group. other research demonstrates greater tlr9 expression in the glomeruli of patients with lupus nephritis, and that the stimulation of glomeruli with endogenous tlr9 ligands augments inflammatory reactions in the kidneys. wong et al. analysed tlr3, tlr7, and tlr9 expression in cd19 b lymphocytes and cd4 and cd8 t lymphocytes among 16 chinese women. this is the only available publication which addressed the same markers as the present study. the results regarding tlr3 and tlr9 expression in cd19 b lymphocytes and cd4 and cd8 t lymphocytes obtained in by both the present study and that of wong et al.. however, these results need to be confirmed by rt pcr on t cells as was done on b cells by nakano et al.. differences between those results concerned only tlr7 population. while our report shows a markedly higher count of tlr7-positive lymphocytes b cd19 in sle patients than in healthy subjects, wong et al. did not find any difference in tlr7 expression for any cell subset between patients and healthy controls. however, after tlr7 stimulation, they observed an increase in the production of the chemokines cxcl10 and ccl5 by pbmcs from patients with sle. the observed inconsistence of the results may be due to heterogeneous nature of the study groups used by the two studies or their different genetic background. similar to our results, although obtained via molecular techniques, are the findings by komatsuda et al., who report that the concentrations of mrna for tlr7 and tlr9 in pbmcs are significantly higher among patients than in healthy controls. there are several publications regarding correlations between the expression of tlrs with sle activity, but the conclusions are contradictory. wong et al. do not report any such correlation in terms of tlr3, tlr7, or tlr9 expression. the lack of any relationship was probably due to the relative predominance of subjects with an inactive disease, according to sledai scale (sle disease activity index). in addition, no such significant relationship was demonstrated, although the majority of patients (63%) presented with active sle. nakano et al. studied 19 subjects in the active sle phase and identified a positive relationship between tlr9 mfi (mean fluorescence intensity) in b lymphocytes and sledai score. analysed a group of 35 newly diagnosed patients and found a negative correlation between the percentage of tlr9-positive b cells and sle activity. they pointed to a possible protective role of tlr9 in the development and propagation of sle. the discrepancy of published data may be explained by heterogeneous study groups in terms of clinical and therapeutic parameters. the presence of anti-dsdna antibodies and tlr expression was also noted in the present study. this type of antibody is a pathognomonic marker of sle, specific for renal involvement. available publications concerning the relationship between the expression of tlr and the presence of anti-dsdna antibodies are inconsistent. there was a positive correlation between the concentration of anti-dsdna antibodies with the percentage of tlr9-positive cd19 b lymphocytes from patients with active sle. however, other studies, as well as our own results, do not reveal any significant relationships between these parameters [17, 21]. on the contrary, komatsuda et al. observed a negative correlation between anti-dsdna autoantibodies and the tlr9 mrna content in cells. this may be explained by the heterogeneity of studied populations in terms of clinical presentation, accompanying diseases, treatment modalities, and occult infections, in particular., unlike other researchers, evaluated an entire pbmc population, including b and t lymphocytes and monocytes, and subjects included in the study were untreated. moreover, while the authors assessed the concentration of anti-dsdna antibodies, the others only noted their presence. a significant part of our study was the assessment of tlr expression with characteristic clinical and laboratory parameters. in the present study, a lower percentage of cd4 cells expressing tlr9 was seen in patients with lymphopenia, compared to those with lymphocyte counts above 1000/l. to our knowledge, there has been only one publication evaluating the relationship between lymphocyte count and tlr expression so far. did not find any relationship between the amounts of tlr2-5, tlr7, and tlr9 mrna in pbmcs and leukocyte, lymphocyte, neutrophil, and platelet counts, although 18 of 21 subjects presented with hematological abnormalities. it may be plausible that the decreased lymphocyte count of cd4 t lymphocytes coexpressing tlr9 may be related to immunosuppressive treatment in this group of patients., who report that methylprednisolone inhibits the survival of activated cd4 lymphocytes activated by specific tlr3 and tlr9 ligands in vitro but has no effect on their expression. there were 8 subjects (23%) with hypogammaglobulinemia in our group and half of them were receiving immunosuppressants. they presented with a significantly higher percentage of cd4 tlr9-positive cells, compared to individuals with gammaglobulin levels above 12%. it may be that the treatment with glucocorticosteroids and/or cytostatic agents led to a decrease of gammaglobulins but did not diminish the number of cd4 t lymphocytes expressing tlr9. this may be due to a low number of subjects with hypogammaglobulinemia with and without immunosuppressive treatment. however, it can not be excluded that the differences in tlr9 expression between these two subgroups (lymphopenia and hypogammaglobulinemia) may be caused by the different numbers of patients receiving immunosuppressive drugs, the number being lower in the case of hypogammaglobulinemia. our findings warrant further studies on tlr expression in t lymphocytes from patients with sle, as they may lead to a better understanding of the complex interactions between innate and acquired immunity in the pathogenesis of sle. one profitable course of action would be to inquire into the molecular level of the cell cycle using rt-pcr. the results of the present study note a lower count of cd19 b lymphocytes with tlr3 in patients with esr>25. glucocorticosteroids have a strong anti-inflammatory potential, caused by the inhibition of cytokine biosynthesis at the genome level treatment with this group of drugs may have led to a decrease in cytokines in the sera of patients with high esr, resulting in a lower percentage of b cells expressing tlr3. despite this, the treatment did not quench the inflammatory process and, therefore, did not lower the increased esr. the only available article by nakano et al., where the authors evaluated the correlation between tlr9s in lymphocytes b and t with increased esr, does not confirm any significant relationship. a higher count of cd3, cd4, and cd19 cells coexpressing tlr7 was found in patients with anemia compared to subjects with hemoglobin above 12 g/dl. in sle, anemia may stem from autoimmune hemolysis or chronic inflammatory process (anemia of chronic diseases, acd). in our group, this type of anaemia develops due to a chronic inflammatory reaction, characterized by increased concentrations of tnf-, il-1, or ifn-gamma, which inhibit the secretion of erythropoietin and availability of iron, essential for efficient erythropoiesis. as a result of tlr activation, numerous proinflammatory cytokines it was indicated that proinflammatory cytokines may regulate tlr expression. moreover, the proinflammatory cytokine-dependent expression of tlr, adaptor proteins, and kinases participating in signal transduction towards the cell interior has been proved. an increased concentration of ifn- in the serum of sle patients, combined with raised ifn-type i dependent gene expression in the mononuclear cells of peripheral blood cells, has been characterized as interferon signature. the continuous, tlr-mediated biosynthesis of ifn- by nucleic acids containing immunologic complexes may be responsible for the interferon signature phenomenon. moreover, it has been revealed that the level of ifn alpha-dependent gene expression is correlated with sle activity and more detrimental clinical disease forms, associated with damage to the kidneys, bone marrow, or cells of the central nervous system [29, 30]. increased inf-alpha concentration is regarded as the response to the continuous activation of tlr pathways. however, komatsuda et al. did not confirm any correlation between tlr and ifn-alpha induced ly6e (lymphocyte antigen 6 complex, locus e) gene expression. the observed higher percentage of cd3, cd4, and cd19 cells with tlr7 among subjects with anemia may reflect the presence of chronic inflammation and increased proinflammatory cytokines. in our study group, a higher count of tlr7-positive b and t cells was seen although 78% of patients received immunosuppressive drugs. this may be due to the majority of patients experiencing active sle (89%). exacerbation of sle may be induced by the usage of oestrogen-based anticontraceptive pills, that may also elevate the risk of a more severe disease course. however, during menopause sle tends to become milder, which is probably due to a decrease in oestrogen levels in peripheral blood. in our study, we demonstrated a statistically significant higher percentage of b lymphocytes cd19 expressing tlr7 in premenopausal women, compared to females after menopause. these observations suggest the influence of female sex hormones on tlr7 expression on lymphocytes b. this effect has been confirmed in other studies. young et al. (2011) indicated the increased in vitro expression of endosomal tlrs, including tlr7, on pbmc cells from normal women ader estradiol stimulation, with no effect after treatment with testosteron. in another study, 17-estradiol treatment of normal postmenopausal women enhanced tlr7/9 pdc production of ifn. however, secretion of ifn by plasmacytoid dendritic cells after tlr7 activation was lower in postmenopausal than in premenopausal females. furthermore, stimulation of tlr7 with a synthetic agonist in lupus-prone mice lacking the alpha oestrogen receptor led to a lower il-6 synthesis by lymphocytes b than in wild type animals. when the clinical symptoms were analyzed, a significantly lower count of lymphocytes b cd19 with tlr9 was found in patients with joint symptoms (75% of subjects) than in patients with no joint symptoms (25%). some publications describe the expression of tlrs in rheumatoid arthritis [38, 39] and note that patients demonstrate higher expression of tlr2, 3, and 4 on fibroblasts from the synovial tissue. the synovial fluid contains various tlr ligands such as peptidoglycan, dsrna released from necrotic cells, lipopolysaccharides, and cpg-rich nucleic acid. their presence stimulates the synthesis of many proinflammatory cytokines and chemokines, which sustain inflammation in joints [38, 39]. the lower percentage of cd19 b lymphocytes expressing tlr9 in patients with joint symptoms in our study group may be related to the presence of immunosuppressive treatment. however, immunosuppressive therapy was found to have no influence on tlr expression in our study, which is consistent with the results of other researchers [16, 17]. reports a significant decrease of mfi for tlr9 in cd20 b lymphocytes in 8 out of 11 patients with sle. no correlation was observed between tlr expression and the degree of organ damage, according to slicc/acr. the lack of any relationship may be explained by the fact that the organ damage reflects the final outcome of the inflammatory process. a significant positive correlation was recorded between tlr3 and tlr9 expression in pbmcs (p<0.00001). however, it is probable that the significant correlation of tlr3 and tlr9, but not tlr7, stems from the higher lability of ssrna (the ligand for tlr7), which undergoes rapid degradation by ribonucleases and is quickly removed from circulation. tlrs are able to recognise endogenous antigens which are released upon cell damage or stress and have been shown to play a key role in numerous autoimmune diseases [40, 41]. these tlr ligands bind tlrs, possibly initiate intracellular signaling pathways, and may initiate autoimmunity processes. tlrs act on the monocyte-macrophage system and activate dendritic cells, which then engage self-antigens, as the first step for the induction of autoimmunity. tlr9 activation induces the expression of membrane-bound b-cell activating factor (baff) on human b cells and leads to increased proliferation in response to both soluble and membrane-bound baff. a sizable body of evidence suggests that the endolysosome-restricted nucleic acid sensing subset of tlrs (na-tlrs) plays an important role in the production of antinuclear autoantibodies. recently, koh et al. documented that na-tlrs promote the induction of antinuclear abs in sle. their data indicates that the presence of na-tlrs in b cells is necessary to drive the initial autoimmune response and to promote the activation and escape of tolerance of self-reactive b cells. in addition, overexpression of tlr7 within the b cell compartment was found to enhance b cell tlr7 expression, permit the specific development of anti-rna autoantibody production, and exacerbate sle disease in an animal model. moreover, the inhibition of both tlr7 and tlr9 reduces autoimmune pathology in experimental sle [48, 49]. this observation suggests that the aberrant activation of a number of tlr pathways may lead to the initiation and/or perpetuation of sle and may indicate the direction for more specific therapy of this disease. in conclusion, our results suggest that tlrs exert an influence on sle development and describe the potential roles played by tlrs in the involvement of specific organs in this disease. even so, more targeted studies concerning the biology and function of tlrs are warranted and may lead to the development of a new class of drugs.

systemic lupus erythematosus (sle) is an autoimmune disease of unknown aetiology. the results of experimental studies point to the involvement of innate immunity receptors toll-like receptors (tlr)in the pathogenesis of the disease. the aim of the study was to assess the expression of tlr3, 7, and 9 in the population of peripheral blood mononuclear cells (pbmc) and in b lymphocytes (cd19 +), t lymphocytes (cd4+and cd8 +) using flow cytometry. the study group included 35 patients with sle and 15 healthy controls. the patient group presented a significantly higher percentage of tlr3- and tlr9-positive cells among all pbmcs and their subpopulations (cd3 +, cd4 +, cd8 +, and cd19+lymphocytes) as well as tlr7 in cd19+b-lymphocytes, compared to the control group. there was no correlation between the expression of all studied tlrs and the disease activity according to the slam scale, and the degree of organ damage according to the slicc/acr damage index. however, a correlation was observed between the percentage of various tlr-positive cells and some clinical (joint lesions) and laboratory (lymphopenia, hypogammaglobulinemia, anaemia, and higher esr) features and menopause in women. the results of the study suggest that tlr3, 7, and 9 play a role in the pathogenesis of sle and have an impact on organ involvement in sle.

PMC3955595

pubmed-477

neuroretinitis (nr) is considered to be an inflammatory condition which is characterized by optic disc edema and, as a result, formation of a macular star figure. nr is an atypical presentation of toxoplasmosis infection, and such cases are quite rare. a 13-year-old girl presented with painless subacute visual loss in her right eye for a week at khatam-al-anbia eye hospital in mashhad, iran. although toxoplasmosis nr is rare, it should be considered in the differential diagnoses of nr. neuroretinitis (nr) is considered an inflammatory condition which is characterized by optic disc edema and, as a result, formation of a macular star figure (1). this disorder is possibly caused by an infectious process affecting the disc; in other instances, a post-viral or autoimmune mechanism seems to be a more probable cause (1). furthermore, some instances of nr have been reported to be accompanied by a wide spectrum of infectious pathogens (1). the most common case reported so far is the result of cat scratch disease (csd), accounting for two-thirds of cases in one study (2). however, there are other infectious etiologies of neuroretinitis, including rubeola, toxoplasmosis, herpes simplex, varicella, tuberculosis, lyme disease, leptospirosis, syphilis, various fungi, and multiple viral illnesses (1). additionally, sporadic cases of nr may occur owing to noninfectious forms of uveitis, such as sarcoidosis and periarteritis nodosa (3, 4). optic disc edema with a macular star may also occur as a result of other factors such as diabetic papillopathy, hypertensive neuropathy, and anterior ischemic optic neuropathy papilledema (1). to determine the relevant history of patients with nr, the practitioner needs to concentrate on plausible risk factors for specific infectious parameters, such as travelling to areas where lyme disease or tuberculosis (for example) are endemic, exposure to waste material (e.g., leptospirosis), animal exposure (especially cats), and sexual contact that may have resulted in the contraction of syphilis. the clinician should investigate patients for systemic symptoms such as lymphadenopathy, headache, fever, and skin rash. laboratory tests ought to be customized for the individuals on the basis of information from both their reported history and the examination. serologic tests for most cases may include the fluorescent treponemal antibody absorption test (fta-abs), cat scratch titers (bartonella species), and a tuberculosis skin test (purified protein derivative, or ppd) (1). in this study, we present a case of unilateral neuroretinitis in which the serology result was apparently negative for acute infection of toxoplasmosis (negative igm titer for toxoplasmosis, but positive for igg). based on anti-toxoplasma treatment that was administered for the positive igg levels, the condition responded well with near optimal visual recovery. a 13-year-old girl was referred to the ophthalmic emergency department of khatam-al-anbia eye hospital, mashhad, iran, which is affiliated with mashhad university of medical sciences (mums), in may of 2013 with painless subacute visual loss in her right eye for one week. she denied any focal neurologic condition, pain on eye movement or ophthalmodynia, or any other systemic symptom. she also stated that she had not engaged in outdoor camping or any other related activity. furthermore, she had no remarkable past medical and ophthalmologic history, and she had no contact or proximity to pets, especially cats. upon examination, the best corrected visual acuity (bcva) was 20/250 in the right eye and 20/20 in the left eye. moreover, the patient had a grade 2+relative afferent papillary defect (rapd) in the right eye. an examination of the anterior segment demonstrated 2+cells in the anterior chamber (a/c) and 1+vitreous reaction with normal intraocular pressure (iop) in the right eye. the investigation of the right fundus revealed optic disc swelling, macular star lipid (hard exudates) deposition, and peripapillary vascular sheathing (figure 1). many tests were performed for the patient, including the erythrocyte sedimentation rate (esr), complete blood count differential (cbc diff.), c-reactive protein (crp), toxoplasmosis serology, purified protein derivative (ppd), angiotensin converting enzyme (ace), fta-abs, brain and orbital mri, chest x-ray, and infectious and rheumatologic consultation. bartonella henselae serology was not available in our ophthalmology center, nor in any other nearby neighborhood. based on our clinical results, neuroretinitis etiology revealed csd as the most common infectious etiology, along with the negative systemic and ocular history, and empirical treatment commenced including azithromycin 500 mg daily, trimethoprim/sulfamethoxazole, and the application of a topical steroid. after 72 hours, all the above-mentioned laboratory indices returned to the normal level, except for the toxoplasmosis serology. furthermore, the serologic test for toxoplasmosis was negative for igm antibody whereas the igg antibody titer was>100 iu/ml. relying on the positive igg toxoplasmosis serology and the absence of any response to initial treatment, we embarked on shifting to classic anti-toxoplasmosis treatment, i.e., sulfadiazine 500 mg qid (four times a day), pyrimethamine 50 mg/day, and folinic acid. after 72 hours had passed, prednisolone 1mg/kg/day was added and thereafter tapered during the treatment. the disc edema diminished over the course of a week. also, visual acuity gradually improved and the a/c inflammatory reaction faded away. after the decrease in disc swelling, a very small hyperpigmented focus appeared in the juxtapapillary position. gradually, over the next two months, the patient s visual acuity improved to 9/10 and the optic disc swelling and macular exudates resolved (figure 2). the clinical syndrome of idiopathic stellate maculopathy accompanied by optic nerve edema was first identified by leber in 1916 (5). later on, this syndrome was renamed as leber s stellate neuroretinitis. as is now known, the most common form of infectious nr results from csd (1). the engagement of the optic nerve suggests the need for urgent intervention. in spite of the lack of evidence either for animal exposure or any other systemic condition for this case, empirical csd treatment was instituted as lab tests were pending and given that cat scratch serology was not accessible. toxoplasmosic retinochoroiditis is rendered as one of the most frequent causes of posterior uveitis, specifically in young patients (6). however, toxoplasmic optic neuropathy is an infrequent condition and is often characterized by subacute visual loss and optic nerve swelling, and is sometimes associated with a macular star (neuroretinitis) (7). the engagement of the optic nerve most commonly encountered in ocular toxoplasmosis is optic nerve edema with a simultaneous distant active lesion. other types of optic nerve involvement include pure papillitis presented as optic disc swelling, associated with peripapillary vascular sheathing close to the healed lesion, and neuroretinitis, i.e., optic disc swelling with macular hard exudate deposition. additionally, monocular involvement has been observed in most cases with favorable visual prognosis (8). considering the positive toxoplasmosis serology in this patient, toxoplasmosis neuroretinitis was suspected, hence prompting the specified treatment. this case disclosed optic disc involvement and neuroretinitis owing to juxtapapillary chorioretinitis reactivation. in some cases, the juxtapapillary chorioretinal scar can not be found in the acute stage of disease due to disc and peripapillary edema. although toxoplasmosis neuroretinitis is rare, it should be still considered in any suspected case of neuroretinitis. this case re-emphasizes the necessity of considering toxoplasmosis in the differential diagnosis of neuroretinitis. awareness of this potential cause and prompt treatment after positive testing may therefore result in a good visual outcome.

introductionneuroretinitis (nr) is considered to be an inflammatory condition which is characterized by optic disc edema and, as a result, formation of a macular star figure. nr is an atypical presentation of toxoplasmosis infection, and such cases are quite rare. case presentationa 13-year-old girl presented with painless subacute visual loss in her right eye for a week at khatam-al-anbia eye hospital in mashhad, iran. following comprehensive evaluation, a diagnosis of toxoplasmic nr was made. the nr favorably responded to classic anti-toxoplasmosis treatment. a juxtapapillary retinochoroidal scar appeared after disc swelling resolution. conclusionsalthough toxoplasmosis nr is rare, it should be considered in the differential diagnoses of nr.

PMC4916323

pubmed-478

neospora caninum is an apicomplexan protozoan, infecting a large range of mammals. in cattle, several studies focussing on the specific immune response to n. caninum in cattle have shown that the time of gestation is important with regard to the outcome of the infection. proinflammatory cytokines, produced by lymphocytes, are crucial for controlling a variety of intracellular pathogens, including n. caninum. these cytokines are produced by natural killer (nk) cells, as well as by cd4 t-cells and cd8 t-cells. cd4 cells mediate the humoral response and their involvement is associated with increased igg1 levels, whereas cd8 cells are involved in the cellular immune response, which is characterized by increased production of igg2a. n. caninum is an intracellular parasite and resides within a specialized compartment, a parasitophorous vacuole, surrounded by a parasitophorous vacuole membrane (pvm). following egress from a host cell, these parasites immediately search for a new host cell to invade, and the direct accessibility for components of the immune system to the parasite within the circulation is rather limited. thus, extracellular tachyzoites have a low chance to be detected by t-helper-2-(th2-) type cells. a humoral immune response is therefore not sufficient to clear a n. caninum infection. for more efficient protection against intracellular pathogens such as n. caninum, the host usually generates a cellular, t-helper-1-(th1-) type response [6, 7]. this is possible since secretory parasite molecules pass through the pvm into the host cell cytoplasm, where they interact with and manipulate host cell functions. once in the cytoplasm these molecules can be processed and corresponding peptides are presented on the host cell surface via major histocompatibility complex (mhc) class i molecules. cd8 lymphocytes will recognize the peptides presented on these mhc-i molecules and activate th1 cells, which then produce cytokines such as interleukin-12 (il-12), interferon gamma (ifn-), and tumour necrosis factor (tnf-). the production of these cytokines leads to the activation of pathways that generate free oxygen radicals and nitric oxide (no) and its metabolites among other factors, which are potentially lethal for many protozoa. however, these processes can also be deleterious to the fetoplacental interface and potentially induce abortion and/or fetal resorption, especially in the first trimester of pregnancy, when levels of pregnancy hormones, such as progesterone, which counteracts these effects, are relatively low. as a result, there is no or only little th2 cytokine polarization and the dam will generate a th1 immune response, which also affects the placental and foetal tissue. since a strong th1 response is incompatible with successful pregnancy, the infection can lead to the loss of the unborn foetus. progesterone promotes th2 cell proliferation the production of il-4, il-5, and il-10 is known to inhibit no and tnf- production and impairs nk cell activity [1113]. however, since the immune response is not capable of controlling the parasite, the risk of transplacental transmission of n. caninum is relatively high. thus, resistance to n. caninum seems to rely, at least partially, on a functional th1 response. in this study, inbred balb/c, cba/ca, and c57bl/6 strains of mice (with different mhc-i haplotypes) were compared for their capacity to cope with a n. caninum infection. in addition, we show that cba/ca mice were the by far most resistant, and protection correlated with the induction of a th1-biased response. the results demonstrate that a meaningful evaluation of the efficacy of, for example, vaccine candidates and/or chemotherapeutically interesting compounds against experimental infection with n. caninum in mice requires standardization with regard to the mouse strains used for such experiments. female balb/c, cba/ca, and c57bl/6 mice aged 812 weeks were obtained from harlan (horst, the netherlands). mice were randomly divided into groups as soon as they came to hand and marked individually. the conditions in the animal housing facilities were negative air pressure, air exchange of 300 m/hr, temperature of 22c 2c, and artificial day/night cycle with 12 hrs light per day. five mice were housed per group in macrolon type iii cages (820 cm) enriched with polyvinyl chloride tubes and tissues. mice received ad libitum standard rodent feed (rmhb2118, ab-diets woerden, the netherlands). water was supplied in bottles and refreshed at least three times a week. prior to challenge, mice were given an acclimatization period of 7 days, during which they were observed daily for normal behaviour, wellness, food and water intake, and posture. n. caninum tachyzoites (nc-liv isolate) were maintained by continuous passages in vero cells grown in rpmi 1640 medium supplemented with 10% foetal bovine serum (fbs), 2 mm l-glutamine and 25 mg/l gentamycin sulphate. cultures were grown in a humidified incubator at 37c with 5% co2. to liberate the tachyzoites from the vero cells, the culture was taken up through a 20 g needle and extruded through a 26 g needle. for the preparation of parasites for infection, tachyzoites were purified on a pd10 column (ge healthcare, diegem, belgium) centrifuged and resuspended in sterile 0.04 m isotonic pbs. tachyzoites were counted in a neubauer chamber. in order to prepare n. caninum tachyzoite antigen lysate, purified tachyzoites were subjected to one freeze-thaw cycle followed by sonication on a branson sonifier s-250a (branson ultrasonics, danbury, usa) for 60 sec, output 4, and a cycle duty of 50%. mice were challenged by intraperitoneal injection of 1 10, 5 10, or 25 10 live nc-liv tachyzoites in 0.5 ml 0.04 m isotonic pbs. mice were then closely monitored daily for neosporosis disease symptoms according to a clinical scoring system that included hunched back, rough hair coat, impaired movement, and spinning. for each feature the average clinical score per group was determined by dividing the total score per group by the amount of mice in that group. as soon as the health status of the animals deteriorated, the frequency of observations was increased to two times a day. blood samples were collected by orbital puncture at day 0 and day 34 postinfection (at the time of euthanasia) or, whenever possible, at the time of inter-current death. latest at day 34 postinfection (pi), surviving mice were sacrificed by cervical dislocation and brains and spleens were harvested. for each mouse, sections of 3.5 m thickness were cut longitudinally from nervus i to medulla oblongata with cerebrum, hippocampus, and cerebellum on a brand microm h355 s microtome (adamas, rhenen, the netherlands), and were placed onto glass slides. serum antibody levels were analysed using a commercially available elisa kit based on antigens of n. caninum (idexx neospora ab test, idexx laboratories, westbrook, usa) according to the instructions of the manufacturer. on each plate, sera were tested in 2log titrations, and negative and positive sera were tested in octaploid. sera of nave swiss outbred mice (charles river, sulzfeld, germany) were used as a negative control. as a positive control for igg1 and igg2a detection, sera of balb/c mice vaccinated with the chimeric antigen r-mic3-1 and challenged with n. caninum (nc-1 isolate) sera of c57bl/6 mice taken 34 days after infection with 5 10 nc-liv tachyzoites were used as a positive control for igg2c analysis. horseradish-peroxidase-(hpr-) conjugated goat antimouse igg1, igg2a, and igg2c were obtained from southern biotechnology (birmingham, usa) and diluted in enzyme immune assay (eia) buffer containing 0.05% polysorbate 80. results were analysed on a tecan sunrise device (breda, the netherlands) using xfluor4 software at 650 nm. antibody titres were determined using caspex software; abendvertical version 0.11 v1 (msd animal health, proprietary software). the cutoff in antibody end titres was defined at bmin2, where bmin is the negative control. spleens from surviving mice were washed twice in rpmi 1640 medium supplemented with 100.000 iu penicillin/streptavidin, 1 mm sodium-pyruvate, and 2 mm l-glutamine and dissociated using the gentlemacs dissociator (miltenyi biotec, leiden, the netherlands) according to the instructions of the manufacturer. suspensions were filtered through a 100 m cell strainer and centrifuged at 300 g for 10 min at 4c. samples were depleted of erythrocytes with erythrocyte lysis solution containing 829 g/l ammonium chloride, 100 g/l sodium hydrogen carbonate, and 3.7 g/l disodium edetate. splenocytes from mice of the same group were pooled in a 1: 1 ratio and a total of 1 10 splenocytes were cultured for 72 hrs in 100 l complete rpmi medium containing 10% fbs in a humidified incubator at 37c with 5% co2. 100 l of an antigenic extract of either 2 10, 1 10, or 5 10 nc-liv tachyzoites prepared as described above was added to the cells. complete rpmi medium and a phorbolmyristateacetate (pma)/ionomycin mixture (9.74 ng/ml/0.26 mm) were used as negative and positive controls, respectively. cytokine levels in supernatants of stimulated splenocytes were analyzed using the bendermedsystems flowcytomix mouse/rat basic kit combined with the cytokine mouse simplex kits for ifn- (bendermedsystems, vienna, austria). the samples of the cytomix assay were interpolated in the standard curve by selecting the 5p logistic fit function: the bendermedsystems flowcytomix pro 2.4 software fits the best curve according to y=d+(( a d)/(1+(x/c))). the mean fluorescence intensity (mfi) of each standard point is blank-corrected by division of the blank-mfi (b_b0=mfi/mfi of blank100). the maximum acceptable bias, which displays the variation for the ideal standard curve defined by the theoretical standard concentrations, was set at 30%. neospora-specific quantitative real-time pcr to determine the number of tachyzoites that has reached the cerebral tissue was performed as previously described [2, 3, 15]. dna extraction from brain tissue was performed using the dneasy blood&tissue kit (qiagen, hilden, germany) according to the manufacturer recommendations. the dna concentration in each sample was determined by uv spectrophotometry (nanodrop, thermo scientific, delaware, usa) and was adjusted to 5 ng/l with sterile dnase-free water. quantitative real-time pcr was performed using the light cycler instrument (roche diagnostic, basel, switzerland). the parasite counts were calculated by interpolation from a standard curve with dna equivalents from 1000, 100, and 10 parasites included in each run. mice survival was analyzed according to kaplan-meier and the survival curves between groups were compared with the logrank test followed by regression coefficient analysis. the weight of the survivors, cerebral parasite burden, lesion score, and the serological data were compared using kruskal-wallis one-way anova followed by the kruskal-wallis multiple comparison z-value test. the p value between two significantly different groups was calculated by mann-whitney u test. the proportion of animals with clinical signs, proportion of animals with brain lesions and data on n. caninum-dna-positive animals were organized in a contingency table and compared by a chi-square test. balb/c mice (figures 1(a) and 1(b)) challenged with 1 10 tachyzoites were not affected, with no decrease in body weight following infection. however, challenge with 5 10 nc-liv tachyzoites resulted in neosporosis symptoms in balb/c mice, starting on day 18 pi. hunched back, ruffled coat, and weight loss of ~25% were observed in these mice. two mice reached the point where severe neurobiological symptoms made it necessary to get them euthanized on day 23 pi, while the other three mice, although exhibiting clinical symptoms, but less severe, survived throughout the observation period of 34 days (figures 1(a) and 1(b)). balb/c mice were not able to control the highest infectious dose of 25 10 tachyzoites, resulting in death of all five animals within that group between days 7 and 12 pi. cba/ca mice (figures 1(c) and 1(d)) exhibited a high degree of resistance against n. caninum at an infection dose of 1 10 and 5 10 tachyzoites, with no mortality and no body weight changes during the entire experimental period (figures 2 and 1(d)). however, none of the cba/ca mice could control the infection at the highest dose and they all were euthanized within 9 days pi (figures 1(c) and 1(d)). in comparison to the other two mouse strains, c57bl/6 mice (figures 1(e) and 1(f)), all mice started to exhibit clinical symptoms including weight loss of ~10% and two out of five mice had to be euthanized at day 29 pi (figure 2). strong variations were observed in the group of c57bl/6 mice challenged with 5 10 tachyzoites. three mice of this group demonstrated severe symptoms of neosporosis and were euthanized on days 7, 8 and 13 pi, while the other two mice showed only very mild symptoms on day 8 pi and returned to being clinically normal 9 days after the challenge. shortly after a challenge with 25 10 tachyzoites, c57bl/6 mice showed a hunched back and ruffled hair coat (figures 1(e) and 1(f)). symptoms progressed to include impaired movements and spinning when picked up by the tail, requiring euthanasia of four of these mice on day 8 pi. the remaining mouse of this group completely recovered by day 11 pi and survived till the end of the experiment. statistically, cba/ca mice exhibited a significantly higher survival rate than c57bl/6 mice at an infection dose of 1 10 tachyzoites (p<0.001, cox regression analysis) and than c57bl/6 and balb/c mice at an infection dose of 5 10 tachyzoites (p<0.001, cox regression analysis). the mean weight of the survivors of the balb/c mice infected with 1 10 tachyzoites was slightly higher than those infected with 5 10 parasites, although the difference was not significant (kruskall-wallis multiple comparison test). the number of symptomatic mice in the cba/ca mice at an infection dose of 5 10 tachyzoites was significantly lower than in the two other groups (p<0.01, chi-square test) while no significant differences between strains were observed for the other infection doses (table 2). the number of mice presenting brain lesions as well as the lesion score per group at an infection dose of 5 10 parasites was significantly lower in the cba/ca mice (p<0.01, chi-square test and p<0.001, mann-whitney u-test, resp.) than in the two other groups (table 2). no significant difference was observed regarding the overall lesion score per strain (table 2). the surviving balb/c mice infected with 1 10 tachyzoites (n=5) and cba/ca mice infected with 5 10 tachyzoites (n=5) were challenged again with 25 10 n. caninum tachyzoites at day 34. however, they all succumbed to infection within 24 hrs, indicating that they had not built up any form of protective immunity. in order to obtain information on the type of immune response induced by n. caninum infection, the production of igg1-type antibodies is primarily induced by a humoral immune response, whereas igg2a subclasses indicate the involvement of a cellular immune response. since c57bl/6 mice poorly produce igg2a antibodies, the igg2c levels were measured in serum of these mice. in none of the groups n. caninum-specific antibodies experimental infection of balb/c mice with 1 10 or 5 10 tachyzoites resulted in high igg2a and igg1 serum levels, indicating the presence of a mixed cellular and humoral immune response (table 1). in cba/ca mice challenged with 1 10 or 5 10 tachyzoites, predominantly igg2a antibodies were detected. igg2a and igg1 were undetectable in cba/ca mice challenged with 25 10 tachyzoites, most likely because mice succumbed to the infection before a humoral response was mounted (table 1). in general, sera of mice surviving until day 34 pi contained higher levels of igg2c compared to sera of their group mates that died at earlier time points (table 1). the difference igg2a/c-igg1 postinfection was highly significantly higher in the cba/ca mice than in the two other strains (p<0.01, mann-whitney u-test) and significantly higher in the c57/bl6 mice than in the balb/c mice (p<0.05, mann-whitney u-test) at an infection dose of 1 10 parasites. at an infection dose of 5 10 tachyzoites, both cba/ca and c57/bl6 mice had a significantly higher igg2a/c-igg1 difference than the balb/c mice (p<0.01, and p<0.05 respectively, mann-whitney u-test), and cba/ca mice also had a higher igg2a/c-igg1 difference than c57/bl6, although not significant (table 2 and figure 4). the production of ifn-, indicating the occurrence of a cellular immune response, was measured in the supernatants of spleen cells derived from survivors of balb/c mice (infected with 5 10 tachyzoites), cba/ca mice (infected with 1 10 tachyzoites), and all surviving c57bl/6 mice. splenocytes were stimulated with either n. caninum antigens or pma/ionomycin for 72 hrs, or were left unstimulated. stimulation with a lysate corresponding to 2 10, 1 10 or 5 10 purified nc-liv tachyzoites resulted in increased production of ifn-. ifn- production was a dose-dependent stimulus in all groups (figure 3). the strongest ifn- response was observed for the cba/ca mice, as was especially evident using low and medium tachyzoite stimulus doses. a lower ifn- production was observed for the balb/c mice, while splenocytes from c57bl/6 mice demonstrated large variations in ifn- production upon antigen stimulation. data from balb/c mice and cba/ca mice infected with 25 10 parasites is missing, since these mice succumbed to infection prior to 34 days postinfection. spleen cells of balb/c mice infected with 1 10 tachyzoites (n=5) and cba/ca mice infected with 5 10 tachyzoites (n=5), which were used for rechallenge, were not assessed. the cerebral parasite load in all groups was measured by real-time pcr and compared with histopathological findings. corresponding results are summarized in table 2. in three out of five balb/c mice challenged with 1 10 tachyzoites, no parasite dna could be detected by pcr, while high numbers were detected in the other two samples. however, in only one of these mice a brain lesion was detected by histology. challenge of balb/c mice with 5 10 tachyzoites resulted in high cerebral parasite load in all mice, and brain lesions were detected upon histopathological inspection in four out of five mice. after infection of balb/c mice with 25 10 tachyzoites, parasites were detected at moderate to high numbers in all brain samples investigated, but histopathology failed to detect any lesions at all. real-time pcr detected n. caninum dna in one of 10 brain samples of cba/ca mice challenged with 1 10 and 5 10 tachyzoites. in contrast, lesions were observed by histopathological means in two mice infected with 1 10 tachyzoites. however, these were samples that were not positive by real-time pcr. in the cba/ca mice infected with 25 10 tachyzoites, lesions in the central nervous system (cns) were observed by histopathology for three out of five animals, and real-time pcr showed high numbers of parasite load in all 5 members of the group, which all died between days 8 and 9 pi. in three c57bl/6 mice challenged with 1 10 tachyzoite lesions in the brain were observed. in two of them infection with 5 10 tachyzoites resulted in parasites and lesions in three mice on days 13 and 34 pi. in the c57bl/6 mice infected with 25 10 tachyzoites, four out of five mice died 8 days after challenge, with real-time pcr-positive brain tissue but no lesions detectable upon histopathology. one mouse survived until day 34 pi, and this mouse exhibited a high cerebral parasite load and also brain lesions. thus, a high cerebral parasite load as determined by real-time pcr did not always correspond with the detection of lesions in the brain and did also not always match with the occurrence of clinical signs of neosporosis. at an infection dose of 1 10 and 25 10 tachyzoites, no significant difference in the number of pcr positive mice between the groups was observed (chi-square test). at an infection dose of 5 10 tachyzoites, the number of pcr-positive mice was significantly lower in the cba/ca mice than in the balb/c and c57/bl6 mice (p<0.01 and p<0.05, respectively, chi-square test). no significant difference regarding the parasite load of infected mice was observed between the groups (kruskal-wallis multiple comparison test). in this study, we compared the capacities of three inbred mouse strains expressing different mhc-i molecules to cope with a n. caninum infection. significant differences in the responses to n. caninum infection in inbred and outbred mice have been demonstrated previously, and these differences have been postulated to be due to varying abilities to present n. caninum antigens on their mhc molecules. when using mouse models to investigate the outcome and immunological parameters of infection, the selection of the n. caninum isolate as well as inoculum doses are important variables that will influence the results. the present study showed high variations in morbidity and mortality in the three inbred mouse strains balb/c, cba/ca, and c57bl/6 after inoculation with different numbers of n. caninum (nc-liv) tachyzoites, demonstrating that the choice of mouse strain plays a crucial role for the assessment of experimental infections and agents that could potentially interfere therein. while balb/c mice did not suffer from clinical signs of neosporosis after a challenge dose of 1 10 n. caninum tachyzoites, infection with 5 10 tachyzoites resulted in severe clinical symptoms starting at day 20 pi. this is in accordance with several vaccination studies, which have used the balb/c and c57bl/6 models to investigate the protective effects of vaccine candidates (reviewed in [2, 3]). in contrast, only limited research has been performed using cba/ca mice as a model to investigate the immune responses against n. caninum infection. our investigations demonstrated that this mouse strain is clearly more resistant against neosporosis compared to balb/c and c57bl/6 mice. previously, these features of cba/ca mice with regard to n. caninum infection had been highlighted by rettigner et al., who described this mouse strain to be resistant against an infection of 5 10 tachyzoites of the nc-1 isolate of n. caninum following treatment with immunosuppressing agents and suitable for the generation of cerebral tissue cysts. this is surprising, since the nc-1 isolate, in contrast to nc-liv, does hardly convert into bradyzoites under in vitro conditions such as high ph treatment or incubation with sodium nitroprusside [19, 20]. with respect to c57bl/6 mice, ramamoorthy et al. observed no clinical signs of neosporosis throughout an observation period of 21 days after an infection with 5 10 nc-1 tachyzoites but also showed these mice to be highly susceptible to an infection with 2 10 nc-1 tachyzoites. others failed to observe clinical signs in c57bl/6 mice after a challenge with 5 10 nc-1 tachyzoites as long as 44 days pi. in this study, after a challenge dose of 1 10 nc-liv tachyzoites disease symptoms occurred after 26 days but only after 8 days when 5 10 tachyzoites were inoculated, with three out of five mice succumbing to infection. this illustrates that, besides the mouse strain used, the selection of the neospora isolate also makes a big difference. in any case, an infection dose of 25 10 tachyzoites of the nc-liv isolate was far too high for any of the mouse strains to cope with, requiring euthanasia of all but one mouse between days 712 pi. since n. caninum is an intracellular parasite, the protective immune response is likely to involve cell-mediated immunity; thus th1-mediated responses are important. however, in balb/c mice it has been demonstrated that besides cellular immunity also humoral immune responses are crucial in controlling n. caninum infection and limiting the pathological changes caused by an excessive proinflammatory response [23, 24]. teixeira et al. described that intraperitoneal injection of n. caninum tachyzoites (nc-1 isolate) in balb/c mice induced a parasite-specific, nonpolyclonal, b-cell response. a strong bias towards a th2-type response resulted in increased susceptibility to n. caninum and enhanced the corresponding pathologic effects. however, vaccination of mice with recombinant rhoptry antigen rop2 emulsified in saponin adjuvants induced an igg1-biased humoral immune response, while formulating the same antigen in freund's incomplete adjuvants resulted in an igg2a-biased antibody response, and both were protective in the nonpregnant balb/c mouse model. protection associated with an igg2a-biased humoral immune response was also demonstrated for recombinant protein disulfide isomerase applied for vaccination intranasally [2729]. vaccination of mice with native ncsrs2 induced a th2-biased protective response that reduced congenital infection of offspring balb/c mice. reduction in congenital infection, also associated with a th2-biased immune response, was demonstrated by vaccination experiments in balb/c mice employing combined ncmic1, ncmic3, and ncrop2 antigens. taken together, it is probably rather a suitable balance in th1/th2-type immune responses than a strict th1 response that allows the host to deal with n. caninum infection. similar to earlier studies in balb/c mice [25, 26], levels of igg1 antibodies in animals infected with 1 10 and 5 10 tachyzoites were generally high, and it is interesting to note that a challenge dose of 5 10 tachyzoites in balb/c mice resulted in more pronounced igg1 responses compared to the igg1 antibody levels found in mice inoculated with 1 10 tachyzoites, while igg2a titres were basically equal in both groups. in the balb/c mice inoculated with 5 10 tachyzoites, titres for igg1 and igg2a were similar, which is in agreement with previous observations reported by lundn et al.. in addition, we found that splenocytes obtained from balb/c mice inoculated with 5 10 tachyzoites and stimulated with n. caninum antigen produced ifn-, albeit at a lower level compared to cba/ca mice. the results of rettigner et al. on n. caninum infection in cba/ca mice showed no evidence of differential isotype secretion but a high expression of ifn-, while in our experiments sera of infected cba/ca mice exhibited a high igg2a response, and splenocytes of cba/ca mice infected with 1 10 n. caninum tachyzoites also secreted high levels of ifn-. this indicated that the induction of a cellular immune response is a prerequisite for successful dealing with the infection. the importance of the humoral immune response during n. caninum infection was demonstrated by eperon et al. who showed increased susceptibility to n. caninum (nc-1 isolate) infection in mt b-cell-deficient mice when compared to wild-type c57bl/6 mice. they also described the predominant presence of parasite-specific igg2a isotypes and an absence of igg1 isotypes in c57bl/6 mice at various time points., we did not observe any igg2a antibody titres in c57bl/6 mice and analysed the igg2c antibody isotype levels instead. our results showed the major parasite-specific igg isotype to be igg2c. in agreement with a shift from a th1- to a th2-biased response, increasing titres of n. caninum-specific igg1 were detected in mice sera collected at later time points after infection. while n. caninum obviously induces differential humoral and cellular immune responses in different mouse strains during the acute phase of infection, one could imagine that chronic infection would be characterized by the production of tissue cysts. therefore, in order to mimic the infection in cattle, a mouse model able to form tissue cysts, and also exhibiting recrudescence during pregnancy, is required. however, only few reports have actually been published on the production of n. caninum tissue cysts in mice. tissue cyst production had been demonstrated in outbred mice and immunosuppressed cba/ca mice inoculated with nc-1 or nc-liv tachyzoites but not all of the mice investigated harboured cerebral tissue cysts and rettigner et al. showed that immunosuppressed female cba/ca mice given 5 10 nc-1 tachyzoites were able to survive and to consistently develop cerebral tissue cysts. more recently, tissue cyst production, marked by positive staining with an antibody directed against the toxoplasma bradyzoite antigen bag5, has also been demonstrated in an experimentally infected carnivorous marsupial, the fat-tailed dunnart sminthopsis crassicaudata. it is also very likely that n. caninum tissue cysts are not necessarily formed predominantly in the cns but also at other locations such as muscle tissue [34, 35], and this should be taken into account in future studies. in conclusion, we comparatively assessed balb/c, cba/ca, and c57bl/6 mice as models for n. caninum infection and demonstrated that cba/ca mice exhibited the highest degree of resistance at low and medium infection doses but rapidly developed acute signs of neosporosis when challenged with the highest dose of n. caninum nc-liv tachyzoites. this mice strain has previously been shown to be suitable for tissue cyst production, which renders them an interesting model for investigations on protective effects and potential tools for vaccination. in contrast, the use of balb/c mice, although extensively employed, is debatable due to their inherent th2 bias, resulting in increased susceptibility to n. caninum. thus, there is clearly a need for a standardisation of experimental murine infection models for n. caninum in order to achieve comparable results between research groups that work with different tools for immunoprevention and chemotherapy.

c57bl/6, balb/c, and cba/ca mouse strains with different mhc-i haplotypes were compared with respect to susceptibility to neospora caninum infection. groups of 5 mice received 1 106, 5 106, or 25 106 tachyzoites of the nc-liverpool isolate by intraperitoneal injection and were observed for disease symptoms. humoral responses, splenocyte interferon- (ifn-) production, cerebral parasite loads, and histopathology were evaluated at human end points or the latest at 34 days postinfection (pi). the mortality rates in c57bl/6 mice were the highest, and relatively high levels of igg1 antibodies were detected in those mice surviving till 34 days pi. in lymphocyte proliferation assays, spleen cells from c57bl6 mice stimulated with n. caninum antigen extract exhibited large variations in ifn- production. in balb/c mice mortality was 0% at the lowest and 100% at the highest infection dose. serologically they responded with high levels of both igg2a and igg1 subclasses, and lymphocyte proliferation assays of surviving mice yielded lower ifn- levels. cba/ca mice were the most resistant, with no animal succumbing to infection at a dose of 1 106 and 5 106 tachyzoites, but 100% mortality at 25 106 tachyzoites. high igg2a levels as well as increased ifn- in lymphocyte proliferation assays were measured in cba/ca mice infected with 1 106 tachyzoites.

PMC4890932

pubmed-479

pityriasis rosea is considered to be a benign cutaneous condition [14]. however, in the setting of pregnancy, adverse effects on the newborn may be observed. craniosynostosis is a congenital abnormality, which can occur as an isolated finding or as part of a syndrome with other associated features. a woman who developed pityriasis rosea during her first trimester of pregnancy and who subsequently delivered a healthy baby with craniosynostosis is described, and observations of infants born to women who are diagnosed with pityriasis rosea during their gestation are summarized. a 28-year-old healthy woman presented at 10 weeks gestation with a skin rash. two weeks earlier, at eight weeks gestation, she had noticed an initial skin lesion on her abdomen (figure 1). her husband, a 29-year-old man, had presented two months earlier with similar-appearing annular lesions on his neck (figure 2). a larger lesion had initially appeared on his left neck. within the next five days, additional lesions appeared on the remainder of his neck and subsequently, a few lesions appeared on his distal upper extremities. a diagnosis of inverse pityriasis rosea was established based on the clinical history and lesion morphology. his lesions resolved over the next two weeks after treatment with triamcinolone 0.1% cream twice daily. cutaneous examination of the patient s lesions showed multiple annular plaques with peripheral scaling on the abdomen and back (figure 1). a diagnosis of pityriasis rosea was established based on the appearance of the lesions and history. cetaphil cream was applied twice daily to the patient s lesions; the lesions resolved during the next eight weeks. she was classified as a high-risk pregnancy and was followed closely for the remainder of her gestation. after 15 hours of labor, the baby had not descended into the pelvis; there was no fetal distress, and a decision for c-section was made. his apgar scores at one and five minutes were 9/9, weight 3827.1 g, and height 50.8 cm. prior to discharge from the hospital, it was noted that the infant had a curved head. neurosurgery consultation confirmed the diagnosis; at 9 weeks postpartum, endoscopic repair was performed successfully with no adverse sequelae. pityriasis rosea classically presents as annular plaques with peripheral scale, typically located between the neck and the groin, and may be seasonal in occurrence. less commonly, it can present with lesions on the neck and extremities (inverse pityriasis rosea) [1011] or during pregnancy. however, associations with human herpes virus (hsv)-6 and hsv-7 have been observed [1418]. several studies have found that patients with pityriasis rosea have higher levels of hsv-6 and hsv-7 detected in their skin, suggesting that infection by these viruses may have a causal effect on the development of pityriasis rosea. occasionally, pityriasis rosea has been documented in siblings or in spouses (table 1) [1921]. in these circumstances, our patient s husband developed and cleared inverse pityriasis rosea two months prior to his wife developing classic pityriasis rosea. similar to our patient, in the majority of cases of pityriasis rosea occurring in couples, the lesions appeared in the husband prior to the wife (table 1). the interval between onset of pityriasis rosea in the wife after occurrence in the husband ranged from seven days to one year (median: 2 months). our review of the literature, including the patient in this report, discovered 54 women who developed pityriasis rosea during their pregnancy (table 2 and table 3 [6,1214,23,24]). the onset of pityriasis rosea ranged from week 8 of gestation (3 patients: cases 7 and 8 in table 2 and case 14 in table 3) to week 32 (1 patient: case 7 in table 3). the median number of weeks of pregnancy at the onset of pityriasis rosea was 19. twenty-five women ages 24 to 34 (median age 29)had no prior pregnancies. however, pityriasis rosea occurred during either the second (20 women) or the third pregnancy (6 women) for the other women. most of the women (66%, n=35) developed pityriasis rosea during the second trimester of gestation (1328 weeks). nineteen percent (10 women) had the onset of their dermatosis during the first trimester (012 weeks). only 10% (5 women) experienced it in the third trimester (2940 weeks). several retrospective studies have observed adverse events affecting the newborn in women who develop pityriasis rosea during pregnancy (table 2). in these individuals, the adverse events predominantly included stillbirth at 11 to 28 weeks (median 16 weeks), premature delivery (< 37 weeks), hypotonia, weak motion, and low birth weight. less common adverse effects were hydramnios and foramen ovale. some investigators have discovered that pityriasis rosea occurring earlier in pregnancy, such as in the first trimester, have been more often associated with a poorer prognosis, compared to women who developed the dermatosis during the second or third trimesters. however, our review of the literature showed that the majority of women (16/25, 64%) who experienced adverse events had the onset of pityriasis rosea that occurred during the second trimester. the onset of pityriasis rosea occurred during the first trimester in 9 women (36%) and none in the third trimester. additional studies looking at the association between hsv-6 and hsv-7 dna and the occurrence of pityriasis rosea in pregnancy have also been performed. some of the studies found reactivation of hsv-6 during pregnancy. however, a positive correlation between viral infection and clinical features of pityriasis rosea was not established. individual case reports, including the patient in this report, have described 29 women who developed pityriasis rosea during pregnancy and have delivered healthy newborns (table 3) [6,1214,23,24]. indeed, the literature shows a ratio of 6:5 with regards to healthy newborns versus newborns with adverse events being delivered to women with gestational pityriasis rosea. however, the number of publications regarding gestational pityriasis rosea on the outcome of the newborn may not accurately reflect the incidence of normal newborns whose mothers had gestational pityriasis rosea, since clinicians may not report these women or journals may elect not to publish the papers. it can occur as an isolated incidental event or as part of syndrome (table 4). as an isolated incidental finding, sagittal craniosynostosis, as observed in our patient s newborn, is the most common form. if left unrepaired, craniosynostosis may lead to a deformed skull, elevation of intracranial pressure, and cognitive impairment. our patient s infant was evaluated shortly after delivery and had repair of the sagittal craniosynostosis at 9 weeks, with no complications and subsequent normal development. the incidence of sagittal craniosynostosis is about 1 in 5,000 live births. to the best of our knowledge, isolated craniosynostosis has not been observed in newborns of women who developed pityriasis rosea during their gestation. indeed, the occurrence in our patient s child may merely be a coincidence and not associated with her episode of pityriasis rosea during her first trimester. however, the true incidence is not known, since gestational pityriasis rosea is not frequently reported. some researchers noted that pityriasis rosea occurring earlier in pregnancy had a greater probability of resulting in adverse events for the fetus, including stillbirth, low gestational weight, hypotonia, and/or premature delivery. however, there are a similar number of reports of women who developed pityriasis rosea during their gestation and delivered normal newborns. our patient developed pityriasis rosea during her first trimester beginning at 8 weeks gestation and lasting through 18 weeks. her son was carried to term and delivered at 40 weeks and 6 days with apgar scores, weight, and height in normal range. whether the presence of craniosynostosis was associated with our patient s development of pityriasis rosea during her pregnancy remains to be determined.

background: pityriasis rosea is a papulosquamous disease. it may occur during pregnancy; in this setting, it has occasionally been associated with adverse outcomes.purpose:a woman who developed pityriasis rosea at the beginning of her eighth week of gestation is described. the outcomes in newborns delivered by pregnant women who developed pityriasis rosea during gestation are summarized. method:a 28-year-old woman developed pityriasis rosea during her eighth week of pregnancy. her husband had pityriasis rosea two months earlier. pubmed was searched for the following terms: conjugal, craniosynostosis, newborn, pityriasis, pregnancy, rosea, sagittal, spouse. the papers were reviewed and the references cited were evaluated. results:our patient delivered a healthy male infant after 41 weeks of gestation. he had normal weight, height, and apgar scores. isolated sagittal craniosynostosis was diagnosed and was successfully treated at nine weeks after birth without complications. conclusion:several retrospective studies have investigated the possibility of adverse outcomes in infants born to women who developed pityriasis rosea during pregnancy, such as stillbirth, low gestational weight, hypotonia, and premature delivery. however, there are also reports of healthy newborns in women who have had pityriasis rosea during gestation. our patient carried the fetus one week post-term and delivered a healthy boy via c-section; isolated sagittal craniosynostosis was later diagnosed and successfully repaired. the occurrence of craniosynostosis in a woman who developed pityriasis rosea during her first trimester of pregnancy may be two coincidental events.

PMC5006551

pubmed-480

the desynchronization of central and peripheral circadian systems contributes to the decline in optimal functioning of bodily systems. this includes changes in neuroendocrine circadian rhythms, insulin sensitivity, altered thermoregulation and acceleration of tumor growth (cincotta et al., 1993; touitou and haus, 2000; hastings et al., 2003; straub and mocchegiani, 2004; cretenet et al., 2010; heller et al., 2011 these are complex interactions and dysfunction can be due to targeted disruption of neurons, neurotransmitter or neuropeptide production, transport or secretion. it is reasonable to expect that the neuronal activity or expression of circadian clock genes be reduced or rhythms phase shifted (kolker et al., 2003). additionally, the connections between central and peripheral oscillators may be degraded or less functional (morales et al., 1987). any one or a combination of these abnormalities may result in the decoupling of the circadian oscillators and the ensuing pathologies. the master central pacemaker is the suprachiasmatic nucleus (scn) which controls circadian rhythmicity. rhythmicity can dampen and/or elongate/shift with age but the number or cell size of neurons in the scn does not change., 1993; madeira et al., 1995). additionally, glucose uptake decreases in the scn in aged animals (wise et al., 1988) and the expression of neuropeptides also diminishes. vasoactive intestinal polypeptide (vip) expressing neurons of the scn are retinorecipient and vasopressin (avp) expressing neurons of the scn modulate rhythmicity (wu et al., 2007). in aged male humans, the number of scn neurons that express vip decreases and in aged female rats the expression becomes arrhythmic (zhou et al. both genders also express less avp protein, less mrna (roozendaal et al., 1987; zhou et al., 1995) and the normal daytime peak of avp is reduced (hofman and swaab, 1994; liu et al., 2000). decreases in the neuroendocrine output of the scn may directly or indirectly affect the coupling of central and peripheral oscillators. in the scn, clock genes constitute the core clock mechanism of the mammalian timekeeping system. though the system is fairly complex, for brevity the simplest model is described. bmal1 and clock proteins dimerize and induce the transcription of period (per) and cryptochrome (cry) genes. in a negative feedback loop, levels of per and cry proteins increase and at a certain threshold form heterodimers which turn off the clock bmal1 regulated transcription of per and cry genes. this process takes roughly 24 h (ko and takahashi, 2006). in aged animals the expression of certain clock genes changes in the scn. per1, per2, and cry1 expression does not change significantly with age, but the normal photic stimulation of per1 expression is reduced (asai et al.. additionally, the free-running period of per1luc rhythmicity is shortened in aged animals (yamazaki et al., 2002) and the amplitude of clock and bmal1 expression is decreased (kolker et al., 2003). changes in clock gene expression in peripheral tissues do not always reflect what is seen in the scn (yamazaki et al., 2000), and may result from a disruption of signals to these tissues or the tissues themselves, which are more susceptible to the aging process. furthermore, projections to and from the scn including peripheral oscillators may change with age. in motoneurons, aging results in a shortening in delay of spike potentials between axon and soma, as well as decreases in axon conduction velocity and increases in input resistance (morales et al. the output from circadian and peripheral oscillators do not only influence the sleep/wake cycle, but regulates metabolism and reproduction. studies indicate that some tissues retain the ability to oscillate, even if connections from the master pacemaker have been degraded. peripheral tissues in vitro that have become arrhythmic can be chemically induced to oscillate (yamazaki et al. however, in aged animals exhibiting a decreased photic response, retinal projections to the scn are not degraded and must be related to either the retina or scn clock functions (zhang et al., it is increasingly evident that determining the source of age-related sleep/wake or circadian dysfunctions is rather complex. another source of sleep/wake changes can likely be attributed to age-related neuronal dysfunction in the arousal and sleep promoting areas of the brain. the scn directly or indirectly communicates with the sleep and wake promoting systems (abrahamson et al., 2001; aston-jones et al., 2001 orexinergic (or hypocretinergic) neurons are known to stabilize or maintain wake (saper et al., 2001, 2005). these cells receive input from the scn via the dorsomedial hypothalamus (dmh) and are localized in the perifornical and lateral hypothalamus (lh). there are two forms of the neuropeptide orexin/hypocretin (a and b) and two receptors. though the neurons are limited to a discrete area, both orexinergic fibers and receptors are widely distributed throughout the brain. in diurnal and nocturnal rodents, orexinergic neurons are most active during the active phase (martinez et al., 2002). hypothalamic microdialysate analysis shows orexin-1 levels increase during wake and rem in adult animals (kiyashchenko et al., 2002). mammals with no orexin or dysfunctional orexin/hypocretin receptors have disrupted sleep/wake cycles and narcoleptic symptoms. when orexin is decreased, the circadian rhythm of the sleep/wake cycle is disrupted. the flip flop model of sleep/wake control suggests that there is a mutual inhibition between the areas that control sleep and the areas that control the wake state (saper et al., 2001). in short, the ventrolateral preoptic area (vlpo) controls sleep and the brainstem cholinergic and monoaminergic systems control waking. flipping weight between these areas blocking or destroying these neurons or the orexin receptor 2 may flip the animal s state quickly from waking to sleep and vice versa, such as what occurs in narcoleptic individuals, orexin knockout mice, and canine narcolepsy (chemelli et al., 1999; lin et al., 1999; peyron et al., 2000; a disruption in orexin function or a reduction in orexin levels leads to less stable sleep/wake cycles such as that seen in many elderly patients with sleep disorders (porkka-heiskanen, 2003). additionally, decreases in excitatory orexin innervation to the noradrenergic locus coeruleus (lc) is thought to be a contributing factor of poor sleep/wake quality in aged cats (zhang et al. orexin b immunoreactive (-ir) axon density was determined to be significantly lower in the lc of aged macaques than that observed in the young or adult animals (43 and 35% decrease respectively; downs et al., 2007). real time pcr studies showed that preprohypocretin mrna does not change in the aged hypothalamus (terao et al., 2002) but in situ hybridization studies show that at the single cell level, preproorexin gene expression does decrease in cell count and optical density (porkka-heiskanen et al., 2004). furthermore, orexin a and b protein expression as measured by radioimmunoassay was decreased in the lh (porkka-heiskanen et al., the number of orexinergic-ir neurons as well as the optical density of respective fibers in the lh is reduced in aged animals (brownell and conti, 2010; sawai et al., 2010). it is interesting to note that orexinergic innervation of the cholinergic basal forebrain, which modulates wake and rem sleep, is reduced in aged guinea pigs (zhang et al., 2005). hypocretin receptor 1 mrna is reduced in the hippocampus and hypocretin receptor 2 mrna is significantly reduced in thalamic areas, hippocampus, and the brainstem (terao et al., 2002). neural activity measured by c-fos immunoreactivity is reduced in orexinergic neurons of mice at 24 months (naidoo et al., 2011). changes are also seen in the cholinergic and monoaminergic wake active areas of aged animals. nicotinic and muscarinic receptors of the acetylcholinergic system decrease in the scn with age (van der zee et al., 1991). in young animals, the noradrenergic neurons of the lc are important in wake promotion, receive direct input from the scn and follow a circadian pattern of activation (aston-jones et al., 2001). in aged rats, lc projections to the frontal cortex and dentate gyrus decrease but axonal branching increases depending on the target and age. this is suggested to be a compensatory mechanism (shirokawa et al., 2000). in the ventral periaqueductal gray (vpag) the wake active dopaminergic neurons we have recently reported a reduction in the neural activity of these dopaminergic neurons of the vpag and the noradrenergic neurons of the lc in aged mice (naidoo et al., 2009, 2011). the wake active histaminergic system originates in the tuberomammillary nucleus (tmn) and sends widespread projections to areas that include the cortex, thalamus and brainstem. histamine levels were found to be increased in middle aged rats when compared to young, and the level of histamine methyl transferase was decreased (mazurkiewicz-kwilecki and prell, 1984). histamine h1, h2 and h3 receptor mrna is decreased in the aging brain (terao et al., 2004). given these changes in the aging wake promoting neurotransmitter systems as well as wake maintaining systems, it is clear that therapies to alleviate or attenuate these changes need to be developed., 1996; szymusiak et al., 1998) and when lesioned results in insomnia (lu et al., 2000). gabaergic and galaninergic inhibitory neurons from this area project to wake active histaminergic neurons (sherin et al., 1996). interestingly, the number of activated vlpo neurons during sleep does not change in old rats (shiromani et al., 2000) although connections between these areas may become dysfunctional or degraded with age. the scn has a minor input into the vlpo, but substantial direct and indirect inputs to the dmh (novak and nunez, 2000; chou et al., 2002). the dmh heavily inputs the vlpo and it would be beneficial if these pathways were examined during aging. age-associated changes in the serotonergic system affect the function of respiratory motor output during sleep. serotonergic input to the hypoglossal nucleus decreases, which is thought to lead to a decline in upper airway muscle performance (behan and brownfield, 1999). in aged rhesus monkeys, serotonin receptor 2 density reduces in the occipital and parietal cortex including the deep layers of the motor cortex (wenk et al., 1989; bigham and lidow, 1995). serotonin levels also decrease in the occipital areas but do not change in the cingulate cortex in aged monkeys (beal et al., 1991). it is likely with normal aging that changes in any neurotransmitter system affecting sleep vary across the brain. in many patients afflicted with neurodegenerative diseases the physical and mental consequences lead to sleep disorders (table 1). for example, sleep fragmentation can occur if the patient can not move well or insomnia may develop due to depression or feelings of helplessness. medications used to alleviate some of the motor or cognitive symptoms such as levodopa in parkinson s disease (pd) can also contribute to disruptions in normal sleep/wake behaviors. however, some research indicates that sleep disturbances may predict manifestation of neurodegenerative diseases (postuma and montplaisir, 2009). sleep disturbance or loss also affects metabolic and immune function (krueger et al., 1998; knutson et al., 2007 chronic sleep loss could lead to neuronal damage resulting in altered hypothalamic pituitary adrenal axis function, cognitive deficits and memory loss. increases in the number of patients with neurodegenerative diseases may be related to or the result of a society that does not sleep. da, dopamine; inos, inducible nitric oxide synthase. sturrock and rao (1985), zhang et al. (1982), foley et al. (2007), magri et al. (1997), touitou (1995), whitehead et al. (( 1998), feinberg et al. (1967), myers and badia (1995), brun and englund (1981), teipel et al. (1985), kremer et al. (1991), arnulf et al. initially there is an increase in nighttime arousals and a decrease in sws (vitiello et al., 1991). in the later stages, circadian disruption, severe daytime wakefulness and a reduction in rem sleep occurs, likely due to a reduction in acetylcholine (dykierek et al., circadian rhythm dysfunction has been proposed to be due to changes in scn and pineal functions (wu et al., 2007). degeneration of cholinergic input from the nucleus basalis of meynert to the cortex may be responsible for some of the sleep/wake changes (montplaisir et al., 1995). neurofibrillary tangles found in the histaminergic tmn of ad patients and amyloid- peptide (a) aggregation also contributes to the ad pathology. normally in the interstitial fluid a has a diurnal fluctuation with low levels during sleep and peak levels during wake. recently one study showed that prolonged wake and/or orexin administration increased levels of the a in the interstitial fluid of the brain in mice (kang et al. administration of an orexin antagonist reduced amyloid deposits in several brain areas suggesting that manipulating sleep or the orexin system in ad patients could improve symptoms (kang et al., 2009). although the research is sparse, melatonin, phototherapy and exercise have all had positive effects in the treatment of circadian and sleep/wake disorders of ad patients (wu and swaab, 2007). as one in three americans develops ad, there is a crucial need for more research in these therapies. rem sleep behavior disorder (rbd) has been associated with pd and thought to be an early manifestation (schenck et al., 1996; boeve et al., 2003; postuma and montplaisir, 2009). sleep attacks and excessive daytime sleepiness (eds) are also commonly seen in patients with pd (factor et al., 1990; diederich et al., the degeneration begins at the brainstem and progresses rostrally, although degeneration of the dopaminergic neurons of the substantia nigra pars compacta is the main contributor to pd characteristics (braak et al., rbd results from pedunculopontine dysfunction and likely explains rbd manifesting previous to pd (rye, 1997; boeve et al., 2007). some studies have successfully seen bright light therapy or sleep modifications reduce the symptoms of pd (hogl et al., 1998; willis and turner, 2007). huntington s disease (hd) is a genetic disorder characterized by a polyglutamine (cag) repeat (scherzinger et al., 1999). neurodegeneration is extensive throughout the brain, affecting cortical and subcortical areas but primarily affects the basal ganglia (vonsattel et al., 1985). sleep and wake regions of the brain including the brainstem, thalamus, hypothalamus and cortex are also affected in hd (kremer et al., 1991). the scn pacemaker is functional in mouse models of hd, so a dysfunction of the circadian circuitry is proposed to contribute to circadian abnormalities (pallier et al., 2007). central and peripheral clock gene expression is altered as well (morton et al., 2005; maywood et al., 2010). the sleep/wake cycle is disrupted in hd patients characterized by self-reported eds, sleep fragmentation at night, and delayed sleep phase (arnulf et al., 2008; videnovic et al., 2009; aziz et al., sleep is lighter with an increase in stage 1 and a decrease in rem sleep (arnulf et al., 2008). disruptions in the circadian and sleep/wake cycles of these patients exacerbate symptoms, increasing depression, cognitive deficits and metabolic dysfunctions (aziz et al., 2010). it is important to note that pharmacological and behavioral manipulation of sleep and wake reduces disease progression and improves cognitive function and circadian gene expression in a mouse model of hd (hockly et al., 2002; pallier et al., 2007; pallier and morton, 2009; maywood et al., 2010) amyotrophic lateral sclerosis (als) is considered an age-associated neurodegenerative disease with the age of onset ranging from 40 to 70. most als cases are sporadic and about 10% are familial. also called lou gehrig s or motor neuron disease (boillee et al., 2006), both upper and lower motor neurons are affected. motor neurons of the motor cortex, brainstem and spinal cord gradually degenerate leading to muscle weakness, sleep disordered breathing (sdb) and paralysis (kimura et al., 1999). additionally, sleep is reduced in both rem and sws stages with resulting eds (barthlen and lange, 2000; lo coco et al., 2011). some patients find relief using assisted breathing such as continuous positive airway pressure (cpap) or bilevel positive airway pressure (bipap; howard et al., 1989; david et al., 1997; barthlen and lange, 2000). amyotrophic lateral sclerosis is a complex disease of many subtypes with various genetic and environmental contributing factors. one such factor is glutamate toxicity which is decreased using the drug riluzole (shaw and ince, 1997). levels of serotonin are decreased in als patients and compensatory increases in glutamate lead to excitotoxicity. it has been suggested that motor neurons with a high density of serotonergic innervation are more susceptible to degeneration (sandyk, 2006). as melatonin has antioxidant properties and inhibits glutamate release, this reduction would further exacerbate degeneration. indeed, melatonin supplements slowed disease progression when given to a mouse model of familial als (weishaupt et al., 2006). normally the scn is coupled to peripheral oscillators, although studies have shown that scn control is not necessary for sustaining oscillatory activity. if signals from central oscillators reduce in strength due to age or neurodegeneration, other cues may entrain the peripheral oscillators (weinert, 2005). unmasking mechanisms within sleep/wake systems is difficult due to the many checks and balances that ensure homeostasis. although compensation and plasticity occurs to a lesser extent in older animals, a relatively high degree is preserved (van someren et al., 2002). this may not always be advantageous as epigenetic methylation of circadian genes has been associated with dementia (liu et al., 2008). understanding how the aging brain can compensate and remain plastic will be beneficial to focus on more effective treatments for sleep/wake and neurodegenerative disorders. the co-morbidity of sleep disorders with neurodegenerative diseases suggests that changes in many of these neural areas manifests in sleep/wake and circadian dysfunction. effects on the sleep/wake and circadian systems may result from, or contribute to, the increasing pathology. some research has shown the benefit of pharmacologically or behaviorally restoring rhythms and sleep/wake for delaying pathologies (table 1). this is important to understand in a society where sleep is not considered a priority. a few points worth considering are as follows: sleep is a basic need that is made secondary to work schedules and some leisure activities for many adults. in developing children and adolescents, early school start times and late night extracurricular meetings contribute to a culture of sleep deprived, cognitively unhealthy americans. if restoring our circadian and sleep/wake cycles can ward off the deterioration of the brain, it is imperative to educate the public about the very real damage of abnormal sleep/wake cycles not only in aging individuals but at every age. the fragmented sleep/wake pattern seen in aging individuals can be due to the degeneration or dysfunction of the circadian and sleep/wake networks. uncoupling of the central and peripheral oscillators may exacerbate dysfunction via altered feedback signals or signaling pathways. it is likely that several brain regions are affected and that there are individual differences in how the sleep/wake and circadian networks degrade. additionally there may be differential plasticity and compensation in the integration of these neural systems, making the identification of applicable therapies very difficult. however, if mechanisms contributing to the normal aging process of these networks are identified, this may elucidate a general therapy for restoring sleep/wake and circadian homeostasis. it is crucial that we immediately invest our energies and resources in understanding these mechanisms as well as in the dissemination and implementation of current knowledge and therapies to the public. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

sleep/wake and circadian rest-activity rhythms become irregular with age. typical outcomes include fragmented sleep during the night, advanced sleep phase syndrome and increased daytime sleepiness. these changes lead to a reduction in the quality of life due to cognitive impairments and emotional stress. more importantly, severely disrupted sleep and circadian rhythms have been associated with an increase in disease susceptibility. additionally, many of the same brain areas affected by neurodegenerative diseases include the sleep and wake promoting systems. any advances in our knowledge of these sleep/wake and circadian networks are necessary to target neural areas or connections for therapy. this review will discuss research that uses molecular, behavioral, genetic and anatomical methods to further our understanding of the interaction of these systems.

PMC3199684

pubmed-481

it is well known that whole breast irradiation (wbi) after breast conserving surgeries for patients with early infiltrating breast carcinoma (bc) significantly reduces the likelihood of local recurrence (lr). there are several evidences that lr is a predisposing factor for systemic metastasis [24] and, within this scope, radiotherapy (rt) is very useful for treating residual tumor cells after the surgery. the most used schedule for wbi is 50 gy delivered in 5 weeks using conventional fractionation. there is no consensus, however, regarding whether the entire breast needs to be irradiated. the accelerated partial breast irradiation (apbi) concept, based on confining the irradiation to the vicinity of the tumour bed, shortening the course of the treatment and allowing more convenience for patients, has contributed to changes in the rt paradigms [6, 7]. a variety of apbi techniques, including low- or high-dose rate brachytherapy, balloon brachytherapy, localized external beam rt (using either three-dimensional or intensity-modulated), and intraoperative electron or photon beam treatments, have been used with encouraging results [813]. nondedicated linear accelerators (linacs) capable of delivering treatment with electrons have been used for intraoperative irradiation of many other tumors [14, 15]. these types of equipment are available in almost every rt facility and are used for daily patients ' treatments. the possibility of delivering intraoperative radiotherapy (iort) with electrons, without dedicated equipment, is very attractive. addressing this issue, the purpose of our paper was to assess the efficacy, toxicity, and cosmetic outcomes of iort delivered by standard linacs, during breast conserving surgeries for the treatment of early breast cancer. a prospective phase ii cohort study started in may 2004 at the sirio libanes hospital in sao paulo, brazil. as of july 2012, 187 women with diagnosis of bc by percutaneous biopsy were enrolled. patients were eligible if they had unicentric invasive ductal carcinoma, with less than 3.0 cm at the largest diameter confirmed by mammography, ultrasonography, and magnetic resonance imaging (mri). patients were considered ineligible if any of the following features were present: skin involvement, history of bc in the contralateral breast, or intraoperative microscopic findings of involvement of surgical margins or sentinel node (sn). invasive lobular carcinoma subtype was also an exclusion criterion due to its high rate of multicentricity and multifocality. the breast conserving surgeries were performed at an operating theater located inside the radiotherapy department, contiguous to the linac suite. the quadrantectomy consisted of an en bloc resection of the parenchyma and pectoralis fascia, with at least a 2 cm macroscopic margin around the tumor. the skin over the tumor was generally removed by a circular incision, with its conservation being possible in small, deeply located tumours (t 1.0 cm). after verification of clear margins by intraoperative histopathologic and cytologic exams, sn radioguided biopsy was generally performed by the unique breast incision, as previously described [16, 17]. as for surgical aspects, the same maneuvers standardized for electron intraoperative therapy (eliot) by veronesi et al. once the wide local excision and the sn biopsy were performed, the glandular tissue was detached from the pectoralis major muscle, to an extension of 3 cm margin around the resected area, and the skin flaps were detached from the parenchyma at the level of the adipose lamina for 2 cm circumferentially. the surgical bed was filled with a wet compress, the wound was covered, and the patient was transferred to the rt room, where all of the materials needed for maintaining anaesthesia, including gases, were available. a three-layer disk made of lead (down), aluminium (middle), and silicon (up) was inserted underneath the gland over the muscle, to protect the normal tissue below the irradiated area and absorb the backscattered radiation. the shielding disks (0.5 cm thick each) were available in three diameters (6, 8, or 10 cm), and the largest one fitting the space was placed. the parenchyma was approximated over the disk by separated stitches, exposing the area to the electron beam. irradiation was performed using one of two standard models of siemens linear accelerators: primus or kd2. both machines produce electrons and are able to generate photon and electron beams with energy ranging between 6 and 21 mev. a single total dose of 21 gy prescribed at the 90% isodosis was delivered directly to the parenchyma at a rate of 300 cgy/min. the electron beam energy was chosen after measuring the gland thickness by inserting a needle perpendicularly to the parenchyma. a sterile, round collimator was connected to the gantry of the linac and gently placed into the surgical bed by appropriated mobilization of the couch and gantry (figure 1). the choice of collimator diameter was made according to each case but was usually up to 6 cm. a portal film was taken placing the film below the accelerator couch, orthogonally to the collimator, to guarantee the exact positioning of the disks. this procedure was repeated, if necessary, until the disk was considered well positioned. afterwards, the staff left the room; the irradiation was delivered during in average 8 minutes, according to the chosen energy, under video surveillance of the vital signs of the anaesthetized patient (figure 2). the breast tissue was then reconstructed using oncoplastic techniques, preferentially outside the linac room, in the operating room [1921]. adjuvant systemic therapy was at the discretion of the physician, in accordance with current guidelines. more than half of the patients received hormone therapy alone (51.3%), 8.5% of the patients received only chemotherapy, 38.1% had both, and 1.9% patients had no adjuvant systemic therapy. follow-up was performed every 3 months in the first year and every 6 months thereafter. lr was considered as a true relapse (tr), which represents regrowth of residual malignant cells in the same region of the primary tumour, or a second primary tumour (spt), representing tumor growth in another quadrant, suggesting a distinct clonal origin. the presence of seromas, hematomas, fat necrosis, wound infections, and dehiscences was investigated at all time points after surgery. cosmesis evaluation was scored by the physician at least 12 months after irradiation, in accordance with the harvard criteria. briefly, the treated breast is compared with the contralateral one and the result is classified as excellent (minimal or no difference in the size or shape); good (mild asymmetry in the size or shape); fair (obvious differences in the size and/or shape); and poor (marked change in the appearance involving more than 1/4 of the breast). the cumulative incidence of lr, overall survival, and bc survival were calculated using the kaplan-meier method. the spss package version 17.0 (chicago ii) medcalc package, 11.3.3.0 version (mariakerke, belgium), was used for statistical analysis. of the 187 enrolled patients, 35 (18.7%) were intraoperatively excluded because of sn positivity (18 patients), difficulty in obtaining clear margins (11 patients), multicentricity/multifocality (3 patients), muscle infiltration (1 patient), t3.0 cm (1 patient), and no sn identification (1 patient). the cumulative incidence of lr as the first unfavorable event was 3.2% (95% ci: 0.88.1) (figure 3). among the 5 cases of lr in the entire cohort 4 were considered to be a tr, and one had a failure in a quadrant other than the index lesion at 30-month follow-up, consistent with spt. regarding other failures, two patients (1.3%) developed distant metastases, three had axillary failure (1.9%), and one patient had a contralateral tumor (0.6%). it is worth noting that among the cases with trs one had sn micrometastasis and one had lobular carcinoma, both identified only in the definitive histopathological analysis. one hundred and nine cases were followed up for at least 36 months with an estimated lr rate of 4.6%. kaplan-meier estimates of efficacy at three years were lr of 4.6%, contralateral breast tumor 0.9%, distant failure 1.8%, cancer specific survival 98.2%, and overall survival 98.0%. the cumulative incidences of first unfavorable events are outlined in table 2, and the 3-year actuarial rates of recurrences are presented in table 3. there were three deaths (1.9%): two related to breast cancer (one secondary to pulmonary metastasis and another due to chemotherapy toxicity) and one nononcologically related death. overall survival is shown in figure 4. in the first month after surgery, 6 cases of skin erythema (3.9%), 2 wound dehiscences (1.3%), and 1 case of hematoma (1.9%) were observed. evidence of late toxicity, observed after at least 6 months of follow-up, was seen in 45 patients (29.6%) in a median time of 8 months (range 824). there were 21 cases (13.8%) of breast fibrosis (13 mild and 8 severe) and 15 cases of fat necrosis (9.8%). among these cases, there were also 3 cases of breast lymph edema and 2 cases of nipple retraction. the esthetic outcomes have shown 70.3% excellent, 14.4% good, 3.9% regular, and 3.2% of bad results. from the entire cohort, cosmetic outcomes are listed in table 4. in figure 5 a case with an excellent esthetic result breast conserving surgery followed by external wbi is a well established treatment for most women with early infiltrating bc [24, 25]. currently, more sophisticated rt techniques are available, allowing better target coverage with better normal tissue sparing. in this context, apbi is a rapidly evolving strategy, with a widespread support for its use [7, 27, 28]. the main biologic rationale for intraoperative partial breast irradiation is that 85% of the lr (almost 100% of tr) occurs in the vicinity of the tumour, next to the scar, as a consequence of the persistence of neoplastic cells that most likely possess aggressive cancer stem cell properties [29, 30]. experimental data indicate hierarchical organisation of bc with a small number of cancer-initiating cells (cics) that have ability to self-renew and exhibit multilineage potential. cics, in contrast to their tumorigenic counterparts, can survive fractions of sublethal doses of rt, retaining self-renewal capacity over several generations [3234]. some properties of cics could make them a more vulnerable target to a single lethal irradiation dose, soon after the breast resection, without allowing postoperative hypoxia and time for cell repopulation. effects of iort on tumor microenvironment could improve outcomes, as it impairs local proliferation caused by surgical manipulation, inflammation, and simulation of the epithelial mesenchymal transition [36, 37]. different rt techniques can be used with this purpose and, given that intraoperative rt with standard linacs has previously been used to treat abdominal tumors, we decided to use this form of treatment during breast conserving surgeries. the surgeries were performed at an operating theatre in the rt department, close to the linac room where the patients were transferred to receive the irradiation. this geographic characteristic by itself turned out to be a feature that helped the better feasibility of the method. however, it is still possible to transport the patient from an operative theatre far from the linac suite (usually out of business hours), previously prepared to be used as an operating room. the patient transport from the operating room to the linac may be regarded as a disadvantage of the method, when compared to the treatment with a dedicated linac. but one must realize that the use of a nondedicated linac, mainly in developing countries, may represent a cost-benefit strategy. we have previously reported our outcomes with focus on technical aspects, and the highlights of the use of nondedicated machine were to explore its capability of producing higher electron beam energies rather than dedicated machines and to check the possibility of misalignment between the collimator and the shielding disks by obtaining portal films using photon beams. other advantages of iort with electrons are accurate targeting of rt and a precise definition of the tumour bed volume under direct guidance, offering very good dose homogeneity and more effectively sparing of the heart and lungs when compared to external beam rt. at the moment there are two published randomised trials focusing on single dose of rt during breast-conserving surgeries. using localized photon beams delivered by the intrabeam device (carl zeiss meditec, oberkochen, germany), concluded that such approach is as efficient as conventional fractioned external beam rt for carefully selected patients. they employed two types of dedicated linear accelerators: novac 7 (hitesys, latina, italy) and liac (info and tech, rome, italy). although they found that the rate of lr in the eliot group was within the prespecified equivalence margin of results, it was observed that this rate was significantly greater than with external radiotherapy, pointing out the necessity of defining the optimal patient selection criteria. by far, the most important benefit of iort with electrons is shortening the rt duration from the traditional 5-6 weeks to 58 minutes, thereby eliminating the delay in receiving rt, alleviating emotional distress, avoiding logistical difficulties in travelling to the radiation facility and ensuring 100% compliance. the rate of undertreated patientsdue to incomplete fractioned adjuvant wbi is far from ideal, especially in developing countries, being such women exposed to a higher risk of bc recurrence [42, 43]. the key feature for the development of eliot by the italian group was the estimation of dose equivalence between the standard 60 gy divided into 30 fractions and the single dose of 21 gy. in a landmark paper, veronesi et al. presented a large phase ii study that included 1,822 cases treated with eliot using dedicated machines. after a mean follow-up period of 36.1 months, 42 women (2.3%) developed a tr, 24 women (1.3%) had a new primary ipsilateral tumour, and 26 women (1.4%) had distant metastases as the first event. compared with conventional rt, eliot was considered a safe procedure for women with tumours measuring less than 2.5 cm, with a slightly higher lr rate. our results presented here are very similar to the results obtained by veronesi et al. the widespread use of apbi motivated the american society of therapeutic radiology and oncology to define a suitable group of patients for whom apbi is acceptable outside of clinical trials, including the following: women older than 60 years, with t1 idc, clear margins, and the absence of multicentricity, multifocality, and axillary nodes involvement. the european society for therapeutic radiology and oncology also proposed suitable conditions for apbi: age 50 years, unicentric and unifocal t1-2 (3.0 cm), pn0 nonlobular invasive cancer, the absence of an extensive intraductal component and lymphovascular invasion, and negative surgical margins of at least 2 mm. currently it is also known that estrogen receptor negativity is associated with increased risk of lr following apbi. this study has started before the publication of these recommendations, and part of our cases should be considered not suitable for apbi. however, some other results have pointed out that even patients who do not meet the ideal conditions may be locally treated with success [47, 48]. anyway, since the publication of the recommendations (2009), women under 50 years of age were no longer accepted in our study. although it might be tempting to offer iort to a large number of patients, at this time, a careful selection of suitable patients is paramount. for this reason we advocate preoperative mri which was performed in all of our patients, to better select the cases for partial breast irradiation. most likely, the traditional wbi reduces the rate of spt in the treated breast solely if they were present and occult at the time of the primary treatment. mri could potentially contribute to the more precise detection of multifocal or multicentric disease, with improvement of operative outcomes and decreased recurrence rates, although, besides the mri high diagnostic accuracy, it is always desirable to have pathological verification of the findings because of the mri high false-positive rates. the confirmation of intraoperative clear surgical margins is also mandatory, since the objective of iort is to reduce lr by treating residual malignant cells that may persist in tumour-bearing areas. with regard to efficacy moreover, complications due to local toxicity were scarce, and this form of iort led to a favorable impact on body image, as already observed by other authors. also, when oncoplastic maneuvers are required, including immediate breast reconstruction with prostheses, they are feasible and safe [19, 21]. we consider as limitations of this study the facts that there was not a control group and that it was performed at a single institution on relatively small number of patients. in spite of these caveats, the technique was demonstrated to be feasible and was successfully implemented, with a very short learning curve. iort with electrons delivered by conventional linacs, immediately after a wide local excision, presented the expected results until now, with very good local control and cosmetic outcomes and a low toxicity rate. selected patients with early infiltrating breast carcinomas may benefit from the technique, which may represent an interesting option for developing countries.

purpose. to assess feasibility, efficacy, toxicity, and cosmetic results of intraoperative radiotherapy (iort) with electrons delivered by standard linear accelerators (linacs) during breast conserving surgeries for early infiltrating breast cancer (bc) treatment. materials and methods. a total of 152 patients with invasive ductal carcinoma (t 3.0 cm) at low risk for local relapses were treated. all had unicentric lesions by imaging methods and negative sentinel node. after a wide local excision, 21 gy were delivered on the parenchyma target volume with electron beams. local recurrences (lr), survival, toxicity, and cosmetic outcomes were analyzed. results. the median age was 58.3 years (range 4085); median follow-up was 50.7 months (range 12101.5). there were 5 cases with lr, 2 cases with distant metastases, and 2 cases with deaths related to bc. the cumulative incidence rates of lr, distant metastases, and bc death were 3.2%, 1.5%, and 1.5%, respectively. complications were rare, and the cosmetic results were excellent or good in most of the patients. conclusions. iort with electrons delivered by standard linacs is feasible, efficient, and well tolerated and seems to be beneficial for selected patients with early infiltrating bc.

PMC4281392

pubmed-482

heart disease, including coronary heart disease, cardiomyopathy, and heart failure, causes functional deterioration and/or failure as well as myocardial cell death. it is one of the leading causes of death in advanced countries because adult cardiomyocytes are highly differentiated and have a limited regenerative capacity; therefore, significant loss of myocardium is mostly irreversible. cell regeneration therapy is a promising new approach for myocardial repair, [1, 2] and in this context, there has been considerable basic research on the mechanisms of cell development into cardiomyocytes using somatic and embryonic stem (es) cells as well as embryonic carcinoma cells [36]. p19 embryonic carcinoma cells are one of the first among such cells to demonstrate differentiation into cardiomyocytes and have contributed extensively to the elucidation of the development mechanisms from stem cells into cardiomyocytes [4, 5, 8]. p19 cells are derived from a teratocarcinoma in ch3/he mice and can differentiate into all 3 germ layers. culture and differentiation of the cells is simple, and this advantage has enabled their extensive application for decades. they continuously grow in serum-supplemented media, can retain an undifferentiated cell state without a feeder cell layer unlike es cells, and their differentiation can be controlled by nontoxic reagents. primarily, cell aggregate formation in suspension culture under 0.51.0% dimethyl sulfoxide (dmso) followed by the reagent application in adhesion culture has been used to induce cardiomyocyte differentiation of p19 cells [7, 8, 10]. we examined the differentiation of p19 cells into cardiomyocytes by the above general method; however, it resulted in large variations in the differentiation rate and low differentiation efficiencies among individual experiments, even though high differentiation efficiencies have been reported previously [7, 8, 10]. p19cl6 cells, clonal derivatives of p19 cells, were established by habara-ohkubo. these subline cells can be differentiated into spontaneously beating cardiomyocytes by treatment with 1% dmso in adhesion culture over a period of 10 days or weeks without cell aggregate formation in suspension culture and more efficiently as compared to the parent cells. therefore, p19cl6 cells may be more useful to examine the differentiation mechanisms of cardiomyocytes in vitro. recently, ohtsu et al. introduced a double stimulation method for cardiomyocyte differentiation from p19cl6 cells. they demonstrated that cells exposed to 10 m 5-azacytidine (5-aza) for 24 hours prior to treatment with 1% dmso differentiated into cardiomyocytes more effectively than the widely used single stimulation method described above. in order to produce a large number of cardiomyocytes, we induced differentiation of p19cl6 cells into cardiomyocytes by the double stimulation method to examine the differentiation efficiency; however, it resulted in large variations in the differentiation rates among individual experiments, as in the case of p19 cells. however, notably, the double stimulation method elicited differentiation from p19cl6 cells most efficiently among the several differentiation methods as long as it was in vitro in our laboratory. we also noticed large variations in the differentiation rates among previous reports on p19cl6 [1214] as well as p19 cells [8, 10, 15, 16] and this may imply a fundamental problem of the cells in themselves and/or the differentiation methods. to overcome this difficulty, we attempted to establish a subline from p19cl6 cells, and accordingly, we could introduce a new subline of cells, that is, p19cl6-a1 cells, that could efficiently and stably differentiate into cardiomyocytes by a differentiation method based on the double stimulation method with many modifications. in the present study, we also introduce a software program, visorhythm, which can analyze the temporal variations in the beating rhythms on moving images and chart correlograms displaying the oscillated rhythms on a windows computer. because the spontaneously beating cardiomyocytes differentiated from p19cl6-a1 cells form large cell clusters, their contractions are large and strong. this feature may prove to be quite suitable for oscillated rhythm analysis based on moving images, and accordingly, we used this software to successfully display the increase in the cardiomyocyte beating rates on correlograms and dose-response curves on treatment with serially diluted cardiotonic reagents. this may indicate that p19cl6-a1 cells and the above software are useful tools for pharmacological screening tests. balb/c mice of either sex maintained under standard housing and feeding conditions were used. the ventricles of the hearts dissected from mice aged 8 weeks and 16.5 days post coitum (dpc) fetuses were examined by reverse transcription pcr (rt-pcr). the cells were cultured in a tissue culture-grade dish and grown in alpha-modified eagle's minimal essential medium (-mem; sigma-aldrich japan k.k., tokyo) supplemented with l-glutamine (4 mm; gibco brl, carlsbad, ca) or dulbecco's modified eagle's minimal essential medium (dmem; sigma-aldrich japan k.k.) containing 510% heat-inactivated fetal bovine serum (fbs; jrh biosciences, lenexa, ka) and penicillin and streptomycin (100 u/ml and 100 g/ml, resp.; sigma-aldrich japan k.k.). several subclones were isolated from the p19cl6 cells by the common cloning ring technique. each clonal colony was expanded increasingly into larger wells and dishes and frozen until use. two types of cell colonies were derived from a single cell: one, growing horizontally to form a single cell layer on a dish and the other, growing both horizontally and vertically to form cell aggregates of multicell layers like embryoid bodies or cell aggregates in suspension culture prepared using bacterial-grade dishes [5, 8, 13]. both cell types were collected for the cardiac differentiation experiment; however, the latter were considerably more efficient in differentiating into cardiomyocytes. among the several differentiation methods, the one proposed by ohtsu et al. elicited most efficient cardiomyocyte differentiation from parent p19cl6 cells as long as the experiment was in vitro in our laboratory; however, it also led to a fundamental difficulty, that is, high variations in the differentiation efficiencies among individual experiments. hence, we modified the above method in order to adapt it to our subclonal cells as well as to the parent cells. the differentiation conditions that we primarily considered were the concentration and/or exposure duration of the differentiation reagents, the concentration of fbs in the culture medium (-mem or dmem), and coating reagents. they were as follows: 5-azacytidine (5-aza; sigma-aldrich japan k.k.): concentration: 5, 10, 15, 20, 30, and 40 m and exposure duration: 24, 48, and 72 hours; dimethyl sulfoxide (dmso, sigma-aldrich japan k.k.): concentration: 0.5, 1.0, 1.5, and 2.0%; fbs in the differentiation medium: concentration: 2.5, 5.5, 6.5, 7.5, and 10%; and the use of a culture dish coated with or without collagen (3.0 g/cm, type i-a; nitta gelatin inc., osaka) or matrigel (3.0 g/cm; bd biosciences, san jose, ca). accordingly, we found a subclone, designated a1, which is the most efficiently differentiated into cardiomyocytes under the differentiation conditions below and used it for further experiments to evaluate the differentiation efficiency. we also found the optimum conditions that could be applied to the parent p19cl6 cell line, and the use of these conditions yielded almost the same results as those yielded by the method of ohtsu et al.; the description has been provided below. the differentiation efficiencies of the subline, p19cl6-a1, and the parent cell line, p19cl6, were compared. the cells were plated at a density of 5.0 10 cells/well on 6-well plates or on a 35-mm tissue culture-grade dish coated with collagen (3.0 g/cm) and containing the growth medium (-mem with 6.5% fbs for p19cl6 cells or dmem with 7.5% fbs for p19cl6-a1 cells) and were incubated in a 5% co2 atmosphere at 37c. the next day, when cells reached ~95% confluence, the medium was replaced with a growth medium containing 10 m 5-aza in order to induce differentiation. the p19cl6 and p19cl6-a1 cells were treated with 5-aza for 24 and 72 hours, respectively, and the 5-aza-containing medium was changed every 24 hours. after treatment with 5-aza, the cells were incubated in the growth medium containing 1.0% dmso for more than 16 days. the dmso-containing medium was also changed everyday in order to remove the cell debris resulting from cell death. the experimental days were numbered consecutively beginning from the day after the first day of treatment with 5-aza (day 0). p19cl6 and p19cl6-a1 cells were cultured on glass coverslips coated with collagen (3.0 g/cm). cells on day 16 were washed twice with hanks ' balanced salt solution (hbss; sigma-aldrich japan k.k.) and then fixed with 4% paraformaldehyde in pbs for 30 minutes at 4c. after the fixation, cells were rinsed twice in cold pbs, treated with 0.1% triton x-100 in pbs for 20 minutes at room temperature and again rinsed twice in pbs. in order to reduce autofluorescence, cells were treated with 50 mm nh4cl in pbs for 10 minutes at room temperature. then cells were blocked in a humid chamber with pbs containing 3% normal goat serum (ngs; sigma-aldrich japan k.k.) for 30 minutes and then incubated with the anticardiac -actin antibody (sigma-aldrich japan k.k.; 10 g/ml in pbs containing 3% ngs) for 16 hours at 4c. after washing with pbs, cells were incubated with alexa488-conjugated goat antimouse igg (molecular probes, inc., eugene, or) for 30 minutes at room temperature, cells were photographed with fluorescence microscopes (ix71, olympus co., tokyo). the specificity of the immunofluorescence staining was verified by incubations without the primary or secondary antibodies and no specific fluorescence was observed in the control. for quantitative analysis of rates of differentiation into cardiomyocytes, we measured ratios of cardiac -actin-positive cell areas to total areas of microscopic fields using image j software (ver. 1.41; national institutes of health, bethesda, md). autofluorescence from multilayer cell regions was rather strong but -actin-specific fluorescence was easily discriminated from the autofluorescence because -actin-positive cells emerged as high-intensity cells in cell clusters (see figure 2). in this way we counted out autofluorescence regions. sixty fields were examined in each cell type and the results from six independent experiments were summarized as mean se. unpaired t-test was carried out to compare the differentiation efficiency between the two cell types. total rna was isolated from the cultured p19cl6 and p19cl6-a1 cells on days 0, 3, 7, and 16, and from the cardiac ventricles of 8-week-old adult mice and 16.5 dpc fetuses using the trizol reagent (invitrogen japan k.k., one microgram of total rna was transcribed into first-standard cdna using m-mlv reverse transcriptase, rnase h (promega, madison, wi), and oligo (dt) primer according to the manufacturer's instructions. for detection of gata4 and nkx2-5, the specific transcription factors for cardiac differentiation, and of the alpha myosin heavy chain (-mhc), which is indispensable for cardiomyocyte contraction, 1 l of the reaction mix (out of the total 25 l) as the reverse-transcribed dna template was amplified by pcr40 cycles for gata4 and nkx2-5 and 25 cycles for -mhc. after amplification, the pcr products were separated on 1.2% agarose gel and visualized by ethidium bromide staining. the expression levels of gata4, nkx2-5, and -mhc mrnas were compared between p19cl6 and p19cl6-a1. they were determined from 3 independent experiments and normalized by reference to the expression levels of gapdh mrna (23 cycles of pcr). all analyses were performed on excel 2007. the values were expressed as the mean se that was represented by ratios to the mean values of the p19cl6 cells in the basal protocol on day 16 as 1.0 according to the differentiation protocol. the following primer pairs were used: gata4, 5-gttgtggtggtgggtttttc-3 (forward) and 5-tttgatgttcctgggagagg-3 (reverse); nkx2-5, 5-tctccgatccatcccactttattg-3 (forward) and 5-ttgcgttacgcactcactttaatg-3 (reverse); -mhc, 5-tacctcatggggctgaactc-3 (forward) and 5-cgaacatgtggtggttgaag-3 (reverse); gapdh, 5-gacttcactcacggcaaatt-3 (forward) and 5-tcctcagtgtagcccaagat-3 (reverse). the 35-mm dish with the differentiated cells were placed in a stage top microscope incubator (oni-inu-f1; tokai hit, shizuoka, japan) mounted on an inverted microscope (dmirb, leica microsystems ltd., heerbrugg, switzerland) and incubated at 37c in a 5% co2 atmosphere. phase-contrast moving images of spontaneously beating cells were recorded through a ccd camera (coolpix4500; nikon co., tokyo) and digitalized to mpeg2 video files (8 bits/channel; 704 pixels 480 pixels/frame; 30 frames/sec) using an encoder (mtu2400 fx; canopus co. ltd., the contraction rhythm of the spontaneously beating cells was analyzed based on the moving images using the following method based on the data analysis algorithm on the calculation of correlation coefficient as reported by yamauchi et al.. a small area of interest where brightness clearly oscillated due to the beating was selected from a moving image, and then, a reference frame was arbitrarily chosen from the moving image frames. temporal variations in the correlation coefficient were calculated between the values of mean brightness of the reference frame and those of other frames in the selected area, and accordingly, a correlogram representing the contraction rhythm of the beating cells in the area of interest was displayed by the vertical coefficient and horizontal time axis. we developed a software program, visorhythm, which calculates the temporal variations in the correlation coefficient up to 6 rectangle areas (150400 pixels/area) simultaneously on a single moving image on a windows computer. the data can then be directly output into excel to chart the correlograms, and the beat-to-beat intervals are represented by the intervals between the upward and downward peaks in each correlogram in the selected areas. we examined the pharmacological reactions of spontaneously beating cardiomyocytes differentiated from the p19cl6-a1 cells to evaluate whether they are useful cellular tools for pharmacological screening tests. we used 3-isobutyl-1-methylxanthine (ibmx; sigma-aldrich japan k.k.) and ouabain (sigma-aldrich japan k.k.), which are well-known potent cardiotonic reagents, for this purpose. phase-contrast moving images of spontaneously beating cells were recorded before and after addition of serial dilutions of each reagent (10 nm, 20 nm, 70 nm, 200 nm, 700 nm, 2 m, 7 m, 20 m, and 70 m) into the medium. the beat-to-beat intervals on exposure to certain concentrations (10 nm, 30 nm, 100 nm, 300 nm, 1 m, 3 m, 10 m, 30 m, and 100 m) were obtained from the correlograms calculated on visorhythm. the median effective dose (ed50) was obtained from the dose-response curve based on the beating rates on which the basal and maximum rates were represented by 0 and 100%, respectively. we selected the differentiation protocol suggested by ohtsu et al. for the p19cl6 cells because this method was the most efficient for cardiomyocyte differentiation from among the several differentiation methods as long as it was in vitro in our laboratory. the undifferentiated populations of the cells, however, considerably proliferated even in the differentiation medium containing 1% dmso as a differentiation inducer and 10% fbs; hence, we reexamined the optimum concentration of fbs. we thus obtained a resultant concentration of approximately 6.5%, and accordingly, the protocol for the cells was as revised as follows. subconfluent p19cl6 cells in -mem containing 6.5% fbs were treated with 10 m 5-aza for 24 hours followed by treatment with 1% dmso for more than 16 days (basal protocol). in case of the p19cl6-a1 cells, we modified the basal protocol by reexamining the conditions in each step according to the criteria of (1) rates of appearance of the beating cells per field and (2) dimensions of the beating area under a microscope. the cells showed a relatively high resistance to 5-aza, and the optimum conditions were as follows. subconfluent p19cl6-a1 cells in dmem containing 7.5% fbs were treated with 10 m 5-aza for 72 hours followed by treatment with 1% dmso for more than 14 days (developed protocol). we also examined the differentiation efficiencies in culture dishes coated with or without collagen or matrigel and found the differentiation to be more effective on collagen coated dishes. the conditions for each protocol and for each cell type are summarized in table 1. we observed the differentiation processes under an inverted microscope to compare the morphological differences between the p19cl6 and p19cl6-a1 cells. both cell types grew similarly in the beginning (days 05) of the differentiation protocols. the p19cl6 and p19cl6-a1 cells reduced in number during treatment with 5-aza due to the damage by the reagent and proliferated slightly everyday thereafter during treatment with dmso. the cells started to grow vertically due to overconfluence, and multilayer cell regions appeared around day 5. on the p19cl6 cells multilayer cell regions were generally small and appeared like scattered islands in monolayer cell regions. however, on p19cl6-a1 cells, they were connected with each other to form mesh-like structures and expanded to comprise approximately 7080% of the total area of the dishes during the procedure. this feature of the multilayer cell regions was similar to that of embryoid bodies or aggregates on culture with dmso in the nonadhesive bacteriological-grade dishes. beating cells first appeared among the p19cl6 cells differentiated by the basal protocol on day 10 and among the p19cl6-a1 cells differentiated by the developed protocol on day 11. the clusters of beating cells increased in number and size thereafter and peaked on day 16 in both cases. the beating cell clusters from the p19cl6 cells were observed generally in the monolayer cell regions and less frequently on the boundary between the monolayer and multilayer regions (figure 1(a)). the clusters, composed of small-sized beating cells densely arranged, were generally small in the monolayer regions (figures 1(c) and 1(e)). on the other hand, the beating cell clusters in the p19cl6-a1 cells appeared mostly in the multilayer and boundary regions (figure 1(b)). they were composed of elongated cells and formed mesh-like sheet structures (figures 1(d) and 1(f)). they were relatively large and sometimes extended beyond the visual field under the inverted microscope through a 10 objective lens, and accordingly, the contractions were large and strong. therefore, the beating cells differentiated from the p19cl6-a1 cells on day 16 or later were suitable for contraction rhythm analyses based on moving images. in order to morphologically identify cardiomyocytes differentiated from p19cl6 and p19cl6-a1 cells clearly and to compare the differentiation efficiencies between the two, we immunocytochemically examined those cells on day 16 using anticardiac -actin antibody under immunofluorescence microscopy. cardiac -actin-positive cells from p19cl6 were various in shape (oval, polygonal, short spindle-shaped) and generally formed round clusters (figures 2(a) and 2(c)). those from p19cl6-a1 were relatively large and elongated, and formed mesh-like sheet structures (figures 2(b) and 2(d)). we measured ratios of cardiac -actin-positive cell areas to total areas of microscopic fields; the positive cell areas were 7.0 1.1% (mean se) of the total areas on p19cl6 while those were 38.3 5.7% on p19cl6-a1. the value on p19cl6-a1 was 5.5 times and significantly higher than that on p19cl6 (p=.002; figure 2(e), table 1). to compare the differentiation efficiencies between the p19cl6 and p19cl6-a1 cells, we examined the expression of the cardiac-specific transcription factors, gata4 and nkx2-5, and of the cardiac-specific myosin heavy chain, -mhc, by rt-pcr on the respective cells with the respective differentiation protocols listed in table 1 on days 0, 3, 7, and 16. both gata4 and nkx2-5 were already expressed in both p19cl6 and p19cl6-a1 cells on day 0, that is, in the untreated cells (figure 3(a)). the expression of the former increased from day 0 to day 16 during treatment in both groups (figure 3(b)), while that of the latter dipped on day 3, but increased thereafter (figure 3(c)). on day 16, when the appearance rates of the beating cells per field and the dimensions of the beating area peaked as observed under a microscope, gata4 expression did not differ significantly between the p19cl6 and the p19cl6-a1 cells; however, nkx2-5 expression in the latter was significantly and 1.45 times higher than that in the former (figure 3(c)). alpha-mhc was not expressed till day 7, although it was clearly expressed on day 16 in both groups (figure 3(d)). its expression in the p19cl6-a1 cells was significantly and 1.77 times higher than that in the p19cl6 cells (figure 3(d), table 1). these results indicated that p19cl6-a1 cells were more efficient than the parent in terms of cardiomyocyte differentiation. we also examined -mhc expression in the ventricles of 8-week-old adult mice and 16.5 dpc fetuses by rt-pcr to compare -mhc expression in the tissues with that in the p19cl6-a1 cells differentiated by the developed protocol on day 16. the expression levels were higher in the adult tissue than in the other tissue, but were almost equal between the fetal tissue and the cells on day 16 (figure 3(e)). we examined the pharmacological reactions of the spontaneously beating cells differentiated from p19cl6-a1 cells by the developed protocol. for this purpose, we employed ibmx and ouabain as cardiotonic reagents, and both of them increased the beating rates. the moving images of the beating cells were recorded before and after addition of serial dilutions of each reagent. to determine the temporal variations in the beating rhythms in detail, we developed the software, visorhythm, to analyze them based on moving images and chart correlograms displaying the oscillated rhythms. figure 4(a) is an example of a series of correlograms corresponding to the temporal variations in the contraction rhythms of the beating cells treated with 10 nm, 300 nm, and 10 m; the beating rate was 1.30 times per second at the basal level and increased up to 2.30 times per second after the addition of 10 m ibmx. figure 4(b) shows the dose-response curve to ibmx corresponding to the data in figure 4(a), and its ec50 was 950 nm. figures 4(c) and 4(d) illustrate another series of correlograms and a dose-response curve to ouabain, respectively. these data indicate that the p19cl6-a1 cells and visorhythm are useful tools for pharmacological screening tests. p19 embryonal carcinoma cells have been extensively studied for cardiac differentiation in vitro and have contributed to the elucidation of the early events in differentiation, such as the involvement of bone morphogenic proteins and their subsequent intracellular signal cascades and oxytocin and its subsequent activation of the transcription factors gata4, nkx2-5, and so on [5, 7, 8, 10, 19]. for their efficient differentiation into cardiomyocytes, prior formation of cell aggregates in suspension culture under 0.51.0% dmso followed by reagent application in adhesion culture is required; however, molecular events occurring during aggregation and the necessity of their aggregation for differentiation have not yet been entirely understood. high cell densities at least can trigger spontaneous differentiation from p19 cells without the formation of cell aggregates and reagent treatment [9, 10]. on the other hand, p19cl6 cells, the clonal derivative of the p19 cells, efficiently differentiate into cardiomyocytes on dmso treatment in adhesion culture without prior formation of cell aggregates, although high cell densities are required for effective differentiation. in the present study, we introduced p19cl6-a1, a clonal derivative of p19cl6 cells, which differentiated into cardiomyocytes most efficiently among the 3 cell lines subjected to double stimulation with 5-aza and dmso in adhesion culture as reported by ohtsu et al. the p19cl6-a1 cells extensively formed multicell layers in adhesion culture under stimulation with dmso after they reached overconfluence. they were highly packed in multicell layers like the cell aggregates of p19 cells in suspension culture. p19cl6-a1-derived cardiomyocytes, which generally formed large clusters of beating cells, almost always appeared in the multilayer regions and on the boundary regions adjacent to the monolayer regions. these findings, in addition to the above differentiation characteristics in the other 2 cell types, may indicate that cell-to-cell contact all around, that is, not only from side to side but also from top to bottom, is a key event in the differentiation of p19 cells and their derivatives. because three-dimensional contact can be achieved by the formation of multicell layers, a clonal derivative differentiating more efficiently into cardiomyocytes than p19cl6-a1 may be established when the dimensions of the multilayer regions is employed as a criterion for clonal selection from p19cl6 cells. further investigations are necessary to elucidate whether multilayer formation is a crucial trigger for differentiation and to determine the molecular events occurring in the multilayer regions in this instance. we employed the double stimulation method using 5-aza and dmso as suggested by ohtsu et al., as the basal protocol for cardiomyocyte differentiation because it elicited differentiation from the parent p19cl6 cells most efficiently among the several differentiation methods as long as it was in vitro in our laboratory. to apply it to the clonal derivative, that is, to p19cl6-a1 cells, we examined the optimum concentration and exposure time of the inducers and accordingly, the protocol for efficient differentiation was modified as summarized in table 1. the optimum concentration of each inducer is the same as that in the basal protocol, but the exposure time of 5-aza is 3-fold longer than that for the parent cells. an almost equal and fair percentage of p19cl6 and p19cl6-a1 cells were necrotic after treatment with 10 m 5-aza for 24 and 72 hours, respectively, in the monolayer culture, and therefore, the p19cl6-a1 cells were more resistant to more prolonged exposure to the dna demethylating reagent than the p19cl6 cells. both cell types were much more resistant to the reagent than the p19 cells because the p19 cells treated with 510 m 5-aza in the monolayer culture became totally necrotic. in the present study, dmem with 7.5% fbs of the composition of the differentiation medium elicited differentiation from both p19cl6-a1 and p19cl6 cells more efficiently than -mem with 6.5% fbs and much more than -mem with 10% fbs (data not shown), which has been generally used for the differentiation of p19 and p19cl6 cells [7, 1115, 2026]. after the 5-aza treatment, p19cl6-a1 cells proliferated slightly under dmem containing 1% dmso and 7.5% fbs and could form multicell layers in adhesion culture more extensively than p19cl6 cells under -mem containing 1% dmso and 6.5% fbs. p19cl6-a1-derived beating cells appeared mostly in the multilayer regions and formed clusters more extensively in the multilayer regions than in monolayer regions. these may indicate that more extensive multilayer formation induces the cardiomyocytes to differentiate more efficiently. however, using 10% fbs instead of 7.5% or 6.5% could not elicit more efficient differentiation of the cells probably due to stable proliferation of the undifferentiated populations in the cells even in the differentiation media containing 10% fbs. in the present study, we found that 1% dmso is optimum for the differentiation of p19cl6 and p19cl6-a1 cells. it is generally used for differentiation of p19 and p19cl6 cells [1115, 20, 2226]. moreover, a previous report has demonstrated that the efficiency of colony formation of p19 cells in the presence of dmso indicates the absence of toxicity at concentrations of less than 1% under -mem with 10% fbs. based on these data, we suppose that the serum concentration in combination with 1% dmso is a considerable factor for efficient differentiation. in fact, the source and lot number of fbs and calf serum used in differentiation are thought to be crucial for successful cardiac differentiation from p19 cells as well as from p19cl6 cells (riken bioresource center, personal communication). in the present study, cardiac-specific transcription factors gata4 and nkx2-5 were expressed in untreated cells. in most previous reports p19cl6 cells untreated with inducers such as dmso and 5-aza expressed neither gata4 nor nkx2-5 [12, 14, 20, 22, 24, 27]. however, there are a few reports in which gata4 and/or nkx2-5 expressed in p19cl6 cells before the treatment [23, 26]. in the present study we used a medium containing 10% dmso for cell cryopreservation and cells cultured for 1 day after thawing as untreated cells (day 0) for the experiments. we suppose this caused the clear expressions because the untreated cells contain cells already exposed to the dmso as a cardiac differentiation inducer probably at a high rate. there were no differences in the differentiation efficiencies under an inverted microscope between p19cl6-a1 cells with and without subcultures after thawing (data not shown) and the same finding was obtained on p19cl6 cells (dr. there are 2 isoforms of cardiac mhcs, namely-and -mhc. the former is characterized by a high atpase activity and a quick contraction velocity as compared to the latter. alpha- and/or -mhc are often used as cardiac-specific markers for determining the efficiencies of cardiac differentiation of p19 and p19cl6 cells [8, 11, 13, 14, 20, 25]. in the present study, we showed that the expression level of -mhc mrna in the p19cl6-a1 cells treated with the inducers was significantly and 1.77 times higher than that in the p19cl6 cells and almost equal to that in the heart ventricles of the mouse fetuses. we also compared the differentiation efficiency between the two cells by using cardiac -actin immunofluorescence staining and showed that the ratio of cardiac -actin-positive cell areas to total areas of microscopic fields in p19cl6-a1 cells was 38.3% and 5.5 times higher than that in p19cl6 cells. this indicates that the differentiation efficiency estimated by the immunofluorescence staining is 3.1 times higher than that by the rt-pcr. this discrepancy is likely to be caused by the multicell layer formation of p19cl6-a1 cells; the two-dimensionally microscopic analysis may overestimate the differentiation efficiency due to the elimination of a factor in rate of formation of multicell layers and the estimation by the rt-pcr may represent the differentiation efficiency close to a real value. p19- as well as p19cl6-derived cardiomyocytes display an electrophysiological phenotype of embryonic ventricular cardiomyocytes based on the studies in which (1) they display a complete set of functional ion channels, but only limited amount of functional sodium channels, (2) the maximal diastolic potential is rather low (approximately 40 to 60 mv), and (3) the shape and characteristics of the action potential of these cells resemble those of primary isolated embryonic cardiomyocytes [5, 21, 24, 29]. thus, p19cl6-a1-derived cardiomyocytes may have similar properties because they are clonal derivatives of p19cl6 cells. they rhythmically and spontaneously beat and form clusters much larger than p19cl6-derived cardiomyocytes, and accordingly their contractions are larger and more obvious as observed under an inverted microscope. therefore, p19cl6-a1-derived cardiomyocytes may be suitable models for studies on the cardiac structural and functional properties during normal physiological and pathological states in vitro, particularly studies on contraction rhythm analyses based on moving images. for this purpose, that is, to figure out the temporal variations in the beating rhythms in detail, we developed the software, visorhythm, to analyze them based on moving images and chart correlograms displaying the oscillated rhythms. to illustrate the availability of p19cl6-a1-derived cardiomyocytes as physiologically functional cardiomyocytes and the software as an analytical tool for detailed analysis of beating rhythms, we employed ibmx and ouabain as potent cardiotonic reagents [30, 31]. additionally, we found that the nonbeating cells started to contract rhythmically after the addition of these reagents (data not shown). this finding is supported by a previous study in which p19-derived nonbeating cells generated action potentials upon electrical stimulation and indicates that the actual ratio of p19cl6-a1-derived cardiomyocytes is higher than that of the beating to nonbeating cells under the microscope. there are largely two different methods to analyze beating rhythms of cardiomyocytes in vitro: one is an electrophysiological method to measure electrical potential of cardiomyocytes and the other is a microscopic method to measure oscillated motions of cardiomyocytes. as for the former method, a culture plate equipped with microelectrode arrays having 64-channels are used to record electrical potentials on cultured cardiomyocytes [32, 33]. it can analyze electrical activities of cardiomyocytes such as conduction velocities and synchronization times. as for microscopic methods, micheletto et al. introduced a free-running scanning near-field optical microscopy setup to observe live cardiomyocytes. weisensee et al. also introduced an image analysis system using the video capture board for real-time subtraction between frames of moving images to analyze motions of beating cardiomyocytes. both tools can analyze beating rates and the relative strength for inotropic force while they monitor only one object at a time. here we have introduced visorhythm to analyze temporal variations in beating rhythms of cardiomyocytes based on moving images and chart correlograms displaying the oscillated rhythms. yamauchi et al. had developed a similar software on which the temporal variations in beating rhythms could be analyzed in 1 fixed area (20 20 pixels) chosen arbitrarily. in contrast, visorhythm can analyze up to 6 such areas (150400 pixels/area) on each frame of one moving image simultaneously. therefore it can also analyze synchronization between cardiomyocytes. in conclusion, we used double stimulation with 5-aza and dmso in this study and showed that p19cl6-a1 cells, a new clonal derivative of p19cl6 cells, differentiated into cardiomyocytes more efficiently than the parent cells. we introduced a new software, visorhythm, that can analyze the temporal variations in the beating rhythms and can chart correlograms displaying the oscillated rhythms up to 6 areas on a single moving image simultaneously. using p19cl6-a1-derived cardiomyocytes and the software, we demonstrated that the correlograms could clearly display the enhancement of beating rates by cardiotonic reagents. these indicate that a combination of p19cl6-a1 cells and visorhythm is a useful tool that can provide invaluable assistance in inotropic drug discovery, drug screening, and toxicity testing .

the p19cl6 cell line is a useful model to study cardiac differentiation in vitro. however, large variations were noticed in the differentiation rates among previous reports as well as our individual experiments. to overcome the unstable differentiation, we established p19cl6-a1, a new clonal derivative of p19cl6 that could differentiate into cardiomyocytes more efficiently and stably than the parent using the double stimulation with 5-aza and dmso based on the previous report. we also introduced a new software, visorhythm, that can analyze the temporal variations in the beating rhythms and can chart correlograms displaying the oscillated rhythms. using p19cl6-a1-derived cardiomyocytes and the software, we demonstrated that the correlograms could clearly display the enhancement of beating rates by cardiotonic reagents. these indicate that a combination of p19cl6-a1 and visorhythm is a useful tool that can provide invaluable assistance in inotropic drug discovery, drug screening, and toxicity testing.

PMC2846686

pubmed-483

in the united states, insufficient participation in leisure time physical activity constitutes a major threat to public health. recent estimates suggest that 25% of americans do not engage in any physical activity at all. even those engaging in physical activity are usually not doing so at recommended levels. in order to promote and maintain health, the american college of sports medicine (acsm) recommends a minimum of 30 minutes of moderate intensity aerobic physical activity five days a week or a minimum of 20 minutes of vigorous intensity aerobic physical activity three days a week. despite these widely disseminated guidelines, the centers for disease control (cdc) report that americans have made no substantial progress towards achieving recommended levels of physical activity with the proportion of 1829 year olds meeting guidelines hovering around 35% and the proportion of adults 65 and older meeting guidelines at about 20%. these numbers are troubling, as aerobic exercise has been convincingly linked to the prevention of myriad negative health outcomes, including several forms of cancer. numerous studies conducted over the past two decades have explored the association between physical activity participation and cancer prevention, consistently implicating strong or probable evidence for reduced risk of colon, breast, and endometrial cancers when physical activity recommendations are followed [36]. likely mechanisms through which physical activity is believed to have an influence on cancer prevention include reduction in adiposity and changes to levels of circulating metabolic hormones and growth factors (e.g., estrogen, testosterone, and insulin-like growth factors) [79] as well as influences on dna methylation [10, 11]. in respect to prostate cancer, because physical activity activates gut motility, gastrointestinal transit time for food wastes is lessened and thus, exposure to carcinogens is attenuated. there is also research to suggest that immune function changes may mediate the relationship between physical activity and cancer development. the promising body of literature regarding the relationship between physical activity and cancer has led the national cancer institute (nci) to regard behavioral primary prevention of cancer (e.g., physical activity) as a top priority. unfortunately, interventions designed to change behavior are typically met with only modest success, even when grounded in empirically supported theory, and behavioral adherence is reported to be a principal challenge faced by exercise promotion programs. indeed, only 50% of individuals who adopt an exercise program stay with it for more than six months [14, 15]. researchers devoted to the goal of improved physical activity participation have suggested that one likely determinant of physical activity behavior is the way in which individuals subjectively experience exercise. in previous work, we organized genetic, physiological, subjective, and motivational factors that may contribute to the initiation and maintenance of physical activity into a conceptual transdisciplinary framework [16, 17]. this framework has received support among both active and inactive samples, and provides the basis for the selection of phenotypes in the current study. briefly, we proposed that genetic factors influence how an individual physiologically (e.g., body-temperature regulation) and subjectively (e.g., affective response, perceived exertion) responds to the experience of exercise. physiological response influences how one subjectively responds to the experience of exercise (e.g., increased lactate during exercise may increase perceived exertion) and these subjective responses influence motivation to exercise (e.g., exercise self-efficacy, exercise intentions). moreover, exercise behavior itself influences both how a person physiologically responds to the experience of exercise and gene expression, thereby recapitulating the framework. importantly, this framework is meant to be dynamic such that the factors selected to represent physiological response, subjective response, and/or motivation can vary depending on the goals of each individual research study. the relationship between physiological changes induced by aerobic exercise (e.g., regulation of body temperature, heart rate, or blood pressure during exercise) and subjective responses to aerobic exercise (e.g., changes in affect during or immediately after exercise, ratings of perceived exertion or pain during an exercise bout) is one that has a clear influence on individual differences in exercise behavior. bryan and colleagues found that physiological factors such as heart rate were related to mood response to exercise, and that mood response was a significant correlate of both motivation to exercise in the future and of current exercise behavior. additionally, subjective experiences during exercise may be influenced by interpretations of exercise-induced physiological responses. for instance, increases in lactate levels during aerobic exercise may be perceived as painful to varying degrees across individuals, and this perceived pain will in turn influence subjective exercise experiences and potentially impact motivation to engage in exercise behavior in the future. understanding potential influences on subjective response to exercise is especially important, given that affective responses to acute exercise have been found to predict long-term exercise behavior [19, 20]. although the heritability of exercise participation in adults has been shown in twin studies to be approximately 50% (with peak heritability of 85%, occurring at age 19-20) [21, 22], there is a surprising lack of research regarding the role played by genetic factors for determining physiological and affective responses to exercise. these responses may serve as promising intermediate phenotypes for the linkage of genes to broader exercise participation phenotypes. also important explained by de geus and de moor as the genetic variance causing differential responses to exercise training, given that the effects of exercise on health and fitness gains appear not to be uniform across individuals [24, 25]. one type of gene-by-exercise interaction that is relevant to the present study is the role of exercise in reducing the phenotypic effects of some detrimental genetic variants. for example, phares et al., showed that sedentary individuals who possess two particular polymorphisms of the adr gene have unfavorable body composition. however, these individuals experience greater loss in percent body fat after 24 weeks of aerobic training in comparison with all other genotypes. it follows that weight loss interventions for individuals with this particular genotype would likely be successful if they focused on aerobic training. thus, identifying particular genetic markers that are related to exercise behavior and physiological and affective responses to exercise may have clear implications for matching individuals to tailored exercise intervention programs. the goal of the current study is to determine whether genotypes predicted subjective physiological and affective responses to a 30-minute bout of aerobic exercise among sedentary individuals. based on the literature and on our prior analysis of the relationships among a range of exercise response phenotypes (see for analysis and detailed information on the rationale for selection of phenotypes), the variables from the physiological responses to exercise domain included in our analysis were temperature, heart rate, systolic blood pressure, lactate, and norepinephrine, all measured as change scores from immediately prior to a bout of exercise to 30-minutes into the bout (just before the end of the bout). genetic associations with vo2 max were also examined, as cardiovascular fitness is highly heritable [2730], and evidence exists for a strong genetic influence on athletic performance. additionally, genetically influenced cardiovascular fitness traits play a role in determining individual experience of exercise intensity and perception of exertion during exercise. the variables selected from the subjective experience of exercise domain were affect (i.e., positive affect and affective valence), perceived pain, and rate of perceived exertion (rpe), which were also change scores measured from prior to the bout to just before the end of the bout. we chose the specific genetic factors for our analyses a priori based on evidence from the literature that they were linked to processes related to physiological and subjective responses during physical activity, general health and fitness traits, or because of evidence that they moderate responses to exercise interventions. a single nucleotide polymorphism (snp) in the fat mass and obesity-associated protein gene (fto; rs9930506) has been associated with obesity traits such as increased bmi and weight and susceptibility to obesity. additionally, physical activity may slow down weight gain associated with the fto risk-allele. in addition, the -opioid receptor gene (oprm1) may be associated with pain sensitivity such that individuals possessing the rare g allele have an increased pressure pain threshold. interestingly, this study also found gender differences in pain threshold among individuals with the g allele when heat pain was tested, such that women with this allele have lowered pain thresholds, and men exhibit higher pain thresholds. snps located within in the slit2 gene(rs1379659), fam5c gene (rs1935881), kcnb2 gene (rs10505543), and rs10498091 (an snp associated with left ventricle mass) have all been found to be associated with echocardiography traits (e.g., left ventricle diastolic dimension, diameter, and systolic dimension) in a genome-wide association study. another genome-wide analysis implicated creb1 in the prediction of submaximal exercise heart rate in response to exercise training [38, 39]. thus, each of these snps was investigated in the current study in order to determine potential relationships with phenotypes related to physiological and affective response to an acute bout of aerobic exercise. participants included in the present analysis were a subset of 238 individuals from a larger intervention study (costride) [17, 40] in which participants were randomly assigned to the stride exercise intervention (costride) or a health-and-wellness contact control condition (hw). participants were men and women (ages 1845) who reported less than 90 minutes on average of at least moderate-intensity physical activity per week for the past three months. individuals were excluded if they smoked cigarettes, were on a restricted diet, were taking psychotropic medications, were receiving treatment for any psychiatric disorder, were diabetic, had a history of cardiovascular or respiratory disease, had the flu or illness within the last month, or were pregnant (if female). all participants were required to be willing to be randomized to an intervention condition, to give informed consent, to be able to engage in moderate-intensity physical activity, to have a body mass index (bmi) between 18 and 37.5, and to have a regular menstrual cycle (if female). all participants were recruited from the denver-metro area and the university of colorado boulder community. the data reported herein are from assessments conducted prior to randomization, and the analysis and questions addressed are unique to this investigation. as described in detail below, we used the illumina human 1 m duov3 dna analysis beadchip to genotype the dna samples. the bead chips accommodate 4 samples each, and we ran a total of 50 bead chips. thus, this experiment allowed for the genotyping of 200 individuals total. due to limitations in funding, we were unable to genotype the remaining 38 individuals in the sample. thus, individuals with the most complete baseline data (baseline dna sample, self-report questionnaire assessments, vo2 max fitness assessment, and submaximal exercise session) were selected to be genotyped out of the full sample. statistical tests revealed no significant differences on demographic variables between participants who were included in genotyping procedures and those who were not included in genotyping procedures (details available from the first author). this reduced sample (n=200) was comprised primarily of females (n=160) and most participants identified as white (n=137), followed by hispanic/latino (n=22), asian american (n=22), african american (n=9), native american (n=5), and mixed ethnicity (n=5), the average age of participants at baseline was 28.68 (sd= 7.86) years old and mean body mass index (bmi; weight in kg/height in m) was 25.18 (sd=4.72). on average participants reported an average of 28.14 minutes of voluntary physical activity in the past week (sd= 50.95), and reached an average vo2 max peak of 34.06 ml/kg/min (sd= 8.11). prior to randomization to intervention condition and after giving informed consent, participants completed three sessions: (1) an orientation (baseline) session in which self-report questionnaire assessments were completed, (2) a vo2 max cardiovascular fitness assessment, and (3) a submaximal exercise session. prior to exercise sessions, each participant was instructed to eat a meal comprised of both carbohydrates and protein and to consume at least 300 calories two hours before coming into the lab (e.g., if a participant is scheduled to come into the lab at 12:00 p.m. a researcher instructed him/her to eat the 300 calorie meal at 10:00 a.m. and no later). participants were also instructed to drink at least 17 oz. of water two hours prior to coming into the lab. participants were instructed not to exercise on their own prior to the laboratory session, and not to consume alcohol during the 24 hours prior to testing. further details regarding recruitment, selection of measures, and study procedures are available elsewhere. consistent with established procedures, maximal oxygen capacity (vo2 max) was assessed during a balke protocol (a graded, incremental exercise test) on a motorized treadmill. vo2 max was assessed with online computer-assisted open-circuit spirometry using the medgraphics cardi02/cp system. prior to the fitness test, saliva samples (5 ml) were collected for dna extraction and measurements of height and weight were taken for calculation of bmi. approximately one week after the fitness test, participants completed a standardized, short 30-minute bout of physical activity on the treadmill at 65% of their previously established vo2 max, calculated during the fitness test (vo2 max test session). prior to beginning activity, an intravenous catheter was inserted by a nurse to collect blood samples during the bout. intensity was maintained by measuring oxygen uptake and expired co2 for two to three minutes at the beginning of exercise and at 10 and 20 minutes during exercise. lactate concentration and catecholamine levels (epinephrine and norepinephrine) were collected via blood samples immediately before activity began (11.5 ml), and 10 (5.5 ml) and 30 (11.5 ml) minutes into activity. tympanic temperature was measured by taking an average of 2-3 temperature readings at each measurement. readings of temperature, blood pressure, and heart rate were taken before activity, at 10 minutes, 20 minutes, and 30 minutes (directly before completion) during activity. subjective experiences during exercise were assessed at six points during the submaximal session: five minutes prior to activity, immediately before activity began, and 10, 20, and 30 minutes into activity (directly before completion of the session). the present study focuses only on change scores created from subtracting the values obtained immediately before the exercise bout began from the values obtained 30 minutes into the bout. for the time points that occurred 10, 20, and 30 minutes into the exercise bout, participants were assessed while they were exercising the bout was not interrupted to make these assessments. while participants continued their session on the treadmill, a research assistant held up cards with the questionnaire items displayed on them. participants indicated the number that they felt reflected their current subjective states, and their responses were manually recorded. physiological measures were obtained at these time points using the iv catheter that was inserted prior to the bout. positive affect was assessed using 3 items from the 12-item physical activity affect scale (paas). participants rated their current state for each item using a 5-point scale (0=do not feel to 4=feel very strongly). the adjectives assessed by the 3-item positive affect subscale were enthusiastic, energetic, and upbeat (= .81). affective valence during exercise was assessed using the 11-point single-item feeling scale (fs), which ranges from 5=very bad to+5=very good. perceived exertion was assessed using the 15-point single-item rating of perceived exertion (rpe) that ranges from 6 to 20 (6=no exertion at all, 20=maximal exertion). perceived pain was assessed using a single-item 12-point borg category ratio-10 scale (cr10) (0=no pain at all, 10=extremely intense pain). samples were genotyped on the illumina infinitum assay platform using the human 1 m duov3 dna analysis beadchip (illumina, inc., san diego, ca, usa) following the manufacturer's protocol at the university of colorado, boulder. in this assay, the dna is then resuspended in hybridization buffer and applied to the bead chip array for an overnight incubation. the amplified and fragmented dna samples anneal to locus-specific 50-mers (covalently linked to one of over 1,000,000 bead types) during the hybridization step. the samples then undergo single-base extension and staining, followed by more washing. the arrays are allowed to dry, and then scanned using the illumina iscan system. genotype calls were made using illumina's genomestudio software in conjunction with the genome studio genotyping module. additionally, we excluded snps with a minor allele frequency (maf) of<10% and snps that showed significant deviation from hardy-weinberg equilibrium (p<1 10). following these quality control checks, although we used a genome-wide approach to genotyping, we only tested a total of 14 snps which were selected for analysis in this study based upon their potential association with aerobic exercise response phenotypes suggested by prior studies (see table 2 for hardy-weinberg p values and minor allele frequencies for each snp tested). our search was conducted primarily using pubmed, and was focused on snps that were directly associated with specific phenotypes of interest and to traits that may be associated with those phenotypes. analyses were run using the snp and variation suite for genetic analysis (svs) (version 7.5.6, golden helix inc., bozeman, mt).the 14 snps selected for inclusion based on our search were tested for associations with the phenotypes using a correlational trend test assuming additive effects of allele dosages for each snp (i.e., homozygous for the minor allele=0; heterozygous=1; homozygous for the major allele=2). these analyses focused on correlations between particular snps suggested by the relevant literature and exercise response phenotypes drawn from our previous work and the existant exercise literature. due to the fact that both exercise phenotypes and candidate snps were selected a priori based on the literature as well as our transdisciplinary framework, critical alpha for all tests was maintained at the .05 level for all analyses. additionally, given that the aim of this study was to examine changes in physiological and subjective responses to exercise over the course of the 30-minute exercise bout, it was not necessary to compare subjects cross-sectionally at the baseline or 30-minute time points. rather, all phenotype values were determined using a change score created by subtracting each subject's baseline values from the values obtained by that subject 30 minutes after the exercise bout began. in order to determine whether allele frequencies for all snps examined in this study were significantly different across racial/ethnic groups, additionally, major and minor alleles, as well as minor allele frequencies (mafs) for caucasians, african americans, asians, and hispanic/latino participants are reported in table 3. significant differences in genotype across racial/ethnic groups were found for three snps, rs1935881 (10,200)=19.25, p=.037, rs1799971 (10,200)=38.13, p<.001, and rs8044769 (10,200)=21.54, p=.018. for rs1935881, the maf is lower among asians. for rs1799971, an maf of 0 was found in african americans. for rs8044769, results of associations between snps and exercise response phenotypes presented below are uncorrected (no pca correction applied). a total of 10 different phenotypes were examined for association with genetic variants in this study. given that many of these phenotypes may have common underlying physiological bases, we tested for associations between these phenotypes. in the following results, all phenotypes tested and reported (except for vo2 max) refer to a change score created by subtracting preexercise values from the values obtained 30 minutes into the exercise bout. vo2 max was significantly correlated with change in lactate (r=.177,<.05), heart-rate (r=.434, p<.01), systolic blood pressure (r=.193, p<.01), and rate of perceived exertion (r=.157, p lactate change was correlated with temperature change (r=.214, p<.01), heart rate change (r=.429, p<.01), systolic blood pressure change (r=.208, p<.01), change in affective valence (as measured by the feeling scale) (r=.173, p<.05), and pain change (r=.215, p<.05). norepinephrine change was significantly correlated with positive affect change (r=.174, p<.05) and pain change (r=.165, p<.05). temperature change was significantly correlated with heart-rate change (r=.172, p<.05) and affective valence change (r=.149, p<.05). heart rate change was significantly correlated with systolic blood pressure change (r=.185, p<.05) and rate of perceived exertion change (r=.231, p<.05). rate of perceived exertion change was significantly correlated with affective valence change (r=.163, p<.05) and pain change (r=.316, p<.01). finally, positive affect change was significantly correlated with affective valence (r=.455, p<.05). the creb1 snps rs2360969 and rs2253206 were associated with temperature change during exercise (rs2360969, r=.17, p=.02; rs2253206, r=.17, p=.02) indicating that for rs2360969, individuals with the t allele had greater changes in temperature over the course of the exercise, and for rs2253206, individuals with the a allele had greater changes in temperature during exercise. these same snps were also significantly associated with vo2 max (rs2253206, r=.17, p=.01; rs2360969, r=.14 p=.049), such that for rs2253206, individuals with the g allele had higher vo2 max, and for rs2360969, individuals with the c allele had higher vo2 max. the oprm1 snp rs1799971 was significantly associated with lactate change during exercise (r=.17, p=.02), norepinephrine change during exercise (r=.16, p=.03), and change in rpe during exercise (r=.14, p=.048), indicating that individuals with the rare g allele had greater changes in lactate, norepinephrine, and rate of perceived exertion change over the course of exercise. the fto snp rs8044769 was related to change in positive affect during exercise (r=.16, p=.03), and individuals with the c allele had greater change in positive affect over the course of the exercise. the fto snp rs3751812 was associated with positive affect change during exercise (r=.14, p=.04), such that individuals with the t allele experienced greater changes in positive affect. the fto snp rs9941349 was significantly related to change in systolic blood pressure during exercise (r=.15, p=.04), and individuals with the t allele experienced greater increases in systolic blood pressure during exercise. the fto snp rs7201850 was significantly related to change in systolic blood pressure during exercise (r=.17, p=.027), with individuals possessing the t allele experiencing greater increases in systolic blood pressure over the course of the exercise bout. the slit2 snp rs1379659 was associated with norepinephrine change during exercise (r=.18, p=.01), with individuals with the g allele experiencing greater changes in norepinephrine during exercise. finally, the fam5c snp rs1935881 was associated with change in norepinephrine during exercise (r=.16, p=.03). individuals with the g allele had greater changes in norepinephrine over the course of the exercise bout (all associations initially reported in the manuscript changed only slightly after applying the pca correction. pca corrected p-values for genotype-phenotype associations are as follows: rs1799971 and norepinephrine change (p=.104), rs1799971 and rpe change, (p=.038), rs8044769 and positive affect change (p=.038), rs3751812 and positive affect (p=.036), rs1935881 and norepinephrine change (p=.059), rs1379659 and norepinephrine change (p=.010), rs9941349 and systolic blood pressure change (p=.053), rs7201850 and systolic blood pressure change (p=.04998), rs2360969 and temperature change (p=.031), rs2253206 and temperature change (p=.066), rs1799971 and lactate change (p=.015), rs2253206 and vo2 max (p=.035), and rs2360969 and vo2 max (p=.032)). due to the fact that several of the variants that were associated with a particular phenotype were in the same gene, it is likely that these snps are in high-linkage disequilibrium with one another. these snp sets within single genes are rs3751812 and rs8044769 in fto, both significantly associated with positive affect change, rs2253206 and rs2360969 in creb1, both significantly associated with temperature change as well as vo2 max, and rs7201850 and rs9941349, both in fto, both significantly associated with systolic blood pressure change. to examine whether these snps were in ld, we ran correlations on each set of 2 snps in the same gene that were associated with the same phenotype. the correlation between rs2360969 and rs2253206 was .805 (p<.01), the correlation between rs3751812 and rs8044769 was .676 (p<.01), and the correlation between rs7201850 and rs9941349 was .938 (p<.01). in order to further explore the direction of the relationship of genotype on exercise response, we graphed the adjusted means for each genotype of three snps which demonstrated particularly robust relationships with exercise response phenotypes. we graphed the relationship between rs2360969 and temperature 30 minutes into the exercise bout, between rs1799971 and rpe 30 minutes into the exercise bout, and between rs8044769 and positive affect score 30 minutes into the exercise bout. as can be seen in figure 1, individuals with the tt genotype of rs2360969 showed the highest temperature after 30 minutes of aerobic exercise, controlling for baseline temperature. in figure 2, we show that individuals with the ag/gg genotypes on rs1799971 show greater rpe after 30 minutes of aerobic exercise than individuals with the aa genotype, controlling for baseline rpe. in figure 3, we show that individuals with the cc genotype in rs8044769 show the highest ratings of positive affect after 30 minutes of aerobic exercise, controlling for baseline positive affect. the present study replicated prior findings suggesting that snps in the creb1, fto, oprm1, slit2, and fam5c genes are all related to phenotypes encompassing various responses to exercise. our study tested conceptually relevant phenotypes that to date had not been explored in this way inanyother exercise research. given that the physiological response to aerobic exercise involves a complex interplay of metabolic, cardiovascular, musculoskeletal, ventilator, and hormonal functions, these genes and snps are likely to explain only a small portion of the variability in individual differences in response to aerobic exercise. subjective responses to exercise may be yet more complex, involving sociocultural factors, effects of previous exercise experiences, and anticipated consequences/rewards of exercise. additionally, our findings suggest that individuals performing equivalent bouts of aerobic exercise may have vastly different subjective perceptions of this exercise (overall experiences which can range from negative to positive), and that these perceptions may be influenced by genotype. giving sedentary individuals information about their propensity to respond to exercise in a particular way could provide useful insight, allowing these individuals to temper their expectations of what aerobic exercise should feel like for them or allowing intervention designers to incorporate external reinforcement contingencies (e.g., social interaction) for individuals who are less likely to experience intrinsic rewards from exercise. despite several inherent limitations, the present study's findings linking genetic variants to exercise responses among sedentary individuals presents promising initial evidence associating genes and exercise behavior. however, it is unlikely that variation at a single genetic locus could fully explain variation in physiological and subjective responses to exercise more possibly, there are many genetic variables influencing this phenotype, each of which contributes only bya small fraction of the observed variation. when combined into a genetic composite, these loci would likely correlate more strongly with phenotypic response. so, although the correlations between genotype and exercise response found in this study are not large, they represent a necessary first step in forming genetic composite scores that are likely to be more highly correlated and significantly predictive of exercise responses. in summary, linking snps to specific physiological and psychological mechanisms that contribute to exercise response will assist in informing individually tailored exercise programs, as well as deepen our understanding of the relationship between genetics, physiology, and psychology underlying health behaviors associated with cancer prevention. our study showed that for rs3751812, the presence of a t allele increased change in positive affect during exercise. this finding is somewhat at odds with previous work suggesting that tt individuals have higher bmi on average. rs3751812 was found to have a strong association with bmi in african-derived populations, with the tt genotype predicting increased bmi. however, the relationship between bmi and positive affective response to exercise is unclear because the hassanein study did not include information about exercise behavior of participants. it is possible that rs3751812 individuals are predisposed to have higher bmi, but if they engage in aerobic exercise, they are likely to have a more positive affective response. this is one example of how knowledge about the effect of a particular genotype could be used to prescribe tailored interventions for overweight individuals with the tt genotype, exercise could be recommended as a more effective weight loss tool, given that these individuals have a more positive affective response to exercise. we also found that for rs8044769, the tt individuals had greater changes in positive affect during exercise. in a hispanic american sample, rs8044769 was found to be weakly associated with waist-to-hip ratio, and the c allele showed an association with variation in bmi. prior research suggests that the c allele of rs8044769 is associated with greater variation in bmi. this snp seems to be related to body fat mass, predisposition to obesity, and response to aerobic exercise yet the nature of this relationship requires further exploration. creb1 is a key component of long-term cardiac memory formation (specific t-wave patterns on an electrocardiogram), as well as long-term memory formation in the brain [54, 55]. our results indicate that for the creb1 snp rs2253206, individuals with the a allele (ag genotypes, and to a greater extent aa genotypes) have a greater change in temperature during exercise. if greater temperature change while exercising translates into a more unpleasant subjective exercise experience, then our findings suggest that the aa individuals (and to a lesser extent ag individuals) may have less pleasant subjective experiences of exercise than gg individuals. rs2253206 was shown to be strongly associated with heart rate (hr) change in response to a 20-week endurance training program, with gg and ag genotypes and showing 57% and 20% better change in hr than the aa participants. our results make sense in the context of the rankinen findings, as the aa individuals may have more unpleasant exercise experiences due to increased temperature, which could influence their ability to exercise effectively (and thus decrease the heart rate improvements they can obtain from an exercise intervention). we also found that this snp was related to vo2 max (an indicator of cardiovascular fitness), such that the gg individuals had greater vo2 max than ag individuals, who had greater vo2 max than aa individuals. these results also coincide with our findings and the findings from previous research, as gg individuals may be more fit to begin with, and also more capable of gaining increased fitness through training, due to the fact that they experience exercise as less painful. additionally, we found that for rs2360969, tt individuals experienced greater change in temperature than did ct and cc individuals. rs2360969 has also been shown to be related to heart rate response endurance training [38, 39], however, these studies did not state direction of effect for this snp. in our analysis, rs1799971 (the a118 g polymorphism) was related to rpe, as well as to lactate change during exercise and norepinephrine change during exercise. for all three of these phenotypes, individuals with the rare g allele showed greater change during the exercise bout. previous research on this snp has found that individuals with the g allele (genotypes of either ag or gg) demonstrated higher pressure pain thresholds than individuals with the aa genotype. this study also found that when heat pain was tested, a sex by genotype interaction emerged, such that the g allele was associated with lower pain ratings among men but higher pain ratings among women. the a118 g variant has greater binding affinity for -endorphin (an exogenous opioid that activates the mu opioid receptor), which is one possible mechanism by which this snp could influence pain sensitivity. the relationship between rs1799971 and subjective responses to pain may extend to the pain and exertion experienced during aerobic exercise. given that our sample was 79.5% female, our findings of greater lactate, norepinephrine and rpe change over the course of exercise for the gg/ag group is in the same direction as the findings for females in the fillingim study. these results lend further support to the idea that individuals (and perhaps particularly women) with the ag/gg genotype have lowered pain threshold, and the present study suggests increases in lactate and norepinephrine as possible physiological explanations, at least in the context of aerobic exercise-induced pain. prior studies have shown that rs1379659 in fam5c is associated with echocardiographic traits, and specifically left ventricular systolic dimension. the results of our study suggest that it is also associated with change in norepinephrine in response to exercise. to date, research has not examined the relationship between fam5c and aerobic exercise response. given the connection between this gene and cardiac function, examining the potential relationship between fam5c and aerobic exercise would provide a logical next step for research in this area. previous research has demonstrated an association between the slit2 snp rs1935881 and echocardiographic traits, specifically left ventricular diastolic dimension. the results of this study suggest that it is also related to norepinephrine change during exercise. further research is needed to elucidate more specific relationships between slit2 and response to aerobic exercise. the genes discussed above represent potential candidates for further explanation in terms of their relationship to exercise response phenotypes. more than a decade's worth of research on the psychophysiological responses associated with exercise has demonstrated that the subjective experience of exercise, how sensations are remembered, anticipated, and interpreted, is closely tied to subsequent exercise behavior [14, 19, 20, 47, 57]. a better understanding of the genetic basis for subjective responses to aerobic exercise may have the potential to lead to more effective and sophisticated intervention designs. eventually, these advances in the basic science of exercise response could lead to the implementation of interventions tailored on the level of individual genetic variants. primary prevention of cancer through behavioral intervention is now a top priority of the nci. this approach is intuitive given that approximately 30% of total cancer deaths are related to energy imbalance (e.g., excessive adiposity) [58, 59]. physical inactivity is not the only contributing factor to energy imbalance, but it is a major contributing factor as trends clearly show that the least physically active regions of the country are also the most obese. the hopeful perspective on behavioral intervention for physical activity is that even small increases in the total amount of participation accumulated per week stands to lead to meaningful differences in cancer risk. for example, found evidence for a 38% reduction in risk for breast cancer with every additional 60 minutes of physical activity engaged in per week. the link between physical activity participation and reduced risk for cancer, especially of the colon, breast, and endometrium is convincing, but also dependent upon good adherence [36, 61]. for this reason, it is imperative that researchers continue to search for ways to improve the likelihood of adherence to behavioral interventions. one way to achieve this goal may be through increasing the amount of focus that is placed on subjective response phenotypes and their underlying genetic variants. developing a better understanding of the link between genes, exercise-relevant physiological mechanisms, and the resulting exercise-response phenotypes is a first step towards tailoring individualized exercise programs that would likely increase adherence and lead to improved health outcomes and decreased rates of cancer and other diseases. as with all research that involves genetic analyses, we can not rule out the possibility that other genetic factors, including rare or common snps, insertions, deletions, or copy number variants, could play a role in determining the physiological responses to exercise that were measured in this study. the phenotypes investigated in this study are likely to be polygenic traits, such that numerous genes and snps other than those examined in the present study may all contribute to these exercise response phenotypes. in contrast, the extent of the pleiotropic effects of the genes and snps investigated in this study are unknown. thus, it is possible that the polymorphisms that influence exercise response may also be more strongly associated with other, possibly unrelated phenotypes that led to our findings. another limitation of note is the present study's lack of power to detect moderation effects of demographic variables. it is possible that variables such as age or ethnicity could moderate the associations between genetic variation and response to exercise. additionally, the results of this investigation are based on one single, standardized, bout of moderate-intensity exercise. for this reason, our results can not be generalized to subjective exercise experiences that occur under less regulated circumstances (i.e., when type of activity, intensity, and duration are individually determined). despite this limitation, there are many examples from the literature in which subjective responses to exercise are measured and analyzed based on a single bout of standardized exercise (e.g., [19, 20, 6265 ]) and therefore, our procedures and analyses are in concert with the approach previously established by the field. importantly, the purpose of the present investigation was to understand how genetic variants are associated with particular subjective responses to exercise when the parameters of the exercise experience are standardized across all individuals. in the present study, this level of standardization was achieved by having all participants perform the same activity (treadmill walking), for the same duration (30 minutes), at the same intensity (65% of each individual participant's previously established vo2 max). further, efforts were made to standardize variables external to the exercise bout as well (i.e., instructions detailing recommended calorie and water consumption prior to the bout described in section 2). it remains to be seen whether the snps and genes reported in this study to be related to exercise response phenotypes would show an association to these same phenotypes in other studies examining different types, duration, and intensity of exercise sessions. however, as noted, the size of the associations changed negligibly after a pca correction, suggesting that the population substructure did not play a major role. overall, replication is needed in order to confirm findings from the present study, and to better understand the functional significance of these genes and snps in relation to physiological and subjective responses to aerobic exercise. the purpose of this paper was to explore the genetic underpinnings of individual physiological and subjective responses to aerobic exercise. one strength of this study was its focus on a sedentary population, a group that has been rarely tested in terms of associations between genetics and exercise phenotypes. the relationship between particular genetic variants and responses to exercise has important implications for the prevention of cancer via increasing exercise behavior in sedentary populations. future studies designed to test genetic influences on a wide range of exercise response phenotypes would help to advance this goal, potentially leading to a panel of markers important for characterizing the physiological and subjective response to exercise. moreover, giving feedback to sedentary individuals regarding the genetic basis for their strengths and weaknesses in fitness/exercise/sports activities could be a potentially useful motivational tool for increasing exercise behavior [13, 66]. in sum, expanding our understanding of the association between genetics and exercise response phenotypes has a myriad of implications for helping to increase exercise behavior in sedentary individuals, an outcome which is crucially important for the reduction of morbidity and mortality associated with cancer .

objective. to determine whether genetic variants suggested by the literature to be associated with physiology and fitness phenotypes predicted differential physiological and subjective responses to a bout of aerobic exercise among inactive but otherwise healthy adults. method. participants completed a 30-minute submaximal aerobic exercise session. measures of physiological and subjective responding were taken before, during, and after exercise. 14 single nucleotide polymorphisms (snps) that have been previously associated with various exercise phenotypes were tested for associations with physiological and subjective response to exercise phenotypes. results. we found that two snps in the fto gene (rs8044769 and rs3751812) were related to positive affect change during exercise. two snps in the creb1 gene (rs2253206 and 2360969) were related to change in temperature during exercise and with maximal oxygen capacity (vo2 max). the slit2 snp rs1379659 and the fam5c snp rs1935881 were associated with norepinephrine change during exercise. finally, the oprm1 snp rs1799971 was related to changes in norepinephrine, lactate, and rate of perceived exertion (rpe) during exercise. conclusion. genetic factors influence both physiological and subjective responses to exercise. a better understanding of genetic factors underlying physiological and subjective responses to aerobic exercise has implications for development and potential tailoring of exercise interventions.

PMC3414053

pubmed-484

dental implants are now a reliable solution for the functional and esthetic rehabilitation of partially and completely edentulous patients; this has been demonstrated by long-term clinical trials, with survival rates of greater than 95% [13]. in order to achieve long-term survival, osseointegration of the dental implant needs to occur; that is, a direct connection must be established between the bone and the implant surface, without the interposition of fibrous tissue; once established, this close bond must be maintained over time, resulting in a clinically asymptomatic fixation of the implant under functional load. osseointegration is a complex phenomenon and depends on many factors; some are related to the implant (material, macroscopic design, and implant surface), others to the surgical-prosthetic protocol (surgical technique, loading conditions, and time), and others to the patient (quantity/quality of bone at the receiving site and the host response) [4, 5]. although survival rates of dental implants are now high, there still remains a seemingly unavoidable number of failures: either cases in which correctly placed implants do not integrate with the bone or cases of peri-implant tissue infection [6, 7]. to be specific, failure to osseointegrate and peri-implantitis are the most frequent causes of early implant failure [3, 6, 7]. such events occur during the early stages of healing (within 2-3 months of implantation) and therefore before the implant is functionally loaded with the prosthetic restoration; these failures are unevenly distributed within the general population and tend to occur in some subjects in particular. in these individuals early failures occur even when optimal materials are used, surgical protocols are strictly followed, and the quantity/quality of bone at the recipient site is sufficient [68]. all these observations would suggest the existence of specific patient-related risk factors; this prompts an investigation into the regulatory mechanisms controlling bone metabolism, bone remodelling, and bone turnover [9, 10]. it is a fat-soluble vitamin which promotes the absorption of calcium in the intestine and regulates calcium and phosphate homoeostasis in the tissues and it is a fundamental element in the mineralization of bones and teeth [1113]. it also acts as a hormone and is vital for the health of the blood vessels and the brain [14, 15]. it has been demonstrated that vitamin d plays a crucial role in the health of the cardiovascular tract, the immune system, and the respiratory tract [18, 19]. vitamin d in an inactive form (cholecalciferol or vitamin d3) is ingested or produced in the skin on exposure to sunlight [11, 12]. this inactive form undergoes double hydroxylation in the liver and the kidneys and is thereby transformed into its active form, known as either calcitriol or 1,25-dihydroxyvitamin d3 [11, 12]. this active form exerts its action on various tissues by binding to the vitamin d receptors and regulating the transcription of specific target genes [1223]. serum levels of vitamin d in the 25(oh)d form are the most accurate way of determining vitamin d status: a subject with<10 ng/ml is considered to be vitamin d deficient; one with 1030 ng/ml is considered to have low levels of vitamin d. the optimal blood level of vitamin d is a value>30 ng/ml [12, 13]. vitamin d deficiency is high in the general population: in italy, for example, it is estimated that about 80% of people can be deficient, particularly in the northern regions where exposure to the sun is lower. this deficiency increases with age and encompasses the majority of the elderly population of italy who are not taking vitamin d supplements. until a few years ago, the guidelines estimated that the daily intake of vitamin d required to maintain adequate blood levels was 200 iu (5 mcg) in adults aged between 19 and 50, 400 iu (10 mcg) in adults aged between 51 and 69, and at least 600 iu (15 mcg) in those over 70 [12, 13]. these guidelines have now been revised upwards and it is currently believed that the amount of vitamin d which should be taken daily is 2000 iu (50 mcg) and up to 4000 (100 mcg) in the case of, for example, pregnant women [12, 13]. there is now substantial literature on the negative effects of low levels of vitamin d, especially in severely compromised patients: vitamin d deficiency seems to be associated with increased mortality, cardiovascular events, and reduced functioning of the immune and musculoskeletal systems [1519, 2123]. on the other hand, normalizing levels of vitamin d can lead to substantial benefits for critically ill patients, with effects on the muscles, the respiratory system, the heart, and the immune system [18, 21, 23]. despite the importance of vitamin d and its effects on bone metabolism [11, 12] few studies have, to date, investigated the effects of its depletion on the osseointegration of dental implants [9, 2435]: almost all these studies have been done on animal models [2432] and very few on humans [3335]. the purpose of this retrospective study was therefore to investigate any possible correlation between low blood levels of vitamin d and early implant failure (failure occurring in the four months prior to the full restoration of the implant, because of a lack of osseointegration or because of infection). all patients who had been treated with morse-taper connection dental implants (leone implant system, florence, italy) [3, 8] inserted to support fixed prosthetic restorations in one single dental centre (gravedona, como, italy), in the period between january 2003 and december 2015, were evaluated for possible enrollment into this retrospective study. patients were enrolled into the study if they were over 18 years of age, had good oral and general health, and had not undergone bone regenerative therapy prior to implant placement. the exclusion criteria were incomplete medical records, the presence of specific systemic diseases (uncontrolled diabetes mellitus, immunodeficient states, and bleeding disorders), and the abuse of alcohol and drugs; patients undergoing radiotherapy and chemotherapy and those who were pregnant were also excluded. all the data used for the study were obtained from the medical records of the patients enrolled. the patient data was evaluated; this included gender (male or female), age at time of surgery, history of chronic periodontal disease, smoking habits, and serum vitamin d levels. vitamin d levels were taken from blood tests, which had been requested two weeks prior to surgery. the medical records also contained a range of information as regards the implant or implants, that is, their site (maxilla or mandible), location (incisor, canine, premolar, and molar), the length and diameter of the implant, the type of prosthetic restoration, and the loading conditions. these included their cause (lack of osseointegration in the absence of infection, infection of the peri-implant tissues or peri-implantitis, or implant failure due to progressive bone loss caused by to prosthetic overload). it also included their classification: early failure, occurring in the early healing period, that is, the four months after implant placement, prior to the placement of restoration and loading, or late failure, occurring after loading. there were also details of any possible biological complications (peri-implant mucositis and peri-implantitis) and/or prosthetic complications (mechanical and/or technical). all implants were inserted under the same strict protocol by the same specialist (c. m.) who had 25 years ' experience in implant dentistry, in the period between january 2003 and december 2015. the implants were inserted after raising a full thickness mucoperiosteal flap; the implant site preparation and implant placement were performed in compliance with modern surgical protocols and in accordance with the manufacturer's instructions. after placement, cover screw was positioned and the implants were submerged. immediately after positioning, patients were prescribed antibiotic coverage with 2 g of amoxicillin (or 600 mg of clindamycin in patients allergic to penicillins) for 6 days. patients were given detailed instructions on oral hygiene and were prescribed chlorhexidine 0.12% mouth rinse twice a day for 6 days. the implants were left to heal submerged for a total period of 4 months, to allow undisturbed healing and achieve osseointegration. after 4 months of undisturbed healing, the patient was recalled for the implant to be uncovered. two weeks later, the sutures were removed and an impression was taken for the manufacture of the temporary restoration. the temporary restoration was maintained in situ for 3 months, in order to monitor the response of the implant, as well as the peri-implant tissues, to masticatory load; at the end of this period, the temporary restoration was replaced with the final restoration. the final restorations were metal porcelain, cemented with a zinc oxide-eugenol cement. a periapical radiograph was taken to check on the sealing of the restoration. all patients were included in a follow-up protocol with an annual check-up at one of the scheduled professional oral hygiene sessions. early implant failure, occurring within 4 months after implant placement and therefore prior to placement of the prosthetic restoration and the functional load of the implant, was the primary outcome studied. early implant failures were divided into two different categories: (a) early failures due to lack of osseointegration and subsequent implant mobility, in the absence of clinical signs of infection; (b) early failures due to infection of the bone tissue around the implant, with inflammation (peri-implantitis) of peri-implant tissues and the presence of fistula, pain, swelling, pus and/or exudate, pocket depth>6 mm with bleeding, and marginal bone resorption>2.5 mm. all the data retrieved from the individual medical records were recorded on a generic spreadsheet (excel, microsoft office, redmond, ma, usa) which was used for the descriptive, qualitative, and quantitative analyses. the mean, standard deviation, median, and confidence intervals were calculated for the quantitative variables (e.g., patients ' age and vitamin d levels in serum). a patient-based technique was used to calculate implant survival. in this analysis, the event was implant failure: thus in patients receiving more than one implant, the occurrence of even a single implant failure led to the patient being classified as a failure. the influence of different variables on implant survival was taken into consideration: gender (male or female), age at time of surgery (three age groups were examined:<40, 4060 years, and>60 years), smoking habits (regardless of the actual number of cigarettes smoked), a history of chronic periodontitis, and serum levels of vitamin d. in the analysis of serum levels of vitamin d, three classes of patients were considered: severely deficient patients (serum vitamin d<10 ng/ml), patients with low levels (serum vitamin d between 10 and 30 ng/ml), and patients with adequate levels (serum vitamin d>30 ng/ml). the influence of each of these variables on implant survival was calculated using the chi square test. the overall implant survival, the survival within the different groups, and the analysis of the influence of the different variables on survival were all made using dedicated statistical analysis software (spss 17.0, spss inc., of the 915 patients originally evaluated for enrollment in this study, 93 presented with conditions corresponding to the exclusion criteria and were therefore excluded from the assessment. by contrast, 822 patients (mean age 57.3 14.2 years; median age 58; range 1890; and 95% ci, 56.358.2), receiving 1625 implants, did not have any of the conditions contained in the exclusion criteria and were therefore enrolled into this retrospective study. the distribution of patients by groups, with relative incidence of failures, was reported in table 1. in total, 27 early failures were recorded (19 due to failure of osseointegration and 8 due to peri-implant tissue infection), with an overall incidence of 3.2%. no differences were observed in the incidence of early failures between males and females (p=0.97) nor according to age at time of surgery (p=0.98). although the percentage of early failures in smokers was slightly higher than that detected in nonsmokers, there was no statistically significant difference (p=0.56) between these two groups of patients. the same was true for patients with a history of periodontal disease; they displayed a slightly higher incidence of early failures than patients who had not been affected by periodontitis, but this difference was not significant (p=0.73). the average serum level of vitamin d in the general population was 29.9 ng/ml (12.1; median 29; range 573; and 95% ci, 29.130.7). in patients in whom early implant failure occurred, the average serum level of vitamin d was 25.5 (13.2; median 24; range 855; and 95% ci, 20.630.4). statistical analysis reported a rather low incidence of early failures (2.2%) in patients with blood vitamin d levels>30 ng/ml. the incidence of early failure was almost double in patients with insufficient serum levels of vitamin d (1030 ng/ml) and became even higher (9.0%) in patients with serious vitamin d deficiency. although the statistical analysis revealed a trend toward an increased incidence of failure in patients with severe vitamin d deficiency, the analysis did not reveal a statistically significant difference (p=0.15) in the incidence of early implant failure in these three groups of patients. similar results (p=0.14) were obtained comparing the incidence of failures in the group of severely deficient patients (2/22: 9.0%) with the incidence of failures in all other patients (25/800: 3.1%). finally, the statistical analysis did not reveal a significant difference (p=0.13) when comparing the incidence of failures in the group of patients with serum vitamin d levels>30 ng/ml (9/394: 2.2%) with the incidence of failures in all other patients (18/428: 4.2%). a relatively small number of experimental studies has attempted to investigate the effects of vitamin d on the osseointegration of dental implants [2432]. the majority of these studies would appear to indicate a positive effect of vitamin d on osseointegration, but it is not yet entirely clear whether supplementation would promote the healing of peri-implant bone tissue clinically [24, 3335]. a recent review of the literature on animal studies has shown that vitamin d supplementation can stimulate new bone formation and increase the contact between the bone and the surface of titanium implants. specifically, kelly et al. demonstrated that vitamin d deficiency could significantly compromise the establishment of osseointegration of ti6al4v implants in rats. in an experimental study on ovariectomized rats, the authors demonstrated that vitamin d deficiency could impair the formation of peri-implant bone; the normalization of blood levels via supplementation of vitamin d stimulated new bone formation. similar results were reported by zhou et al., who found an increase in osseointegration in osteoporotic rats given vitamin d supplements, and wu et al., who demonstrated an increase in the percentage of contact between bone and implant in diabetic rats given vitamin d supplements. finally, liu et al. reported that the administration of vitamin d could increase the fixation of dental implants in mice suffering from chronic kidney disease. a further possibility for study, in order to understand the effects of the administration of vitamin d on bone healing of the peri-implant tissues, is that of coating the implant surface with vitamin d [3032]. evaluated the effect of the topical application of vitamin d to the surface of implants inserted in postextraction sockets in dogs, with histological and histomorphometric analyses of tissues removed at 12 weeks. topical application of vitamin d increased the percentage of bone to implant contact of 10%. similarly promising results were reported by cho et al. in a histological and histomorphometric study on rabbits, where the coating of anodized implant surfaces with a solution of poly(d, l-lactide-co-glycolide) plga and 1,25-dihydroxyvitamin d3 (1,25-(oh)2d3) stimulated the apposition of new bone on fixtures. finally, in a further experimental work in rabbits, implants with a surface coated in 1,25-(oh)2d3 have shown an improved tendency to osseointegrate compared to noncoated implants; however, this difference was not statistically significant. unfortunately, very few clinical studies have so far investigated the effects of vitamin d deficiency on osseointegration and on bone regeneration in dentistry [3335]. this is probably due to the fact that there are many factors which can determine the success or failure of dental implants; the attention of clinicians has been mostly focused on drawing up surgical and prosthetic protocols and identifying new materials and implant surfaces to improve osseointegration, rather than on the analysis of patient-related risk factors [69]. in a recent clinical work, alvim-pereira et al. found no relationship between polymorphism of the vitamin d receptor and implant failure. in a randomized, investigated the effects of supplementation with a combination of vitamin d3 (5000 iu) and calcium (600 mg) on the formation of new bone following maxillary sinus lift. ten patients were assigned to the test group and given vitamin d and calcium; ten other patients were assigned to the control group and received only calcium. six to eight months after surgery for bone regeneration, bone samples were taken for histological analysis during implant placement. although supplementation with vitamin d3 would have increased the serum levels of vitamin d with potentially positive effects on bone remodelling at the cellular level, no statistically significant difference was demonstrated between the two groups at the histological level. the results of our study would appear to suggest that a severe deficiency of vitamin d in the blood might be related to an increase in the incidence of early implant failure. in fact, the incidence of early implant failure was rather low (2.2%) in patients with normalized levels of vitamin d in the blood (> 30 ng/ml), rose to almost double (3.9%) in patients with insufficient serum levels (1030 ng/ml), and were rather high (9.0%) in patients characterized by severe deficiency states. however, despite the tendency to an increased incidence of early failure in patients characterized by deficiency states, the differences between the three groups of patients were not statistically significant (p=0.15). our study also confirms that the serum values of vitamin d in the local population are rather low: we found that the proportion of patients with insufficient levels was 49.4% and that the percentage with a severe deficiency was 2.7%. this is not surprising, as most of the patients treated came from northern italy and southern switzerland, regions where exposure to sunlight is somewhat reduced for long periods of the year. in the light of this, the administration of vitamin d in the weeks prior to placement of a dental implant could be useful, particularly in patients with severe deficiency states; in these patients, vitamin d supplementation should be maintained for the whole life, in order to guarantee a good remodelling of the bone around the implant. this study has the distinction of being one of the first clinical studies carried out on a large number of patients to investigate the possibility of an association between low blood levels of vitamin d and the incidence of early failure in implantology. by restricting the analysis to early failures, occurring in the first period of healing and therefore prior to placement of the prosthetic restoration, we were able to focus our research and avoid a range of factors (linked to the restoration itself and the prosthetic load) which could have confused the issue in the study. it is, in fact, well known that implant survival and thus osseointegration depend on a large number of factors (related to the surgical and prosthetic protocol, the materials used, and lastly the patient) [4, 5] and it can be difficult to identify which of them might be determining the success or failure of the treatment. in order to avoid this and to limit the confounding factors, the same materials were used for all the patients in this study (the same implant system for all the patients) [1, 3, 8]. in addition, the same surgical protocol was used, involving submerged healing in the absence of prosthetic loading. thus the only possible confounding factors were the different quantity and quality of bone at the implant receiving sites and the patients ' responses: these are unavoidable factors. however, some of those categories of patients most at risk of implant failure (patients undergoing bone regeneration to create the conditions for the positioning of the implant fixtures or those with particular medical conditions which might increase the risk of treatment failure) were excluded in the present study. it is a retrospective work, in which the number of patients having a severe deficiency of vitamin d in the blood was low (only 22); thus the presence of even just one less failure in this group would have led to quite different results. it is possible that some residual confounding may have biased the association between vitamin d and implant failures that we observed. for instance, this study did not investigate the influence of other patient-related factors (e.g., the bone quality) which can affect implant survival in the period immediately following implant placement. in addition, if subjects with low levels of vitamin d were also likely to receive more than 1 implant, their risk of being classified as failures may increase. however, no patient in this study experienced more than 1 failure, and the probability of implant failure was not higher (1.5% versus 2.1%) in presence of another implant. therefore, randomized, controlled clinical trials are needed to confirm the presence of an association between low serum levels of vitamin d and an increase in the incidence of early failure in implantology. it would be appropriate to assess whether supplementation of vitamin d in the weeks before the operation could lead to a reduction in early failures, whether due to lack of osseointegration or implant infection. further scientific studies with an appropriate design and a more rigorous statistical analysis will therefore be required in order to thoroughly investigate this issue. until now, very few studies, and those mainly on animal models, have involved assessing the influence of blood levels of vitamin d levels on the osseointegration of dental implants. although most of these studies have shown that the administration of vitamin d can improve the healing of the peri-implant bone tissue, it is not yet clear whether vitamin d supplements can promote the osseointegration of dental implants. our retrospective clinical study aimed to investigate if there is a link between low levels of vitamin d in the blood and an increased risk of early implant failures. although the incidence of early implant failures was higher in patients with low serum levels of vitamin d, our study failed in proving an effective link between low levels of vitamin d in the blood and an increased risk of early implant failure. further higher level studies (prospective controlled trials or, even better, randomized controlled clinical trials) with a more rigorous statistical analysis are therefore needed to investigate this issue. if an association between low serum levels of vitamin d and higher risk of early implant failure could be demonstrated, the clinician could give a set dose of vitamin d in the weeks before surgery, in order to normalize serum levels and obtain a positive effect on the healing process.

aim. to investigate whether there is a correlation between early dental implant failure and low serum levels of vitamin d. methods. all patients treated with dental implants in a single centre, in the period 20032015, were considered for enrollment in this study. the main outcome was early implant failure. the influence of patient-related variables on implant survival was calculated using the chi-square test. results. 822 patients treated with 1625 implants were selected for this study; 27 early failures (3.2%) were recorded. there was no link between gender, age, smoking, history of periodontitis, and an increased incidence of early failures. statistical analysis reported 9 early failures (2.2%) in patients with serum levels of vitamin d>30 ng/ml, 16 early failures (3.9%) in patients with levels between 10 and 30 ng/ml, and 2 early failures (9.0%) in patients with levels<10 ng/ml. although there was an increasing trend in the incidence of early implant failures with the worsening of vitamin d deficiency, the difference between these 3 groups was not statistically significant (p=0.15). conclusions. this study failed in proving an effective link between low serum levels of vitamin d and an increased risk of early implant failure. further studies are needed to investigate this topic.

PMC5055956

pubmed-485

osteoarthritis (oa) is characterized by cartilage breakdown, synovial fibrosis, and osteophyte formation. the main clinical symptom of oa is chronic joint pain, which is typically treated using analgesic drugs, such as nonsteroidal anti-inflammatory drugs and corticosteroids [1, 2]. however, due to potential adverse effects [3, 4] and variable efficacy of these drugs for providing symptomatic pain relief, more effective analgesics are needed to improve oa treatment and patient care. in addition, because only a few effective disease-modifying drugs are available for oa [5, 6], a better understanding of the mechanisms that drive oa pain is required to guide drug development. nerve growth factor (ngf) is a widely known pain mediator and plays a critical role in the modulation of oa pain [811]. the neutralization of ngf with tanezumab, an anti-ngf monoclonal antibody, has robust analgesic effects on oa pain [8, 10, 11]. due to these effects, the regulation of ngf has been investigated in several in vitro and in vivo studies [12, 13]. evidence from these studies suggests that the activity of ngf is mediated by inflammatory cytokines. for example, in an experimental arthritic mouse model, il-1, but not tnf-, increased ngf levels in knee joints. however, the mechanism regulating ngf expression in synovial tissue (st) remains unclear. st contains macrophage- and fibroblast-like cells in the lining layer [1416]. the treatment of cultured synovial oa fibroblasts with il-1 and tnf- induces the production of ngf. more recent studies have shown that macrophages produce a number of inflammatory cytokines, including il-1, il-6, and tnf-, which contribute to oa progression and associated joint pain [1721]. despite these findings, the regulation of ngf expression in synovial macrophages is not fully understood. str/ort mice are a well-characterized oa model that spontaneously develop oa with a progression resembling that of humans [2224]. synovial hyperplasia is also observed in str/ort mice, and we previously showed that the cd11b+ macrophage population in st str/ort mice is higher than that found in c57bl/6j mice [17, 18]. in addition, we reported that compared to synovial fibroblasts (cd11b), synovial macrophages produce high levels of il-1 and tnf- in str/ort mice. here, we characterized the expression profiles of several inflammatory cytokines and ngf in the st of str/ort mice. in addition, the regulation of ngf expression by inflammatory cytokines in synovial macrophages and fibroblasts was also examined. nine-month-old male str/ort (average body weight, 40.6 4.1 g) and c57bl/6j (control; average body weight, 32.8 1.2 g) mice (charles river laboratories, inc., specific pathogen-free colonies of each mouse line were maintained at nippon charles river laboratories (kanagawa, japan) in a semibarrier system with a controlled environment (temperature: 23 2c; humidity: 55% 10%; lighting: 12 h light/dark cycle). all experimental protocols were approved by the kitasato university school of medicine animal care committee. c57bl/6j and str/ort mice were sacrificed by the intramuscular injection of a mixture of medetomidine, midazolam, and butorphanol tartrate. after removing skin with a scalpel, st was harvested, and total rna was then extracted from the harvested tissue using trizol (invitrogen, carlsbad, ca, usa), according to the manufacturer's instructions. the extracted total rna was used as a template for first-strand cdna synthesis using superscript iii rt (invitrogen) in pcr reaction mixtures consisting of 2 l cdna, specific primer set (0.2 m final concentration), and 12.5 l sybr premix ex taq (takara, kyoto, japan) in a final volume of 25 l. the primers for il6 and ngf were designed using primer blast software and were synthesized by hokkaido system science co., ltd. the other primers used in this study were designed based on previously published primer sequences. the sequences of the pcr primer pairs used in this study are listed in table 1. quantitative pcr was performed using a cfx-96 real-time pcr detection system (bio-rad, hercules, ca, usa). the pcr cycles consisted of an initial denaturation step at 95c for 1 min, followed by 40 cycles of 95c for 5 s and 60c for 30 s. mrna expression was normalized to the levels of glyceraldehyde-3-phosphate dehydrogenase (gapdh) mrna. the gene expression levels in the st of str/ort mice were compared with those in the st of c57bl/6j mice (str/ort/c57bl6j). in addition, the gene expression levels in cd11b+ cells were compared with those in cd11b cells (cd11b+/cd11b). st samples were collected from both knees of five str/ort mice, and mononuclear cells were then isolated from the collected tissue by digestion with type i collagenase for 2 h at 37c. the obtained mononuclear cells were suspended in 500 l phosphate-buffered saline (pbs) containing biotinylated anti-cd11b antibody. following a 30 min incubation at 4c, the cells were washed with pbs, mixed with streptavidin-labelled magnetic particles (bd imag streptavidin particles plus-dm; bd biosciences, tokyo, japan), and incubated for 30 min on ice in an imag separation system (bd biosciences). warmed (37c) -minimum essential medium (mem) was added to the cell suspension to collect unbound (cd11b-negative) cells, and an additional 3 ml -mem was added to collect cd11b-positive cells after removing the tub from the magnetic support. the cd11b-positive and cd11b-negative cells were collected by centrifugation at 300 g for 10 min, and the obtained cells were cultured in -mem in six-well plates. tnf-, il-1, il-6, and ngf expression in the cells was analyzed by reverse transcription-polymerase chain reaction (rt pcr). st-derived mononuclear cells, including synovial macrophages and fibroblasts, which were isolated as described above, were cultured in -mem in six-well plates. after a 1-week incubation at 37c in a 5% co2 incubator, synovial fibroblasts were incubated with either mouse recombinant tnf- (25 ng/ml), il-1 (50 ng/ml), or il-6 (100 ng/ml) (biolegend, san diego, ca, usa) for 24 h. cells that were not treated with any cytokines were used as controls. to examine the effect of tnf- on synovial macrophages and fibroblasts, cd11b-positive and cd11b-negative cells were collected from st, as described above, and were then cultured in -mem in six-well plates. after a 1-week incubation at 37c in a 5% co2 incubator, cd11b-positive and negative cells were incubated with mouse recombinant tnf- (25 ng/ml) or il-1 (50 ng/ml) for 24 h. cells that were not treated with tnf- were used as controls. the treated and control cells were harvested for total rna isolation, as described above, and ngf expression was analyzed by rt pcr. ten l3 and l4 drg samples were harvested from the 9-month-old c57bl/6j and str/ort mice and were then immersed in a buffered paraformaldehyde fixative at 4c overnight. the samples were further incubated in pbs containing 20% sucrose for 24 h at 4c. after freezing in liquid nitrogen, each specimen was sectioned at 10 m thickness on a cryostat (leica microsystems, cm3050s, wetzlar, germany) and was then treated for 90 min at room temperature with a blocking solution of pbs containing 0.05% tween-20 and 1% skim milk. the sectioned drg specimens were stained with rabbit antibodies against calcitonin gene-related peptide (cgrp; 1: 1000; immunostar, hudson, wi, usa) and transient receptor potential vanilloid 1 (trpv1; 1: 500; calbiochem, san diego, ca, usa) by incubation for 18 h at 4c. the drg sections were then incubated with alexa 488-conjugated goat anti-rabbit igg (for cgrp immunoreactivity, 1: 1000; molecular probes) and alexa 488-conjugated goat anti-rabbit igg (for trpv1 immunoreactivity, 1: 1000; molecular probes). the immunostained sections were visualized using a fluorescence microscope (axiovert 200, zeiss, jena, germany) in a treatment-blinded manner. the numbers of cgrp- and trpv-positive cells were counted, and the proportion of these cells to the total number of nucleated cells in drg was calculated for each drg sample. differences between c57bl/6j and str/ort mice were examined using the t-test. all statistical analyses were performed with spss software (version 11.0; spss, inc., chicago, il, usa). a p value of<0.05 was considered statistically significant. real-time pcr analysis of the genes encoding tnf-, il-1, il-6, and ngf showed that the expression levels of these genes were significantly elevated in the st of str/ort mice compared to those in control c57bl/6j mice (figures 1(a)1(d)). activated macrophages reportedly produce higher levels of inflammatory cytokines, including tnf-, il-1, and il-6, than tissue-resident macrophages [18, 26, 27]. to determine whether macrophages in st also produce inflammatory cytokines, expression of the genes encoding tnf-, il-1, and il-6 in cd11b-positive cells isolated from the st of str/ort mice was examined by real-time pcr. tnf-, il-1, and il-6 gene expression in cd11b-positive cell fractions was higher than that in cd11b-negative cell fractions (figures 2(a)2(c)). in contrast, no differences in the gene expression of ngf were detected between the two examined cell fractions (figure 2(d)). real-time pcr analysis revealed that the gene expression of ngf increased significantly in isolated synovial cells in the presence of exogenously added tnf- and il-1 compared to untreated control cells (figure 3). in contrast, the gene expression of ngf was not affected in il-6-treated synovial cells (figure 3). ngf expression was also significantly increased in the presence of exogenously added tnf- in isolated populations of synovial fibroblasts and macrophages compared to untreated control cells (figure 4(a)). ngf expression was also significantly increased in the presence of exogenously added il-1 in isolated populations of synovial fibroblasts, but not in synovial macrophages (figure 4(b)). to investigate pain-related sensory innervation by ngf, the proportion of trpv1- and cgrp-positive cells in the drg of str/ort mice was investigated by immunohistochemistry. the number of trpv1- (figures 5(a), 5(b), and 5(e)) and cgrp-positive cells (figures 5(b), 5(d), and 5(f)) in the drg of str/ort mice was significantly higher compared to that found in the drg of c57bl/6j mice. in the present study investigating the mechanisms underlying the regulation of ngf and development of oa pain, higher expression of the genes encoding tnf-, il-1, il-6, and ngf was observed in the st of an oa str/ort mouse model compared to that in c57bl/6j mice. in addition, ngf expression was specifically detected in the cd11b-positive and cd11b-negative cell fractions isolated from st, whereas higher tnf-, il-1, and il-6 gene expression was observed in the cd11b-positive cell fractions. notably, the treatment of cultured synovial cells with tnf- and il-1 stimulated ngf expression and tnf- also stimulated ngf expression in cd11b-positive and cd11b-negative cell fractions. the number of trpv1- and cgrp-positive cells in the drg of str/ort mice was significantly higher compared to that found in the drg of c57bl/6j mice. taken together, these findings suggest that tnf- regulates ngf expression in both synovial fibroblasts and macrophages and that il-1 regulates ngf expression in synovial fibroblasts and that these factors may contribute to the development of pain in oa patients by innervation of the peripheral nervous system. ngf is upregulated in human synovial fibroblasts, suggesting that this factor plays an important role in oa pathology [12, 13, 28]. ngf elevated inflammatory knee joint and tnf- and il-1 stimulate synovial fibroblasts to produce ngf. reported that ngf expression is upregulated in the knee joint of a carrageenan-treated inflammatory arthritis model and is further stimulated by the intraarticular injection of tnf- and il-1. further, tnf- and il-1 treatment of cultured synovial fibroblasts derived from a human oa patient also increased ngf production. however, because these studies consisted of analyses of whole knee joints or cultured synovial fibroblasts exogenously stimulated with tnf- and il-1, the cell populations responsible for regulating ngf and producing tnf- and il-1 in st were not conclusively determined. here, elevated expression of the inflammatory cytokines tnf-, il-1, and il-6 was observed in the st of str/ort mice, and tnf- and il-1 were increased in synovial macrophages isolated from this oa model. in addition, ngf expression was stimulated by tnf- and il-1 in the synovial fibroblast fraction. taken together, these findings suggest that tnf- and il-1 stimulate ngf expression in synovial fibroblasts. a recent immunohistochemical analysis revealed that ngf not only is localized to fibroblasts, but is also produced by a specific population of macrophages in human st. however, the factor regulating ngf in macrophages was not determined. in the present study, isolated synovial macrophages and fibroblasts from str/ort mice expressed ngf at similar levels to each other. ngf expression in the macrophage fraction was also stimulated by tnf-. taken together, these findings indicate that tnf- stimulates ngf expression in both synovial fibroblasts and macrophages. ngf plays a key role in the generation of acute and chronic pain and peripheral sensitization [3033]. recent evidence suggests that the trpv1 ion channel and cgrp are involved in the development of ngf-induced persistent mechanical and thermal hypersensitivity in rats [30, 31]. ngf acutely modulates trpv1 activity [32, 33] and also stimulates cgrp expression in drg neurons in vitro and in vivo. consistent with this speculation, inhibition of ngf by treatment with tanezumab was recently shown to have high analgesic efficacy in oa knees compared with placebo. in the present study, cgrp- and trpv1-positive cells, which are localized in drg neurons in monoiodoacetate- (mia-) treated oa animals [35, 36], were increased in the drg of str/ort compared to c57bl/6j mice, suggesting that the elevation of synovial ngf in str/ort mice contributes to peripheral sensitization. however, several studies have shown that human oa chondrocytes also produce ngf by inflammatory cytokines [37, 38]. further investigations are needed to clarify the contribution of chondrocytes derived ngf on peripheral sensitization. first, the mechanism underlying the development of oa pain that was proposed based on the present results was based on cross-sectional analysis and therefore no data on the effects of the observed changes on the progression of oa are available. second, although ngf was found to be elevated in the st of oa mice, it remains to be determined if the elevation of synovial ngf levels contributes to peripheral sensitization. finally, present study did not confirm that the protein levels of these cytokines correlated with the expression levels. in conclusion, the present results support the hypothesis that tnf- regulates ngf expression in synovial fibroblasts and macrophages and il-1 regulates ngf expression in synovial fibroblasts in oa mice. tnf- and il-1 may therefore regulate ngf signaling in oa joints and be suitable therapeutic targets for treating oa pain.

to investigate the role of macrophages as a regulator and producer of nerve growth factor (ngf) in the synovial tissue (st) of osteoarthritis (oa) joints, the gene expression profiles of several inflammatory cytokines in the st, including synovial macrophages and fibroblasts, of oa mice (str/ort) were characterized. specifically, real-time polymerase chain reaction analysis was used to evaluate the expression of tumor necrosis factor- (tnf-), interleukin- (il-) 1, il-6, and ngf in cd11b+ and cd11b cells isolated from the st of a murine oa model. the effects of tnf-, il-1, and il-6 on the expression of ngf in cultured synovial cells were also examined. the expression of tnf-, il-1, il-6, and ngf in the st of str/ort was higher than that in c57/bl6j mice. compared to the cd11b cell fraction, higher expression levels of tnf-, il-1, and il-6 were detected in the cd11b+ cell fraction, whereas no differences in the expression of ngf were detected between the two cell fractions. notably, tnf- upregulated ngf expression in synovial fibroblasts and macrophages and il-1 upregulated ngf expression in synovial fibroblasts. il-1 and tnf- may regulate ngf signaling in oa joints and be suitable therapeutic targets for treating oa pain.

PMC5007361

pubmed-486

hepatitis b virus (hbv) is a major health problem, with approximately 400 million chronically infected people worldwide, and 1560% of the normal population in many african countries may be positive for one or more of the serological markers of hepatitis b virus infection. these chronically infected patients not only are at an increased stage of developing liver cirrhosis and hepatocellular carcinoma, but also serve as a potential reservoir of infection. the major structural protein of virus envelope, hepatitis b surface antigen (hbsag), is universally considered as a diagnostic marker of hbv infection. the absence of hbsag in the serum and the presence of antibodies to core antigen (anti-hbc) usually indicate resolved infection. occult hbv infection (obi) usually has a serological evidence of previous hbv infection that has been described in a few cases. hiv coinfection has been reported to modify the natural history of hbv with potential consequences on morbidity and mortality. data on obi in art untreated hiv patients is limited from a vast number of african countries like nigeria, where prevalence of hbv monoinfection, mode of transmission, viral genotype, and mutational pattern varies considerably in different parts of the country. no previous study from nigeria on prevalence on obi among any groups has been carried out. this is particularly important as exposure to hbv is common among hiv-infected cases because of shared routes of transmission. notably, there is considerable variation in prevalence of hiv/hbv coinfection according to geographic regions and exposure risk. successful implementation of art leads to immune reconstitution that can potentially result in immune mediated liver injury in the setting of hbv coinfection. some studies have reported an association between obi and elevated transaminase; therefore identification of obi is of importance. of the 1,200 hiv-infected patients enrolled in the haart clinic of the specialist hospital, ikole, ekiti state, nigeria, from october, 2012, to april, 2013, we identified 980 hbsag negative patients (art-nave subjects). among them, 188 were selected for the study by a simple random method. informed consent was obtained from the patients, and the institutional committee approved the study protocol. all samples were tested for hbsag, anti-hbs, anti-hbc, anti-hcv, and anti-hiv using elisa (drg diagnostics, marburg, germany). all anti-hbc positive samples were retested for hbsag as well as for anti-hbc, and only repeat positive samples were included in the study. dna was extracted from all the serum samples using qiaamp dna blood mini kit (qiagen gmbh, hilden, germany) following the manufacturers ' instructions. briefly, samples (200 l) were incubated with protease and lysis buffer. after incubation, there were two washing steps, and the nucleic acids were eluted in a volume of 50 l of elution buffer. the presence of hbv dna was examined in all samples using a routine diagnostic pcr. primer pairs were designed from the highly conserved overlapping regions of the s and p genes of the hbv genome. a nested pcr was performed: outer primer pairs were hbpr134 (sense) 5-tgctgctatgcctcatcttc-3 and hbpr135 (antisense) 5-cagagacaaaagaaaattgg-3 and the inner primer pairs were hbpr75 (sense) 5-caaggttatgttgcccgtttgtcc-3 and hbpr94 (antisense) 5-ggtataaagggactcacgatg-3. pcr amplifications were carried out in 25 l reaction volumes with 5 ng of genomic dna, 10x pcr buffer (20 mm tris-hcl ph 8.4, 50 mm kcl; qiagen), 2 mm of dntps, 50 ng of each primer, and 1 u ampli taq gold dna polymerase (applied biosystems) on a ptc 200 cycler (peltier thermal cycler watertown, massachusetts, usa). thermal cycling parameters were initial denaturation at 94c for 2 min, followed by 35 cycles of 30 sec at 94c denaturation, 30 sec at 52c annealing temperature, and 45 sec at 72c extension, followed by a final extension of 5 min at 72c. thermal cycling parameters remained the same as in the first pcr round except for the number of cycles that is increased to 40 cycles of amplification. each pcr product (5 l) was analysed by electrophoresis in 2% agarose gels. a positive control (hbv plasmid dna) and a negative control of the master mix only were integrated to each run to validate the pcr products that yielded a 340 bp fragment. quantification of hbv dna was performed with quantitative real-time pcr using a previously described procedure in a geneamp 7300 sequence analyzer (applied biosystems, perkin-elmer, foster city, ca). hbv-plasmid dna was used to generate a standard curve following a serial 10-fold dilution. mean age and all the numerical data were analysed using student's t-test. the chi-square test and fisher's exact test were used to compare categorical data. for the purpose of our study, the demographic, biochemical, and virological parameters of the study group are summarized in table 1. the mean age was 35 (range: 367) years. the majority (45%) had multiple sexual partners and 25% of the subjects had a history of concomitant alcohol use. overall, 29/96 (29.2%) of patients were reactive for anti-hbc, an indication of prior exposure to hbv dna, and majority 6/8 (75%) of the patients were female (table 2). thus, in the total study population, 21/188 (11.2%) of patients were identified as obi and 62.5% of the obi patients had cd4 count less than 200 cells/mm. averagely the hbv viral load was<50 copies/ml in the obi samples examined by quantitative pcr. serum levels of ast and alt were higher among patients with obi in comparison to anti-hbc positive hbv dna negative individuals, but the difference failed to reach standard significance (p=0.13 and p=0.07), respectively. the comparison of different demographic, biochemical, and virological factors between hbv dna positive and negative cases was illustrated in table 3. the distribution of the study participants as per the 1993 revised classification system for hiv infection and expanded surveillance case definition for aids among adolescents and adults was as shown in table 4. the present study represents a comprehensive cross-sectional analysis of prevalence of obi in an art nave hiv positive cohort comprising various risk groups. most previous studies looking at the clinical effects of obi in hiv include a large number of patients on anti-hbv drugs as a component of art. this study describes the risk factors associated with obi, frequently of anti-hbc positivity and its possible values as a serological marker for identifying hiv-infected patients who benefit from hbv dna assay. we found the prevalence of occult hbv to be 11.2% among a random selected group of hiv-infected patients. the prevalence of obi in hiv positive individuals varies worldwide between 0 and 90%, depending on the geographic regions, risk factors, and the exposure involved. in the present study, the prevalence of anti-hbc (29.2%; 28 of 96) and obi (28.6%; 8 of 28) among the art nave hiv positive cohort was higher compared to previous report on blood donors from studies done in areas of india, areas which reported 21.3% obi among the hbsag negative anti-hbe positive donors. within nigeria, hbv and hcv coinfection among hcv-infected patients most of the previous studies on hbv/hiv co-infection are aimed at detecting hbv prevalence in the hiv population are based on hbsag positivity (prevalence 9.9 to 11%), but reports on obi are scarce. a previous study on intravenous drug users in northeastern india detected a prevalence of 15.9%. among the obi cases, the rate of anti-hbs was lower (2/8; 25%), which may be due to the fact that hiv-infected patients are prone to lose anti-hbs immunity at a higher frequency than the general population. previous reports suggested that the lower hbv replication was associated with milder hepatic damage. among the subjects with obi, elevated alt or ast however, our study is cross-sectional; therefore evaluation of long term clinical significance of obi should be better addressed by follow-up studies. the low level of viral load obtained in this study buttress the findings in another study that showed that showed that almost all obi cases are infected with replication incompetent hbv, revealing a strong suppression of overall replication activity and gene expression, thereby resulting in a significant reduced viral load. hiv patients are screened for concomitant chronic hepatitis b using hbsag elisa, and it is not considered cost-effective to perform hbv dna testing for all hiv patients in our resource-poor setting. our study tried to identify possible clinical and serological markers which could guide dna testing in these patients. obi is reported to be common among hcv infection, but we found its prevalence to be low among our study group. however, anti-hcv was not tested among the other hiv positive samples attending the specialist hospital, ikole, ekiti state, nigeria. furthermore, none of the risk factors were found to be statistically significant markers of obi and can not be used as an independent marker for identifying patients who should benefit from hbv dna estimation. however, as one third of the anti-hbc positive negative patients were positive for hbv dna (8 of 28), it is recommended that hiv positive patients with hbsag negative/anti-hbc positive patterns should be tested for the presence of hbv dna irrespective of their anti-hbs status. nevirapine is commonly included in the first line art regimens at most treatment centers in nigeria. our study identified only 21 subjects with obi, and all the samples were collected from a single center indicating that results might differ in setting with significant different demographic characteristics. a main implication of the presently viable data is therefore further emphasizing the need for efficient hbv vaccination programs. overall, the present study highlights the need for screening hbv before the initiation of any haart containing anti-hbv regimens in hbv/hiv coinfected patients. it necessitates the use of nat for effective laboratory diagnosis of occult hbv infections in hiv positive patients, especially in developing countries where these assays are not widely available.

hiv has been known to interfere with the natural history of hepatitis b virus (hbv) infection. in this study we investigate the prevalence of occult hepatitis b virus infection (obi) among hiv-infected individuals in nigeria. overall, 1200 archived hiv positive samples were screened for detectable hbsag using rapid technique, in ikole ekiti specialist hospital. the hbsag negative samples were tested for hbsag, anti-hbc, and anti-hcv by elisa. polymerase chain reaction was used for hbv dna amplification and cd4 counts were analyzed by cytometry. nine hundred and eighty of the hiv samples were hbsag negative. hbv dna was detected in 21/188 (11.2%) of patients without detectable hbsag. cd4 count for the patients ranged from 2 to 2,140 cells/l of blood (mean=490 cells/l of blood). hcv coinfection was detected only in 3/188 (1.6%) of the hiv-infected patients (p>0.05). twenty-eight (29.2%) of the 96 hiv samples screened were positive for anti-hbc. averagely the hbv viral load was<50 copies/ml in the obi samples examined by quantitative pcr. the prevalence of obi was significantly high among hiv-infected patients. these findings highlight the significance of nucleic acid testing in hbv diagnosis in hiv patients.

PMC4020157

pubmed-487

in august of 2005, hurricane katrina devastated the new orleans area with high wind, heavy rainfall, and a storm surge of about 7 m which caused the collapse of the levee system surrounding the city. approximately 80% of the city was flooded to varying depths for many weeks before the us army corps of engineers was able to implement temporary levee repairs and install emergency pumping capacity. in the aftermath of the flood event, the infrastructures of the city along with residences and commercial buildings were grossly contaminated with sediments deposited by the floodwaters and subsequently by microbial overgrowth supported by the residual moisture, high humidity, and elevated temperatures in the area. after floodwaters had receded, various surveys were conducted for measurement of indicators of microbial contamination in air, dust, and damaged building materials, including total and culturable mold spores, fungal fragments, mycotoxins, 13--d-glucan, and bacterial endotoxin. generally, observed levels of microbial contaminants in these surveys were elevated, often extremely so, and were relatable to the depth and duration of flooding, and indoor levels were typically higher than those in the surrounding outdoor environment [15]. subsequent to the posthurricane flooding event, there has been extensive rebuilding in the new orleans area. residents who personally performed repairs of their properties as well as various skilled and unskilled laborers working in the construction and building maintenance trades were at risk for inhalation exposures to dust containing microbial and other agents during demolition, removal, and repair of flood-damaged and contaminated infrastructure and building materials [68]. exposures to microbial contaminants in agriculture, waste management, and in water-damaged and moldy buildings have been linked to various upper and lower respiratory illnesses and adverse effects including rhinitis, hayfever, toxic pneumonitis (tp), hypersensitivity pneumonitis (hp), and respiratory infections including pneumonia and exacerbation or initiation of asthma [912]. the potential for respiratory illness arising from inhalation exposure to bioaerosols and microbial contaminants during restoration activities in the post-hurricane katrina environment was of particular concern. as a part of a 5-year longitudinal study investigating the risk of respiratory illness associated with work in and around flood-damaged structures in post-hurricane katrina new orleans, baseline findings from initial cross-sectional survey are reported. the study cohort consisted of 791 adults residing or working in the greater new orleans metropolitan area. study participants were recruited from several sources: (1) employees of three large institutions in the city of new orleans, two of which are academic and the third is a branch of local government (n=488 total). all three institutions experienced heavy flood damage to their buildings and facilities and utilized their regular staff as well as contract labor to perform restoration work. recruiting from the academic institutions focused primarily on workers from departments normally engaged in maintenance, custodial, and facilities services. support personnel (clerical, managerial, etc.) from the targeted departments were included in the recruitment. (2) members of a local union hall for the skilled and unskilled building trades (n=63). (3) private building contractors and self-employed tradesmen (n=95). (4) other residents of the new orleans area (n=145), many of whom performed restoration work on their own properties. overall, 54% of the study cohort reported a skilled or unskilled trade as their primary occupation, including carpentry (n=50), electrician (n=27), plumbing (n=12), paint/drywall (n=21), hvac (n=12), groundskeeping (n=20), general construction (n=102), general maintenance (n=36), operating/building engineering (n=18), and being mechanic/machinist (n=15). an additional 15% of study participants worked in custodial or janitorial services (n=115). testing was conducted in a mobile laboratory van outfitted with spirometry and interview work stations and ancillary equipment. the mobile laboratory van was moved to the work locations or union hall of the study participants for the duration of their respective testing period, generally 2 to 3 weeks, and to the parking lots of several large building supply stores, in order to allow private contractors and self-employed construction tradesmen to participate. spirometry testing procedures and equipment have been previously described and comply with both the original and updated american thoracic society spirometric test criteria. spirograms were collected with a sensormedics model 1022 dry rolling seal spirometer interfaced to a laptop computer running omi spirometry software version 5.05.9 (occupational marketing, inc., all spirometric testing was conducted by the same individual who is a member of the research staff and is a certified pulmonary function technician; in addition, all spirometric test results were quality assured and interpreted by senior study investigators. predicted lung function parameters and lower limit of normal (lln) lung function values for forced expiratory volume in one second (fev1), forced vital capacity (fvc), and fev1/fvc ratio were computed from predictive equations developed by hankinson et al.. separate predictive equations were used for caucasians, african americans, and latinos. predicted values for study participants of asian heritage were calculated using the equations for caucasians. in addition to race, the predicted values were based on age, gender, and height. the lln values were calculated by subtracting 1.645 see from the predicted values, where see was the standard error of the estimate and 1.645 is the 95th percentile of a standard normal distribution. those participants with chronic obstructive pulmonary disease (copd) were identified according to the gold criteria, that is, fev1/fvc% predicted less than 70% and fev1% predicted less than 80%; however, only prebronchodilator lung function values were available and thus may not have adequately differentiated asthma (with reversible obstruction) from copd. ever asthma on questionnaire were therefore excluded from the analyses of copd prevalence as a function of exposure. a demographic, medical, smoking, and occupational questionnaire it was based on a modified version of the standardized questionnaire reported by burrows et al., which accounts for a variety of putative and established risk factors and potential confounders for the development of airways disease including asthma, allergic disease, historical confounding exposures, serious childhood respiratory illness, cigarette smoking history, environmental tobacco smoke, and age, gender, and race. additional questions were designed to capture the development of specific symptoms after hurricane katrina that might be associated with living and working in the post-katrina environment. these included post-hurricane katrina onset of asthma, sinus symptoms, pneumonia, and transient fever and cough absent infection, with the latter used as an indicator of possible hypersensitive (hp) or toxic (tp) reaction. asthma was defined dichotomously and required a positive response to both of the following questions: have you ever had asthma or attacks of shortness of breath with wheezing in the chest when not having a cold? followed by the response to the question how old were you when your asthma started? was used in conjunction with the participant's date of birth to determine whether asthma onset was after september 30, 2005 (post-katrina new onset asthma). dyspnea was also defined dichotomously and required a positive response to the question do you have shortness of breath when hurrying on level ground? the interview also included queries on pre- and post-katrina work and occupation, and detailed information was gathered on time spent after hurricane katrina performing five specific types of hurricane/flood remediation work: demolition and ripout, trash removal, landscape restoration, sewer line repair, and mold remediation. participation in any of these work activities, herein identified as restoration work, was assumed to result in occupational or vocational exposure to flood-related contaminants. participants were asked to report the number of hours spent in each of the five restoration work activities, for each year since the hurricane up to the point of interview, and the type and relative frequency of any respiratory protective equipment that may have been used during the work. restoration of personal property was included in the total time spent in restoration work along with any from the subject's regular employment. the study protocol was approved by the authors ' institutional review board and all study participants provided a written informed consent. for the current hayfever compared to current trouble with pollen, grass, or fur, analysis indicated that the parallel question significantly enhanced the positive response rate for these symptoms (22% claiming current hayfever versus 39% claiming sensitivity to pollen, grass, or animal fur; p<0.0001). similar but nonsignificant results were observed for ever and current asthma versus attacks of dyspnea and for chronic bronchitis versus copd. the unadjusted prevalence rate ratios for each symptom or condition for those doing any restoration work versus those not doing any restoration work were calculated within smoking categories based on 2 2 contingency tables. the prevalence rate ratio was defined as prr=p1/p2, where p1=a/n1 and p2=c/n2 represent the sample proportion of exposed (n1) and unexposed (n2) individuals with disease. if a and b represent the number of exposed subjects who do and do not have disease, respectively, and c and d represent the number of unexposed subjects who do and do not have disease, respectively, the asymptotic 95% confidence interval for prevalence rate ratio is calculated using the following standard logarithmic transformation: ln(prr)1.96b/an1+d/cn2. exponential transformations on the confidence limits of this log transformed interval provided the asymptotic 95% confidence intervals for prevalence rate ratio. multiple logistic regression analyses were used to compute adjusted prevalence odds ratios for each symptom or condition per 100 hours of restoration work as well as to compute asymptotic 95% confidence intervals for prevalence odds ratios. due to significant interactions between gender and total hours of restoration work, logistic regression analyses were performed separately by gender and adjusted for age (because of a significant correlation with prevalence of pneumonia) and smoking categories. all interactions between age, smoking category, use of respiratory protective equipment (ever versus never), and total hours of restoration work were considered and were not significant. multiple linear regression related% p fev1, fvc, and fev1/fvc to restoration work hours, use of respiratory protection, gender, asthma classification, and smoking category. all possible interactions were considered and were not significant, and no significant exposure associations were detected. the majority of the study cohort was african american and male (table 1). current smokers comprised 28% of the cohort, whereas 18% were ex-smokers and 54% had never smoked. 3.7% of the study cohort reported having pneumonia after hurricane katrina and almost half reported newly developing sinus symptoms. among those reporting never having had asthma prior to hurricane katrina (n=539), about 4.5% reported new onset asthma. episodes of transient fever and cough occurring after hurricane katrina were reported by about 29% of the study cohort. multiple episodes were also common in this reporting group: the median number of episodes was 3, and 10% of the group reported having 12 or more such occurrences. overall, lung function parameters were somewhat depressed in the cohort (tables 1 and 4) and correlated with cigarette smoking and presence of current asthma symptoms. percent predicted (% p) fev1 averaged 93.4% (sd: 16.0) for current smokers, while ex- and never smokers had a mean level of 96.0% (sd: 15.4), p=0.037 by t-test. the proportions of the cohort falling below lln for fev1 and fvc were also somewhat elevated (5% being the expected proportion based on the definition of lln), particularly for the current and ex-smokers, as expected. participants reporting current asthma symptoms had a mean% p fev1 of 89.2% (sd: 21.8) and% p fvc of 91.8% (sd: 16.8), while asthmatics without current symptoms had a mean% p fev1 of 95.8% (sd: 18.0) and% p fvc of 96.7% (sd: 15.2). participants who never had asthma had mean% p fev1 of 96.1% (sd: 13.6) and% p fvc of 96.4% (sd: 13.6). almost 75% of the study participants reported having performed some restoration work activity after hurricane katrina (n=587), and details on the actual time spent in these activities were self-reported by 474 or 81% of this group (table 2). demolition/ripout was the most commonly reported restoration work activity, followed by landscape restoration and trash/debris removal. the distributions of time spent in these activities were highly skewed because many study subjects worked as much as 16 hours per day, seven days per week, for extended durations after hurricane katrina. the majority of the study subjects also reported time spent in more than one type of restoration work activity. for the total combined hours spent in any of the specific restoration work activities, the mean and median values reported by 474 subjects with complete data were 1646 and 620 hours, respectively. among those who reported performing restoration work, 80.1% reported some use of respiratory protective equipment (i.e., filtering facepiece or air-purifying cartridge respirator). 202 study subjects reported no time spent in restoration work. the prevalence rates for post-hurricane katrina episodes of transient fever and cough and new onset sinusitis were significantly elevated for those reporting any restoration work (prr: 1.7 and 1.3, resp.; table 3). the prevalence rate ratios were statistically significant for ex- and never smokers but not current smokers. the prevalence rate ratios for post-hurricane katrina new onset asthma were elevated for the overall cohort (prr=2.2) and especially for ex- and never smokers (prr=2.7) but were not statistically significant (p=0.09); the overall lack of significance may have been due in part to the low incidence (29 cases) and the reduced size of the base population; that is, only those who never had asthma prior to hurricane katrina. statistically significant elevations in prevalence rate ratios for those having done any restoration work were not observed for pneumonia, dyspnea, copd, and being below lln for any of the lung function parameters. figure 1 illustrates the prevalence of respiratory symptoms and conditions with quartiles of reported time spent in post-hurricane katrina restoration work activities (proportions for new onset asthma and pneumonia are multiplied by 10 in the figure for purposes of scaling). the unadjusted proportions for several post-hurricane katrina symptoms and conditions, including transient fever and cough, dyspnea, new onset sinus symptoms, new onset asthma, and pneumonia, show trends of increasing prevalence with time in restoration work. statistically significant increases in prevalence odds ratio with restoration work time were observed only for transient fever and cough, for new onset sinus symptoms, and for dyspnea. when analyzed by logistic regression, the prevalence of fever and cough was statistically significantly associated with restoration work time, but only for men, with a prevalence odds ratio of 1.016 per 100 hours. likewise, for new onset sinus symptoms in men, the prevalence odds ratio was 1.042 per 100 hours. in contrast, only women exhibited a statistically significant association between the prevalence of dyspnea and restoration work time, the odds ratio being 1.031 per 100 hours. the restoration work time-gender interactions observed in the logistic regression analysis of the prevalence odds ratios for these symptoms may be confounded with job type since women in the study tended to be in the custodial/janitorial occupations, whereas men were more likely to be in the building, construction, or maintenance trades. the exposures associated with restoration work done within these two broad categories of occupation are likely to be qualitatively and quantitatively different. the prevalence odds ratios for post-katrina new onset asthma and for pneumonia with restoration work time were elevated but were not statistically significant by logistic regression analysis. for new onset asthma the crude, unadjusted proportions in each of the restoration work time quartiles (figure 1) were all higher than for participants with no restoration work time (1.99.1% versus 1.7%). for pneumonia, the unadjusted prevalence among those with no restoration work was 3.0% whereas subjects in each of the first three quartiles of restoration work time exhibited higher prevalence rates (3.25.1%); however, those in the highest quartile of restoration work time had a pneumonia prevalence rate of only 2.5%. the group mean values for% p fev1, fvc, and fev1/fvc ratios generally were depressed but were within 5% of the normal except for smokers performing restoration work. current smokers who did restoration work showed lower overall predicted lung function compared to smokers who did not; however, multiple linear regression analysis yielded no statistically significant correlations of any of the lung function parameters with restoration work time after adjustment for smoking, gender, asthma status, and use of respiratory protective equipment. the results of this study suggest moderate adverse impact on respiratory health from time spent in post-hurricane katrina flood restoration activities. published reports and public health surveillance systems generally did not show increases in emergency room visits or hospitalizations resulting from exposures in the post-hurricane katrina environment, although there have been a few reports of increased risk for respiratory effects. in a survey of 525 new orleans firefighters, 79% had contact with floodwaters following hurricane katrina, and 38% reported new onset respiratory symptoms including sinus congestion, throat irritation, and cough. the prevalence rate ratio for those who had contact with floodwater versus those who did not was 1.9 and was statistically significant. first responders would have had significant exposures to flood sediments and associated contaminants, in addition to microbial agents. inhalation exposure to aerosolized sediment collected in the aftermath of hurricane katrina was also shown to elicit significant pulmonary inflammation, increased airways resistance, and airway hyperreactivity in a mouse model. there were widespread anecdotal reports of persistent nonproductive cough, often with sore throat and rhinorrhea, in the population residing in new orleans in the fall of 2005. an investigation of this phenomenon by the louisiana department of health and hospitals concluded that visits to medical facilities for respiratory complaints in the population of new orleans were not related to exposure to dust or molds at the residence or at work. it is likely that katrina cough was an irritant phenomenon resulting from a dry fall season with high levels of airborne particulate matter, coinciding with the start of the regular allergy and flu seasons [21, 24]. the prevalence of episodes of fever and cough in the present study population is clearly elevated for those who have done restoration work. however, given the overall strong correlation with restoration work time, the common reports of multiple and distinct episodes of fever and cough, and the inclusion of the febrile component in the symptom complex, tp is likely to be underlying many of these reports and appears to be a common adverse effect of restoration work exposures in the post-hurricane katrina environment. unlike hypersensitivity pneumonitis, it is uncertain whether toxic pneumonitis and inhalation fevers result in significant lasting decrements in lung function, and functional parameters are expected to return to baseline upon recovery from an episode [25, 26]. this study did not identify any restoration work-related decrements in functional parameters, nor in the prevalence of being below lln. the world health organization, in its report on guidelines on indoor air quality related to damp indoor spaces and mold, concluded that the evidence is inadequate to identify an association between damp indoor environments or the presence of mold with risk of alterations in lung function. however, in a recent study of 6,443 individuals in the european community respiratory health survey, lung function measurements across 9 years showed statistically significant excess declines in fev1 of 2.25 ml/year and an additional 7.43 ml/year for women who reported dampness in the home and visible damp spots in the bedroom, respectively. annual excess declines of such small magnitude are difficult to detect over a short time period and are unlikely to result in a detectable group difference in function, measured cross-sectionally, after only a few years in the post-hurricane katrina environment, as in this baseline study. however, our study population is being evaluated annually over the course of a 5-year period, and currently undetectable decrements in lung function may reach the level of significance when measured directly over this extended period of time. it is generally accepted that exposure to flood-related microbial contaminants can exacerbate existing asthma, and there is a mounting evidence that such exposures increase the risk of development of new asthma [11, 12]. in a recent extensive review and meta-analysis of the literature from 1980 to 2010, overall odds ratios of 1.49 (c.i.: 1.281.72) and 1.68 (c.i.: 1.481.90) were found for the associations of asthma and wheezing, respectively, in children living in homes with visible mold. in this study, there was an observable elevation in prevalence of new onset asthma after hurricane katrina which increased with increasing quartiles of restoration work time but was not statistically significant. the lack of significance may be due in part to a self-selection process occurring in the cohort, with some study subjects who developed new onset asthma in the wake of hurricane katrina avoiding or terminating further restoration work exposure because of personal health concerns. furthermore, the reported restoration work time is at the time of the interview, not at the time of development of new onset asthma, which always preceded the interview. some of the study subjects may have continued to engage in and increase their time in restoration work to varying extent after developing asthma, but there is no information on the magnitude of this confounding nor its effect on the analysis. this study has several additional limitations: as noted, health outcomes were assessed cross-sectionally, and there was a significant time element over which the baseline information was collected for the entire study population. those subjects evaluated later in the study period could therefore have opportunity for greater amounts of time spent in restoration work with concomitant increase in risk for development of respiratory effects. as the longitudinal component of the study moves to completion, additional reports of development of respiratory symptoms and conditions are coming to light, and the ability to detect ongoing small excess decrements in lung function will also increase. exposure to flood-related contaminants was assumed to be related to reported time spent performing restoration work. however, there is no information as to a particular individual's exposure intensity nor can it be assumed that all exposures associated with restoration work activities were qualitatively or quantitatively similar. finally, the reliance on self-reporting of respiratory symptoms and conditions could have led to misclassification due to recall bias. this study provides further evidence that workers performing restoration work on flood-damaged structures are at risk of respiratory health impacts from exposure to microbial-contaminated dust and debris. moderate adverse respiratory health effects including toxic pneumonitis and sinusitis were commonly reported in the study cohort, and the prevalence of new onset asthma among restoration workers was noticeably elevated. while it is unclear from this cross-sectional analysis whether restoration work exposures have adversely affected pulmonary function in the population, the functional parameters overall are depressed in the cohort. ongoing longitudinal health surveillance of this study cohort, along with a quantitative exposure assessment, will examine whether there is an increased risk for long-term or irreversible effects on respiratory health and how the risks relate to the nature and magnitude of the exposures occurring during posthurricane flood restoration work.

background. this study examines prevalence of respiratory conditions in new orleans-area restoration workers after hurricane katrina. methods. between 2007 and 2010, spirometry and respiratory health and occupational questionnaire were administered to 791 new orleans-area adults who mostly worked in the building construction and maintenance trades or custodial services. the associations between restoration work hours and lung function and prevalence of respiratory symptoms were examined by multiple linear regression, 2, or multiple logistic regression. results. 74% of participants performed post-katrina restoration work (median time: 620 hours). symptoms reported include episodes of transient fever/cough (29%), sinus symptoms (48%), pneumonia (3.7%), and new onset asthma (4.5%). prevalence rate ratios for post-katrina sinus symptoms (prr=1.3; ci: 1.1, 1.7) and fever and cough (prr=1.7; ci: 1.3, 2.4) were significantly elevated overall for those who did restoration work and prevalence increased with restoration work hours. prevalence rate ratios with restoration work were also elevated for new onset asthma (prr=2.2; ci: 0.8, 6.2) and pneumonia (prr=1.3; ci: 0.5, 3.2) but were not statistically significant. overall, lung function was slightly depressed but was not significantly different between those with and without restoration work exposure. conclusions. post-katrina restoration work is associated with moderate adverse effects on respiratory health, including sinusitis and toxic pneumonitis.

PMC3529447

pubmed-488

road traffic injuries are responsible for the death of 1.23 million people around the world annually (1). in terms of disability adjusted life year (daly (( 1). road traffic injuries ranked ninth in 1999, and are expected to rise to the third place by 2020 (2). these events account for 1-2% of gross national product (gdp) of different countries (3). in iran, cost of road traffic injuries is 2.19% of gdp that is significantly higher than the global average (4). more than 20% of deaths caused by traffic crashes belong to pedestrians. according to the estimates, about 273000 pedestrians around the world lost their lives due to traffic crashes in 2010 (5). in 2013, 22% of all fatalities as a result of road traffic crashes in the european union(eu) were fatalities of pedestrians (6). studies have shown that most traffic crashes related to pedestrians, particularly in high-income countries, occur in interurban streets and roads (7). for example, in the european union countries, 70% of these crashes occur in cities, and this figure is 76% in the u.s. pedestrians are associated with the highest contribution of deaths caused by traffic crashes in the world s densely populated cities. for instance, in mumbai and delhi, pedestrians accounted for 78% and 53% of traffic fatalities, respectively (9). according to the statistics of tehran traffic police, more than 6,000 traffic crashes causing injury and more than 100 traffic crashes causing death annually occur in tehran. in fact, more than 40% of deaths from traffic crashes in tehran occur for pedestrians (11). given the importance of this issue, in the recent years, many studies have been conducted in different parts of the world, especially in densely populated cities, on the role of environmental and demographic factors in frequency, spatial distribution, and severity of traffic crashes related to pedestrians in urban areas, using different methods of spatial analysis and a variety of statistical models (12-21). in the recent years, due to the increase in automobile production, traffic load has dramatically increased in urban and suburban streets and roads of iran, while these transport infrastructures have not been developed qualitatively and quantitatively commensurate with car production. since a major proportion of traffic crashes in iran occur due to environmental factors, especially the quality of roads and streets and as the capacity of streets and roads, studying the urban and interurban roads and streets, particularly in densely populated cities with heavy traffic such as tehran, in terms of the environmental factors involved in traffic crashes can be helpful to more accurately identify the environmental factors of such crashes and their contribution. the results of such studies can be used in better planning for reduction of this type of risk factors. hence, this study aimed to determine the high-risk areas and spatially analyze the traffic crashes causing death to pedestrians in tehran in 2013 and 2014. tehran is the largest city and the capital of iran, with an area of more than 612 square kilometers (22). tehran is divided into 22 districts, 370 parishes, and 560 traffic zones, with more than 535 km of highway and 445 arterial streets (24). statistical analyses were performed by descriptive and inferential statistics in spss and arc-gis. analytic surveys were conducted in the following steps: first, the exact location of traffic crashes causing death to pedestrians was extracted by reviewing the files of crashes in the database of tehran traffic police. second, according to the universal transverse mercator (utm), geographical coordinates of the traffic crashes locations with the accuracy of one to three meters were recorded and saved in a computer. the utm system divides the earth into 60 zones, each 6 of longitude in width and uses a secant transverse mercator projection in each zone. the point of origin of each utm zone is the intersection of the equator and the zone's central meridian. third, using arc-gis, different layers of geographic information were put on each other and mapping was extracted. finally, global moran s index with euclidean distance method was used to study and analyze the distribution pattern of traffic crashes related to pedestrians in terms of being either cluster or scattered. moran s i index is the most common index used for measuring the spatial autocorrelation to determine how close are the objects compared to other close objects. a strong autocorrelation occurs when the values of a variable that are spatially close to each other are correlated. in other words, if the spatial objects or the values of variables related to them are randomly distributed in space, there must apparently be no relationship between them. moran s index studies the distribution pattern of these spatial objects by considering the values of the studied variable in terms of being cluster or scattered. if the value of moran's index is equal to+1 or near+1, it implies a complete cluster pattern or existence of autocorrelation. if this value is zero, it means that the pattern is random or multipolar accumulation. finally, if moran's index is equal to -1 or near -1, it shows that the crashes follow a scattered pattern. hot spots analysis: for this analysis, global getis-ord g index with euclidean distance method was calculated for each feature. z-score of this index shows that to what extent the studies feature has been distributed cluster-like, which may be statistically significant. in this study, this index was used to display the areas with high, low or moderate frequency of traffic crashes associated with pedestrians. getis-ord g index values were interpreted based on comparing the observed values and the expected values. if the observed values of getis-ord g index in an area are more than the expected values, that area is considered among the hot spots, and if the observed values are less than the expected values, that area is considered among the cold spots. one hundred ninety-eight incidents were studied; 92 of which, (46.4%) occurred in april 2013 to march 2014 and other 106 cases (63.6%) occurred in april 2014 to march 2015. the highest and the lowest frequency of crashes belonged to january (26 cases) and june (10 cases). figure 1 demonstrates the trend of the traffic crashes causing death to pedestrians in 2013-2015 in tehran. distribution of fatal crashes associated with pedestrians in tehran by different months: 2013-2015 overall, 158 cases (79.8%) of crashes have occurred in tehran highways. azadegan highway, which stretches from northwest to southeast of tehran, had the highest frequency of crashes (25 cases). during the study period, fatal road traffic injuries that caused the death of pedestrians occurred most frequently in traffic zones located in the west, east and south of tehran. figure 2 depicts the distribution of studied crashes in tehran and their position to highways, and urban to interurban bus terminals. mapping of fatal crashes associated with pedestrians in tehran and their position relative to highways and urban and interurban passenger terminals: 2013-2015 among the 22 districts of tehran, districts located in the south, east, and northwest of tehran, as well as districts located in the center of the city, have shown the highest and the lowest frequency of traffic crashes, respectively. figure 3 displays the distribution of studied crashes in the 22 districts of tehran in 2013 and 2014. mapping of fatal crashes associated with pedestrians in the 22 districts of tehran in 2013-2015 result revealed that the density of events in the areas with dominant transportation, industrial and military land use was higher than the areas with dominant residential land use (fig. 4). during the study period, fatal road traffic injuries that caused the death of pedestrians occurred most frequently in traffic zones located in the west, east and south of tehran. mapping of fatal crashes associated with pedestrians in tehran and their position relative to land use in 2013-2015 figure 5 depicts the distribution pattern of traffic crashes causing death to pedestrians in traffic zones. moran s i index value showed that the studied crashes had a cluster-like distribution (p<0.001). distribution pattern of fatal crashes associated with pedestrians in tehran by traffic areas in 2013-2015 the hot spots in tehran in terms of traffic crashes causing death to pedestrians included the western, southern, northern, partly eastern suburbs, and cold spots were mainly located in the central areas of tehran (fig. getis-ord general g index showed that the distribution of hot and cold spots of the studied crashes was statistically significant (p<0.001). low and high-risk traffic areas in terms of frequency of fatal crashes associated with pedestrians in tehran in 2013-2015 the results of this study revealed that occurrence of the traffic crashes causing death among pedestrians in tehran during different months of 2013 and 2014 does not follow a specific and fixed trend. for instance, the highest and the lowest frequencies of studied crashes in 2013 have been recorded in january-february and october, respectively, while the highest and the lowest frequencies belonged to may and december, respectively. however, it can be generally concluded that the number of crashes in this period during the summer months (especially june and august) is fewer than the winter months (especially january and march). since some studies have shown that the frequency of traffic crashes associated with pedestrians increases with increased pedestrian volume (10,25,26), these seasonal differences can be attributed to changes in pedestrian volume. since some studies such as those conducted by cloutier, cottrill, green, miranda-moreno, mcarthur et.al found that the number of schools or students is associated with the frequency of traffic crashes associated with pedestrians in urban areas (17,26-29), school holidays and reduced traffic of students in june and august can be one of the reasons for the reduced frequency of such crashes during these months. therefore, better organization of students traffic and promotion of the safety of the streets near schools can be considered as one of the strategies for reducing the deaths from traffic crashes related to pedestrians in tehran. this study showed that about 80% of the studied crashes have occurred in highways and freeways. highways located at the main western and southern entrances and exits of tehran such azadegan highway (from northwest to southeast of tehran) and karaj special road, which connects karaj to 36 km west of tehran had the highest share of such crashes. these results are consistent with the findings of studies conducted by wier et.al in san francisco and mueler et al. in washington that showed that frequency of traffic crashes associated with pedestrians since traffic load in the western and southern highways of tehran is higher than other areas and there is an intense congestion of different means of transport in some of these areas, one reason for the high frequency of fatal crashes in these areas would be the high traffic load and congestion of different means of transportation. therefore, the reasons of this problem need to be identified by conducting more studies. in addition, the necessary measures should be taken to reduce the rate of traffic crashes through organizing the passage of vehicles and pedestrians and separating the passage of light and heavy means of transportation in the western and southern highways of tehran. distribution of studied crashes in terms of proximity to or remoteness from the west (azadi) and south (khazaneh) terminals showed that the density of traffic crash was relatively high at the end of the roads leading to azadi passenger terminal such as tehran-karaj freeway, karaj special road, afsarieh bridge, and the eastern part of azadegan highway. thus, one of the reasons for the high frequency of traffic crashes causing death to pedestrians in south and west of tehran is the location of azadi and khazaneh passenger terminals in these areas. on the other hand, distribution of the studied crashes in terms of proximity to or remoteness from urban passenger terminals does not follow a specific pattern and the frequency of traffic crashes associated with pedestrians is very low near these passenger terminals, especially those far from interurban passenger terminals. therefore, it can be concluded that the streets and roads around the urban passenger terminals are of low risk in terms of traffic crashes associated with pedestrians. comparison of the 22 districts of tehran in terms of the frequency of fatal crashes involving pedestrians shows that the highest frequency of this type of crashes has occurred in district 2, which accounts for 13.6% of the total cases, while this district covers only 8% of the total area of tehran. given that most crashes in district 2 have happened on highways, the relative high length of highways could be mentioned as one of the reasons for the higher frequency of traffic crashes associated with pedestrians in district 2 of tehran. highways in this district, with a length of about 61 km, account for about 11% of all highways of tehran. results of this study revealed that high-risk traffic areas in terms of crashes causing death to pedestrians during the studied years are mostly located in the west and south of tehran. high value of moran s i index indicated the distribution of crashes by the 560 traffic areas of tehran followed a cluster-like pattern. in other words, the location of occurrence of these crashes in tehran did not follow a random distribution. in addition, findings demonstrated that the hot spots of the studied crashes were mostly located in the west and south of tehran, while the cold spots were mainly focused in the central parts of the city. since getis-ord general g index showed that the distribution of hot and cold spots of the studied crashes is statistically significant, it can be stated that some certain factors were involved in non-random distribution traffic crashes causing death to pedestrians in tehran and high frequency of these crashes in high-risk areas. due to the environmental and structural differences between the high-risk and low risk areas, which are mainly located in the center of tehran, environmental factors similar studies in other parts of the world, including the studies conducted by slaughter et.al in new york, anderson in london, taquechel et.al in atlanta, wang and kockelman in texas, siddiqui et.al in florida and kuhlmann et.al in colorado, have also shown that environmental factors are highly involved in non-random distribution of traffic crashes related to pedestrians (12,14,16,31-33). it seems that one of the factors determining the distribution of the studied crashes in tehran, which is the higher frequency of crashes in the suburban areas than the central part of the city, is the difference between drivers and pedestrians in terms of traffic culture. the drivers and pedestrians in central parts of the city have higher education levels and respect traffic regulations more than those in the suburban areas. higher traffic load on the outskirts of the city compared to the downtown was another reason for this issue. enforcement of regulation of traffic limits and alternative passage of vehicles with even or odd license plates in the central parts of tehran causes the central parts of the city to have a lighter traffic load than the margins of tehran. this was one of the few studies in iran that has used the files drawn by police experts for conducting an applied research to determine the high-risk areas in a metropolis. using these files, we recorded the geographical coordinates of the location of crashes and we prepared a map to determine the distribution pattern of traffic crashes causing death to pedestrians and their position relative to other spatial objects. this study was conducted using only the data of crashes registered by tehran traffic police. comparison of the statistics provided by the police and forensic medicine organization showed that the actual number of fatal crashes in tehran is more than those mentioned in the database of tehran traffic police and the information related to some crashes causing death does not exist in this database. therefore, it is not possible to acquire information on their location of occurrence through the police files. in such studies, it is better that the relevancy of the death of the injured by traffic crashes to these crashes be defined based on the time of death after crash. this time in different countries varies between 6 days to one month, and in iran, this time has been defined to be one month. however, due to the lack of consistency between the data provided by tehran traffic police and forensic medicine organization, relevancy of the death of the injured by traffic crashes to these crashes in this study was determined based on the data extracted from data based on tehran traffic police. various districts of tehran, excluding central districts, are considered high-risk areas. therefore, the majority of traffic crashes causing death to pedestrians have occurred on highways located at the main entrance; that is, districts located in south, east and northwest of tehran. significant cluster-like distribution of crashes by the 560 traffic zones of tehran and existence of hot spots in terms of the studied crashes in the western and southern parts of tehran indicates the decisive role of environmental factors in occurrence of traffic crashes causing death to pedestrians. moreover, it is necessary to organize the pedestrians traffic in high-risk areas. this study was part of a thesis conducted in shahid beheshti university of medical sciences. we would like to sincerely thank all who helped us in this study, especially gen. hosseini, the commander of tehran traffic police, personnel of crashes department of tehran traffic police, particularly lt. soroush, headquarters of public health faculty and members of the epidemiology department of shahid beheshti university of medical sciences.

background: more than 20% of deaths from traffic crashes are related to pedestrians. this figure in tehran, the capital of iran, reaches to 40%. this study aimed to determine the high-risk areas and spatially analyze the traffic crashes, causing death to pedestrians in tehran. methods: mapping was used to display the distribution of the crashes. determining the distribution pattern of crashes and the hot spots/ low-risk areas were done, using moran s i index and getis-ord g, respectively. results: a total of 198 crashes were studied; 92 of which, (46.4%) occurred in 2013 to 2014 and other 106 cases (63.6%) occurred in 2014 to 2015. the highest and the lowest frequency of crashes was related to january (26 cases) and june (10 cases), respectively. one hundred fifty- eight cases (79.8%) of crashes occurred in tehran highways. moran s index showed that the studied traffic crashes had a cluster distribution (p<0.001). getis- ord general g index indicated that the distribution of hot and cold spots of the studied crashes was statistically significant (p<0.001). conclusion: the majority of traffic crashes causing death to pedestrians occurred in highways located in the main entrances and exits of tehran. given the important role of environmental factors in the occurrence of traffic crashes related to pedestrians, identification of these factors requires more studies with casual inferences.

PMC5307606

pubmed-489

since its first application in 1980, extracorporeal shock wave lithotripsy (swl) has become the preferred treatment method in many ureteric and kidney stone diseases. advances in stone treatment in the endoscopic age, such as the ability to perform retrograde intrarenal surgery more frequently and almost independently of the size of the stone and percutaneous nephrolithotripsy (pnl) gaining less invasive features defined as mini and micro, can be listed as developments that have hindered the preference of swl. the wide use of swl is due to its higher efficacy in selected cases while its low morbidity rates is one of the most important advantages of this method, making it the first treatment choice in many cases today, despite the other treatment alternatives that are available. however, there are conditions that limit the use of the method and affect its success. among the factors that affect the success and results of swl are; the type of lithotripter; stone-related factors such as the size, structure, number, and localization; the anatomy and the functioning of the kidney; and patient specific structural features [3, 4, 5]. al ansari et al. investigated the prognostic factors affecting the success of swl in 427 patients and they demonstrated that in cases with renal stones larger than 30 mm, the stone's size, localization, number, radiologic renal features and congenital renal anomalies were significant factors, while ureteral stent use, age, gender, and the nature of the stone (de novo or recurrent) had no effect.. reached the same conclusions in their study of 2954 cases with renal stones that were smaller than 30 mm. in addition, factors such as the presence of additional interventions pre- and post-swl, complications, and costs can also affect the efficacy of the treatment [2, 3, 4, 7]. the routine use of ureteral stent prior to swl is not recommended in renal stone cases despite the lack of any defined criteria in the guidelines [2, 8]. while the use of ureteral stents can reduce post-swl complications such as obstruction and renal colic, it does not prevent steinstrasse formation and infectious complications, and does not increase stone-free rates [8, 9, 10]. patient discomfort, pain in bladder, and issues related to urination that are associated with ureteral stent use can often be experienced. according to kirkali et al., pre-swl ureteral stent use should be preferred only in solitary kidney patients. the goal of our retrospective study of over 1361 patients was to compare the stone-free rates, steinstrasse formation, treatment efficacy and complications between patients with renal pelvic stones with and without pre-swl stent and to contribute to medical literature based on real life experiences. this is a retrospective study conducted by scanning the medical data of 1378 patients treated with swl for renal pelvic stones at our clinic between 1995 and 2011. seventeen patients who had percutaneous nephrostomy tube placement prior to swl were excluded from the study. the median age of the 1361 patients included in the study was 40 (1-85) years. all patients had routine renal function tests, urinalysis and urine culture, coagulation tests, kidney-ureter-bladder x-ray (kub), intravenous pyelography (ivp), and ultrasonography (usg) before swl. an abdominal contrast-free computerized tomography (ct) patients with urinary system infections were treated with antibiotics according to their culture results prior to swl. uncontrolled infections, coagulation dysfunctions, ureteropelvic junction obstruction, and pregnancy were considered contraindications for swl. the swl procedure was carried out via siemens lithostar lithotripter (siemens medizinische technik, erlangen, germany). the size of the stone was calculated in squared centimeters, by multiplying the widest width and length observed in kub. when multiple stones were observed, the sum of their sizes was calculated. in order to avoid statistical bias in this study, the patients were separated into 3 groups based on the size of the stone: 1 cm (group 1), 1.12 cm (group 2), and>2 cm (group 3). table 1 displays the patient characteristics, prior interventions for stones on the same side, stone characteristics, and treatment features of the 1361 patients. specialized in swl and the energy and shock wave count for each patient were determined by the same physician. prior to the procedure, an ureteral stent (percuflex plus 4.8 f x 26 cm, boston scientific, quincy, ma, usa) was placed in solitary kidney patients, patients with renal ectasia of grade 2, and patients with obstruction symptoms lasting a long period of time (> 1 month). swl was performed on all patients in the supine position, under fluoroscopic control and as an outpatient procedure. fourteen patients had the procedure under anesthesia where 0.10.2 mg/kg midazolam and 0.5 mg alfentanil were used for analgesic sedation. the treatment was initiated with 13 kilovolt (kv) and was increased with 0.3 kv increments up to the highest level that the patient could tolerate. the procedure was ended when full fragmentation of the stones was observed in the fluoroscopic control. the procedure was considered unsuccessful in cases where fragmentation was not achieved at the end of the 3 session and/or in patients who wanted to try another treatment. patients were given hydration, analgesic and antispasmolytic treatments during the sessions and the first post-treatment week. patients were evaluated with kub and usg at the end of the first week after the procedure and at 3-month follow-ups. ct was not performed on any patients who did not display significant symptoms or if hydronephrosis was not detected in their usg. size of the stone(s), auxiliary procedures (with or without stent use), radiologic evaluations at 3-month follow-up, and complications were evaluated retrospectively. statistical analyses were performed using chi-square, fisher's exact and mann-whitney u tests. using the efficacy coefficient equation (eq): stone free%/(100%+re-treatment+auxiliary procedures %) x 100. characteristics of patients, stones and treatments the grouping of 1361 patients according to the renal pelvis stone size was as follows: 514 patients in group 1, 530 in group 2, and 317 in group 3. there were 178 patients (13%) who had stent placement prior to the procedure according to the aforementioned criteria. the number of patients in groups 1, 2 and 3 who had pre-swl stent placement was 30 (6%), 44 (8%), and 104 (33%), respectively. patient and stone characteristics by groups and stent placement are presented in table 2, while treatment features by groups are shown in table 3. the average number of sessions was found to be significantly higher among the patients with stent placement in all groups (group 1: p=0.022; group 2 and 3: p=0.000), while the proportion of stone-free patients was similar across patients with and without stent placement in group 1 (86.4% and 73.3%; p=0.06), a significant difference was observed in group 2 (without stent 80.2% vs. with stent 56.8%; p= 0.000) and group 3 (without stent 75.1% vs. with stent 64.4%; p= 0.047). treatment eq for patients without and with stent placement, and for all patients within the groups were 64%, 46%, and 63.7%, respectively, for group 1; 52%, 30%, and 49%, respectively, for group 2; and 43%, 33%, and 40%, respectively, for group 3. the distribution of patients with and without stent placement according to steinstrasse formation was 6.7% and 5.8% (p=0.692) in group 1, 11.4% and 15.8% (p=0.431) in group 2, and 26% and 22.5% (p=0.500) in group 3, respectively. according to these findings, no significant difference was observed between the patients with and without stent placement within the groups in terms of steinstrasse formation. gender distribution and side of the stone localization yielded similar results across groups, as well. while the rates of solitary kidney patients were significantly different across patients with and without stent placement in groups 1 and 2 (p=0.000), no such significant difference was observed in group 3 (p=0.209). none of the major complications such as stent migration, infection, pyelonephritis or stent breakages were observed in any of the patients with stent placement in this study. of the 178 patients with stent placement, 68 (38%) complained of frequent urination and pain in the bladder and kidney area that was associated with the stent but these issues were resolved via symptomatic therapies. comparison of characteristics of patients and stones according to the groups treatment characteristics according to groups ureteral stents are mostly used to enable continuation of drainage in the presence of complications such as stone, tumor or obstruction between the kidney and the bladder. the pieces of the stones broken down still have a risk of causing obstruction in the ureter following swl and this condition is associated with the size of the stone. use of ureteral stent in swl is generally not recommended and there are various studies on the matter. although ureteral stent is useful to prevent complications such as obstruction and renal colic following swl, it does not protect from steinstrasse formation or infectious complications and does not increase the proportion of stone-free patients [8, 9, 10]. in our study, the stone-free rate following swl was found to be higher for renal pelvis stones>1 cm in patients without stent placement, although no difference was observed regarding steinstrasse formation. as for the demographic characteristic of our patient series, the female/male ratio, side ratio, stone load, and the number of swl sessions were in line with the literature findings. libby et al. demonstrated that ureteral stents reduced morbidity in case of stones of sizes>2.5 cm. on the other hand, reported that the swl morbidity with or without stent placement is similar to that of pnl in patients with stones of>2 cm. groeneveld recommended ureteral stent placement in case of stones that are greater than 3 cm, if swl with prior debulking or swl alone will be performed. however, the benefits of ureteral stent use prior to swl are still disputable [17, 18]. low et al., with their 179-patient retrospective series, determined that there was no difference between the stone-free rates of patients with or without stent placement in their 1-month and 3-month evaluations. sulaiman et al. reported that ureteral stents did not make a difference in steinstrasse formation in cases of stones that greater than 2 cm. preminger et al. also failed to detect a difference between patients with and without stent placement in terms of their stone-free rates, independent of the stone load and shock strength. reported that stent use did not cause a significant difference in the stone-free rates of large kidney stones or reduce post-swl morbidity and that their use in routine care is not necessary. however, in hollowel's inquiry of urologic specialists in the united states, it was determined that most of the urologists declared that they use stents in case of stones that are greater than 2 cm in size although there was no objective data about their usage. we believe that the urologists desire to stay on the safe side in terms of colic-type pain or obstructions following swl for relatively larger stones play a role in the decision. in sfoungaristas's study investigating stent use in ureter stones that are 4-10 mm in size, stent use was reported to reduce stone-free rates and that it negatively affected the post-swl quality of life. in pettenali et al. 's retrospective study, stent use was reported to reduce swl success in cases of proximal ureteral stones that are larger than 8 mm.. 's retrospective analysis showed that, in cases of kidney stones larger than 30 mm, steinstrasse formation was more common among patients with stent placement, and that the sole removal of the stent would be sufficient for these patients; and in cases of stones that are between 2030 mm, they reported no difference between patients with and without stent placement in terms of steinstrasse formation. they do not recommend stent use in the case of stones smaller than 20 mm in size. mustafa et al. also reported that the placement of a ureteral stent for the purpose of improving stone free rates or enhancing the passage of fragments during swl is unnecessary in renal stones with diameters less than 2.5 cm. though medical literature specifies that stents generally do not affect swl results or at least do not have a negative effect on stone-free rates, we state in our series that ureteral stents were observed to have a negative effect on the stone-free rates among stones greater than 1 cm in size. according to this finding, while stone-free rates do not vary across groups with or without stent placement in case of stones up to 1 cm, stent use was observed to have a negative impact on the stone-free rates in groups 2 and 3. again, the eq values were higher among patients without stent placement in all groups. this is contradictory with the predisposition of stent use with increase in stone size and in fact shows that ureteral stents may reduce the efficacy of swl as stone size increases. in all 3 groups of our series, we believe that the difficulty the stent creates in focusing on the stone and the additional sessions performed for the stones traveling down the ureter plays a significant role in these findings. furthermore, in such cases stents can also block the particles that could migrate downwards under normal conditions. in terms of steinstrasse formation, no significant difference was observed between the patients with and without stent placement in any of the 3 groups and ureteral stents were observed to be unable to prevent steinstrasse formation. literature reports migration, stent breakage, encrustation, infection, pyelonephritis, and stone formation as stent-related complications [26, 27]. 's evaluation of 290 cases with stent placement that had uretero-renoscopy and swl, the 12-week rates for encrustation was 76.3%, migration was 3.7%, and breaking of stent was 0.3%. joshi et al. also reported that 60% of the patients had stent- related symptoms of overactive bladder such as increased urination frequency and urge incontinence. while we did not observe complications such as migration, encrustation, breaking or pyelonephritis in our series, about 38% of the patients had symptomatic (frequency, urgency, hematuria, dysuria, and in some patients a colic-like pain) complaints. we believe that urinary reflux related to stent may play a role especially if the patient with a full bladder experiences colicky pain after urination. these complaints were generally controlled via symptomatic treatments and none of the patients required stent removal due to these symptoms. we believe that informing the patients about the possible symptoms that can be experienced following stent placement will allow them to tolerate the ureteral stents better. the retrospective nature, lack of randomization in terms of ureteral stent use prior to swl and that only 13% of patients had stent placement may be considered the limitations of this study. on the other hand, these limitations are avoided by the sole use of specialists with 25 years of swl experience (n.t.) in the performance of all procedures, which contributed significantly to our study with respect to the standardization provided in ureteral stent preference and adjustment of the number and strength of shock waves. we believe that our patient series with cases of various stone loads has a lot to contribute to the medical literature on this matter. the usage of kub and usg for monitoring the treatment results, but not ct could also be argued. kub and usg are preferred in the aim to avoid the risk of high radiation levels and financial burden on the health system especially in symptom-free patients. 's study on this topic with 2759 patients between the ages of 1876 years reported that usg, compared to ct, does not increase complications, pain score, admission rates to the emergency department, or hospitalization but does reduce exposure to radiation. in our study, the stone-free rate following swl was found to be higher for renal pelvis stones>1 cm in patients without stent placement, although no difference was observed regarding steinstrasse formation. our results showed that ureteral stents were observed to have a negative effect on the stone-free rates among stones greater than 1 cm in size. the results of this study support the idea of limiting the application of stents during swl and prevent possible complications as well as reduce the financial cost of treatment. considering that ureteral stents reduce stone-free rates, we believe that they should be preferred in special cases such as solitary kidney patients or those with long-term obstruction.

introductionthe aim of our study was to determine the efficacy of ureteral stents for extracorporeal shock wave lithotripsy (swl) treatment of pelvis renalis stones and to compare the results and complications in stented and non-stented patients. material and methodsbetween 1995 and 2011, 1361 patients with pelvis renalis stones were treated with swl. patients were subdivided into three groups according to stone burden: 1 cm2 (group 1; n=514), 1.1 to 2 cm2 (group 2; n=530) and>2 cm2 (group 3; n=317). each group was divided into subgroups of patients who did and did not undergo ureteral stent implantation before swl treatment. the efficacy of treatment was evaluated by determining the effectiveness quotient (eq). statistical analysis was performed by chi-square, fisher's exact and mann-whitney u tests. resultsof the 514, 530 and 317 patients in groups 1, 2 and 3 respectively, 30 (6%), 44 (8%) and 104 (33%) patients underwent auxiliary stent implantation. steinstrasse rates did not differ significantly between stented and non-stented patients in each group. the eq was calculated as 62%, 33% and 70% respectively in non-stented, stented and totally for group 1. this ratio calculated as 58%, 25% and 63% for group 2 and 62%, 26% and 47% for group 3. stone-free rates were significantly higher for non-stented than for stented patients in groups 2 and 3. conclusionsstone free rates are significantly higher in non-stented than in stented patients with pelvis renalis stones>1 cm2, whereas steinstrasse rates are not affected.

PMC4643708

pubmed-490

noncommunicable diseases, including type 2 diabetes mellitus (t2 dm), are considered the leading cause of death in the world and, in brazil, constitute the main cause of diseases. t2 dm assumes an important role in this context given that it is considered a worldwide epidemic disease; in 2011 it appeared among the ten leading causes of death in the world and it is prospected that number of cases will continue to increase. the ones most frequently being discussed are the increase in population life expectancy, changes in lifestyle (including unbalanced diet and physical inactivity), and obesity. diabetes can be defined as a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. the underlining cause of t2 dm can be attributed to a combination of resistance to the action of the hormone, increased production of insulin in a compensatory manner, and inadequate secretory response. on the other hand, according to a study conducted by kaprio et al., the heredity has an input around 47% in susceptibility to t2 dm; thus, genetic factors play an important role in the development of the pathology. some facts corroborate the importance of heritability in t2 dm: the greater concordance between monozygotic twins than among dizygotic twins and a wide variation in the prevalence of t2 dm in epidemiological studies with different ethnic groups as well as positive results in numerous other genetic studies. in this regard, it should be noted that even as more than 30 genes associated with t2 dm have been identified, the contribution of each individual gene in the disease susceptibility is very small. additionally, most of these genes identified are related to dysfunction of pancreatic cells. several reports suggest that the characteristics of each individual, including genetic polymorphisms, may confer differences in the occurrence of diabetes and other complex diseases such as cancer and heart disease. withal, in most cases, a positive association between polymorphisms of these genes and t2 dm are not reproducible in the analysis of a different population. two polymorphisms in the promoter region of the tnfa gene have been described, one present at position -308. in this regard, swaroop et al. demonstrated an association between the adipocytokine tnf- and development of insulin resistance. this systematic review was conducted to provide a comprehensive assessment of the association between the polymorphism in the gene tnfa -308g/a and the presence of t2 dm. the literature search was made through the virtual health library (vhl) which uses as bibliographic databases the publications found in sources such as lilacs, medline, and other information bases such as open educational resources, websites, and scientific events. any publications with case-control studies, on january 21, 2016, that show the relationship between polymorphisms of tnfa gene -308g/a and susceptibility to type 2 diabetes mellitus (t2 dm) were surveyed. the search terms used were as follows: diabetes and tnf or tumor necrosis factor, and polymorphism. the filters are selected in accordance with the main subjects of the following terms: type 2 diabetes mellitus, tumor necrosis factor alpha, single nucleotide polymorphism, and genetic polymorphism. the filter base on the type of study included solely the term case-control study. inclusion criteria were as follows: (1) the study must have been case-control; (2) the study should have assessed type 2 diabetes mellitus and -308g/a polymorphism of tnfa gene, even if associated with other comorbidities; and (3) the study should have provided sufficient data, including the number or frequency of alleles and genotypes. studies were excluded if (1) there were commentaries, case reports, or non-case-control or meta-analyses studies; (2) they did not report sufficient data; (3) they presented data from other types of diabetes or did not specify what kind patients had; and (4) they showed polymorphism in another coding region of the tnfa gene or (5) if the sample was composed of only dm patients. data from eligible studies were extracted independently in accordance with the inclusion and exclusion criteria. for each study the following characteristics were collected: the authors, the study title, the year of publication, where it was published, the sample size, the tnfa -308g/a genotypes with their corresponding simple frequencies and percentages for the control and the case group, and the study's p value and odds ratio, when calculated. the literature search found articles published starting from 2000, given that the year the complete mapping of the human genome was made available. advances in molecular biology technology since this project have been quick and, thus, increasing the number of genetic studies published for the association of certain genetic polymorphisms with various pathologies. in the case of literature search for articles with case studies that related polymorphism position -308 of the tnfa gene with diabetes, the result presented forty-three publications between the years 2003 and 2015, ten of which met the criteria inclusion. thirteen studies presented results for other types of diabetes; eleven related to type 1 diabetes and two to gestational diabetes. two others did not specify what type of diabetes had patients in the case group. two of these studies did not show enough genetic data for analysis. in relation to tnfa gene, seven studies presented polymorphisms in region other than the -308 position and three publications had single nucleotide polymorphism in a gene that was not tnfa. other studies were excluded due to different methodological designs in their inclusion criteria: two meta-analysis, one being observational and the other prospective and, relative to the sample. furthermore, there was a publication that presented data of diabetic patient's polymorphisms in both the control and the case group. the number and homogeneity of the included studies presented were too limited to allow a meta-analysis. relate the polymorphism in gene studied with t2 dm and coronary heart disease, while another two (buraczynska et al., dabhi and mistry) relate association frequencies in diabetic patients with nephropathy or renal failure. finally, five studies reported data that directly relate polymorphism in question with t2 dm (shiau et al., bouhaha et al., guzmn-flores et al. one of which also relates it with obese t2 dm patients (bouhaha et al.). the general characteristics of the studies included in this review are summarized in table 1. in three studies conducted in spain, hungary, and morocco significant associations between the tnfa -308g/a polymorphism and the risk of t2 dm were identified [11, 17, 20]: two of them in patients with other comorbidities such as coronary heart disease and atherosclerosis. in the present study, therefore, we infer that although there was no evidence of heterogeneity in the association between polymorphisms in the tnfa -308g/a gene and increased risk for development of t2 dm, the presence of positive results may mean that few studies have not been enough to clarify this relationship, and besides there is no documented evidence in various ethnic groups. due to contradictory results, this systematic review is aimed at providing a comprehensive assessment of the association between tnfa -308g/a and phenotype involving type 2 diabetes. the concern regarding the quantification and qualification of the genetic impact on phenotypic characteristics started after the first complete human sequence after genomic era. as a consequence, health research delved into the genetic information as a way to find answers to complex disease questions. this type of research has shown high prevalence and increasing prospects in a number of cases, such as diabetes. for this purpose, various portions of the human genome are now analyzed in search for genetic variations among individuals that could explain these diseases. studies seeking interactions between these variations, in particular, have been very promising. in the case of diabetes, a syndrome whose etiology is complicated and involves several risk factors for its development and progression, some studies on single nucleotide polymorphisms were suggested as causing the phenotypic characteristics and responsible for the increased risk of developing pathology [2527]. to study the risk factors for a disease is to study the possibility of a certain event happening. within the epidemiological concepts, the term is used to predict the likelihood of healthy individuals, exposed to certain factors, developing a given disease. this does not necessarily mean that it would occur; nevertheless the presence of these factors makes the individual more vulnerable to manifest the disease. a risk factor x may be the trigger of various diseases, and several risk factors can cooperate for the genesis of a common disease. environmental factors such as eating habits [2931] and physical inactivity [3234], in addition to the concomitant presence of diverse mutations in many genes, need to be considered so that the contribution of each of them is understood. linkage research studies genetic markers and specific polymorphisms in a family group that has the disease. this, thus, allows the researchers a way to better study the regions of a chromosome that is affected. after the connection is made to the chromosome, one can test for association of polymorphisms for identifying a genotype specific to a given disease in groups of people who have the targeted disease [34, 35]. several association studies have discussed the importance of clarifying the relationship between genetic polymorphisms and the development of type 2 diabetes. the search for candidate genes was initially focused on possible genes encoding proteins directly involved in the pathophysiology of t2 dm: related encoders, for example, production, secretion or activity of insulin, or development of the pancreas [3638]. gradually, however, emerging studies suggested that several other factors could contribute to the development of t2 dm, as the expression of proinflammatory cytokines and other molecules that act in the inflammatory process and that appear to play a critical role in the development of chronic complications of dm. several studies have shown the influence of various polymorphisms in proinflammatory cytokines genes as a risk of development of obesity, diabetes, and metabolic syndrome. one of these cytokines, tumor necrosis factor (tnf-), has been investigated since diabetics have elevated levels of it circulating. tnf- has also been implicated as an insulin resistance-causing factor associated with the pathogenesis of t2 dm. tnf- is a proinflammatory cytokine that acts in the regulation of cell proliferation, differentiation, and apoptosis. among the various polymorphisms describing the tnfa gene, the -308g>a variant located in the promoter region excels by affecting the expression of its gene. the presence of the a allele at nucleotide -308 increases transcription of tnfa gene approximately twofold, therefore increasing the production of this cytokine. under this circumstance, it is expected that the polymorphic genotype may be associated with an increased frequency of diabetes and, for the proper association, of case-control studies can be used. in this review, several studies with the frequency of genotypes in control and case group were observed for a better understanding of the population genetic profile and a possible association between polymorphism of tnfa gene and t2 dm in groups with different ethnicities. the influence of genetic polymorphisms on certain diseases may be found in a population and yet not in another; this may impact the frequencies and distribution of a given polymorphism in distinct population. the difference in influence may be due to racial variantion as well as other factors.. in addition, some studies have included a proportionally small sample size in relation to the country's population and most of them define the case of groups in t2 dm patients who have concomitant comorbidities. case-control studies were selected in this review and were made with the people of spain, taiwan, poland, india, tunisia, mexico, hungary, and morocco; nonetheless a positive association was only found in the spanish, hungarian, and moroccan population. sefri et al. in their meta-analysis, which considers 21 case-control studies published until august 2013, argue that no significant associations were found between the polymorphism in the studied region of the tnfa gene and risk of developing t2 dm. their finding was consistent with a previous meta-analysis, which included data from 18 association studies. in these studies, publications with different ethnic groups were included; among them were africans, asians, caucasians, and other populations. so, when comparing the frequencies of polymorphic genotypes (ga and aa) in the groups of case-control study (described in table 1), it is possible to observe that these genotypes are presented more frequently in the group that has the disease. the exceptions to this were the studies in tunisia and india; the most frequent genotype in type 2 diabetic patients was the gg. in all other countries this evidence supports the assumption that polymorphisms in the tnfa gene and its association with other aspects, both genetic and environmental, may represent an important risk factor for type 2 diabetes mellitus. risk factors not modifiable or irreversible in nature, such as genetic profiles, refer to the individual characteristics. even though these factors may not be changed, the identification of their presence in individuals and families may enable health professionals to advise change in the lifestyle of these patients, avoiding early manifestation of t2 dm.

diabetes mellitus (dm) is considered to be a worldwide epidemic disease and its type 2 form comprises more than 95% of all cases. tumor necrosis factor-alpha (tnf-) is a proinflammatory cytokine. its dysregulation has been implicated in a variety of human diseases, including type 2 diabetes mellitus (t2 dm). the control of expression of this cytokine is associated with insulin resistance and has a strong genetic influence. in order to understand this relationship, the literature from all case-control studies since 2000 to date was reviewed. the genotypes frequency results presented in ten publications with different ethnicities were compared. the correlation between the tnfa promoter genotypes and the risk of developing t2 dm remains controversial due to the many discrepancies between the different studies available. ethnic differences may play a role in these conflicting results, since the distribution of tnfa promoter polymorphisms is distinctive between individuals of dissimilar racial origin. hence, although the relationship between t2 dm incidence and presence of polymorphisms at position -308 of the tnfa gene is not entirely clear, the results of these studies suggest the need for further investigation.

PMC5086378

pubmed-491

of the 130 million babies born worldwide every year, approximately 4 million are stillborn, more than 98% of these occur in developing countries. stillbirth accounts for more than half of perinatal mortality in developing countries. in sub-saharan africa, while countries in south-east asia report the highest overall numbers of stillbirth, countries in africa report the highest incidence rates per 1000 live births. the average stillbirth rate in developing countries has been reported to be 26 per 1000 live births, about five times higher than in developed countries (5 per 1000). one fourth to one third of all stillbirths is estimated to take place during delivery [5, 6]. stillbirths occurring in the intrapartum period generally have a normal appearance and are often called fresh the skin not being intact implies death more than 24 hours before delivery (antepartum), often called macerated stillbirths. stillbirths have not been widely studied, have been under-reported, and rarely have been considered in attempts to improve birth outcomes in developing countries [5, 6]. there are many factors associated with stillbirth including inadequate access to obstetric care, inadequate care, malaria, hypertensive disease, poor nutritional status, history of stillbirth, congenital anomalies, sickle cell disease, and high burden of infectious comorbidities [5, 810]. first, maternal infection may cause severe illness, leading to fetal death [12, 13]. also, an infection in the uterus or anywhere else in the mother's body may precipitate preterm labor. last, the placenta may be directly infected, leading to reduced blood flow to the fetus, a likely cause of stillbirth associated with malaria infection. when malaria parasites infect the placenta, placental insufficiency results because of lymphocyte and macrophage accumulation, and increased expression of pro-inflammatory cytokines; these impede maternal blood flow through the placenta [16, 17]. intestinal helminths, including hookworms and trichuris trichura, have been associated with anemia [18, 19]. it has been suggested that low hemoglobin concentrations can cause a state of chronic hypoxia, which is presumably exacerbated in pregnancy when oxygen demands are particularly high because of the metabolism of the mother and the fetus, and that oxygen transfer to the fetus is probably reduced in anemic women. a strong association has been observed between maternal plasma, cord plasma, and placental folate concentrations, suggesting that transplacental folate delivery depends on maternal plasma folate concentrations. according to the world health organization's opportunities for africa's newborns 2006 report, the stillbirth rate for ghana is 24 per 1000 deliveries. even though stillbirths represent a large proportion of perinatal deaths, causes of stillbirths are poorly understood in ghana. to our knowledge, the association between malaria and intestinal helminth coinfection in pregnancy and stillbirth has not been studied. few studies have studied the association between malaria and helminths in pregnancy, with conflicting results. given that 98% of stillbirths occur in developing countries, especially sub-saharan africa, which also has a high burden of malaria and intestinal helminth infections, it is important to investigate the role of these infections in contributing to stillbirth. the study was conducted in kumasi, the capital of the ashanti region of ghana. the climate in kumasi is humid and tropical, with two rainy seasons, april to june and september to october. helminth infection is endemic in the ashanti region, which also has an intense perennial malaria transmission, the predominant parasite being plasmodium falciparum. the institutional review board of the university of alabama at birmingham and the committee on human research, publications and ethics, school of medical sciences, kwame nkrumah university of science and technology, kumasi reviewed and approved the study protocol before its implementation. a cross-sectional study of women presenting for delivery at two hospitals in kumasi, the komfo anokye teaching hospital (kath), and the manhyia polyclinic was conducted in november and december 2006 after informed consent was obtained, a questionnaire was administered to collect sociodemographic information, and medical and obstetric histories. body weight and mid upper arm circumference (muac) were measured for each woman. obstetric information was also obtained from the mothers ' antenatal care (anc) charts. anc charts provided information on number of antenatal care visits, gestational age as assessed by palpation at first anc visit or ultrasound at first anc, tetanus shots, malaria prophylaxis, antihelminth medications, hemoglobin level, and illnesses and treatments during pregnancy. blood was drawn by venipuncutre for determination of hemoglobin levels, serum folate level, and malaria antigen tests. state of the newborn (alive or stillbirth), sex, weight, and length were obtained as recorded by the midwives. determination of malaria antigen in plasma was done using the malaria antigen celisa (cellabs, brookvale, australia). the malaria antigen celisa kit is a monoclonal antibody-based assay specific for p. falciparum malaria. the assay detects a merozoite antigen that circulates in the blood for up to 14 days postinfection. determination of hookworms, ascaris lumbricoides, and trichuris trichura was done using the kato-katz thick smear technique (who, 1991). stool samples were processed within 12 hours of collection and examined microscopically within one hour of preparation to avoid missing hookworm ova. for strongyloides stercoralis, samples were processed using the baermann method. hemoglobin level was measured in an automatic cell counter (sysmex m-2000; digitana ag, hamburg, germany) about 30 minutes after blood sampling. uncomplicated pregnancy: absence of hypertension, pre-eclampsia, history of a previous caesarean section and hemorrhage, and a normal presentation of the fetus. malaria infection: presence of malaria antigen in the mother's peripheral blood at the time of delivery. intestinal helminth infection: presence of helminth eggs or larvae in stool collected at the time of delivery. anemia: hemoglobin levels<11 g/dl of blood, and severe anemia: hemoglobin level<8 g/dl. stillbirth: an intrauterine death of a fetus weighing at least 500 grams after 20 completed weeks of gestation occurring before the complete expulsion or extraction from its mother. an intrauterine death of a fetus during labor or delivery was considered a fresh stillbirth, and an intrauterine death of a fetus sometime before the onset of labor, where the fetus showed degenerative changes was considered a macerated stillbirth. induced abortion: the purposeful interruption of an intrauterine pregnancy with the intention other than to produce a live-born infant, and which does not result in a live birth. sample size was calculated using unpublished reports on stillbirth from the two study hospitals, which estimated that at least 1%1.5% of 1000 births would be stillbirths. we made the assumption that if we obtained 10 stillbirths, and that 1025% of women with normal births had both malaria and intestinal helminth infections, at a 5% significance level, we would have 80% power to detect an odds ratio of 7.59.0; assuming 15 stillbirths, we would be able to detect an odds ratio of 6.07.5. data analysis was performed using sas software version 9.1 (sas institute, cary, nc). differences in socio-demographic and obstetric characteristics by stillbirth were assessed by chi-square or t-test. correlation analyses were performed to identify potential multicollinearity between independent variables. to determine factors associated with stillbirth, we used multiple logistic regression. variables that were significant (p<.05) on bivariate analysis and those that are known to be associated with stillbirth based on previous studies were entered into a regression model. through this procedure, we calculated odds ratios (or) and 95% confidence intervals (ci). seven hundred and eighty five (785) women were recruited into the study before delivery in the two hospitals in kumasi. we obtained both malaria and intestinal helminth results from 746 women, and data analysis was limited to these women. overall, the mean age of the women was 26.8 years (range: 15 to 48 years); 21.1% were single, 30.2% were primigravidae, 30.6% were anemic, 29.5% had a prior induced abortion, and 5.2% had a history of stillbirth (table 1).. a higher proportion of women who were single did not receive sp during pregnancy, had fewer than 5 anc visits, had low folate levels, were anemic, had had a prior induced abortion or a prior stillbirth and delivered a stillborn infant compared to their counterparts (table 1). of the 746 women, 407 (54.6%) had neither infection, 147 (19.7%) were infected with p. falciparum only, 68 (9.1%) were infected with helminths only, while 124 (16.6%) were coinfected. a higher proportion of women with either organism presented with stillbirth than women with neither infection. women who were coinfected had a modestly higher rate of stillbirth than women with a single infection (table 2). low serum folate, severe anemia, prior induced abortion and prior stillbirth were each strongly, independently associated with stillbirth, with increased odds ranging from over 3-fold to a 6-fold increase (table 3). women with malaria irrespective of whether or not they had intestinal helminths had a 90% increased odds of stillbirth. although intestinal helminth infection had a stronger association, it was not statistically significant (table 3). this study demonstrated that the study population had a relatively high rate of stillbirth (5% of all deliveries). factors associated with stillbirth were malaria, severe anemia, low serum folate concentration, past induced abortion, and history of stillbirth. many stillbirths were fresh (75.7%), an indication that a proportion of these cases could likely have been prevented. it has been suggested that stillbirths occurring in the peripartum period could be prevented through appropriate cesarean section, improved obstetric care, and improved emergency response to obstetric complications. in this study, women who had fewer antenatal care visits had an increased risk of stillbirth, suggesting that stillbirths are closely linked to use and quality of maternal services. malaria is endemic in many african countries, and is thought to play a role in contributing to stillbirth. intestinal helminths, especially hookworms and trichuris can cause anemia [18, 19], which in turn leads to adverse birth outcomes including stillbirth. we did not observe an association between intestinal helminths and stillbirth, a finding that has been previously reported. however, our observation could be the result of small numbers, that is, malaria was more common than intestinal helminthes. coinfection with malaria and intestinal helminths did not increase the risk for stillbirth but as in the case of intestinal helminths, this could be a matter of numbers. malaria contributes to anemia by hemolysis or destruction of parasitized cells and causes shortened red cell survival [36, 37], while hookworms and trichuris cause anemia through direct blood loss [19, 38]. since the mechanisms by which malaria and intestinal helminth infections cause anemia differ, it is possible that their impact on anemia are additive and could exacerbate adverse birth outcomes. a strong association has been observed between maternal plasma, cord plasma, and placental folate concentrations, suggesting that transplacental folate delivery depends on maternal plasma folate concentrations. some studies [40, 41] have reported higher rates of stillbirth in women with megaloblastic anemia than those without. abortion is legal in ghana only for medical reasons, and is not available upon request (ministry of health, ghana). most women seeking abortion therefore sometimes attempt illegal abortions, and then go to the hospital for treatment of complications. removal of retained products of conception in the hospital setting is usually performed by cervical dilation and curettage. there has been concern that this may result in cervical insufficiency, hence future adverse birth outcomes. the tendency to repeat pregnancy outcomes in successive births is well known and includes risk of stillbirth. previous studies have demonstrated that women with a history of stillbirth may have a 6 to 10-fold increased risk of stillbirth [46, 47]. the causal mechanism may involve impaired placental development and function due to compromised vascular support system. a methodological weakness of our study lies in limited power due to the number of stillbirths, therefore our findings should be interpreted with caution. the fact that it was a cross-sectional study also limits our ability to draw causal or temporal associations. the study however has several strengths including new findings, high participation and consistency with other studies in some risk factors of stillbirth, which strengthens confidence in the new findings. another strength of the study lies in the fact that the hospitals in which the study was conducted are secondary and tertiary hospitals which cater to large numbers of women of all socioeconomic status from kumasi and surrounding areas. the 2008 demographic health survey for ghana (dhs, 2008) reported that 82.4% of women in urban areas in ghana deliver in a health facility. the fact that most of the stillbirths were fresh suggests that higher quality intrapartum care could reduce stillbirth rates. more studies need to be conducted to further assess the association between stillbirth and malaria and intestinal helminth coinfection. it is important to conduct further studies to investigate risk factors of stillbirth to determine which stillbirths are preventable so that targeted interventions can be developed and tailored for resource-poor settings .

objective. the objective of the study was to assess plasmodium/intestinal helminth infection in pregnancy and other risk factors for stillbirth in ghana. methods. a cross-sectional study of women presenting for delivery in two hospitals was conducted during november-december 2006. data collected included sociodemographic information, medical and obstetric histories, and anthropometric measures. laboratory investigations for the presence of plasmodium falciparum and intestinal helminths, and tests for hemoglobin levels were also performed. results. the stillbirth rate was relatively high in this population (5%). most of the stillbirths were fresh and 24% were macerated. when compared to women with no malaria, women with malaria had increased risk of stillbirth (or=1.9, 95% ci=1.29.3). other factors associated with stillbirth were severe anemia, low serum folate concentration, past induced abortion, and history of stillbirth. conclusion. the fact that most of the stillbirths were fresh suggests that higher quality intrapartum care could reduce stillbirth rates.

PMC2850132

pubmed-492

periodontal diseases are polymicrobial immune-inflammatory infectious diseases that can lead to the destruction of periodontal ligaments and adjacent supportive alveolar bone. the subgingival plaque contains more than 700 bacterial species, and some of these microorganisms have been shown to be responsible for initiation/progression of periodontal diseases [1, 2]. the red complex, which includes porphyromonas gingivalis, treponema denticola, and tannerella forsythia (formerly bacteroides forsythus), encompasses the most important pathogens in adult periodontal disease. additionally, fusobacterium nucleatum, prevotella species, eikenella corrodens, peptostreptococcus micros, and campylobacter rectus are increased in deep periodontal pockets and are implicated as possible periodontopathogens [14]. these bacteria are not usually found alone, but in combination in the periodontal pockets, suggesting that some bacteria may cause destruction of the periodontal tissue in a cooperative manner. studies using animal models have reported the synergistic pathogenicity of mixed infections with p. gingivalis-t. furthermore, coaggregation, nutrient effects, and modulation of virulence factors by periodontopathogens or by interspecies interactions between periodontopathogenic and nonpathogenic organisms have been reported to contribute to oral microbial pathogenesis. this paper focuses on interspecies pathogenic interactions within the red complex, in particular the combinations of p. gingivalis-t. potential therapies using normal inhabitants of the oral microbiota that have an antagonistic relationship with the red complex are discussed. p. gingivalis possesses many virulence factors, such as fimbriae, lipopolysaccharides, and proteases [1315]. the arg-gingipain (rgp) and lys-gingipain (kgp) cysteine proteinases are important for the virulence of p. gingivalis as they elicit dysfunction of inflammatory and immune responses and can degrade various connective tissue proteins [16, 17]. rgp is encoded by two separate genes (rgpa and rgpb), whereas kgp is encoded by a single gene (kgp). in a murine abscess model, rgpa and rgpb double and kgp single-mutants induced smaller abscesses than did the wild type, and the rgpa-rgpb-kgp triple (gingipain-null) mutant showed negligible lesion formation. these findings indicate that gingipains play an important role in abscess formation in mice. compared to the abscess formation induced by monoinfection with each bacterium in the abovedescribed murine model, mixed infection with wild-type p. gingivalis atcc 33277 and t. forsythia atcc 43037 showed a synergistic effect on abscess formation. on the other hand, mixed infection with p. gingivalis mutants devoid of gingipain (rgpa rgpb, kgp, and rgpa rgpb kgp) with t. forsythia showed only just an additive effect on abscess formation. these findings suggest that the gingipains of p. gingivalis play an important role in the pathological synergism between p. gingivalis and t. forsythia. a combination of t. forsythia strains isolated from periodontitis patients and p. gingivalis also showed synergistic pathogenesis in a rabbit abscess model. the researchers found 100% abscess formation in rabbits with mixed infection using p. gingivalis and t. forsythia but 0% in rabbits with monoinfection with each bacterium. recently, verma et al. injected p. gingivalis fdc 381 and t. forsythia atcc 43037 into the oral cavity of rats and evaluated the synergistic effect of mixed infection on periodontal disease. mixed colonization by p. gingivalis and t. forsythia in the rat oral cavity was confirmed by polymerase chain reaction (pcr). the induction of moderate periodontal inflammation and pronounced apical migration of junctional epithelium, generation of a specific immunoglobulin g (igg) antibody response, and stimulation of both th1- and th2-like immune responses as reflected by the serum igg subclass profiles were evident. mixed infection with p. gingivalis and t. forsythia resulted in a significant increase in interproximal alveolar bone resorption compared to that in rats infected with a single bacterial strain and controls, but there was no synergy between the bacteria. the researchers indicated that the virulence of p. gingivalis and t. forsythia mixed infection resulted from the immune-inflammatory responses and the lack of humoral immune protection during periodontitis in rats. mice that were first infected with a wild-type strain and subsequently reinfected with the same wild-type strain showed significantly smaller lesions than control mice that were mock-infected with medium only and then re-infected with the wild-type strain. assessed the serum igg antibody response in a murine abscess model after mixed infection with various levels of p. gingivalis atcc 33277 or t. forsythia atcc 43037 whole bacteria cell antigens. after mixed infection, igg antibody responses to p. gingivalis increased in proportion to the level of t. forsythia injected. in contrast, igg antibody responses to t. forsythia did not correlate with the level of p. gingivalis injected. reasons for this difference may be that p. gingivalis and t. forsythia induced different antibody responses after mixed infection in mice; alternatively, these bacteria may exhibit different interactions in terms of growth at the injected sites. t. forsythia, which is a fastidious anaerobic gram-negative rod, is frequently isolated together with p. gingivalis, especially from the active state of periodontitis [2427]. it is well known that the growth of t. forsythia is accelerated on blood agar when cocultivated with p. gingivalis or f. nucleatum, suggesting that a form of symbiosis occurs with respect to nutrition. sonicated cell extracts of t. forsythia atcc 43037 stimulate growth of p. gingivalis atcc 33277 in nutrition-decreased medium in a dose-dependent manner, whereas the cell extract of t. forsythia had no stimulatory effect on the growth of the rgpa rgpb kgp triple (gingipain-null) mutant of p. gingivalis. these results suggest that gingipains of p. gingivalis play an important role in the digestion or uptake of the growth-promoting factor derived from t. forsythia. the growth-promoting interaction between p. gingivalis and t. forsythia may be partly related to synergistic virulence in a murine abscess model. t. forsythia is a member of the polymicrobial flora that invades buccal epithelial cells taken directly from the mouth. epithelial cell invasion by periodontopathogens is considered to be an important virulence mechanism for evasion of the host defense responses and for forming reservoirs important in recurrent infections. t. forsythia possesses some putative virulence factors, such as a trypsin-like protease, a sialidase, hemagglutinin, components of the bacterial s-layer, and a cell surface-associated and secreted protein (bspa). bspa has been recognized as a virulence factor important for alveolar bone loss in mice. inagaki et al. investigated the epithelial cell adherence and invasion abilities of t. forsythia and reported that these are dependent on bspa. additionally, they found that p. gingivalis fdc 381 or its outer membrane vesicles enhance the attachment and invasion of t. forsythia atcc 43037 to epithelial cells. t. denticola, a small oral spirochete, is frequently found with p. gingivalis in progressing periodontitis lesions [1, 3335]. t. denticola is located within the surface layers of the subgingival plaque, whereas p. gingivalis is observed predominantly beneath the spirochete layer; a symbiotic nutrient utilization relationship between these two periodontopathogens has been shown in vitro. the growth-stimulating factors produced by p. gingivalis atcc 33277 and t. denticola atcc 35405 have been identified as isobutyric acid and succinic acid, respectively. coculture of p. gingivalis fdc 381 and t. denticola atcc 35405 induced synergistic biofilm formation and coaggregation. confocal microscopy demonstrated that p. gingivalis attaches to the substratum first as the primary colonizer followed by coaggregation with t. denticola to form a mixed biofilm. the t. denticola flagellar mutant and cytoplasmic filament mutant exhibit significantly reduced biofilm formation with p. gingivalis. similarly, the p. gingivalis gingipain mutant and major fimbriae mutant exhibited significantly reduced biofilm formation with t. denticola. using two-dimensional electrophoresis followed by a ligand overlay assay with p. gingivalis fimbriae, hashimoto et al. determined that dentilisin, a chymotrypsin-like proteinase of t. denticola, was the p. gingivalis fimbriae-binding protein. these results support the hypothesis that these two organisms assist each other's survival in subgingival sulcus and explain why they are frequently isolated together from subgingival plaque. synergistic virulence of mixed p. gingivalis and t. denticola infection has been assessed in several animal lesion models. using a murine abscess model, one group reported that high doses of p. gingivalis w50 (1-2 10 cells per dose) together with t. denticola atcc 35404 (1 10 or 1 10 cells per dose) had no effect on the formation and size of the spreading lesion caused by p. gingivalis. however, at low p. gingivalis doses (1-2 10 cells), addition of t. denticola (1 10 cells) significantly enhanced the virulence of p. gingivalis compared with monoinfection. investigation of the synergistic virulence of p. gingivalis and t. denticola using a murine experimental model of periodontitis found that a 1: 1 ratio of p. gingivalis w50 and t. denticola atcc 35405 coinoculum at 5 10 or 1 10 total bacterial cells induced the same level of bone loss as four doses of 1 10 p. gingivalis. coinoculation induced strong p. gingivalis-specific t cell proliferative and interferon- (ifn-) cytokine responses and induced a strong t. denticola-specific ifn- cytokine response. another study using a rat model of periodontal disease reported that a mixed p. gingivalis fdc 381 (5 10 cells) and t. denticola atcc 35404 (5 10 cells) infection produced significantly more interproximal and horizontal alveolar bone loss compared to monoinfections (1 10 cells); however, there was no synergy between p. gingivalis and t. denticola. furthermore, colonization of these bacteria was observed in the rat oral cavity during 7 weeks of periodontal disease, resulting in the generation of a specific serum igg antibody response that reflected oral infection and the induction of an inflammatory response consistent with the established characteristics of periodontitis. these results suggest that p. gingivalis and t. denticola act synergistically (with no synergy between the bacteria) to stimulate the host immune response and to induce alveolar bone loss in a rat model of periodontitis. epithelial cells and macrophages play a major role in the host response to periodontopathogens, and secretion of inflammatory mediators and matrix metalloproteinases (mmps) by these host cells contribute to periodontal tissue destruction. investigated the inflammatory response of an in vitro macrophage/epithelial cell coculture model following mono- or mixed infections with whole bacterial cells of the red-complex and their lipopolysaccharide (lps). mono- or mixed infections of the coculture model induced the secretion of interleukin-1 beta (il-1), il-6, il-8, prostaglandin e2 (pge2), and mmp-9. all lps mono- or mixed infections induced an increase in chemokine, mmp-9, and pge2 production. compared to mono-infections with individual bacterial species, no synergistic effects on cytokine, pge2, or mmp-9 production by the bacterial mixtures tested were observed. p. gingivalis and t. forsythia induced the secretion of rantes (regulated and normal t cell expressed and secreted), whereas t. denticola alone or in combination with p. gingivalis resulted in a significant decrease in rantes levels. rantes degradation by mono- or mixed infections with red-complex bacteria resulted in massive proteolytic degradation of rantes by t. denticola. the ability of periodontopathogens to degrade cytokines and chemokines in vivo may play an important role in their pathogenicity by disrupting the host inflammatory response. recently, polymicrobial infection with p. gingivalis, t. denticola, and t. forsythia in a rat model of periodontal disease was investigated. a 1 10 cell mixture (1 ml) containing an equal number of cells of each bacterium was injected into the rat oral cavity. pcr of the bacterial dna in the oral sample revealed that polymicrobial infection enhanced colonization by p. gingivalis, t. denticola, and t. forsythia compared to their levels in monomicrobial infections. oral infection of rats with a polymicrobial consortium comprising p. gingivalis, t. denticola, and t. forsythia induced significant increases in maxillary and mandibular alveolar bone resorption compared to those resulting from any of the monomicrobial infections (p<0.001). the levels of serum igg against all of the bacteria in the polymicrobial infection were lower than the respective levels induced by monomicrobial infections. this suggested that the host response to the polymicrobial infection was altered, resulting in enhanced evasion of protective immune responses by the bacterial consortium. most therapeutic modalities for treatment of periodontitis aim to remove pathogens and kill all bacteria in the periodontal pocket. understanding the effect of interbacterial interactions on the pathogenesis of periodontitis may facilitate development of novel treatment modalities, such as the inhibition of adherence using antagonists, passive immunization, replacement therapy, regulation of levels of nonpathogenic bacteria to modulate virulence, probiotics, and interference with signaling mechanisms. the disruption of the harmonic relationship between the host and commensal microorganisms is considered to be an important factor for the development of oral pathologies., we discuss the significance of interbacterial antagonism for maintenance and recovery of a healthy oral microbiota. moreover, antagonistic bacteria have the potential for probiotic action, which may protect against periodontitis. staphylococcus aureus and streptococcus mutans isolates inhibited the growth of t. denticola and p. gingivalis. s. aureus strains produced a bacteriocin-like inhibitory substance, whereas the inhibitory effect of s. mutans was related to the production of lactic acid. s. sanguinis, s. cristatus, s. salivarius, s. mitis, actinomyces naeslundii, and haemophilus parainfluenzae inhibited the adhesion of standard p. gingivalis strains in vitro. s. cristatus arginine deiminase repressed fima, a major subunit protein of the long fimbriae, and inhibited biofilm formation by p. gingivalis. an analysis of the ability of clinical isolates from healthy and periodontitis patients to inhibit the growth of periodontopathogens showed that the number of isolates from healthy volunteers that inhibited either p. gingivalis or p. intermedia was significantly higher than that from diseased patients. these isolated growth-inhibiting strains included some viridans group streptococcus, actinomyces, and bifidobacterium strains. compared to these isolates, commercial dietary probiotics showed stronger inhibition of the periodontopathogens. in a study that compared oral lactobacilli from patients with chronic periodontitis and periodontally healthy subjects, the most prevalent species in healthy subjects were lactobacillus gasseri and l. fermentum, whereas the most prevalent species in subjects with periodontitis was l. plantarum. furthermore, the greatest antimicrobial activities were associated with l. paracasei, l. plantarum, l. rhamnosus, and l. salivarius. the international guidelines for the evaluation of probiotics confirm that these four organisms exhibit both high antimicrobial activity and high tolerance to environmental stress. some oral bacteria possess the potential for antagonism towards periodontopathogens, which highlights the therapeutic potential of stimulation of oral health using beneficial effector strains. most evidence indicates that probiotics in the gut do not populate the gastrointestinal microbiota permanently, and they disappear from feces soon after cessation of probiotic ingestion. probiotic bacteria used in the human oral cavity include bifidobacterium and lactobacillus species, and most of them were not derived from the oral cavity. l. reuteri and l. salivarius colonized the oral cavity of patients; however, the study was of only 2 weeks duration [5759]. another study reported that oral administration of l. salivarius decreased the proportion of l. salivarius in saliva during the 4- and 8-week intervention periods, although the sampling and analysis methods differed. organisms antagonistic to periodontopathogens that are derived from typical representatives of the oral microbiota and have probiotic potential may overcome the weaknesses associated with exogenous probiotic bacteria.

the red complex, which includes porphyromonas gingivalis, treponema denticola, and tannerella forsythia (formerly bacteroides forsythus), are recognized as the most important pathogens in adult periodontal disease. these bacteria are usually found together in periodontal pockets, suggesting that they may cause destruction of the periodontal tissue in a cooperative manner. this article discusses the interspecies pathogenic interactions within the red complex.

PMC3606728

pubmed-493

uterine cancer is the most common diagnosed gynecologic cancer among women in the united states, with 43,470 estimated new cases and approximately 7,950 estimated deaths in 2010. although uterine cancer is found mainly in the older patient population, up to 14% of uterine cancers are found in women younger than 45 years of age. most studies have demonstrated that younger women have a lower risk of death from uterine cancer than older women independent of stage at diagnosis [4, 5]. surgical management of uterine cancer entails a staging procedure which includes a total hysterectomy, salpingooophorectomy, and possible lymph node dissection depending on the spread of the disease and histological type. a high index of suspicion must be maintained if the diagnosis of uterine cancer is to be made in the young patient. young women with or without risk factors may present with abnormalities in their menstrual periods, which can be initially treated with hormonal therapy, thereby delaying the diagnosis of cancer. some risk factors for uterine malignancy include prolonged exposure to unopposed estrogen either endogenous, (e.g., early menarche, late menopause, nulliparity, polycystic ovarian syndrome, obesity), or exogenous (e.g., estrogen therapy or hormone replacement without progesterone). uterine cancer in young women is often associated with early-stage disease, well-differentiated tumors, and a good prognosis [68]. the actual care delivered to women diagnosed with uterine cancer appears to depend upon a number of variables including surgeon training, hospital volume, extent of disease, patient characteristics, and physician access. these women are mostly managed by gynecologic oncologists as well as by nongynecologic oncologists (general gynecologists and general surgeons). gynecologic oncologists are specifically trained to perform the required staging and cytoreductive surgical procedures for uterine cancer. however, patterns of care studies have shown that gynecologic oncologists provided care in 48.8% of the patients and gynecologists in 50.2% of the cases. roland et al. have shown that the gynecologic oncologist completed the surgical staging two times more frequently than the general gynecologist (94.0% versus 45.2%, p they concluded that women with uterine cancer managed by gynecologic oncologists are more likely to receive comprehensive surgical staging resulting in an efficient use of health care resources and minimizing the potential morbidity associated with adjuvant radiation therapy. numerous studies have evaluated the association of surgeon case volume with clinical outcomes for various procedures and have shown that higher surgeon volume is associated with improved outcomes [1013]. while specialty training is critically important to quality cancer care, recent attention has also focused on the positive relationship between surgeon and hospital case volume and clinical outcomes for malignancies treated with technically complex surgical procedures. recently, there has been an interest in elucidating patterns of care for patients treated with uterine cancer. most studies evaluating age-based outcomes for uterine cancer have been single institution analysis containing only a small portion of patients. more specifically, prior studies have not evaluated whether care provided by high-volume surgeons at high-volume centers leads to differences in resource allocation and short-term survival among younger patients. therefore, the goals of this study were to evaluate volume-based care for women with uterine cancer aged 50 years and younger using a statewide population database. approval to conduct this study was obtained from the johns hopkins medical institutions clinical research committee and joint committee on clinical investigation, and the requirements for informed patient consent were waived. the study design was a cross-sectional analysis of hospital discharge data from the nonfederal acute care hospitals in maryland collected by the maryland health services cost review committee (hscrc). the hscrc database provides information regarding the index hospital admissions and is limited to 30 days of followup. the main study objective was to characterize volume-based care among women 50 years when compared to women>50 years. a subgroup analysis among women 50 years with regards to surgeon and hospital uterine cancer volume was performed to evaluate the risk of in-hospital related death and associations with the length of hospital stay, hospital-related costs, and icu length of stay. all adult female patients, 18 years of age and older, who underwent a surgical procedure including a hysterectomy for a malignant uterine neoplasm in maryland between january 1, 1994 until december 31, 2005 were included in the study. the international classification of disease, 9th revision (icd-9) code 182.0 (malignant uterine neoplasm) was used for sorting. the surgical procedures included in the analysis were limited to those incorporating hysterectomy as this was felt to be the most likely to capture those patients undergoing initial surgery for uterine cancer. patient age was modeled as a categorical variable (patients 50 years of age and younger compared to patients over 50 years of age). this age cutoff was designated based on the average age of menopause as 51.4 years [15, 16]. frequency distribution for variables like ethnicity, insurance payer status, hospital volume, surgeon volume, hospital type, inpatient death, and concordance between attending and operating physician was tabulated among the two groups. information regarding international federation of gynecology and obstetrics (figo) or american joint committee on cancer (ajcc) stage of disease, tumor grade, histological subtype, extent of disease, or residual disease was not available from the hscrc database. in addition, the hscrc database only provides clinical information for the index hospital admission, so that data on clinical outcomes beyond this time period could not be assessed. based on previous research in volume-based care in uterine cancer, surgeons performing 100 cases of uterine cancer per study period were categorized as high volume, and those performing<100 cases of uterine cancer per study period as low volume. surgeons were included in the analysis if they performed at least one uterine cancer surgery during the entire study period. based on previous research in volume-based care in uterine cancer, hospitals with 200 cases of uterine cancer per study period were categorized as high volume, while those with<200 cases of uterine cancer per study period were categorized as low volume. similarly, hospitals were included in the analysis if at least one uterine cancer surgery was performed during the entire study period. a community teaching hospital was defined as a nonuniversity hospital with a residency program in obstetrics and gynecology. hospital-related charges for each index admission were converted to the organizational cost of providing care using cost to charge ratios for individual hospitals. cost to charge ratios were calculated from data from the health services cost review commission by dividing the average inpatient expense by the average inpatient revenue of each hospital during each year of the study interval. this ratio was then multiplied by each patients ' charge to obtain the cost per admission. t-test for simple linear regression was employed to evaluate adjusted cost of hospital-related care, intensive care unit length of stay, and admission length of stay among the two age groups. t-test for simple linear regression was done for the subset group of women aged 50 years and younger to evaluate the variables of adjusted cost of hospital-related care, admission length of stay, and intensive care unit length of stay among hospital's and surgeon's volume. all statistical computations were performed using the sas system, and all reported p values are two sided. a total of 6,181 women who met the criteria for a primary surgical procedure for a malignant uterine neoplasm were identified in the state of maryland during the period of 19942005. women aged 50 years and younger comprised 13.6% (n=844) of the cases. most of the women aged 50 years and younger were white (78.0%), had insurance coverage through an hmo (40.3%), and were treated in a community-based hospital (67.4%) (table 1). young women were more likely than older women to have concordance between the operating physician and the attending physician (p=0.03) (table 1). in other words, young women were more likely to have an operating surgeon that was the same clinician of record. younger women were more likely to have shorter length of hospitalization stay (p<0.0001) and lower adjusted cost of hospital-related care (p=0.0002). there were no significant differences between the lengths of intensive care stay between groups (table 2). four of the reported deaths occur among the group of women 50 years of age and younger. in total, 894 different surgeons provided primary uterine cancer surgical care, although not all surgeons provided continuous care for the entire duration of the study period. only 9 (1.0%) of the surgeons were categorized as high-volume surgeons. women aged 50 years and younger were less likely to be managed by high-volume surgeons when compared to women older than 50 years (31.6% versus 35.1%, resp., p=0.02). in the subgroup of women aged 50 years and younger, those that were managed by high-volume surgeons were more likely to have longer hospitalization stays (p=0.003) and higher adjusted cost of hospital-related care (p<0.0001) (table 3). no difference was observed in terms of icu length of stay between high- and low-volume surgeons. no deaths were reported among high-volume surgeons during the immediate 30-day period. due to the lack of events on one of the groups, a statistical comparison of death rates between high- and low-volume surgeons could not be performed. a total of 49 hospitals provided care for uterine cancer patients during the study period. only 8 (16.3%) hospitals meet the criteria of high-volume hospitals. no difference was observed among groups in terms of access to high-volume centers (52.0% versus 54.0%, p=0.22). in the subgroup of women 50 years of age and younger, those admitted to a high-volume hospital were more likely to have higher adjusted cost of hospital-related care (p=0.01) (table 4). no deaths were reported among high-volume hospitals during the immediate 30-day period. due to the lack of events on one of the groups, a statistical comparison of death rates between high- and low-volume surgeons could not be performed. several studies have demonstrated that age is an independent predictor of overall survival [4, 5]. our analysis of the hscrc database from 19942005 confirms that younger women have lower short-term mortality than older women diagnosed with uterine cancer. the fact that younger women have better outcomes can be explained by a number of hypotheses. it may be that younger individuals have a better health profile, with less morbidity and concurrent medical problems. younger body/tissues may respond to a surgical insult with less postoperative complications and accelerated healing. also, uterine cancer in young women is often associated with early-stage disease, well-differentiated tumors, and a good prognosis [68]. even though the lack of events did not allow performing a statistical analysis, no in-hospital related deaths were reported among the young group of women treated by high-volume surgeons or at high-volume centers. in addition to reduced mortality, lengths of hospital stay and hospital costs for younger women with uterine cancer were lower. the reduced rate of medical comorbidities at baseline in younger women likely contributed to this difference as preoperative comorbidities are an independent predictor of length of postoperative hospitalization and therefore cost. younger women were more likely to be managed at university-based hospitals and more likely to have an operating surgeon that was the same attending physician of record decreasing the opportunity for miscommunication and repetition of testing that could occur when multiple providers are involved in patient care. rogers and curtis had shown that continuity of care leads to decreased hospital admissions, decreased length of stay, reduced duplication of diagnostic testing, increased patient satisfaction, and improved compliance. the high level of concordance in the younger women may account for the improved outcomes such as the decreased cost and shorter length of stay. the analysis suggests that younger women were less likely to be managed by high-volume surgeons. for women a perceived lower risk of cancer risk or mortality, limited financial resources, unwillingness to travel long distances for care, and the absence of medical comorbidities may lead fewer younger women to seek out the expertise of a high-volume specialist on their own. several studies have shown the benefit of women that have been cared for by a gynecologic oncologist [9, 24, 25]. although the overall peri-operative outcomes were similar, the subspecialists were more likely to perform comprehensive surgical staging, if necessary, and the patient was less likely to receive adjuvant radiotherapy. the overall survival favored those patients managed by the general gynecologist, with no difference in disease-free interval. this may reflect the patient population that is usually managed by the general gynecologist, as compared to the gynecologic oncologist. similar to the differences in costs of care between younger and older women, younger women treated at low-volume settings had lower costs of care and shorter hospital stays. the reduced rate of medical comorbidities of women treated at these settings is likely to contribute to these cost differences for the same reasons as mentioned previously. the hscrc database can not be queried to clarify whether high-volume surgeons are located exclusively at high-volume hospitals; therefore it is unclear whether higher costs are impacted by more frequent care by gynecologic oncologists. low-volume hospital costs may also be reduced due to the different resources of these facilities. surgeons may be less inclined to perform aggressive procedures if these low-volume facilities do not have adequate support (i.e., access to consultants or high-acuity icu care) and thus refer complex patients to high-volume centers. improved efficiency at low-volume hospitals can not be excluded as an alternative cause for lower costs. the strength of this study lays in the relatively large number of patients included form institutions across the state of maryland. unfortunately, the maryland hscrc database is limited in ways typical of large population-based studies as it does not provide followup beyond 30 days from the index admission and contains no information on either ajcc or figo stage of disease, tumor grade, or histological subtype. given these restrictions, a meaningful analysis of long-term surgical outcome was not practical from the available data. also, the hscrc does not provide information in terms of performance status, asa scores, and comorbidities. these factors may have had an impact on the overall morbidity and mortality between the two populations studied. furthermore, additional information regarding the baseline medical condition of the populations was not examined, and this could be a confounding factor on the results. a second limitation is that there is a possibility that the grouping by case volume could be too selective and there could be subgroups in the lower-volume level that still are able to achieve excellent outcomes. due to the lack of previous reports of case volume among uterine cancer, a third limitation of the current study is that our selected volume criteria have not been widely validated as outcomes measures. despite these limitations, the current study provides the most extensive evaluation on volume-based care among young women that underwent surgery for uterine cancer. the information obtained in this study increases our current knowledge of volume-based care of young women diagnosed with uterine cancer and may be useful in developing strategic planning in order to improve the care offered to this population.

purpose. to characterize volume-based care of uterine cancer among women aged 50 years. methods. the maryland health service cost review commission database was accessed for uterine cancer surgical cases from 1994 to 2005. cross-tabulations and logistic regression models were used to evaluate for significant associations among volume-based care and other variables comparing women 50 years with those aged>50 years. results. women 50 years comprised 13.6% of the cases. women 50 years were less likely to be managed by high-volume surgeons (31.6% versus 35.1%, p=0.02). for women 50 years, there was a trend toward management at low-volume hospitals (52.0% versus 54.0%, p=0.22). no deaths were reported among the group of women 50 years treated by high-volume providers or at high-volume centers. women 50 years managed by high-volume surgeons had longer length of stay (p<0.001) and higher adjusted cost of hospital-related care (p<0.00). women 50 years managed at high-volume centers had higher adjusted cost of hospital-related care (p=0.01). conclusion. primary surgical care of young women with uterine cancer is often performed by low-volume providers.

PMC3236364

pubmed-494

parvovirus b19 is a small, uncoated dna virus belonging to the family parvoviridae (1). parvovirus b19 associates with a wide range of clinical symptoms and their pattern is under effect of age and immune status of the host (4). most of b19 infections are asymptomatic or only associate with mild non-specific illness. this virus can also be transmitted through blood transfusion and blood products (5). b19 infects humans widely and is almost endemic in all parts of the world (2). parvovirus b19 can also be transmitted through vertical transformation (from mother to fetus) (6). its prevalence in pregnancy is about 15%, but in epidemic situation it receives to 10% (78). its incidence in pregnancy is about 15% and it can cause complications in 3% of infected pregnant women (9, 10). the virus is transmitted through vertical transmission to the fetus and creates some complications to fetus including anemia, spontaneous abortion, hydrops fetalis, intrauterine fetal death and congenital anomalies (11). parvovirus infection of mothers is diagnosed using serologic or an immune assay enzyme b19 igm and b19 igg (12). b19 igg positive serologic evidence of previous infection with the virus that leads to lifelong immunity. in this study, we tried to determine prevalence of serum parvovirus infection in pregnant women and its association with some of the parameters such as number of lived children, number of family members, number of commensalts and amount of hemoglobin. the risk of adverse fetal outcome increases if maternal infection occurs during the first two trimesters of pregnancy but may also happen during the third trimester. it is significant cause of fetal loss throughout pregnancy, but has a higher impact in the second half of pregnancy when spontaneous fetal loss from other causes is relatively rare (13). for example, the risk of infection in pregnant women with one child are 3 times more than nulliparous women, but this risk for women with three or more children are 7.5 times more. the other risk factors are working in the school, care centers and other full stress jobs (1416). igg antibody levels elevated with the increase of age (17, 18) at the age of 15, about 50% of persons have detectable igg levels and its amount in older people, has increased to more than 90% (2). the importance of b19 investigation in pregnant women is due to vertical transmission of this virus. the complications related to this virus are anemia, hydrops fetalis, fetal death, and unintended abortion (11). intrauterine growth retardation, myocarditis, pleural effusion, pericardial effusion and brain involvement of the fetus may occur following infection with the virus. although, parvovirus b19 is not related to congenital malformations (19, 20), in case of pregnant women infection, the prevalence of transmision to the fetus is 30% (21). acute parvovirus b19 (b19v) infection is a proven risk for pregnant women and their fetus (22). regarding to complications related to b19 virus infection during pregnancy and lack of study about its infection prevalence in ardabil county, this study was conducted with the aim of determination of the prevalence of parvovirus and igg in pregnant women in ardabil. the numbers of samples were calculated 350, based on statistical formula for sample size. these numbers were divided into 39 prenatal care centers, according to population covered by each center. 5 ml of blood was taken from participant in care centers and was referred to the reference laboratory, immediately. the serum of blood samples were isolated and were stored at 20 c, until analysis. the questionnaire consisted of two parts: part i including mother s age, location, number of family members (father, mother, sister, brother), number of children, number of commensalts, and in part ii information about blood test of pregnant women, were recorded. for measurement of igg antibodies in serum samples against b19 virus, the elisa kit euroimmune, (germany) was used as follows: 100 l of serum 1/201 diluted patients and 100 l of each of the standards were added to micro plate wells in which the viral antigen was coated. the samples were incubated for 1 h at room temperature, then the wells were washed and 100 l goat, anti-human, anti-igg antibody binded to the peroxide was added to each sample and they kept at room temperature for one more hour. then, after washing the wells, 100 l of substrate (3, 3, 5, 5 tetra-methyl benzidine) was added to each well and allowed to create color within 30 minutes at room temperature. subsequently, 100 l of fixing solution (sulfuric acid) was added to each well and a maximum light absorption of the double wavelengths of 450/630 nm were read using a plate reader. the chi-square and t-test and descriptive methods were used for data analysis. the significant levels of 0.05 were used for all statistical analysis. in order to consider the principles of medical ethics, in this study, of 350 pregnant women, 64.6% (226/350) were ardabil citizen and the rest were from rural area (124/350). the youngest participant was 15 years old and the oldest was 34 years (average of 23 3.92 years). overall, 242 (69.1%) had positive serology b19 igg and 124 (30.9%) were seronegative. distributions of seroprevalence of parovirus b19 among pregnant women in residence location are shown in table 1. of 108 people that living in rural areas, 85 (68.5%) were seropositive and 39 (31.5%) were negative. there was no significant relationship between residence location and prevalence of serum parvovirus b19 (p=0.475). distribution of serology parvovirus b19 prevalence among pregnant women based on residence location the average age of participants with positive serology was 24.774.26 years, and the mean age of those with negative serology was 22.983.58 years. result showed there was a significant correlation between age and the prevalence of parvovirus (r=0.268) and the immunity against the virus was higher in older pregnant women (table 2). distribution of serology parvovirus b19 prevalence among pregnant women according to age group the average number commensalts seropositive pregnant women was 3 1.74 and those with negative serology 2.83 1.16 that showed no significant relationship between the number of commensalts and prevalence of parvovirus (p=0.377). the average family members of participants with positive serology was 6.86 2.4 and those with negative serology was 6.61 2.23, which showed no significant relation between family size and prevalence of parvovirus (p=0.369) (table 3). relationship between the number of people breaking bread together, family size and the number of children living with an outbreak of parvovirus b19 the average number of lived children in participants those with positive serology was 0.57 0.655 and average number of lived children in those with negative serology was 0.51 0.634 which presented the relationship between family size and sero prevalence of parvovirus was insignificant (p= 0.417). the average amount of hemoglobin in participants with positive serology was 12.51.12 (p=0.177) and the average amount of hemoglobin in those with negative serology was 12.261.06 (p=0.169) which showed no significant relationship between hemoglobin and serum parvovirus. results of this study showed that the prevalence of b19 in pregnant women in ardabil was 69.1%. (2005) in shiraz showed that parvovirus infection providence was 69.01% (17). in a cross-sectional study, sohrabi et al. (2007) reported that 55.7% of pregnant women referred to ahvaz imam khomeini hospital were b19 positive (had specific igg of b19). according to their findings, more than 40% of pregnant women had not igg b19 and were at the risk of the virus infection and its fetus complications (23). in a study conducted in nigeria by emiasegen et al. (2011) parvovirus infection prevalence among pregnant women attending to prenatal care clinics and its relation to occupation, number of children and transfusion history were studied. they reported that 273 pregnant women were studied and 27.5% of them had igg antibody for b19 and the relationship between the numbers of living children, occupation and achieved transfusion history were significant (24). in another study, elnifro (2009) found that its prevalence in libya was 69% (25). the other hand, khameneh (2014) reported that in orumiyeh prevalence of parvovirus was 75.6% (26), that was further from the results of our study. for example, sohrabi in a study in ahvaz showed that its incidence was 55.7% (19); abiodun (2013) reported 20% in nigeria (27), and cohen (1995) declared 53% of pregnant women are immune to the virus (28). the difference between rural and urban areas in the prevalence of b19 virus was not significant. of 226 people lived in urban area, 157 patients (69.5%) were positive while 68.5% of people (85 of 108) lived in rural areas were positive in serology. the significance of difference among mean age between positive serology and negative serology indicates that relationship between age and the prevalence of parvovirus. therefore, its incidence was higher in older women. in the ziyaeyan (2005) and sohrabi (2007) studies, there was no significant correlation between age and the prevalence of parvovirus, that is contrast to our results (17,23). in this study, the relations between seropositive incidences with number of children, number of family members, number of commensalts, and hemoglobin content were not significant. (2001) (29). in a study from nigeria, emiasegen et al. (2011) showed that the number of children was related to the outbreak of parvovirus (24) that is not consistent with the results of our study. comparison between the average number of sero-positive children and seronegatives revealed that the number of children did nt related to the prevalence of parvovirus that is in accordance with the shahraki et al. the relation between the number of children and outbreak of parvovirus was significant (24) that is not consistent with our results. the results of this study showed a high susceptibility of pregnant women to parvovirus b19 in ardabil region and immunity to the virus increases with age. in our study, a significant correlation was not found between the prevalence of parvovirus and the number of living children, number of family member, number of commensalts and hemoglobin content. considering that a high percentage of infected pregnant women with parvovirus b19 in the ardebil region, health education and screening for the virus, especially in pregnant women with anemia is recommended to prevent fetal complications.

background and objectives: trans-placental transmission of parvovirus b19 during pregnancy can causes adverse outcomes. regarding its importance in prenatal care, we decided to study prevalence of parvovirus b19 infection among pregnant woman in ardabil, iran. materials and methods: in a community based study with a cluster sampling, 350 pregnant women that attended in health care centers in ardabil were selected. serum samples were collected and anti-b19 specific igg was detected using commercial enzyme-linked immunosorbent assays (euroimmune elisa kit, germany). furthermore, a questionnaire filled for all participants during samples collection. results:64.6% (226/350) of participants were ardabil citizen and the rest were from rural area (124/350). anti-b19-specific igg antibody was detected in 69.1% of pregnant women (242/350). participants ages ranged from 15 to 34 years with average of 23 years. according to our study, seroprevalence of igg antibodies had positive significant correlation with the participants age (r=0.268) but there were no significant relations between b19 seropositivity and living area, family member, number of commensals, number of living children, and the amount of hemoglobin (p>0.05). conclusion: approximately, one-third of the participants were at risk of primary b19 infection. therefore, health education of pregnant women and screening of infected pregnant women is recommended to prevent fetal complications.

PMC5139926

pubmed-495

left ventricular noncompaction cardiomyopathy (lvnc) is a very rare cardiomyopathy characterized by an increase in the noncompacted, trabeculated myocardium adjacent to compacted myocardium in the left ventricular (lv). though current research suggests a developmental arrest in embryogenesis as the underlying pathology, the etiology of lvnc are not fully understood. the patients will often present with a spectrum of disease severity ranging from no symptoms to cardiac arrhythmias, cardiac failure, thromboembolism or even sudden cardiac death. historically, lvnc has been diagnosed by echocardiography when the ratio of noncompacted to compacted myocardium is>2. echocardiography may not visualize the apical region optimally, leading to underestimation of the degree of lv noncompaction. however, cardiac magnetic resonance imaging (cmri) provides a comprehensive depiction of cardiac morphology in any imaging plane. recent cmri reports suggest a ratio of the noncompacted myocardium to compacted myocardium of>2.3 yield the highest sensitivity (86%) and specificity (99%) in diagnosis. we present a case of lvnc in an adult patient with its clinical and imaging findings. a 24-year-old female patient was initially referred to our vascular department for investigation of bilateral lymphedema. her echocardiogram showed mildly impaired lv with evidence of segmental wall motion abnormality and hypertrabeculation in the apex suggestive of noncompaction. the history of the patient started at birth when she had difficulty swallowing and several delayed milestones such as walking and talking at the age of 5. due to socioeconomic issues as the patient matured, there was an additional history of palpitations, which were occasionally associated with syncopal attacks. lower limbs bilateral nonpitting edema up to the knees, proximal bilateral upper and lower limbs weakness with intact sensation, waddling gait, and easy fatigability with few steps were noted. computed tomography coronary angiogram was performed to exclude coronary artery disease. results showed segmental wall motion abnormality and revealed normal coronaries, lv dilatation, and lateral wall and apical noncompaction with the noncompacted myocardium to compacted myocardium ratio in the range of 2.53.5 [figure 1]. her cmri showed mildly dilated lv with mild global hypokinesis and a marked decrease in longitudinal shortening. end-diastolic volume was 120 ml, end-systolic volume was 61 ml and the ejection fraction was 50%. in addition, multiple areas of noncompacted myocardium, particularly in the apex and most of the lateral wall extending from apical lateral wall to the mid-to-basal segment were seen. the ratio of maximum thickness of the noncompacted myocardium to compacted myocardium was more than 2.5 in multiple areas [figure 2]. gated cardiac computed tomography scan was performed to evaluate the coronary arteries and showed patent coronary arteries of normal anatomy (not shown). axial image shows left ventricle lateral wall noncompaction with prominent trabeculation (arrows) noncompaction cardiomyopathy in a 24-year-old female. a four chambers view dark blood using horizontal long axis t1 fast spin echo-magnetic resonance imaging. (a) apparent thickening of the left ventricle lateral wall (arrows). a static image of four chamber view from cardiac magnetic resonance imaging cine, using the balanced steady state free precession technique. (b) shows mild dilatation of the left ventricle, prominent left ventricle wall trabecular network (arrowheads) and only a thin compacta (arrows) further investigation showed elevated creatine kinase and electromyography findings consistent with myopathy. thus, the clinical impression was proximal myopathy mostly mitochondrial or congenital, congenital lymphedema, lv noncompaction with lv ejection fraction of 40%, and normal rv function. as her lv impairment is mild, the decision was made to keep her on beta-blockers and on angiotensin-converting enzyme inhibitor in the form of metoprolol and lisinopril, respectively. on subsequent follow-up visits, she reported a history of interval improvement apart of occasional palpitations with no history of shortness of breath, syncope or dizziness. she has been advised to do regular physiotherapy and to wear compressive stockings for her lymphedema. bellet and gouley described the first case of noncompaction in an autopsy of a newborn infant with aortic atresia and coronary ventricular fistula. lvnc without other cardiac abnormalities (isolated noncompaction cardiomyopathy) was first described by an echocardiogram performed by engberding and bender. historically, the prevalence of lvnc has been underestimated due to the lack of knowledge about this rare condition and its similarity to other diseases of the myocardium and endocardium. a study of pediatric patients with primary cardiomyopathy showed that 9% of the patients had lvnc. the median age at diagnosis of isolated noncompaction cardiomyopathy in the initial case series of isolated noncompaction was 7 years ranging from 11 months to 22 years. the prevalence of lvnc in the adult population ranges between 0.01% and 0.3% of all adult patients referred for echocardiography studies. these numbers are mostly extracted from the referred patient for abnormal echocardiographic findings or congestive heart failure (hf). therefore, because of this potential selection bias, the true prevalence is not clear. male patients are predominately affected accounting for 63%, 70%, and 74% of cases in three different reported series. in 2001, jenni et al. evaluated seven patients with lv noncompaction with pathologic correlation (four postmortem patients and three cardiac transplant patients). histologic analysis demonstrated ischemic lesions in the thickened endocardium and thickened trabeculae with accompanying fibrosis. clinical manifestations are highly variable, ranging from no symptoms to disabling congestive hf, arrhythmias, and systemic thromboemboli. according to a french registry, the diagnosis of lvnc diagnosis was confirmed in 105 cases through the use of echocardiogram performed in a laboratory between 2004 and 2006. in that study, lvnc was first detected in 12 patients with rhythm disorders, 45 patients with hf symptoms, and eight patients through familial screening. during the follow-up study, patients suffered several complications including hf occurring in 33 of the patients, ventricular arrhythmia in seven, embolic events in nine, and nine of the patients received heart transplantation while death occurred in 12 of the patients. a swiss registry recorded a total of 34 cases over 15 years among a patient population who underwent echocardiogram. in this study, major complications included hf (53%), ventricular tachycardia (41%), thromboembolic events (24%), and death (35%). six of the reported 12 deaths were sudden and four due to end-stage hf in four and the other two cases were due to unrelated causes. the presented case had a family history of sudden death in two brothers, with one of them presumed to have an unknown type cardiomyopathy. sporadic and familial forms of noncompaction have been described. in the original report of isolated noncompaction ventricular cardiomyopathy that predominantly affected children, half of the patients had a familial recurrence. however, a larger reported adult population with isolated noncompaction ventricular cardiomyopathy showed only 18% familial recurrence. lower percentage compared with the earlier report might be attributed to incomplete screening of siblings in oechslin et al. this patient has a progressive myopathy, which raised the question of other association with her left ventricular noncompaction cardiomyopathy. about 82% of lv noncompaction cases were found to be associated with neuromuscular disorder including becker muscular dystrophy, metabolic myopathy, myotonic dystrophy, barth syndrome, and other rare genetic disorders. the ratio of noncompacted myocardium to compacted myocardium at the end of systole is>2:1 is the most often used echocardiographic criteria that were proposed by jenni et al. the criteria also include presence of segmental thickening of the myocardial wall of the lv with two layers: prominent trabeculations and deep recesses with a thin epicardial layer and a thick endocardial layer. the absence of coexisting cardiac abnormalities is required to fulfill the criteria. lv diastolic dysfunction, reduced global lv systolic function, abnormal structure of papillary muscles, and lv thrombi are nonspecific findings that can be seen on echocardiography. cardiac magnetic resonance (cmr) can be used in correlation with an echocardiogram to localize and quantify the extent of noncompaction. cmr offers a detailed view of the cardiac morphology of the noncompacted myocardial layer in the lv in any image plane including the apical and lateral segments-segments, which are not well visualized by echocardiogram. recent reports suggest that echocardiography diagnostic criteria are strict and mri enhances the detection of more subtle forms of noncompaction. further, cmr identifies a higher rate of two-layered structures in segments such as the anterior, inferior, anterolateral, and inferolateral segments compared with echocardiogram. however, contraindication after implanted devices, cost and availability of cmr are considered barriers on the way of implementing cmr. according to the american heart association, 1.5 tesla is the minimum acceptable magnet strength to perform cardiac imaging in order to be able to visualize both short and long axis views in approximately 17 segments. acquiring three diastolic long axis views best identifies the nc/c ratio of the most prominent myocardial trabeculations segment. the nc/c ratio in cmr is measured at the end of diastole and should be more than 2.3, more than echocardiography, which is 2.1. stagnant blood flow, within the myocardial trabeculae may be detracted as a high signal in cmr black blood imaging, supporting the diagnosis lvnc. the efficacy of cmr was evaluated in a report of seven patients with lvnc in whom other features supported the diagnosis; the results were compared with 170 healthy volunteers, athletes, or patients with dilated cardiomyopathy or hypertrophic cardiomyopathy, aortic stenosis, and hypertensive heart disease. the most distinguishing feature was a ratio of noncompacted to compacted myocardium during diastole (sensitivity 86% and specificity 99%). direct imaging of myocardial fibrosis is possible with the use of an inversion recovery prepared t1-weighted gradient-echo sequence and the extracellular fluid tracer gadopentetate dimeglumine. this technique has been termed delayed hyperenhancement and shows nonviable tissue as hyperenhanced or bright. nevertheless, the early and precise diagnosis is mandatory to rule out other underlying diagnoses and to allow a timely start of standard hf and anticoagulation therapy, thus preventing further complications. some trabeculae show delayed hyperenhancement despite having a normal compacted to noncompacted myocardial ratio, suggesting that lv noncompaction may be a more diffuse disease process than previously suspected. the use of delayed hyperenhancement sequences improves the correlation between cmri and the parameters of the clinical stage of the disease. cmri follow-up examinations would be helpful to assess a potential change of noncompacted or compacted mass in a chronological sequence. in conclusion, cmr can distinguish lvnc from other cardiomyopathies and normal hearts with high sensitivity and specificity.

left ventricular noncompaction cardiomyopathy is a very rare condition, yet believed to be often overlooked. it is thought to be caused by the developmental arrest in embryogenesis and characterized by an increase in the noncompacted, trabeculated myocardium adjacent to compacted myocardium in the left ventricular. the clinical presentations of this type of cardiomyopathy are of variable severity. echocardiography used to be the diagnostic modality, but recent reports suggest that cardiac magnetic resonance imaging has higher sensitivity and specificity by showing a ratio of the noncompacted myocardium to compacted myocardium of>2.3.

PMC4738500

pubmed-496

lung cancer is the most common cause of cancer mortality in the western world, accounting for approximately 5% of all deaths in many countries. five-year survival for those with pathological stage ia non-small cell lung cancer (nsclc) is 73% whereas metastatic disease has a dismal prognosis (13% 5-year survival). results from several studies suggest that frequent chest radiographic screening does not result in reduced lung cancer mortality, a conclusion reinforced by the prostate, lung, colorectal, and ovarian (plco) cancer screening trial [3, 4]. in fact, some studies suggest that frequent chest radiographic screening is associated with an 11% relative increase in lung cancer mortality compared with less frequent screening. randomized trials of screening low-dose computed tomography (ldct) scans demonstrate that computed tomography (ct) is far more sensitive than chest radiography. the national lung screening trial (nlst) showed that, in heavy (30 pack-years or more) current or former (within 15 years) smokers between the ages of 55 and 75, three annual ldct screens reduced lung cancer-specific mortality from 309 to 247 deaths per 100,000 person-years. there are still many unanswered questions about the benefits and harms of those programs that could determine the ultimate success of the mass screening implementation. additionally, despite expert guidelines for screening high-risk populations, most national health service providers have not implemented (and probably will not implement in the near future) mass lung cancer screening programs. one of the main concerns is that the extrapolation of findings from tightly controlled trials to real-life mass screening programs requires uniform standards and high quality controls not easily achievable in most institutions. consequently, the current practice is that the patients themselves or their physicians may choose early lung cancer detection on an individual basis. there is little information, however, on the clinical characteristics and outcomes of patients with incidentally detected early stage lung cancer from strictly controlled randomized trials. the objective of this study was to analyze the clinical records of lung cancer patients who underwent surgical resection to evaluate the clinical characteristics and outcomes of patients with incidentally detected lung cancer and patients with symptomatic lung cancer. all patients undergoing pulmonary resection with a curative intention for non-small cell lung cancer (nsclc) in the british hospital in buenos aires between january 1986 and july 2009 were eligible for inclusion in this retrospective study. our thoracic oncology centre keeps a database of all patients evaluated, with data entered prospectively at the time of their initial evaluation. patients were excluded if they had exhibited small cell lung cancer or a rare histological result. preoperative data included methods of diagnosis and a symptoms questionnaire, tobacco exposure history, and medical history. preoperative staging was performed according to the 7th tnm classification system of the international association for the study of lung cancer using chest computed tomography (ct) and abdominal ct or ultrasonography in all patients. brain computed tomography or magnetic resonance imaging was done only in case of clinical suspicion of brain metastases. in cases of uncertain clinical or radiologic findings, pet was included only during the last 3 years and not on a routine basis. mediastinoscopy has not been performed routinely in this series unless the ct scan demonstrated mediastinal lymph node enlargement, pet suggested a malignant involvement of hilar or mediastinal nodes, or high- risk criteria of n2 were present. patients were classified into two groups: group 1 (asymptomatic): patients who had no symptoms attributable to lung cancer at the time of imaging (patients whose cancer was detected by a medical checkup or under evaluation for other diseases), and group b (symptomatic): patients with lung cancer-related symptoms. the charts of patients classified as having asymptomatic incidentally detected lung cancers were reviewed to check if the indications for imaging really were not based on any potentially cancer-related symptom. postoperative follow-up included office visits, quarterly chest x-rays, and yearly chest-ct. operative or in-hospital mortality was defined as death occurring within 30 days after the operation or during hospitalization, respectively. the analysis of differences in categorical outcomes was determined using the chi-squared test or fisher's exact test. probabilities of survival rates were estimated using the kaplan-meier method and asx and sx patients were compared by using the log-rank test. of 593 patients included in this study (68.3% male, median age 60.9, and range 2386 years) 320 patients were asymptomatic (asx) (53.9%). two hundred and thirty (71.8% of the asx patients) were diagnosed incidentally on chest x-ray and the remaining on ct scan. amongst the patients with symptoms, the leading complaints that resulted in the indication for imaging were the appearance of new cough or the increase of a previously manifested clinical picture suggestive of pneumonia and haemoptysis (table 1). amongst the 320 asx patients, once the initial chest-x ray (71.8%) or ct scan (28.2%) showed an abnormal image, the usual workup for pulmonary nodules was started. patients in the asx group were older than patients in sx group (median age 61.9 9.9 versus 59.51 years/old 10.2, p=0.007), without differences in sex (men 66 versus 73.5%, p=0.084). they had a higher prevalence of previous malignancy (13.2 versus 4.8%, p=0.002). the frequency of presentation as spn (49.5 versus 19.4%, p<0.001) or peripheral location (80.3 versus 63.7%, p<0.001) was higher in this group, without differences in clinical suspicion of n2 (8.8 versus 12.9%, p=0.146). patients with incidentally detected lung cancer were more likely to have earlier-stage disease, smaller cancers (3.00 2.2 versus 4.3 2.9 cm, p=0.0001). clinical characteristics of both groups are shown in table 3. when the last ten years were analyzed, a higher prevalence of incidental detection compared to previous years was observed (51.7 versus 39.8%, p=0.008). the overall 5-year survival rates were higher for asx patients: 66.2% and 46.0% for asx and symptomatic patients, respectively (p=0.001) (figure 1). amongst the stage i patients, the 5-year survival rates were 81.2% in asx patients and 58.6% in sx patients (p=0.014) (figure 2). when only stage ia was considered, 5-year survival rates were not different (71.2 versus 84.1%, p=0.191) (figure 3). when analysis was restricted to t1a tumors there were no differences either in 5-year survival (94.7 versus 93.2, p=0.489). median survival times in pathological stages iiib (41.6 m in asx versus 22.0 m in sx patients, p=0.065) and iv (13.7 versus 12.7 m, p=0.964) were not significantly different. our study shows that the incidental finding of non-small cell lung cancer occurred more frequently in smokers and in patients with a history of previous malignancy. the mortality of patients with nsclc as an incidental diagnosis was lower, and this difference persisted into stage i. more than a half of patients who underwent surgical resection of lung cancer at our institution had incidentally detected cancers and the most common indication for the initial imaging was a routine checkup. that proportion of asx patients is higher than that reported by raz et al. in san francisco but far lower than that published by hanagiri et al. in japan. in the absence of a uniform policy appertaining to the role for screening in clinical practice, the indication of imaging asymptomatic patients relies on the preferences and beliefs of both patients and physicians. different levels of awareness and access to healthcare may justify differences amongst different studied populations. also, our asx patients were slightly older (contrary to the study by raz et al.) and more frequently smokers which may mean a higher degree of awareness of their risk for lung cancer as the higher prevalence of previous malignancy may have been one of the reasons for routine radiological surveillance. however, it is noticeable that more than 70% of patients in our group (as in other series) were studied by chest-x-ray, a method that has proved to be ineffective and that is not recommended as a screening tool by any major medical organizations. the proportion of early stages of lung cancer was higher amongst our patients with incidental findings. that stage i cases accounted for 65.3% and stage iii cases for 22% in their mass screened group, while, in the symptom group, there was only half that percentage of stage i cases (32.2%). similarly, in the two more recently published series [7, 8] and in the korean lung cancer registry study asymptomatic patients had higher proportions of stages i-ii. however, there were still 20% of our patients that did not have any symptom and had a stage iii or iv lung cancer. interestingly, in a retrospective review of coronial autopsies even when the median tumor size of previously undetected cancers was 3 cm, the range was 110 cm and there were several tumours over 5 cm and even some large endobronchial and hilar tumours undetected before death. we found that survival time in symptomatic cases was worse than in incidentally detected lung cancer patients. the 5-year overall survival was lower for the whole group and for pathological stage i. that better outcome has been consistently demonstrated in all the previous reports; however, the causes for those differences are still unclear. the korean registry has shown that absence of symptoms at diagnosis significantly reduced the risk of death from nsclc, regardless of age, gender, stage, smoking history, or whether treatment was performed. similarly, hanagiri and colleagues showed that their patients with incidentally diagnosed nsclc had significantly better prognoses than the symptomatic group even in stage ii even when they had a larger proportion of stage iv amongst asymptomatic patients than our series (6.7 versus 3.4%) they were still a small number of patients (n=18) and exact figures of survival rates for those advanced patients were not provided. in our series, whilst pathological stage i patients had a better survival in asx patients, median survival times in stages iiib and iv were similar. it resembles the results of raz et al. who found that their patients with completely resected incidental lung cancer had similar long-term survival rates as patients with symptomatic lung cancer, after adjusting for stage. in the present study, stage ia disease was diagnosed less frequently in the symptomatic group, similar to what has been reported in other studies [7, 8, 10]. a study by kashiwabara et al. (published in 2002, before the publication of the 7th tnm edition) compared the outcomes in patients with one-year delayed detection of lung cancer on mass screening with chest-x-ray and in patients with no delay (patients with tumours which could versus could not be detected on past chest roentgenograms). they found that one-year delayed detection of lung cancer on mass screening did not affect outcome, but that, according to the maximum dimension of the tumours on the overlooked chest roentgenogram, the 5-year survival rates in patients with missed tumours were different and that survival in early stages (i-ii) for missed tumours>20 mm was worse than that in patients with missed tumours<10 mm. we had previously shown that tumors over 15 mm are associated with shorter 5-year survival in all tnm stages and several studies have reported tumor size may have an independent predictive value on survival in stage i patients [14, 15]. the impact of the tumor size was finally made evident by the analyses of the database of the iaslc and generated the reclassification of t1 in t1a and t1b and t2 in t2a and t2b. when we analyzed separately the pathological stage ia cases, differences in survival in stage i between the two groups disappeared, suggesting that the size was the most important factor in determining survival. our study shows that patients with incidentally detected lung cancer had a better survival because they had smaller cancers and earlier-stage disease. the conclusions of clinical studies like this or any of the previously published studies should not be extrapolated to the potential value of mass screening. this sort of study design does not allow demonstrating if there is a survival benefit of treatments in asymptomatic patients or how large the proportion of invasive procedures for benign lesions is performed. one of the main concerns about any screening program is that a proportion of screen-detected cases will be overdiagnosed simply because of competing mortality, a hypothesis that can not be excluded by a population study like this one. on the other hand, many of the patients in this and the other clinical series were not represented in the clinical trials about mass screening programs: 15% of our asymptomatic patients were never smokers and many of them were under 55 years old and would have not filled criteria for being included in a screening program. this study shows the importance of identifying risk at an individual level as many subjects different from the nlst participants may have a risk similar to or greater than the level of risk observed in nlst. several studies have previously recognized that there is wide variation in lung cancer risk even amongst those who are smokers [18, 19] and we do not know yet how to identify other risk factors for lung cancer that could potentially justify extending screening to those individuals. future research to develop clinically useful risk model might include molecular or genetic indicators of risk in order to answer these questions. the national lung screening trial (nlst) showed that, in heavy (30 pack-years or more) current or former (within 15 years) smokers between the ages of 55 and 75, three annual low-dose computed tomographic (ldct) screens reduced lung cancer-specific mortality from 309 to 247 deaths per 100,000 person-years (relative risk of 0.8). but at the moment to make individual decisions (such as screening of certain nonsmokers) it is necessary to take into account the potential effectiveness of such measures. whilst the number needed to screen (nns) to prevent one death for the entire nlst population was calculated as 320, according to bach and gould for very low risk individuals (defined by the authors as a 40-year-old former smoker) the nns was over 35,000 to prevent one lung cancer death. in order to minimise the potential for harm when screening large populations for a condition that is very rare (derived not only from the costs associated with screening but also from the impact on quality of life of the potential for invasive procedures for incidental findings) but at the same time not to miss other high-risk subjects out of the nslt criteria, better risk models must be developed to have the greatest predictive accuracy for lung cancer risk. firstly, the classification of a cancer as incidentally detected is a potential bias, once the spontaneous patient consultation may not exclude the presence of some nonspecific symptom that prompted the patient to seek medical consultation. it is remarkable that, in the study by kashiwabara et al. about patients that did not consult a physician after the discovery of a shadow in a radiological screening (almost 25% of the asymptomatic screened patients in their series), when asked about the reason why patients did not consult a doctor, two-thirds answered that it was because they did not have any respiratory symptoms. it shows that a screening program must assure that the health care system can provide all the necessary resources to treat the incidental findings and also the education to guarantee the availability of well-qualified primary care providers trained to encourage patients to follow diagnosis and treatment recommendations once a suspicion of lung cancer is raised from the imaging studies. in summary, our study shows that lung cancer as an incidental finding is not uncommon even amongst nonsmokers and that the better survival of patients with asymptomatic nsclc is related to the greater number of patients with earlier-stage disease. future research is needed to prospectively identify those patients not represented in the nslt who might benefit from ldct screening.

objective. to evaluate clinical characteristics and outcomes in incidentally detected lung cancer and in symptomatic lung cancer. material and methods. we designed a retrospective study including all patients undergoing pulmonary resection with a curative intention for nsclc. they were classified into two groups according to the presence or absence of cancer-related symptoms at diagnosis in asymptomatic (asx)incidental diagnosis or symptomatic. results. of the 593 patients, 320 (53.9%) were asx. in 71.8% of these, diagnosis was made by chest x-ray. patients in the asx group were older (p=0.007), had a higher prevalence of previous malignancy (p=0.002), presented as a solitary nodule more frequently (p<0.001), and were more likely to have earlier-stage disease and smaller cancers (p=0.0001). a higher prevalence of incidental detection was observed in the last ten years (p=0.008). overall 5-year survival was higher for asx (p=0.001). median survival times in pathological stages iiib-iv were not significantly different. conclusion. incidental finding of nsclc is not uncommon even among nonsmokers. it occurred frequently in smokers and in those with history of previous malignancy. mortality of incidental diagnosis group was lower, but the better survival was related to the greater number of patients with earlier-stage disease.

PMC4320896

pubmed-497

hip arthrodesis as a solution for hip-related problems is mainly superseded by total hip arthroplasty (tha) and, in the current treatment of hip disorders, there is hardly room left for arthrodesis of the hip. however, we must realise that in certain scenarios in young adults joint preserving therapy is not an option and total hip arthroplasty is a difficult procedure with an increased risk of failure [13]. in these selected young patients with severe hip pathology due to trauma or infectious causes an arthrodesis may be a suitable solution with an acceptable outcome [14]. there has been considerable literature in the past about the technique and outcome of a hip fusion. several studies report of pain in adjacent joints in the long term; the most frequent being lower back pain based on degenerative changes reported between 15% and 100% of cases [59]. following in percentage are complaints of the ipsilateral knee and less frequent complaints of the contralateral hip and knee [911]. the frequency of these complaints seems to be predicted by the alignment of the fusion which should be optimal [3, 6, 12]. in our clinic, we perform hip fusions with our standard technique using a cobra plate; however, in those complex cases where optimal alignment and optimal fixation can not be achieved by this method we use the technique with a subtrochanteric pendulum osteotomy to achieve the correct alignment. even in the most difficult cases this method enables the optimal combination between a stable connection of the remaining femoral head neck and pelvis and an optimal alignment of the leg. although a hip fusion is not the most ideal solution it remains an important option for selected scenarios. apart from the short-term results, attention should be paid to the long-term results in adolescents and young adults. when performing an arthrodesis the main objective of our study was to show the long-term effects of fusion on the adjacent joints using our technique. in the period between 1974 and 1994, 47 hip arthrodesis were performed by the senior author (rkm). the average age at the time of the arthrodesis was 29 years (range 1255). the hip disorder which led to arthrodesis was septic arthritis in ten patients (including tuberculosis), aseptic necrosis in one, post-traumatic in 21, congenital luxation in three patients, four patients were secondary to childhood hip disorders and there were other indications in eight. we prefer to perform a cobra plate technique, which was initially developed by schneider and later modified by several others, eliminating the need for the pelvic osteotomy [13, 14]. the technique we use is similar to this reported technique; however, we pay extra attention to the abductor muscle since it is mandatory to preserve it if a later total hip arthroplasty is a possibility. the abductors are spared by performing a trochanteric osteotomy which is fixed on/over the cobra plate after the arthrodesis is performed. for complex cases in which the optimal combination of fixation of the arthrodesis and perfect limb alignment is not possible, for instance in septic arthritis with severe destruction of the joint or severe osteonecrosis, we performed an arthrodesis with a subtrochanteric pendulum-osteotomy. the arthrodesis is performed in such a position that optimal contact between femoral head and acetabulum is present, regardless of the position of the leg. the arthrodesis is then relieved by a subtrochanteric osteotomy which also allows optimal positioning of the extremity. after-treatment consisted of traction immobilisation for two weeks followed by six weeks of plaster immobilisation. in this study 33 hips were fused with the cobra plate technique and 14 with the arthrodesis with subtrochanteric pendulum-osteotomy technique. the clinical follow-up consisted of a thorough clinical investigation and the short musculoskeletal function assessment (smfa) questionnaire. the smfa is a well-validated, universal, self-reported health status questionnaire. the smfa questionnaire is developed for clinical assessments of the impact of treatment in groups of patients who have musculoskeletal disease or injury. the smfa questionnaire consists of the dysfunction index, which has 34 items for the assessment of patient function, and a bother index, which has twelve items for the assessment of how much patients are bothered by functional problems. since a hip arthrodesis can have a negative influence on the contralateral hip joint, the back and knees, we included these adjacent joints in this analysis. the clinical investigation consisted of: range of motion of hips and knees, mobility of the back, laxity of the knee joint, leg length discrepancies and quadriceps strength. the mobility of the back was measured using a standardised mildenberg finger-floor method and the schber test. the schber test was conducted with the patient standing upright; a mark was made at the height of l5. a second mark was made 10 cm above and a third mark 5 cm below the first mark. the patient was asked to bend forward with the knees straight, reaching as far as possible towards his/her toes. the increase between the upper and lower mark was taken as a measurement of lumbar flexion. the finger-floor measurements were taken upon the first attempt of the patient to bend forward with extended knees. quadriceps strength was graded from 0 to 5 according to the british medical research council scale. subjective outcome measurement visual analogue scores (vas) were obtained for pain in hips, knees and back. vas scores were also obtained for satisfaction with the results of the arthrodesis and for the eagerness to have the arthrodesis converted to tha. additional questions concerning work, walking distance, problems with sitting and problems with socks and shoes were posed. for both knee joints, the international knee documentation committee (ikdc) knee examination form was used to evaluate secondary knee problems. this form results in four groups: group a is normal knees, group b nearly normal knees, group c abnormal knees, and group d severely abnormal knees. for the radiological analysis, weight bearing x-rays of pelvis, both knees and lumbar spine were used. the contralateral hip and both knees were scrutinised for signs of osteoarthritic changes which were scored according to kellgren and lawrence for the medial compartment, the lateral compartment and the patello-femoral compartment. for the knees, the ahlback score was also used for the medial and lateral compartment separately. on the lumbar spine x-rays the presence of osteophytes as an indicator for osteoarthritic changes were scored; the percentage of joint-space narrowing between the vertebrae and the presence of olisthesis and scoliosis were documented. after an average follow up of 18.9 years (range 5.631.4), six patients had died. seven hips were eventually converted to tha of which one was performed to facilitate a total knee arthroplasty on the same side. the remaining 30 patients were invited for follow-up and intensively screened. in this group of difficult hip disorders one patient suffered from a deep venous thrombosis which was treated with anticoagulants. in two patients a deep infection occurred, of which both received the arthrodesis for a destructive septic arthritis. in one of them a revision operation was performed with debridement of the infection and surgical drainage; the infection resolved but resulted in a nonunion. the second infection was treated conservatively, and after 24 years of follow-up a fistula and deep infection were still present. besides the nonunion after infection, in five other patients a nonunion was diagnosed, and in three patients one re-arthrodesis was sufficient to achieve union, while in one patient two re-arthrodesis were performed to achieve union. one patient was left untreated because of co-morbidity for which this arthrodesis was performed for severe post-traumatic destruction; however, the same trauma led to a post-traumatic ankylosis of the ipsilateral knee, a paresis of the contralateral arm and permanent brain damage (mainly cognitive). clinical and radiological follow-up was obtained for 30 patients after an average of 18.2 years (range 6.230.5) (fig. 1). clinically, the smfa showed a mean total score of 31.2 (range 9.070.0), the subscore dysfunction an average of 29.1 (range 8.070.5) and the subscore limitations an average of 37.3 (range 9.084.0). the clinical and radiological results for the arthrodesis and adjacent joint will be discussed separately. h x-rays of pelvis, knees and lumbar spine 28 years postoperatively. the arthrodesis was converted to a total hip replacement (thr) 20 years postoperatively (on the contralateral hip a shortening at intertrochanteric level was performed 1 year after the fusion) a a 17-year-old boy with a central hip luxation. h x-rays of pelvis, knees and lumbar spine 28 years postoperatively. the arthrodesis was converted to a total hip replacement (thr) 20 years postoperatively (on the contralateral hip a shortening at intertrochanteric level was performed 1 year after the fusion) the clinical and radiological investigation of the fused hip joint showed an average vas for pain of 1.9 (range 0.08.0). the average alignment of the arthrodesis was 19 flexion (5; 30), 1 of abduction (10; 10) and 2 external rotation (10; 15). an average leg length discrepancy of 3.3 cm (range 11.5 to 2) was present. walking distance averaged 115 minutes (range 10unlimited) for which one patient needed two crutches and two patients needed one crutch for support. twenty-three patients experienced difficulties in putting on their shoes, in 17 of them this was caused by the inability to tie their shoelaces. of these, three had no complaints of the fused hip (vas pain 0,0,3), and one patient with a nonunion after infection had a vas of 6. the two remaining patients reported vas of 6 and 7 for pain; however, they performed full time heavy labour (north sea fisherman and factory worker). the vas for eagerness to have their fusion converted to a tha was 5.4 average (range 010). the contralateral hip joint on clinical examination showed an average vas for pain of 2.0 (range 08). one contralateral hip underwent an intertrochanteric shortening osteotomy and one was converted to a tha. the average range of motion was: for flexion 104 (range 30140), extension 0.2 (0 5), abduction 25 (1040), adduction 17 (030), external rotation 27 (560) and internal rotation 16 (0 30). on clinical examination the ipsilateral knee joint showed an average vas for pain of 2.0 (range 08). average range of motion for flexion was 125 (20160) and for extension 2 (010). on radiological examination three knees showed severe osteoarthritic changes with a grade 4 k-l and iv ahlback in both compartments, and in one knee only in the lateral compartment. a grade 3 or 4 k-l oa in the patello-femoral compartment was present in four knees. the ikdc knee examination form showed a grade a in nine knees, grade b in 14 knees, grade c in four knees and grade d in eight knees. on clinical examination the contralateral knee joint showed an average vas for pain of 1.8 (range 08). quadriceps strength was optimal (grade 5) in all. average range of motion for flexion was 140 (100160) and for extension 1 (10 to 20). on radiological examination one knee showed severe osteoarthritic changes with a grade 4 k-l and a grade iv ahlback in both compartments, and in one knee only in the lateral compartment. a grade 3 or 4 k-l oa in the patello-femoral compartment was present in three knees. the ikdc knee examination form showed a grade a in 23 knees, grade b in five knees, grade c in five knees and grade d in three knees. there was a significant differences in the outcome of the ikdc examination form (laxity) for the ipsilateral and contralateral knees (chi-square p<0.001). clinical investigation of the lower back showed an average vas for pain of 3.6 (08) (table 1). the average schber test was 6.1 (310.5), and the standardised mildenberg finger-floor method averaged 32.5 cm (1551). radiological examination of the sacro-iliacal joint showed three joints with severe arthrosis (one grade 3 and two grade 4 k-l) on the ipsilateral side, and two (both grade 3 k-l) on the contralateral side. the lumbar spine showed signs of oa in the form of osteophytes in 12 backs. in two spines an olisthesis was present, one grade 1 and one grade 2. ten ap lumbar spine showed a scoliosis, of which eight had the convexity on the opposite side of the arthrodesis. results per operation technique sfma short musculoskeletal function assessment, avg average, vas visual analogue score although arthrodesis has been forced into the background by the successful tha, it still remains a valid option for specific difficult problems. this descriptive study of our fusion techniques shows a good outcome measured by the smfa and a good vas for pain for all related joints after an average of 19 years of follow-up. especially lower back pain seems to occur frequently after a hip fusion in the long term, since 42% of our patients had a vas for pain of 3 and higher for lower back pain. the cobra head plate technique is our first choice since it provides a high fusion rate and allows early mobilisation. for some indications, including severe unilateral destruction of the femoral head in young patients with severe loss of bone stock, which can occur in chronic septic arthritis or osteonecrosis, a cobra head plate arthrodesis does not provide the necessary stability. this method enables the optimal combination between a stable connection of the remaining femoral head-neck and pelvis and an optimal alignment of the leg even in the most difficult cases. the fact that an arthrodesis is only indicated in difficult cases is probably the best explanation for the relatively high complication rate in this group of patients. a second important complication is the occurrence of nonunions in our patient group. at follow-up, six hips showed signs of a nonunion, although in three of them a real nonunion was unlikely due to the absence of symptoms. two other patients with signs of a nonunion indicated pain in the fused hip (vas 6&7), but were able to perform full time heavy labour. using these two techniques for these selected patients our nonunion rate of 6% it is known that an arthrodesis of any joint has influence on the adjacent joints. these effects can be minimized by performing an optimal alignment of the affected extremity. in an ideal arthrodesis this alignment is adjusted to the patient's preferences. in patients with a mainly sitting occupation more flexion both knee joints did not suffer from the arthrodesis in a way that they caused pain or discomfort. however, we noticed that significantly more knees on the ipsilateral side showed an increased laxity (ikdc knee examination form). no differences were found in the vas scores for the contralateral and ipsilateral knees. in 2000, karol et al. showed in a detailed gait analysis that excessive motion of the lumbar spine and the ipsilateral knee were present in patients with a fused hip. these excessive movements caused complaints in the ipsilateral knee and lower back in their group of seven patients. our data supports the fact that a fusion causes more lower back complaints in the long term (42% with a vas higher than 3); however, we could not confirm the occurrence of more complaints in the ipsilateral knee. in our study seven arthrodeses (15%) were eventually converted to a tha (fig. 1), all with a good result. in the literature several studies can be found regarding conversion of an arthrodesis to a tha, showing identical results to primary tha in patients older than 50 [1, 19, 20]. although primarily tha may not be a solution for these patients, an arthrodesis can bridge the gap to a future tha, while generating a good quality of life in the meantime. we asked our patients how eager they were to convert their arthrodesis to a tha and 13 (43%) saw no reasons for conversion at long-term follow-up, indicating that these patients were content with their current quality of life. we found that the mobility and quality of life were good for patients with an arthrodesis. the average walking time was almost two hours (115 minutes), while ten patients were able to walk for at least three hours, and one patient even participated in marathon running. although hip arthrodesis has lost it popularity it still is an option for young patient with severe hip disorders, while leaving the possibility to perform a tha at a later stage. if the arthrodesis is performed with an optimal alignment of the leg, complaints from the adjacent joints are minimal, even in the long term, and an acceptable quality of life can be obtained.

even in current orthopaedic practice, some cases are still not suitable candidates for hip replacement and hip fusion remains the only option in these highly selected patients. in this retrospective study we describe the long-term clinical outcome, quality of life and radiological evaluation of all adjacent joints in a cohort of 47 hip fusions. the main objective of our study was to show the long-term effects of a fusion. thirty patients were analysed after an average of 18.2 years (range 6.230.5 years) with a mean smfa of 31.2 (range 970). the vas for pain for the fused hip was an average 1.9 (range 08), for the contralateral hip 2.0 (08), for the ipsilateral knee 2.0 (08), for the contralateral knee 1.8 (08) and for the lower back 3.6 (08). average walking distance was 115 minutes (range 10unlimited). although the hip arthrodesis has lost popularity, it still is an option for the young patient with severe hip disorders, while leaving the possibility to perform a tha at a later stage. if the arthrodesis is performed with an optimal alignment of the leg, complaints from the adjacent joints are minimal, even in the long-term, and an acceptable quality of life can be obtained. we believe that in highly selected cases a hip fusion, even in current practice, is still a valid option.

PMC2989023

pubmed-498

leukotrienes (lts), first described by samuelsson's group [1, 2], are a class of lipid mediators involved in several diseases but classically known for their effects on asthma and allergy. the generation of leukotrienes (lts) is dependent upon the action of 5-lipoxygenase (5-lo) in association with membrane-bound 5-lipoxygenase-activating protein (flap) on arachidonic acid (aa). aa is derived through the action of cytosolic phospholipase a2 (cpla2) and/or secreted phospholipase a2 (spla2) on membrane phospholipids. lta4, an unstable precursor of all leukotrienes, is quickly metabolized to one of the two different classes of lts, ltb4 (by lta4 hydrolase) or ltc4 (by ltc4 synthase) and its metabolites (ltd4 and lte4). collectively, ltc4, ltd4, and lte4 were previously known as the slow-reacting substance of anaphylaxis (sr-a) and are currently termed the cysteinyl lts (cyslts) [3, 4]. the receptors for ltb4 (btl1 and btl2) and cysteinyl lts (cyslt1 and cyslt2) are cell surface g protein-coupled receptors. additionally, some studies support the existence of other cyslt receptors [5, 6]. some cells express both btls and cyslts; however, the expression of these receptors differs in different cells types. in addition, these receptors are also expressed on peripheral blood leukocytes [7, 8]. lt receptors and 5-lo are expressed mainly in immune cells, and lts play important roles in innate and adaptive immune responses and are involved in several inflammatory and infectious diseases [4, 9]. for example, cyslts increase vascular permeability and edema, and ltb4 is involved in leukocyte chemotaxis, lysosomal enzyme secretion, neutrophil degranulation, adhesion molecule expression, defensins and nitric oxide (no) production, phagocytosis, and other functions. lts are produced during the interaction of phagocytes and microorganisms in vitro and experimental infections in vivo. pharmacologic or genetic approaches to reduce or block the lt biosynthesis pathways decrease the phagocytic and antimicrobial activities against bacteria, fungi, and parasites [12, 13]. in addition, immunodeficient individuals, such as hiv patients, are characterized by low lt production, which has been associated with impaired immune responses and infection control. lts play important roles in both th1 and th2 immune responses, which are involved in the defense against protozoan and helminth infections, respectively. in light of the current research on the role of lts in infectious diseases, we have divided the current review into two sections focusing on (1) protozoan infection and (2) helminth infection. each year, protozoan parasites infect many people worldwide, mainly in developing countries, causing serious health, political, social, and economic problems. the major protozoan parasites with clinical importance for human diseases are plasmodium ssp, leishmania ssp, trypanosoma cruzi, toxoplasma gondii, trichomonas vaginalis, and entamoeba histolytica [1517]. the first three of these organisms are obligate intracellular protozoan parasites that are transmitted to vertebrate hosts by insect vectors. t. gondii is also an obligate intracellular protozoan parasite; however, its transmission to human hosts occurs by ingestion of raw or undercooked meat containing tissue cysts or food or water contaminated with oocysts. t. vaginalis is transmitted sexually (trophozoites) and e. histolytica is transmitted through food and water contaminated with cysts [1517]. protective immunity against protozoans is mediated mainly by t helper 1 (th1) responses which are characterized by the production of inflammatory cytokines, such as il-12, which is required for the development of the th1 immune response, and interferon gamma (ifn-) and tumor necrosis factor alpha (tnf-), which activate macrophages to produce no, which is involved in the control of parasite replication [16, 1820]. reiner and malemud [21, 22] conducted the first studies to demonstrate the role of leukotrienes in protozoan infection (leishmania spp). the main effects of lts, in both innate and adaptative immune responses, during the protozoan infections are illustrated in figure 1. mouse strains resistant (c57bl/6) to leishmania infection mount th1 immune responses against leishmania. in contrast, infection of susceptible mouse strain (balb/c) is associated with the development of a th2 immune response. in vitro studies have demonstrated increased ltc4 production in splenocytes and macrophages from l. donovani-infected or uninfected balb/c mice upon stimulation with nonspecific (phytohemagglutinin) or specific (l. donovani amastigotes) stimuli [21, 22]. in another study, splenocytes from balb/c mice stimulated with antigens from l. major promastigotes displayed increased ltb4 and il-4 production with concomitant decreases in ifn- and tnf- production.. demonstrated an increase in the parasite burden of balb/c macrophages infected with l. amazonensis when compared to macrophages from the resistant mouse strain c3h/hepas. this effect was associated with lower levels of ltb4 in macrophages from balb/c mice. in agreement with this finding, macrophages from either susceptible or resistant mice treated with mk0591 (flap inhibitor) and u75302 (blt1 antagonist), but with not mk571 (cyslt1 antagonist), as well as macrophages derived from 5-lo-deficient mice, exhibited decreased leishmanicidal activity. interestingly, treatment with exogenous ltb4 or ltd4 favored parasite killing by macrophages from balb/c mice. supporting these in vitro results, susceptible and resistant mice treated with zileuton (inhibitor of 5-lo) or 5-lo-deficient mice infected with l. amazonensis displayed larger footpad lesions than nontreated or wild type animals. the success of lutzomyia longipalpis, an insect vector of the leishmania ssp, at blood feeding on mammals depends on the inhibition of the immediate inflammatory response (e.g., increased vascular permeability, swelling, pain, and itching). it is well known that active substances in the saliva of hematophagous arthropods facilitate the uptake of blood by counteracting host hemostatic, inflammatory and immunological defenses [2528]. mixed lysates from the salivary glands of l. longipalpis significantly increased the cutaneous lesions and/or parasite loads in the footpads of mice infected with l. major or l. braziliensis when compared to infected animals not exposed to the saliva lysates [29, 30]. in addition, the modulation of infection by saliva was il-4-dependent. in agreement with these results, the salivary gland extract of l. longipalpis exhibited anti-inflammatory activities by decreasing tnf- and ltb4 production, neutrophil numbers, and ltb4-induced chemotactic activity in a murine ovalbumin-induced peritonitis model., these findings suggest that lts, and particularly ltb4, play a role in immune response to leishmania infection by promoting leishmanicidal activity and consequently, control of infection. therefore, the modulation of ltb4 during infection in association with the modulation of the immune system during leishmania transmission (by saliva from the insect vector) in synergism with genetic factors (susceptibility; th2) could markedly affect leishmania infection in humans. the components derived from the saliva of the arthropod vector of malaria (e.g., anopheles stephensi) have also pharmacologic effects, such as inhibition of inflammation and coagulation, similar to those observed in the saliva of insect vectors of leishmania. in addition, these proteins also have the ability to neutralize inflammatory small molecules by rapid binding. the anst-d7l1 protein produced by a. stephensi binds cyslts (ltc4, ltd4, and lte4) but does not chemically modify them. anst-d7l1 effectively inhibited ltc4-induced ileal contraction by binding ltc4, thereby preventing interactions between this molecule and its appropriate cellular receptor. the effects of ltc4 inhibition on the course of malaria infection as well as the influence in the malaria pathogenesis are not known. in the experimental cerebral malaria model, mice infected with plasmodium berghei showed increased ltb4 production in the serum. interestingly, treatment with aspirin, which may direct arachidonic acid metabolism away from the cyclooxygenase (cox) pathway and toward the lo pathway, induced increased parasitemia and death of infected mice. this effect was associated with the overproduction of ltb4 in the serum. in agreement with these results, children with cerebral malaria treated with salicylate demonstrated complications of severe malaria (metabolic acidosis, hypoglycemia, and death). although ifn- plays a protective role in malaria infection, it has also been associated with the immunopathology of cerebral malaria [36, 37]. besides playing a role in initiating the th1 immune response mediated by dendritic cells, therefore, the overproduction of ltb4 after aspirin treatment in experimental and human cerebral malaria could be associated with the overproduction of ifn-. further studies are needed to evaluate this hypothesis. eryptosis, or suicidal death of erythrocytes, which occurs in a wide variety of diseases including malaria, is characterized by cell shrinkage, membrane blebbing, and exposure of phosphatidylserine (ps) at the cell surface. like apoptotic cells, ps-exposing erythrocytes are identified by macrophages and are engulfed, degraded, and removed from the circulation. demonstrated increased phagocytosis of mutant red blood cells infected with trophozoites of p. falciparum, which may represent a protective mechanism against infection. remarkably, an in vitro assay demonstrated that erythrocytes were able to produce cyslts upon energy depletion. these effects were inhibited by cyslt1 receptor antagonists and by the 5-lo inhibitor (bw b70c). these results suggest that ltc4 might confer protection during the course of malaria by accelerating the clearance of infected erythrocytes. on the other hand, excessive eryptosis might favor the development of anemia; thus, ltc4 might have a dual effect in malaria pathogenesis. during t. gondii infection, an efficient immune response is important to contain dissemination of the parasite and to prevent mortality of the host. ltc4, ltd4, and free aa were detected when murine macrophages from swiss mice were cultured with viable t. gondii. in contrast, when macrophages from resistant mice (balb/c; major histocompatibility complex haplotypes h2) or human macrophages were cultured with viable t. gondii, no 5-lo products were observed. accordingly, prior incubation of human macrophages with viable t. gondii decreased the ltb4 release induced by the calcium ionophore a23187, suggesting that t. gondii inhibits ltb4 production. treatment with zileuton (an inhibitor of 5-lo) decreased the toxoplasmacidal activity of ifn- in human macrophages, whereas exogenous ltb4 promoted intracellular killing of ingested t. gondii in human monocytes. this effect might be associated with the effect of ltb4 on the induction of cytotoxicity (surface membrane vesiculation, extravasation of cytoplasmic contents into a space between the intermembrane spaces and cytoplasmic vacuolization) in t. gondii tachyzoites [47, 48]. in agreement with these results, 5-lo-deficient mice infected with t. gondii displayed decreased survival as a consequence of an excessive inflammatory response characterized by elevated il-12 and ifn- concentrations in the serum and cd4 and cd8 t-cell infiltration in the brain tissue and not of increased parasitic burden. the increased inflammation in the absent of lts might indicate a compensatory mechanism to control the parasite infection. taken together, these findings suggest that the downregulation of lts production, and particularly of ltb4, by t. gondii might be considered an evasion mechanism, as this lipid mediator can promote cytotoxicity and toxoplasmacidal activity studies by our group and others have demonstrated reduced lt synthesis (e.g., ltb4) in hiv-infected subjects [14, 50]. although the clinical manifestation of t. gondii infection is usually asymptomatic in immunocompetent individuals, immunocompromised individuals, such as hiv-seropositive patients, exhibit reactivation of latent tissue cysts (bradyzoites become tachyzoites) and consequent toxoplasmic encephalitis or retinochoroiditis [51, 52]. interestingly, in agreement with these results, the ltb4 and ltc4 concentrations in the cerebrospinal fluid of hiv-1-seropositive patients with toxoplasmic encephalitis but not those of hiv-1-seropositive patients without inflammatory disease or encephalitis were below the detection limit. these results support those described above and suggest that the reduced basal production of lts in hiv-1-seropositive patients synergizes with the suppression of lts by t. gondii. moreover, this synergistic decrease in lt production might contribute to the pathogenesis of cerebral toxoplasmosis through the increased reactivation of bradyzoites from tissue cysts and the reduced control of the parasitic infection. protective immunity against toxoplasmosis and chagas disease is mediated by th1 cells, cd8 t cells, and ifn-. chagas ' heart disease is a severe clinical manifestation of trypanosoma cruzi infection. in chronic chagas disease, cardiomyopathy is observed as an inflammatory process characterized by the infiltration of t cells and macrophages, resulting in myocarditis, fibrosis, and heart fiber damage. treatment with lt inhibitors has demonstrated beneficial effects in cardiovascular pathologies [55, 56]. t lymphocytes from patients with chronic chagas ' heart disease or from chagasic mice show increased contractile activity (positive inotropic and chronotropic effects) of heart (atrial) in an in vitro assay. interestingly, pretreatment with lipoxygenase inhibitors (ndga) or a cyslt receptor antagonist (fpl 55712) decreased this effect. in a separate study, ltc4 production was observed in the supernatants of murine atria cocultured with t lymphocytes from chagasic mice. in accordance with these results, ltb4 induces chemotaxis of lymphocytes (cd4/cd8 t cells) [8, 59]. therefore, lts might modulate the cardiac pathology of chagas disease by modulating the immune response profile during this infection. ltb4 and ltc4 also increased the phagocytic and trypanocidal activity of murine macrophages incubated with t. cruzi trypomastigotes in vitro. in addition, ltb4 restored no and tnf- levels, which were decreased by an ltb4 receptor antagonist (cp-105,696). cp-105,696 treatment also decreased the trypanocidal activity of ifn- in murine macrophages. with the use of pharmacologic (ltb4 receptor antagonist and lo inhibitors) and genetic approaches (5-lo-deficient mice), in addition, the following anti-inflammatory profiles were observed in t. cruzi infection: (1) decreased leukocyte infiltration in the heart; (2) reduced numbers of cd4, cd8, and ifn--producing cells in the heart; (3) decreased fibrosis in cardiac tissues; (4) decreased inos expression and no production in the heart; (5) decreased tnf- and ifn- in the heart; (6) increased il-10 in the heart; and (7) decreased oxidative stress in erythrocytes [6365]. the survival of 5-lo-deficient mice was greatest when the animals were infected with low number of parasites when compared to animals infected with higher number of parasites. taken together, these findings suggest that lts, and specifically ltb4, play important roles in the control of chagas disease. the supernatant of viable t. vaginalis induced increased ltb4 production in neutrophils in an igg- and complement-(c5-) dependent manner. this effect was decreased by sc-41930 (ltb4 antagonist) treatment. in the vaginal discharges from patients with vaginal trichomoniasis, shaio and lin demonstrated a positive correlation between neutrophils and ltb4 production in symptomatic patients when compared to asymptomatic patients. peritoneal and splenic macrophages from nave mice incubated directly with e. histolytica trophozoites or with their excretory/secretory products show increased ltc4 production. on the other hand, peritoneal and splenic macrophages from e. histolytica-infected mice produced low levels of ltc4. the downregulation of ltc4 by e. histolytica in inflammatory but not nave macrophages might be associated with the pathogen's evasion mechanisms. over one-third of the human population is infected with one or more species of helminths [69, 70]. although host immune responses attempt to control or expel the parasites, these organisms can develop evasion strategies to modulate the innate and adaptive immune responses, allowing them to survive. the most prevalent human helminthiases are caused by nematodes (e.g., ascaris lumbricoides, strongyloides spp., enterobius vermicularis, and trichuris trichiura), including filarial worms (e.g., brugia malayi and wuchereria bancrofti), hookworms (e.g., ancylostoma duodenale and necator americanus), and trematodes (schistosoma spp). asthma and helminthiasis present similar features and are both controlled by a cd4 t-cell immune response. initial exposure of the immune system to allergic or parasitic antigens leads to the activation of a subset of t cells known as th2 cells, which orchestrate the immune response to these exogenous antigens by secreting cytokines, including il-4, il-5, and il-13 [7174]. in addition, the accumulation of eosinophils in the blood (eosinophilia), as well as in different organs and tissues, is a hallmark of both diseases. eosinophils are multifunctional cells that are involved in tissue damage as a consequence of the release of cationic proteins [7679]. in addition, eosinophils are important sources of various inflammatory and regulatory cytokines, chemokines, and lipid mediators, such as lts [78, 80, 81]. during a helminth infection such as a nematode infection, most of the ige produced binds to mast cells and basophils through their high-affinity ige fc receptor (fcri) [82, 83]. subsequent exposure of immune cells to parasitic antigen induces the degranulation of ige-sensitized mast cells and the release of both preformed and newly generated mediators. these mediators, such as lts, function alone or in conjunction with th2 cytokines to increase the contractility of smooth muscle cells, the permeability of epithelial cells and the production of mucus, thereby contributing to worm expulsion. the experimental gastrointestinal infection of rats with the nematode trichinella spiralis demonstrated that preimmune rats (previously infected with t. spiralis) expelled the nematode t. spiralis more rapidly than nonimmune rats. this effect was associated with the increased production of ltb4 and ltc4 in the gut homogenate as well as the release of rat mast cell protease ii (rmcpii) in the serum [85, 86]. ltc4 causes smooth muscle contraction, increases vascular permeability, and stimulates mucus hypersecretion, and ltb4 recruits and activates inflammatory cells such as eosinophils to favor the expulsion of helminths. therefore, leukotrienes released from mast cells may effectively participate in protective immune responses resulting in the rapid expulsion of t. spiralis and possibly other helminths. the main effects of lts, in both innate and adaptative immune responses, during the helminth infections are illustrated in figure 3. parasitic worm survival in the host for longer periods depends on the ability of the parasite to evade the host immune system. the aba-1 protein from ascaris lumbricoides (human parasite) and ascaris suum (pig parasite) the interaction between aba-1 and leukotrienes might be associated with an evasion mechanism; however, further studies are needed to evaluate the ability of this interaction to inhibit the biologic effects of lts in vitro or in vivo. brugia malayi is a nematode (roundworm) that can cause lymphatic filariasis in humans. the infective larvae (l3) of brugia malayi are transmitted to a vertebrate host by an insect vector and undergo two molts to develop into adult worms and complete the life cycle. interestingly, treatment with inhibitors of lipoxygenases (aa861) or cyslt biosynthesis (ethacrynic acid or acivicin) or with a cyslt1 antagonist (zafirlukast) inhibited the brugia malayi l3 larvae from molting to the l4 stage without altering their survival or motility. in contrast, u-75302, an antagonist of the ltb4 receptor btl1, failed to inhibit molting. the -glutamyl transpeptidase, the enzyme that converts ltc4 to ltd4, has been cloned from brugia malayi (adult worms). in another filaria that causes human infection, dirofilaria immitis, the glutathione s-transferase, which can function as an ltc4 synthase, these results demonstrated that a lipoxygenase pathway involved in the generation of cyslts could be required for molting of the infectious larvae and may possibly have some role in the adult worm. in vivo models of infection with b. malayi could be used to better understand the role of cyslts in the pathogenesis of filariasis. it is widely known that some types of infections in immunocompromised individuals are critical in determining the severity of the disease. the immunosuppression observed in hiv-seropositive subjects has been associated with strongyloides spp infections of abnormally high intensity. interestingly, reduced lt production was observed in hiv-seropositive patients. in an experimental model that mimics human strongyloidiasis (mice infected with strongyloides venezuelensis), an increase in the concentration of ltb4 but not of ltc4 was observed in the lung and small intestines. in addition, increased larvae recovery in the lung and/or increased worm burdens in the intestines were observed in animals treated with mk886 (a selective inhibitor of 5-lipoxygenase-activating protein (flap)) and in 5-lo-deficient mice than in control animals. moreover, treatment of animals with mk886 resulted in decreases of igg1 and ige levels in serum, eosinophil numbers in the blood, peritoneal cavity and bronchoalveolar fluid volumes and il-5 concentrations in the lung homogenate as well as increased levels of il-12, which is involved in the th1 response. il-5 is the major cytokine involved in the accumulation of eosinophils in the blood during allergic inflammation and parasitic infections. this cytokine is essential for eosinophil migration from the bone marrow to the blood [72, 95] and specifically supports the terminal differentiation and proliferation of eosinophil precursors as well as the activation of mature eosinophils [9699]. ltb4 regulates il-5 production by human t lymphocytes and consequently contributes to parasite elimination. these findings suggest that lts, and specifically ltb4, might be necessary to control s. stercoralis infection. thus, the reduced levels of ltb4 observed in hiv-seropositive subjects might favor opportunistic hyperinfection with s. stercoralis; however, further human studies are needed to evaluate this association. toxocara canis is an intestinal parasite of dogs and is the etiologic agent of toxocariasis, also known as visceral larva migrans syndrome (vlms). infection of both humans and animals with t. canis is characterized by eosinophilia in the blood and tissues, increased total serum ige, and inflammation of the upper respiratory system [72, 95, 101104]. during the inflammatory response, leukocyte recruitment is directly related to the expression of adhesion molecules, which allows the transmigration of these blood cells to the tissues. it has been proposed that the 2 integrin mac-1 (cd11b/cd18) and the 1 integrin vla-4 (cd49d/cd29) adhesion molecules are the major molecules involved in cytokine- and chemokine-induced adhesion and migration of eosinophils in vitro [106, 107]. t. canis infection causes early upregulation of mac-1 with late changes in vla-4 profiles on both peritoneal cavity fluid and bronchoalveolar lavage fluids, whereas mk886 treatment promoted the opposite effect. in addition, lt inhibition had a clear impact on eosinophil recruitment to tissues and on blood eosinophilia throughout the course of infection. in another study, in addition to increased eosinophil numbers, the researchers showed increased numbers of mast cells in the peritoneum, lungs, and small intestines of t. canis-infected rats. interestingly, these animals increased the concentration of ltb4 in the serum and this was correlated with mast cell and eosinophil accumulation and/or recruitment. thus, lts might play an important role in eosinophilic inflammation during toxocariasis by inducing leukocytes recruitment and modulating the expression of adhesion molecules. in schistosomiasis, lts can control parasite infection by modulating immune responses and through direct cytotoxic effects on the parasite. ltb4, but not cyslts (ltc4 and ltd4), enhanced the ability of neutrophils and eosinophils to kill the schistosomula of s. mansoni in a complement-dependent manner. the cytotoxicity of eosinophils against helminths has been associated with the expression of cellular receptors (high affinity ige receptor, fcri) and adhesion molecules and with degranulation and the release of cationic proteins. in an in vitro assay, ige-coated schistosomula induced eosinophil adherence, resulting in the death of the parasites. in addition, the release of ltc4 was observed during this interaction. in agreement with this finding, schistosomula can produce ltb4 and ltc4. the function of lts in schistosomula is not known; however, their production might accelerate parasite elimination and/or modulate the pathogenesis of schistosomiasis. in addition to the proteolytic enzymes produced by cercariae, host-derived skin essential fatty acids and lts including ltb4 also play important roles in the penetration of the skin by the parasite. in an in vitro assay, increased penetration rates were correlated with increased lts levels. in addition, penetration was reduced upon treatment with a 5-lo inhibitor [116, 117]. hepatic stellate cells (hscs) are involved in liver remodeling due to collagen production and deposition of extracellular matrix as a consequence of proliferative and fibrogenic phenotypes induced by several mediators (cytokines, lipid peroxide, and others). mrna for 5-lo, flap and ltc4-synthase and 5-lo expression was observed in hscs from schistosomal granulomas of s. mansoni-infected mice. consequently, these cells produced cyslts, but not ltb4, and the production of cyslts was increased upon treatment with transforming growth factor beta (tgf-, a fibrogenic cytokine). the proliferation induced by tgf- in hscs from schistosomal granulomas of s. mansoni-infected 5-lo-deficient mice or wild type mice treated with zileuton (5-lo inhibitor) was reduced. in addition, ltc4 induced tgf- production, suggesting a synergic effect in schistosomal granulomas. in another study, dipeptidases were isolated from extracts of hepatic granulomas of mice infected with s. mansoni; these enzymes increased the hydrolysis of ltd4 to lte4, potentially accelerating the metabolism of lts and decreasing their effects on liver remodeling. moreover, ltb4 and ltc4 are produced by schistosomula and adult females, while males produced only ltb4. together, these results suggest that cyslt inhibition might influence liver remodeling in s. mansoni infection. in this way, cyslt1 antagonists (such as montelukast, zafirlukast, and pranlukast) [4, 122, 123], which are currently used in asthma treatment, could be evaluated for their effects on schistosomal granuloma remodeling in experimental or human schistosomiasis. similar to schistosomiasis, fasciolosis causes liver alterations, which can range from fibrosis to cirrhosis. fasciolosis is considered both a human health concern and a veterinary problem (zoonoses). during the course of f. hepatica infection in sheep, a reduction in serum ltb4 was observed when compared to control animals. interestingly, ltb4 was produced in both the culture supernatant and the homogenate of f. hepatica adult parasites recovered from the bile duct 20 weeks after infection. moreover, recruitment of leukocytes consisting mainly of eosinophils, macrophages, and lymphocytes was observed in the livers of goats infected with f. hepatica. in this way, ltb4 produced by host inflammation in synergy with that produced by the parasite could contribute to liver alterations and consequent pathology. lts are associated with the control of helminth and protozoan infections through their ability to modulate inflammatory processes and/or to promote direct cytotoxicity of protozoans. in addition, lts may also be associated with exacerbated pathogenesis in protozoan diseases, such as cerebral malaria, and helminthic diseases, such as schistosomal granulomas. interestingly, some helminths (b. malayi) might use the lts to complete their development to adult worms. in addition, other parasites produce lts (s. mansoni and f. hepatica) or produce enzymes involved in lt biosynthesis (dirofilaria immitis). taken together ,

leukotrienes (lts), formed by the 5-lipoxygenase-(5-lo-) catalyzed oxidation of arachidonic acid, are lipid mediators that have potent proinflammatory activities. pharmacologic or genetic inhibition of 5-lo biosynthesis in animals is associated with increased mortality and impaired clearance of bacteria, fungi, and parasites. lts play a role in the control of helminth and protozoan infections by modulating the immune system and/or through direct cytotoxicity to parasites; however, lts may also be associated with pathogenesis, such as in cerebral malaria and schistosomal granuloma. interestingly, some proteins from the saliva of insect vectors that transmit protozoans and secreted protein from helminth could bind lts and may consequently modulate the course of infection or pathogenesis. in addition, the decreased production of lts in immunocompromised individuals might modulate the pathophysiology of helminth and protozoan infections. herein, in this paper, we showed the immunomodulatory and pathogenic roles of lts during the helminth and protozoan infections.

PMC3337730

pubmed-499

the term replication stress describes the slowing or stalling of replication forks by endogenously or exogenously derived impediments to dna polymerization (zeman and cimprich, 2014). replication stressors can be local factors, such as dna damage or secondary structures that affect forks randomly as they are encountered, or global ones, such as nucleotide pool depletion or imbalance that simultaneously slows all forks (poli et al., 2012, it is now recognized that replication stress induced by nucleotide pool imbalance is an important consequence of the activation of some oncogenes, which drive cells into s phase without upregulation of nucleotide supply (bester et al., 2011). the resulting loss of polymerase processivity is thought to lead to localized uncoupling of the replicative helicase and polymerase and formation of tracts of single-stranded dna (byun et al., 2005, pacek and walter, 2004). while this normally induces checkpoint activation and senescence (bartkova et al., 2006,, in cells that can bypass the checkpoint, such replication stress provides a fertile source of genetic instability, particularly in the vicinity of fragile sites and sites capable of forming secondary structures (de and michor, 2011, tsantoulis et al., 2008). in addition to the extensive genetic changes that have been well documented in many types of cancer, there are also extensive local and global alterations in histone and dna modifications. the consequent changes in chromatin structure are accompanied by significant dysregulation of gene expression (timp and feinberg, 2013, berdasco and esteller, 2010), which, since it is not accompanied by changes in the dna sequence, may be considered epigenetic (berger et al., 2009). these epigenetic changes could act alongside genetic instability to produce clonal variation within a tumor, upon which selective pressure can act, and so may contribute to tumor evolution. mutations in histone and dna-modifying enzymes, and even histone proteins themselves, have been found in several cancers and are likely to explain at least some of the observed epigenetic instability (timp and feinberg, 2013). however, it is not clear that mutations in histone-modifying enzymes account for all the alterations observed in different cancer types. we recently provided evidence that deficiencies in enzymes responsible for replicating g quadruplex (g4) structures, such as the specialized dna polymerase rev1 and helicases fancj, wrn, and blm, can lead to localized changes in histone modifications and gene expression (sarkies et al., 2010, sarkies et al., 2012, schiavone et al., 2014). g4s can form within motifs comprising four short runs of dg bases, separated by linker sequences. the dg bases in the motif form planar hoogsteen-bonded quartet structures that can stack on top of each other, resulting in an often highly thermodynamically stable secondary structure, the g4 (reviewed in maizels and gray, 2013). we proposed that persistent replication fork stalling at g4s in mutants such as rev1 or fancj leads to pathologically long daughter strand gap formation, resulting in local uncoupling of dna synthesis from parental histone recycling. this, in turn, leads to loss of the histone modifications present on the parental chromatin, which, if in the vicinity of a gene promoter, results in changes in transcription (sarkies et al., 2010, sarkies et al., 2012, schiavone et al., a prediction of this model is that global replication stressors that lead to loss of processive dna polymerization with uncoupling of the replicative helicase and polymerase also should promote epigenetic instability by dissociating dna synthesis from histone recycling. here we test this hypothesis by examining the effect of hydroxyurea (hu)-induced nucleotide pool depletion on the epigenetic stability of a sensitive reporter locus, bu-1, in chicken dt40 cells (sarkies et al. we show that chronic treatment with low-dose hu induces stochastic instability of bu-1 expression, characterized by loss of the chromatin marks h3k4me3 and h3k9/14ac seen in the normally active locus. this instability depends significantly on the presence of a g4 motif 3 of the promoter, oriented to stall the leading strand of a fork heading toward the transcription start site (tss). the presence of this g4 motif not only increases the rate at which bu-1 expression is lost, but is additionally associated with phosphorylation of h2ax and appearance of the heterochromatic mark h3k9me3. this is consistent with the g4 acting to focus dna damage induced by the global replication stress imposed by hu, with the damage leading to repression of the locus. further, we show that, across the genome, chronic exposure to hu results in an altered pattern of gene expression similar to that seen in cells lacking the g4-unwinding helicases fancj, wrn, and blm, and that affected genes are enriched in g4 motifs. together, these observations indicate that nucleotide depletion can combine with naturally occurring dna secondary structures to promote epigenetic instability. we first sought conditions in which we could culture dt40 cells in low-dose hu such that replication is slowed but completed (alvino et al., 2007). we therefore exposed wild-type dt40 cells to a range of hu concentrations and monitored their doubling time. the cells were able to proliferate for over a week in up to 150 m hu (figure 1a). at this dose, their doubling time increased from 12.3 to 32.7 hr, recovering when the hu was washed out (figure 1a). to determine the effect of low-dose hu on replication dynamics, we performed dna molecular combing after pulse labeling the cells with halogenated nucleotides (figure s1a) 3 days after initiating culture in hu. average fork velocity decreased from 1.26 to 0.71 kb/min (figure 1b), with a compensatory decrease in average interorigin distance from 72 to 40 kb (figure 1b). consistent with these perturbed replication dynamics, cell-cycle profiles revealed a significant accumulation of cells in s phase while in hu (figure s1b). we have reported previously that replication-dependent transcriptional instability associated with g4 motifs can be monitored by following expression of a surface marker, bu-1a, in dt40 cells (sarkies et al., 2012, schiavone et al., 2014). the bu-1 locus contains prominent g4 motifs 3.5 kb downstream of the tss and 3 kb upstream. both are orientated to be g-rich on the feature strand with respect to the bu-1 transcript (figure 1c). epigenetic instability of bu-1 in rev1 cells is entirely dependent on the +3.5 g4 motif, and it requires the motif to be orientated such that its g-rich strand forms on the leading strand of a replication fork entering the locus from the 3 end (figure 1c; schiavone et al., 2014). we have reported previously that the bu-1 locus is bidirectionally replicated, meaning that during any given s phase there is a 50% probability of the +3.5 g4 being replicated on the leading strand template (schiavone et al., 2014). growth of wild-type dt40 cells in 150 m hu resulted in the appearance of a bu-1a population as cells divided over the course of 7 days (figure 1d). surface expression of bu-1a correlates closely with transcript abundance (sarkies et al., 2012), and this held true for bu-1a clones recovered after hu treatment (figure s1c). to estimate the rate at which bu-1a variants are formed in hu, we performed a fluctuation analysis by expanding multiple parallel populations of 10 bu-1a cells in hu for 7 days, after which we monitored the appearance of bu-1a variants. this revealed a striking degree of expression instability despite the small number of cell cycles through which the cells had passed (figure 1e). using our previously described monte carlo simulation of bu-1a loss as a replication-dependent phenomenon (schiavone et al., 2014), we estimated a per-division probability of generating of a bu-1a state during culture in hu of c. 0.15. to obtain additional evidence that this induced transcriptional instability of bu-1 reflected decreased dna polymerase processivity, we asked whether bu-1 variants could be induced by aphidicolin. aphidicolin slows replication by directly inhibiting dna polymerases, particularly pol (oguro et al., 1979), and a low dose induces replication stress (pacek et al., 2006). dt40 cells were able to proliferate in up to 150 m aphidicolin for 10 days and, as with low-dose hu, this resulted in substantial instability of bu-1a expression (figure 1f). we have shown previously that removal of the +3.5 g4 motif from both alleles of bu-1 in rev1-deficient cells results in complete stabilization of expression of the locus (schiavone et al., 2014). we therefore examined the extent to which this motif also accounted for the observed hu-induced instability of bu-1a expression in wild-type dt40. we grew wild-type cells lacking the +3.5 g4 on both alleles, bu-1 (schiavone et al., 2014), in hu and assessed the frequency of bu-1a variants after 7 days by fluctuation analysis (see figure e3 in schiavone et al this revealed that removal of the +3.5 g4 motif resulted in a significant reduction in the rate at which bu-1a variants were generated. however, it did not result in complete stabilization of the locus (figures 2a and 2b, i and ii). we considered the possibility that the residual instability could be due to the 3.0 g4 upstream of the tss. however, deleting this motif (figure s2) had no impact on hu-induced instability (figure 2b, iii). to confirm the contribution of the +3.5 g4 motif, this resulted in the return of the high-level hu-induced instability of bu-1 expression observed in wild-type cells (figure 2c, i). however, this was not seen if the motif was mutated to render it incapable of forming an intramolecular g4 (figure 2c, ii) or when it was inverted so that the g4 structure would form on the lagging strand template (figure 2c, iii). thus, hu treatment alone can induce instability of bu-1a expression, but its effect is significantly potentiated by the presence of a g4-forming sequence orientated to stall the leading strand replication of a fork heading toward the tss. we next investigated the basis for the hu-induced generation of bu-1a variants. to test whether the bu-1 state is permanent, we isolated five bu-1 clones at the end of 1-week growth in hu and cultured them for a further 3 weeks in hu-free medium. the clones remained stably bu-1 with no evidence of reversion to bu-1, suggesting that this was a permanent change. we considered the possibility that bu-1 cells resulted from genetic changes in the locus, although the observed rate of mutation would be extraordinarily high for this to be the case. we therefore sequenced the region around the +3.5g4 to look for mutation of the motif and used pcr with restriction digestion to detect larger deletions (figure s3). neither assay revealed any evidence of genetic instability consistent with the formation of bu-1 variants being an epigenetic event. we therefore examined the pattern of histone modification at the bu-1 promoter by chromatin immunoprecipitation (chip) from bulk populations of cells exposed to hu. bu-1 is a transcriptionally active locus characterized by high levels of h3k4me3 around its tss. after 48-hr treatment with hu, we observed a small but not significant loss of h3k4me3 at the bu-1 promoter (figure 3a), consistent with the size of the population of bu-1a cells generated by this time point (figure 1d). however, after 7-day treatment, we observed a more significant loss of h3k4me3 correlating with the much larger population of bu-1a cells at this time point (figures 1d and 2a). the loss of h3k4me3 was accompanied by a reduction in h3k9/14 acetylation (figure s4). seven days of hu treatment also induced a marked increase in h3k9me3 at the bu-1 promoter (figure 3a). it has been proposed previously that hu-induced displacement of parental h3/4 and its buffering by the histone chaperone asf1 may lead to unscheduled heterochromatinization by ectopic deposition of pre-marked histones upon their release from asf1 (jasencakova et al., 2010, schwab et al., 2013). alternatively, the appearance of h3k9me3 may result from dna damage-induced heterochromatinization, which has been observed following double-strand breaks (ayrapetov et al. breaks can arise from fork collapse in hu (petermann et al., 2010), and thus, if unscheduled incorporation of h3 with k9 methylation was responsible, then an increase in h3k9me3 would be observed irrespective of whether the +3.5 g4 motif was present. however, if localized g4-induced dna damage was responsible, then the appearance of h3k9me3 would be dependent on the +3.5 g4 motif. we therefore examined h3k4me3 and h3k9me3 at the promoter of bu-1 in cells lacking the +3.5 g4 motif. after 7 days in hu, h3k4me3 was reduced (figure 3b), but to a lesser extent than in wild-type cells (figure 3a), consistent with the reduced rate at which bu-1a variants are generated in cells lacking the +3.5 g4 motif (figure 2b). however, we observed no associated increase in h3k9me3 (figure 3b). to monitor the extent to which hu induced dna damage in the two situations, we performed chip for phosphorylated h2ax (h2ax) (rogakou et al., 1998) at the bu-1 promoter. h2ax was enriched 2.5-fold at the bu-1 promoter in wild-type cells after 7 days in hu, but not enriched in cells lacking the +3.5 g4 motif grown under the same conditions (figure 3c). this favors a model in which heterochromatinization of bu-1 in hu is promoted by dna damage, likely from fork collapse associated with the +3.5 g4 motif. we next examined the extent to which hu-induced changes in histone modifications were permanent by performing chip at the bu-1 promoter in the five stable bu-1a clones discussed above. this revealed that promoter h3k4me3 remained low, showing that loss of this mark was permanent (figure 3d). thus, while h3k9me3 is induced by hu in cells containing the +3.5 g4 motif, this mark is not essential to maintain the bu-1a state. this may be because it is installed only transiently during repair of hu-induced dna damage in the locus, or because cells in which h3k9me3 persists are growth disadvantaged and are lost from the population. the data thus far were consistent with a working hypothesis that reduced polymerase processivity increases the probability of g4 formation at the +3.5 g4 motif through exposure of more single-stranded dna within the replisome, which, in turn, focuses replication stalling at this site. implicit in this model is the idea that the +3.5 g4 can form during replication but that it is usually rapidly resolved to maintain fork progression. we therefore reasoned that trapping the g4 structure using a g4-binding ligand also might induce instability of bu-1a expression in otherwise wild-type cells further, we predicted that g4 ligands and hu would act synergistically to destabilize expression of the locus. to test these ideas, we treated cells with the g4 ligand n-methyl mesoporphyrin ix (nmm) (nicoludis et al., 2012). we first identified the maximum dose at which the cells retained normal viability and global replication dynamics. at 2 m nmm, the fork rate, as assessed by molecular combing, was 1.13 kb/min compared with 1.26 kb/min in wild-type cells, with no significant change in the inter-origin distance (figure s5). nonetheless, fluctuation analysis for bu-1a loss in wild-type and bu-1 cells cultured for 7 days in 2 m nmm revealed instability in bu-1a expression in wild-type cells, but not cells lacking the +3.5 g4 motif (figure 4a). since 2 m nmm does not in itself significantly reduce global fork rates and the agent will only interact with the formed g4 structure, not with just the linear dna sequence (ren and chaires, 1999), this observation is consistent with transient formation of g4s during normal replication. we next asked whether combining nmm-induced g4 stabilization with hu-induced reduction in polymerase processivity led to a further destabilization of bu-1 expression. interestingly, use of both drugs together resulted in significant toxicity, meaning that we had to reduce the dose of each drug by 50% in order to carry out the fluctuation analysis. as expected, growth of cells in hu at 75 m or nmm at 1 m individually had little effect on stability of bu-1a expression (figure 4b). however, the combination of nmm and hu at these doses resulted in a significant increase in bu-1a instability, revealing a marked synergy between hu-induced replication stress and g4 stabilization. we replaced the natural +3.5 g4 motif with a series of four g4 motifs of varying in vitro thermal stabilities (schiavone et al., 2014). all four motifs (g4 14) potentiated the formation of bu-1a variants upon treatment with hu (figure 5a). interestingly, we observed no correlation between the degree of potentiation by the motifs and the in vitro melting temperature of the equivalent oligonucleotides (figure 5b). however, there was a significant trend toward greater potentiation of bu-1a loss being associated with longer non-g loops in the range of 1 to 9 bp (figure 5c, solid line). to explore this further, we also tested a single repeat of the g4 motif containing human ceb1 mini-satellite (piazza et al., 2012), which has 18 bp between its first three and last run of dgs. this g4 motif, but not a mutated form that is incapable of forming a g4 structure in vitro (piazza et al., 2012), also potentiated bu-1a instability after treatment with hu. however, this was not to a greater extent than the natural +3.5 g4 dna with its central 9-bp loop, suggesting that there may be a limit after which lengthening the loop has no further effect. finally, we asked whether we could detect genome-wide evidence of an interaction between hu and g4s. we therefore performed affymetrix expression microarray analysis on cells before and after culture in 150 m hu. three parallel cultures of dt40 were treated with 150 m hu for 7 days, or mock treated, and then recovered into normal medium for 7 days, after which rna was prepared for array hybridization. despite this relatively short treatment, a total of 2,937 of 12,920 unique genes exhibited a change in expression of>0.25 log2 units with p <0.05, with an approximately equal number of genes being upregulated and downregulated (figure 6a). we previously have observed a similarly large number of dysregulated genes in cells deficient in the 5-3 g4-unwinding helicase fancj and in double mutants for the 3-5 helicases wrn and blm (sarkies et al., further, we found a highly significant overlap in the identity of dysregulated genes in the two sets, the direction in which their expression changed, and the association of the dysregulated genes with g4 motifs (sarkies et al. we anticipated that if transcriptional dysregulation by hu was linked with g4s that there might be a significant similarity in the gene set altered by hu and the sets altered by loss of fancj and wrn/blm. indeed, the overlap in the identities of the genes dysregulated in all three conditions was highly significant (figure 6b), as were the pairwise correlations in the direction of the change in expression (figure 6c). nearly 68% of the 6,061 genes within the venn diagram in figure 6b have a g4 motif within 1 kb upstream of the tss and the end of the body of the gene in comparison with 59% of the 6,859 genes in the remainder of the array (p <1 10) (table s1). to ascertain whether the overlaps in the identity of dysregulated genes reflected the perturbation of common pathways in the three datasets, we analyzed the functional annotation terms associated with the genes in each set and in the overlap sets using david (https://david.ncifcrf.gov; huang et al. while treatment with hu resulted in dysregulation of genes with gene ontology (go) terms associated with cellular stress and nucleotide metabolism, a large number of miscellaneous go terms also were enriched to a similar degree (table s2). significantly, despite the large number of genes overlapping in the three datasets, there was no evidence of their being members of common pathways (figure s6). this is consistent with much of the dysregulation of expression resulting from processes that are not related to a coordinated physiological response either to treatment with hu or ablation of fancj or wrn and blm helicases. nonetheless, the degree of overlap in the dysregulated transcriptomes in these three conditions suggest that cells treated with hu and cells lacking fancj and wrn/blm face similar challenges. however, the enrichment of g4s in affected genes, while statistically significant, is still relatively modest, suggesting that other factors, such as secondary effects or other dna secondary structures, may be contributing as well. numerous lines of evidence have linked replication stress with genetic instability (halazonetis et al., 2008, zeman and cimprich, 2014). imbalanced or depleted nucleotide pools during replication are an important cause of such stress and can arise from the expression of oncogenes uncoupling entry into s phase from upregulation of nucleotide supply (bester et al., 2011). importantly, the dna damage resulting from replication stressors like hu or aphidicolin that act directly on the replicative dna polymerases is not randomly distributed across the genome, but is instead focused on sites that often have features that make them potentially problematic to replicate even under ideal conditions (tsantoulis et al., 2008). many of these hotspots also correspond to classical fragile sites, in which chromosome breaks are observed after replication stress. such sites have been linked to regions depleted in replication origins, meaning that single forks have to traverse long distances (letessier et al., 2011). thus, a combination of regions of low fork density and problematic structures may focus sites of fork collapse under conditions of global replication stress (wickramasinghe et al., 2015). the mechanisms by which replication stress leads to epigenetic changes are less well explored. alterations in chromatin composition and structure are common features of cancer cells (berdasco and esteller, 2010, hansen et al., 2011, timp and feinberg, 2013) and are particularly associated with g4-dense breakpoint hotspots (de and michor, 2011). although the cell line we used in this study, dt40, is itself transformed, we found no evidence of significant stress to the dna replication program under normal growth conditions. however, growth of the cells in low-dose hu recapitulated the key features of the acutely stressed replication observed in oncogene-expressing primary cells (bester et al. we have been able to explore directly the interaction between global replication stress induced by nucleotide depletion and a dna secondary structure to demonstrate how they conspire to exacerbate replication-dependent epigenetic instability. we have provided evidence that two parallel epigenetic perturbations contribute to permanent and transient epigenetic changes following an episode of replication stress. the first mechanism relates to the uncoupling of the activity of replicative helicase and polymerase (byun et al., 2005, pacek and walter, 2004) during hu treatment. this has been shown to lead to interruption of the normal flow of histones from the parental to the nascent daughter strands, with the histone chaperone asf1 buffering the displaced h3/h4 (jasencakova et al., 2010). groth and colleagues suggested that their release from asf1 might lead to local alterations in epigenetic state of chromatin due to unscheduled incorporation of inappropriately marked histones (jasencakova and groth, 2010, jasencakova et al., 2010). (2013) invoked this model to explain an increase in heterochromatin formation in cells deficient in fancj, suggesting that failure to unwind lagging-strand template g4 structures in fancj-deficient cells led to unscheduled deposition of histones bearing marks that would lead to h3k9me3 and heterochromatin formation. however, this model does not adequately explain the bidirectional changes in gene expression changes seen either in fancj cells or in wild-type cells treated with hu (sarkies et al. in contrast, the model we have developed previously, in which loss of processive replication at g4s leads to localized loss of parental histone mark recycling, could explain both derepression of loci, such as -globin (sarkies et al., 2010), and loss of activation, as can be observed in the bu-1 locus (sarkies et al. however, since the mechanism by which h3k4me3 is maintained during replication is poorly understood, the precise mechanisms by which replication impediments disturb the maintenance of this mark remain to be fully elucidated this mark is induced alongside the loss of h3k4me3 when the +3.5 g4 motif is present and cells are exposed to hu. importantly, the appearance of this mark of heterochromatin is accompanied by h2ax phosphorylation, a marker of dna damage (rogakou et al., 1998). g4 motifs have been linked to hotspots of genetic instability and translocation (de and michor, 2011). further, the g4 ligand pyridostatin, which acts similarly to nmm, leads to localized h2ax accumulation at g4 motifs across the genome, suggesting the formation of dna breaks (rodriguez et al., dna breaks have been shown to induce transcriptional repression (shanbhag et al., 2010) and to induce h3k9me3 even in a normally euchromatic locus (ayrapetov et al., 2014). thus, the appearance of h2ax and h3k9me3 in the bu-1 locus only in hu-treated cells containing the +3.5 g4 is consistent with collapse or incision of replication forks, already stressed by nucleotide depletion, that have stalled at the g4. we therefore propose that nucleotide depletion can give rise to loss of parental h3k4me3 and the appearance of h3k9me3 by distinct mechanisms. h3k4me3 is lost stochastically as a result of interruption of parental histone recycling, a mechanism that is locally exacerbated by the presence of a g4 motif. in contrast, we propose that h3k9me3 may reflect protective transient heterochromatinization of the locus during repair of breaks resulting from hu-induced fork collapse at g4 structures (figure 7). in the case of loci like bu-1, in which hu-induced epigenetic instability is linked to g4 formation, an interesting question is whether hu results in a greater opportunity for g4 formation during replication or diminished g4 resolution. it is not currently possible to formally distinguish these possibilities and indeed it is likely that elements of both are true. notably, the observation that the g4-binding ligand nmm can induce bu-1a expression instability at a dose that does not significantly impact on global replication dynamics provides strong evidence that g4 structures form readily during normal replication and that they are usually promptly resolved. the synergy between replication stress caused by nucleotide depletion and structured dna is of considerable potential importance to understanding the development of cancer. epigenetic changes are prevalent in many cancer types, although their origin is unclear, and likely complex (berdasco and esteller, 2010, timp and feinberg, 2013). recently, several instances of epigenetic instability in cancer have been linked to mutations in histone or dna-modifying enzymes. however, the widespread and often apparently random nature of epigenetic changes in tumors suggests that other processes also may be at work. we have suggested previously that delayed replication of g quadruplex structures could contribute to the epigenetic diversity of cancer (sarkies and sale, 2012). however, mutations in enzymes that may cause this form of epigenetic instability, for example rev1, fancj, wrn, and blm (sarkies et al., 2010, sarkies et al., 2012), are rarely observed in sporadic cancers. replication stress, on the other hand, is emerging as an important feature of cancer cells, particularly in the early stages of their evolution (bester et al., 2011, di micco et al., 2006, thus, we suggest that some of the epigenetic changes seen in tumors may be explained by problems managing replication blocks. consistent with this idea, both copy number variations and changes in dna methylation patterns in cancer have been linked to g4 motifs (de and michor, 2011). finally, it is worth noting that hu is used extensively in treatment of hemoglobinopathies, such as sickle cell disease and thalassaemia, as it can re-induce expression of the fetal -globin gene, ameliorating the effects of the defective adult globins found in these disorders (platt et al., 1984). importantly, the effect of hu on -globin expression is unlikely to be specific, since chronic exposure to the drug leads to quite widespread changes in erythroid gene expression (flanagan et al., 2012). although the -globin locus in humans has no g4 motifs in the immediate vicinity of its promoter, its key transcriptional regulator, bcl11a (bauer et al., 2013), has a high density of g4 motifs on both sides of its tss. it will, therefore, be interesting to explore whether the mechanisms we propose here could help explain the action of hu on fetal globin expression. dt40 cells culture and the strategy for removing and replacing the +3.5 g4 motif in the bu-1 locus have been described previously (schiavone et al., 2014). genetic manipulation of the +3.5 g4 motif was performed in the bu-1a allele of cells in which the motif had been removed from the bu-1b allele to avoid the transvection-like effect between the alleles (schiavone et al., 2014). oligonucleotides are listed in table s3. for fluctuation analysis, 150 m hu (sigma-aldrich, h8627) after 7 days, cells at a concentration between 0.2 and 1 10 were stained for 20 min at 37c with anti-bu-1a-phycoerythrin (1:100, santa cruz biotechnology clone 5k98, 70447). bu-1a expression was assessed by flow cytometry using an lsrii cytometer (becton dickinson). experiments with aphidicolin (sigma-aldrich, a0781) and nmm (frontier scientific, nmm580) were conducted in 96-well plates starting with ten cells expanded for 10 days. bu-1a cells were isolated after hu treatment using a moflo sorting cytometer (dako-cytomation). chip was performed as described previously (nelson et al., 2006) with modifications. following a 10-min incubation at room temperature with 1% (v/v) formaldehyde, glycine was added to 0.2 m for 5 min. the extracted nuclei were sonicated at 4c using a bioruptor water bath sonicator (diagenode) with 30 cycles of 30 s separated by 30-s intervals. sheared chromatin samples were resuspended in dilution buffer (1.1% triton x-100, 1.2 mm edta, 16.7 mm tris [ph 8.0], 167 mm nacl supplemented with pmsf, and a protease inhibitor cocktail). for immunoprecipitation, lysates were incubated overnight with the following antibodies at 4c: histone h3 (1:100, cell signaling technology, 2650), h3k4me3 (1:100, cell signaling technology, 9727), h3k9/14ac (1:200, millipore, 17-615), h3k9me3 (1:200, abcam, ab8898), h2ax (1:50, abcam, ab2893), and the negative control normal rabbit igg (millipore). following overnight incubation at 4c with tumbling and four washing steps, the qpcr was performed with power sybr green master mix (applied biosystems, 4367659) on an abi prism real-time cycler with the following cycle times: 50c for 2 min, 90c for 10 min, 45 cycles of 90c for 15 s plus 60c for 1 min. the cdna was made from 5 mg mrna with super rt (ht biotechnology) and oligodt primer in a final volume of 40 l. dna molecular combing was conducted 3 days into culture with 150 m hu. it was performed and analyzed as previously described (guilbaud et al., 2011). rna was extracted from three independent wild-type cell populations treated for 7 days with hu and allowed to recover for another 7 days, as well as from three untreated parallel controls. microarray analysis was performed using r (http://www.r-project.org/) and its bioconductor packages (gentleman et al., 2004). raw cel files were processed using the robust multichip average (rma) algorithm available in the affy package (gautier et al., 2004). genes that showed a change of>0.25 log2 units relative to the mean wild-type intensity, with a p value of <0.05 (t test), were identified as exhibiting statistically significant transcriptional dysregulation. custom written r scripts were used to identify and plot genes co-dysregulated between different mutants. venn diagrams were generated with the limma package (smyth, 2004), and significance for the overlaps was calculated using fisher s hypergeometric distribution.

summarynucleotide pool imbalance has been proposed to drive genetic instability in cancer. here, we show that slowing replication forks by depleting nucleotide pools with hydroxyurea (hu) can also give rise to both transient and permanent epigenetic instability of a reporter locus, bu-1, in dt40 cells. hu induces stochastic formation of bu-1low variants in dividing cells, which have lost the h3k4me3 present in untreated cells. this instability is potentiated by an intragenic g quadruplex, which also promotes local h2ax phosphorylation and transient heterochromatinization. genome-wide, gene expression changes induced by hu significantly overlap with those resulting from loss of the g4-helicases fancj, wrn, and blm. thus, the effects of global replication stress induced by nucleotide pool depletion can be focused by local replication impediments caused by g quadruplex formation to induce epigenetic instability and changes in gene expression, a mechanism that may contribute to selectable transcriptional changes in cancer.

PMC4695339

pubmed-500

heat shock proteins (hsps) are a group of proteins that repress the denaturation of molecules by various stressful circumstances such as exercise, gravity, heat, oxygen, ca etc. hsp family has a structurally common chaperone subset controlling the form of proteins folding (misfolding, facilitating, or reconstructuring), which maintains homeostasis of proteins to stressful circumstances (dimauro et al., 2016). heat-induced heat shock factor 1 controls transcriptional upregulation of hsps (brinkmeier and ohlendieck, 2014). small hsp (shsp), hsp70, and hsp90 exist in the cytoplasm and glucose regulated proteins (grp) 78 (a homologue of hsp70), bip, hsp47 are located in mitochondria to transmit each of specific functional proteins to mitochondria. tcp1 (cytoskeleton forming chaperones) is associated with neogenesis of actin and tubulin and shsp are involved in intracellular dynamics (polymerization and depolymerization). a transcript (unfolded protein response element, ure; ccaaan9 ccacg) binds to the sequence of the chaperone gene. hsp families are made up by range from 10 to more than 100 kda in molecular size, and are located in various cellular compartments. the expressional locations of homologue or cognate members of hsps (such as hsc70 vs hsp70 or hsp90 vs hsp90) are arbitrary according to the accumulated evidences indicating cell or tissue restricted expressions in vivo systems. accumulated studies show that hsp families are located in various sites within cell; hsp10, hsp60, and hsp75 are located in mitochondria, however, others are present in the cytoplasm, cytosol, endoplasmic reticulum and nucleus in physiological conditions (xu, 2002). the following general description involving molecular chaperones shows the outline regarding the maintenance of physiological homeostasis of living body. based on comprehensive studies of hsps, this review offers important information regarding the mechanism related with the characteristic procedure of signal pathway according to the specifically cared substrate of each hsp, structural characteristics, specifically expressed location and those roles. molecular weight of shsp varied from 15 to 43 kda are also called heat shock protein (hspbs) and have a chaperoning function in the process of embryonic development. hspbs are also interacting with cytoskeleton proteins to maintain the homeostasis of cytoskeleton proteins by preventing those from damages, which results in conservation of the cell function. hspb1, hspb6, hspb7, and hspb12 are involved in the development of respiratory morphology such as cardiac muscles and the lethal myopathy in diaphragm and cardiomyopathy is caused with the lack of those hspbs (juo et al., 2016; ke et al., 2011; rosenfeld et al., hsp10 (chaperonin) is considered as a suppressor of maternal immune response via releasing from fetal placental unit (noonan et al., 1979). this includes 101 amino acids and is used as a plausible biomarker in endometrial cancer (dub et al., 2007). hsp 10 in mitochondria plays a role in protein folding supplied by adenosine triphosphate (atp) (table 1) (meyer et al., 2003). it has been reported that hsp10 is a by-product during the process of neoplastic cell proliferation and is considered as a growth factor in the cell (quinn and morton, 1992). hsp10 is also known as an obligatory autocrine growth factor in tumor cells (quinn et al., 1990). molecular size 1530 kda hsps are related with shsp and it has been know that there are 9 shsp in mammals (mounier and arrigo, 2002): hsp27 (denoted hsp25 in mice), a- and b-crystallins, hsp20, hspb2, hspb3, cvhsp or hspb7, hsp22 or hspb8, and hspb9. human shsp has 105205 amino acids and -crystallin domain as a homologous 80 sequenced residues (ingolia and craig, 1982). this domain shows highly conserved structure and 38%60% of amino acid identity (mounier and arrigo, 2002). alpha-crystallin structure is influenced by factors such as ph, temperature, calcium ions, and ionic strength (mounier and arrigo, 2002). each of the shsp is phosphorylated via specific kinase to involve teleological signal pathway (fig. hsp16.5 has been structurally studied that it has dimeric -crystallin domain as a basic structural domain unit and this common domain is possibly related with the basic function as a molecular chaperone (bertz et al., 2010). (2006) reported a function of shsp on myosin enzymatic activity that b-crystallin cares atpase activity of myosin and the comparing rate shows 58% in relation to control (8%, without b-crystallin) under 43c for 30 min. alpha b-crystallin maintaining atpase activity of myosin prevent aggregation of the protein and this is probably related with myofibrilogenesis without myosin unfolding that induces muscular performance under stress condition or vigorous exercise (melkani et al., 2006). forty-three kda sized hsps has various amino acidic sequences that results in different n-terminal length. those hsps also comprise conserved -crystallin core domains and c-terminal extension domain including highly conserved i-x-i/v motif (ghosh et al., 2005). hsp47 acting as a chaperone for procollagen has been also found to be involved in atherosclerosis. heat shock and oxidized low density lipoprotein stimulate the expression of hsp47 mrna in smooth muscle cells. these findings identify hsp47 as a novel constituent of human coronary atheroma, and selective upregulation by stress raises the possibility that hsp47 may be a determinant of plaque stability. collagen, an extracellular matrix substrate, requires hsp47 to be transported within the cell and folded from three helices. hsp47 located in endoplasmic reticulum and functions to support collagen folding and its release to extracellular environments. hsp60 forms a large (970 kda) hetero-oligomeric protein complex called the tcp1 ring complex (containing tcp1 and several other proteins), which is essential for protein assembly. the hsp60 family has been shown to be involved in the development of many diseases, such as adjuvant arthritis in rats, rheumatoid arthritis in humans, insulin-dependent diabetes mellitus in mice, and systemic sclerosis in humans. hsp70 is in the cytosol and has family of grp78 which plays role in helping protein folding assembly and refolding, transporting and blocking protein degradation in endoplasmic reticulum. hsp70 and hsp90 expression levels are variant according to different individual athletic abilities and seem to be upregulated in response to heat acclimation (banfi et al., 2004; mcclung et al., 2008; shastry et al., 2002). as a chaperone, hsp70 plays a role in the assembly and transport of newly synthesized proteins within cells, as well as in the removal of denatured proteins. downhill (eccentric contraction exercise induced muscle damage by increased delayed onset muscle soreness) in hot conditions elevates the largest expression of hsp72 and hsp90 mrna level (tuttle et al., in related with myocyte formation, hsp90 plays a key role in myosin folding and sarcomere formation (du et al., 2008; etard et al., 2007; hawkins et al., 2008; srikakulam et al hsp90 consists of n-terminal atp binding domain, substrate interacting middle domain, and c-terminal dimerization domain (jackson, 2013). by atp binding to the n-terminal atp-binding domain, hsp90 regulates myosin thick filament formation and muscle myofibrilogenesis (hawkins et al., 2008). hsp90 binds steroid receptors, protein kinases, intermediate filaments, microtubules, and actin microfilaments in a specific manner. hsp90 is an essential component of the glucocorticoid receptor, assembled in a complex of several proteins. hsp90 is divided into hsp90 and hsp90. hsp40 and hsp70 are cochaperones with the hsp90s. hsp90s are located in cytoplasm and endoplasmic reticulum to play roles in folding newly made proteins. hsp100 is located in the cytoplasm and cochaperones with hsp40, hsp70, and hsp90. hsp110 is in the cytosol and nucleus (dimauro et al., 2016). hsp110 and hsp70 super family commonly have the presence of a loop structure and hsp110 helps immune response (zuo et al., 2016). hsp110 is also in working with hsp70 or grp78 to fold proteins and counter stress for cell survival (gething and sambrook, 1992; hartl, 1996). (2016) vaccinated with purified hsp110 results in reduced cancer metastasis thus large hsps amplify inflammatory signals in the cellular environment, suggesting potential endogenous immuno-stimulation during injury such as infection (zuo et al., 2016). there are comparatively very rare studies regarding large hsps especially in the effect of exercise on the expression of large hsp and its function. over several decades, hsps have been known as a potent factor playing a role in maintaining the homeostasis of living body. versatile functions of hsps according to those wide ranges of molecular weight that care those each substrate have been increasingly attentive with other research subjects such as exercise, carcinogenesis, muscles, etc. the broad ranges of hsps are organelle-specifically expressed within the body to play physiological roles via interacting with various signal pathways. provoking these pathways in a positive way (e.g., physical exercise) enhance hsps signal pathway and maintain/improve vital function of hsps in the living body.

molecular chaperones are ubiquitous and abundant within cellular environments, functioning as a defense mechanism against outer environment. the range of molecular chaperones varies from 10 to over 100 kda. depending on the size, the specific locations and physiological roles of molecular chaperones vary within the cell. multifunctionality of heat shock proteins (hsps) expressed via various cyto-stress including heat shock have been spotlighted as a reliable prognostic target biomarker for therapeutic purpose in neuromuscular disease or cancer related studies. hsp also plays a critical role in the maintenance of proteins and cellular homeostasis in exercise-induced adaptation. such various functions of hsps give scientists insights into intracellular protective mechanisms in the living body thus hsps can be target molecules to know the defense mechanism in cellular environment. based on experimental results regarding small to large scaled hsps, this review aims to provide updated important information regarding the modality of responses of intracellular hsps towards extracellular stimulations. further, the expressive mechanisms of hsps data from tremendous in vivo and in vitro studies underlying the enhancement of the functionality of living body will be discussed.

PMC5031383