New considerations in the prevalence of periodontal disease.

International surveys demonstrate that the prevalence of pocketing of 6 mm or more is between 5% and 20% for much of the world's population. A recently completed national survey of employed adults found the prevalence of gingival bleeding was 44%, the prevalence of pocketing of 4 mm or more was 14%, and the prevalence of attachment loss of 3 mm or more was 44%. Risk indicators for a higher prevalence of periodontal disease include increasing age, poor education, lack of professional dental care, previous periodontal destruction, tobacco use, and diabetes. African-Americans show a higher prevalence of juvenile periodontitis and adult periodontitis than whites. The female-to-male ratio in juvenile periodontitis may be close to 1:1. Another report during the review period suggests that periodontal disease in adults may have a strong genetic component. A doctoral dissertation demonstrated that the sensitivities of methods used in a national survey to detect pocketing and attachment loss range from 0.24 to 0.87 in a high-prevalence population.


Occlusal philosophies and gnathologic instrumentation.

The field of occlusion continues to be prolific in publications. While no major advancement in the field was noted in the 1-year period of review, existing concepts and instrumentation continued to be refined. Topics covered in this review include occlusal tooth contacts, choice of alternate occlusal schemes, evaluation of gnathologic instruments and techniques, vertical dimension of occlusion, and mandibular flexure eliminate occurring on opening.


Considerations for planning for and fabricating complete dentures.

During the past year, studies have been completed to advance our knowledge of the diagnosis and treatment of the edentulous patient. The need for pretreatment radiographs on every edentulous patient is not always indicated. Advances in preprosthetic surgery help to improve the predictability of these procedures. New techniques for establishing the vertical dimension of occlusion may allow this to become a more objective measurement. Evaluation of dental materials for use with complete dentures continues, with soft liners and the use of microwave energy for curing denture resin just two areas of interest. A recent study showed a very high prevalence of signs and symptoms of craniomandibular disorders among complete denture wearers. Methods for its treatment are discussed. The use of increased infection control procedures in the dental office and laboratory have created a need to evaluate the effects of disinfecting agents on the dental prosthesis. The removable complete denture remains an effective alternative for the replacement of missing teeth.


The potential role of controlled-release delivery systems for chemotherapeutic agents in periodontics.

Following advances in periodontal disease etiology and pathogenesis, treatment strategies have evolved to eliminate specific pathogens or suppress the destructive host response. Research indicates that chemotherapeutic agents such as antimicrobials and antimetabolites can alter disease progression; consequently, three delivery systems have been investigated: systemic, topical, and controlled release. Using polymers to control drug administration, controlled-release delivery systems theoretically produce concentration profiles that are more constant and longer lasting than those of other systems. Furthermore, patient compliance can be maximized, and systemic complications avoided. During the past two decades, numerous investigations have been conducted to evaluate the potential role of controlled delivery in periodontal treatment. These investigations fall into two distinct categories: those documenting release kinetics, and those documenting clinical effects. Accordingly, pharmacologic agents can be released from drug polymers at therapeutic levels within the periodontal pocket. Many researchers have demonstrated that controlled delivery of antimicrobial agents such as tetracycline, metronidazole, and chlorhexidine can be effective in reducing the signs of periodontitis. In addition, controlled release of antimicrobial agents can alter the periodontal flora with a decrease in total bacterial mass and pathogenic species. Although the majority of these studies are proof-of-principle trials, many agents have been evaluated with undefined hypotheses, ill-suited outcome variables, unrepresentative patient populations, poor controls, and less-than-ideal therapy applications. The true test of controlled delivery will be the demonstration of its slowing disease progression. While future research will concentrate on developing more ideal polymers and introducing new agents, controlled delivery offers clinicians a potential adjunct or alternative to traditional treatment modalities.


A predoctoral honors program in prosthodontics.

Over the last 25 years, the advent of new disciplines in dental education and the increasing body of knowledge in various dental specialties have led to a struggle for curriculum hours within many dental schools. At the same time, the amount of time available for teaching clinical skills in dental schools has not increased appreciably, and fewer patients require (or can afford) sophisticated prosthodontic treatment. As a result of these trends, there has been a general decline in the depth and range of clinical skills of recent dental school graduates, particularly in prosthodontics. New York University College of Dentistry has attempted to address this problem by establishing a predoctoral honors program in prosthodontics.


Diagnostic procedures for evaluating pulpally involved teeth.

Pulpal and periapical problems are often difficult to diagnose because of seemingly conflicting or unclear symptoms. This difficulty increases when there is an emergency situation, and improper diagnosis can lead to improper treatment, causing distress to both the patient and clinician. Because the histopathologic condition of the pulp cannot be determined with clinical means or other diagnostic data, the clinician should focus on making an accurate clinical diagnosis using the patient's history and description of pain, clinical examination, and various diagnostic tests. This review discusses these factors and stresses the importance of making an accurate diagnosis.


Current endodontic instruments, materials, and drugs, and new techniques for their application.

Technical improvements in endodontic instruments, materials, and drugs and the development of new techniques for their application present a problem for dental practitioners in the selection of materials and methods appropriate to their needs. Claims of "superior" products and "state-of-the-art" treatments only add to the confusion. A review of the dental literature reveals that there is seldom a consensus of opinion on any given subject. Preferences among individuals may reflect a desire for a less complicated, more rapid approach to endodontic treatment. Trends in thought among researchers may offer some direction in the choice between changing concepts and traditional methodology.


The potential contributions of cell and molecular biology to periodontal tissue regeneration.

Periodontal disease results in the gradual loss of supporting periodontal tissues, and ultimately, the teeth. Although conventional periodontal therapy can arrest the progression of the disease and allow healing, the resulting tissue is unlike that of the normal, healthy periodontium. Cellular and molecular biologic techniques may facilitate our understanding of the regeneration of the periodontium. This review discusses recent findings in cellular and molecular biology relevant to the biology of periodontal tissue regeneration.


Pulpal response to restorative procedures and materials.

This paper reviews the pulpal response to common restorative procedures and dental materials. Pulpal inflammation and dentin hypersensitivity are commonly associated with routine restorative procedures. Although it is not possible to avoid injury to the pulp completely, in order to prevent unnecessary adverse effects, the practicing dentist should be aware of the potential dangers involved. This article provides a current review of recent findings of the pulpal response to glass ionomer cements and acid-etch composite resin materials.


Post and core restoration of endodontically treated teeth.

This review discusses the multifactorial nature of tooth strength and concludes that endodontic treatment alone does not weaken intact anterior teeth. Therefore, restoration of such teeth does require post placement or full-coverage restorations. Posterior teeth, however, require full-coverage protection. It appears that the full-crown restoration "covers up" some of the disadvantages or deficiencies of some post and core restorations. The literature supports the use of the amalgam coronal-radicular core or the post-retained amalgam core, as well as the composite post and core. The literature does not support use of the glass ionomer post and cores. As to which post system to select, the literature indicates that the parallel-sided, serrated Para-Post or Para-Post plus is a safe post when seated passively in the canal and cemented with zinc phosphate cements or composite resin cement, or when retained with amalgam. Dentin-retained, stress-producing posts such as the threaded posts should be avoided.


[Neurosarcoma associated with Von Recklinghausen disease: apropos of 25 cases observed at the Gustave Roussy Institute from 1967 to 1990].

In the absence of systematic immunohistochemistry investigations, only 25 cases (out of 69) clearly diagnosed as neurosarcoma due to the association with Von Recklinghausen disease (ie, neurofibromatosis type 1) and treated at the Institut Gustave Roussy were included in the present study. Neurosarcoma consists of a neurocristopathy whose cells migrate to several parts of the body in order to constitute neuroglia, Schwann cells, pigmented and endocrinal tissues. From 1967 to 1990, 25 cases of such neurosarcomas associated with a neurofibromatosis type 1 were seen at our institute. Three different histological terms exist for this tumour: malignant schwannoma, neurosarcoma, and more recently, malignant peripheral nerve sheath tumours (MPNST). The median age (23 years) of the patients with neurofibromatosis type 1 is lower than that of patient with isolated neurosarcoma. Their sex ratio is 2/1. Primary tumour surgical exeresis was performed in all cases, with poor results in 7. Post-operative radiation therapy was not systematically used in this series. It was administered only in cases with incomplete surgical exeresis or when a local recurrence occurred. Adjuvant CYVADIC (doxorubicin, procarbazine, cyclophosphamide, vincristine) chemotherapy was administered in 5 cases, of in cases of relapse (8). All cases but 2 (the most recent ones) relapsed within 1 to 226 months (median 7 months). In addition, 13 patients developed metastases. Overall, the 2-year and 4-year survival rates were 41% and 18%, respectively. Our observations confirm the very poor prognosis for the association of neurofibromatosis type 1 and neurosarcoma. This finding should lead to systematically associate radical surgical exeresis, post-operative irradiation and adjuvant chemotherapy in the treatment of these patients.


[Characterization of autoantigen (p105) in a model of colonic adenocarcinoma in the rat].

Sera from BDIX rats bearing the syngeneic colon tumors PROb or REGb were analysed by Western blotting in order to detect a possible humoral response against the grafted tumor. The PROb clone grows progressively in syngeneic hosts and metastasizes, whereas the REGb clone grows slowly and then is rejected. This model was developed by F Martin and his group in Dijon, France. We observed that rats bearing PROb tumors only develop an antibody response against a water-soluble protein of 105 kDa (p105) which is expressed by both tumor clones. This antibody response has never been detected in rats bearing REGb tumors. The antigen p105 was also expressed by normal adult colon as well as some other foetal or adult tissues. It is also present in extracts from several tumor cell lines including human colorectal carcinoma cell lines. Moreover, the titer of detected antibody at day 30 was inversely correlated with the survival of rats after tumor inoculation, suggesting a possible facilitating role of this antibody response.


[Risks of cancer among workers of the nuclear industry].

The epidemiological studies on cancer incidence or mortality among workers in the nuclear industry have been reviewed in a comparative manner. The results concerning workers monitored for external radiation or internal contamination and those on female workers have been analysed separately. A lower cancer risk was found in most studies, which is explained by the "healthy worker effect"; no difference was found between the workers with or without a radiation record. A high rate was found for a few cancers at specific sites, but results were variable in the different studies and no relationship was found between cumulative dose and excess mortality due to cancer. A link between ionizing radiation, multiple myeloma and prostatic cancer was indicated in more than one study; in the latter case, there might be a relationship with internal contamination, especially by tritium. In spite of the difficulties and limitations involved, it is important to pursue these studies and pool them, in order to directly evaluate the risks of cancer following exposure to low-dose radiation and low dose rates, instead of calculating these by extrapolation from high doses.


[Analysis of changes in serum levels of TNF, IL-1 and IL-6 during administration of IL-2. Correlation with clinical response to treatment].

We have investigated the serum concentrations of TNF, IL-1 and IL-6 in 49 patients with metastatic renal carcinoma receiving interleukin 2 (IL-2) or a combination of IL-2 and interferon alpha (IFN). Our results demonstrate that IL-2 and/or IFN induce an increase of serum concentrations of IL-1 and TNF in 95% and 75% of the patients respectively. Serum IL-6 levels increase in 44% of the patients. Serum concentrations of IL-1 and TNF remain elevated 48 hours after the end of IL-2 infusion. IL-1 and TNF levels are higher in patients receiving a combination of IL-2 and IFN. TNF and IL-1 levels in serum are significantly higher in responders to IL-2 treatment 48 hours after the end of IL-2 infusion. These two biological criteria enable a subgroup of patients with a very low response rate to IL-2 to be defined. The persistent increase of these cytokines in serum indicates a persistent activation of the immune system lasting after the end of IL-2 treatment which could be involved in the antitumor response.


[Cytology in the positive diagnosis and grading of prostatic cancers: which indications do remain at the time of automatic biopsies and endorectal echography?].

The diagnostic sensitivity (Se) and specificity (Sp) of fine-needle aspiration cytology (FNAC) of the prostate can be evaluated by comparing its results to a histological reference: rates of reported Se range from 65-98%, Sp being equal or superior to 95%. Published series are heterogeneous in terms of cancer prevalence, with a 25-85% proportion of histologically proven adenocarcinomas, irrespective of the anatomical stage of the disease. The overall accuracy of screening by core biopsies or FNAC is lower than 5%, and does not justify wide-scale application of these tests. In 75%, cytological assessment of the tumor grade correlates with Gleason's histological score and grade. Severe intraductal dysplasias (DIC 3) are probably involved in some of the cytological grade I cases. Ultrasonographic guidance of FNAC is not recommended in comparison with histologically obtained data. The indications for performing FNAC of the prostate should be different from those of standard biopsies: the former should be carried out on suspicious lesions revealed by digital rectal examination or ultrasonography, or in a staging attempt. FNAC should be reserved for early diagnosis of prostate cancer in patients presenting with non-specific urologic symptoms. Samples should be obtained by digitally-guided transrectal bilateral FNAC.


[Cytotoxic effects of the combination of a new nitrosourea, fotemustine, combined with 5-fluorouracil and folinic acid depend on the sequence of their administration].

The present study was designed to analyse the cytotoxic effects of the combination of fotemustine plus 5-fluorouracil (5-FU) and folinic acid (FA). Two human tumor cell lines were used; one line was derived from colon cancer (WIDR) and the other from a non-small cell lung cancer (CAL 12). Cytotoxic effects were assessed by the MTT semi-automated test in 96-well incubation plates. The effects of various drug combinations were evaluated by the isobologram method. The drug combinations tested included fotemustine concentrations of 20, 30, 40, 50 and 70 micrograms/ml, 5-FU concentrations of 5, 15 and 30 micrograms/ml, and a constant FA concentration of 10(-5) M. A total of 180 different experimental conditions were tested. When cells were exposed to fotemustine before 5-FU the final cytotoxic effects for both cell lines were additive or synergistic in the majority of cases (P less than 0.001). The 5-FU concentration was a determinant factor affecting modification of the effects of the drug combination from antagonism (with low 5-FU concentrations) to synergism (high 5-FU concentrations) (P less than 0.001). Addition of FA (10(-5) M) resulted in a significant shift towards synergistic associations for both cell lines. Administration of 5-FU before fotemustine caused a marked antagonism which 10(-5) M FA was unable to significantly shift towards simple additivity.


[Extramammary carcinoid tumor metastatic to the breast. Apropos of a case].

We report the case of a 42-year-woman treated for an appendicular carcinoid tumor with bilateral ovary metastases and mesenteric node involvement. After a systematic mammography, an infraclinical lesion of the breast was detected. Mammographic and echographic images revealed nodular mass with possibly malignant features. Fine-needle aspiration biopsy indicated malignancy. The definitive diagnosis of breast metastasis of a carcinoid tumor was made by biopsy. This case is related to 14 previously published cases. The authors emphasize the importance of making a precise histological diagnosis to avoid overtreatment of a metastatic lesion.


The characterization of two new low molecular weight proteins (LMPs) from Streptococcus pyogenes.

Two novel extracellular mitogenic substances were isolated from Streptococcus pyogenes strain NY-5 and characterized. The purification steps involved an initial enrichment of the proteins from culture supernatant by silica gel adsorption, followed by ion exchange chromatography and gel filtration. The purified materials were homogeneous in SDS-PAGE, showed estimated molecular weights of 12 kD and isoelectric points of 4.7 and 4.3, respectively. Both proteins (LMP-12k-4.3pI and LMP-12k-4.7pI) demonstrated lymphocyte transformation activity at a concentration of 0.1 microgram/ml. The LMP-12k-4.7pI showed a 69.2% homology of the amino acid sequence with that of a phosphocarrier protein of Staphylococcus aureus and with a total identity in the active centre. The same protein was also isolated from streptococcal group C strain H46A with an N-terminal amino acid sequence being identical. The LMP-12k-4.7pI demonstrated biochemical properties identical with those of the earlier described streptococcal pyrogenic exotoxin type D. The LMP-12k-4.3pI did not show such a clear relation to other functional proteins.


Characterization of the virulence regions in the plasmids of three live Salmonella vaccines.

Three live Salmonella vaccines, Zoosaloral "Dessau", Bovisaloral "Dessau", and Suisaloral "Dessau" successfully used in veterinary medicine in eastern Germany were analysed for their plasmid DNA content. Plasmids of 60 MDa (S. typhimurium), 55 MDa and 26 MDa (S. dublin) and 34 MDa (S. choleraesuis) were found. The three large plasmids contained a virulence region as shown by restriction enzyme analysis and hybridization studies with virulence-region-specific DNA probes. Restriction enzyme pattern analysis of the whole plasmids revealed differences between vaccine strains and parental strains. We wanted to know if there were alterations within the virulence region of the vaccine strain plasmids compared with parental strains caused by the attenuation procedure. Therefore, the 6 kb-Cla I fragments of several plasmids were cloned and characterized by restriction enzyme fingerprinting. The expression of the Cla I fragment-encoded proteins was analysed in the minicell-producing strain DS 410. The experiments revealed that the restriction pattern of the 6 kb-Cla I fragments of Zoosaloral and Bovisaloral as well as the PAGE pattern of virulence-region-encoded proteins were in complete accordance with one another, between vaccine strains and the parental strain of Zoosaloral, and with a wild-type strain of S. typhimurium.


In vitro activity of antifungal agents against clinical isolates of aspergilli.

The growth-inhibitory effect of 9 antifungal agents against 32 strains of aspergilli belonging to 10 species was studied. MIC values were determined in a microtiter dilution method using Brain Heart Infusion Broth (polyenes, azoles) and a medium with Yeast Nitrogen Base (5-flucytosine). Saperconazole was the most active agent against all isolates tested, the growth of 90% being inhibited at a concentration of 0.09 mg l-1. The susceptibility profile of individual species differed only little, with the exception of high MIC values of polyenes in A. terreus. Nine isolates (28%) were resistant to 5-flucytosine. The intra-species variation of susceptibility studied in A. fumigatus was only little pronounced.


Human giardiasis: correlation of specific secretory IgA levels in duodenal fluid to the severity of disease and infestation by Giardia lamblia.

Giardia lamblia specific secretory immunoglobulin A (sIgA) levels in the duodenal fluid of adult giardiasis cases are reported for the first time. The sIgA levels in the study group were found to be significantly higher (p less than 0.01) than in the 20 age- and sex-matched controls comprising cases classified as non-ulcerative dyspepsia who did nor reveal any G. lamblia in their stools and the duodenal fluid. An inverse relationship between the clinical severity of giardiasis and the level of sIgA in the duodenal fluid was noted. Cases with a higher trophozoite load in duodenal aspirate tended to be associated with envanescent G. lamblia-specific antibodies.


Varying incidence of Blastocystis hominis in cultures from faeces of patients with diarrhoea and from healthy persons.

A study was performed on the frequency of Blastocystis hominis in the faeces from 100 patients suffering from diarrhoea and from 100 healthy persons. Surprisingly, an increased detection rate was observed in samples from healthy persons after anaerobic cultivation. This increased frequency is obviously not dependent on the kind of serum used as a culture supplement and raises the question whether the protozoa morphologically described as B. hominis represent a homogenous species. When rabbit and horse sera were used instead of human serum for cultivation, in both groups the share of positive cultures increased and more large forms of B. hominis cells were observed. Biological implications are being discussed.


Diagnosis, prevalence and drug resistance of mycobacteria in HIV-positive and HIV-negative patients of an urban population in Germany.

In a survey of 3004 clinical specimens from 1112 HIV-negative and 679 clinical specimens from 140 HIV-positive patients which were submitted to our mycobacterial laboratory, we have analysed the different diagnostic approaches concerning mycobacterial disease in these two populations. We have assessed specimen-type, culture result, microscopical diagnosis and prevalence in relation to HIV status. Isolation rates of mycobacteria were highest in blood cultures from HIV-positive patients. 10 out of 140 HIV-positive patients had positive mycobacterial culture results. All 10 had positive blood cultures, but only three of them were positive for M. tuberculosis.


Salmonella enterica: infection, cross infection and persistence within the environment of a broiler parent stock unit in Denmark.

A total of three hundred and ninety nine isolates of Salmonella enterica were recovered from 3106 cloacal swab cultures, chicken carcasses and environmental samples taken from a broiler breeding farm over six successive generations. S. enterica serovars Berta (S. Berta), 4,12:b:-, Taksony, Binza and Typhimurium were identified. One hundred and seventy one strains of S. Berta were analysed by plasmid profiling to determine the epidemiological relationships between isolates from each generation and from each house section of the production unit. All isolates contained plasmids; plasmids of eleven different molecular weights were identified. Eighteen different plasmid profiles were identified throughout the observation period. An increase in the diversity of plasmid profiles in each successive generation, the restriction of particular plasmids to particular houses and their persistence within that house were taken to indicate that contamination within the unit was due to persistence and cross-infection rather than to a reintroduction of serovar Berta into new generations of stock.


Identification of aerobactin genes in clinical isolates of E. coli using a non-radioactive DNA probe.

A digoxigenin-labelled gene probe was used for the identification of aerobactin genes in 21 E. coli strains from clinical isolates by means of a colony hybridization test. The results were compared with those obtained by previous hybridization experiments with radiolabelled DNA probes as well as with a crossfeeding-assay. It could be demonstrated that only after an additional proteinase K treatment of the filters before hybridization and after a repeated blocking during the immunological detection procedure, specific hybridization signals could be observed.


Polymorphism of outer surface proteins of Borrelia burgdorferi as a tool for classification.

A total of 23 isolates of Borrelia burgdorferi were characterized by SDS-PAGE and immunoblot analysis. One isolate came from the CSF of a Lyme neuro-borreliosis patient in Valais (Switzerland) and 22 were tick isolates (2 from I. dammini of Shelter Island, USA and 20 from I. ricinus of Valais, Switzerland). Based on the electrophoretic mobility of outer surface proteins (OspA and OspB), four groups of B. burgdorferi could be defined. Group I isolates possess an OspA of 31 KD and an OspB of 34 KD. The group II isolate showed an OspA of 32 KD and OspB of 35 KD. Group III isolates have a 33 KD OspA and group IV a 33.5 KD OspA. This classification was confirmed by the reactivity of a monoclonal antibody (D6) to a 12 KD antigen that was recognized in group III only. A Lyme patient's serum showed a 2-band pattern (10 and 13 KD) for group I and a one-band pattern (12 KD) for the other 3 groups. Therefore OspA, OspB and other proteins of low molecular weight (10, 12, and 13 KD) seem to be important keys for the classification of B. burgdorferi isolates. This typing system correlates with genetic analysis.


Lack of immunoglobulin A1 protease production by Haemophilus ducreyi.

A collection of 20 strains of Haemophilus ducreyi including freshly isolated, low-passage and multi-passage reference strains was examined for immunoglobulin A1 protease production by SDS-PAGE and immunoblotting. None of the strains demonstrated IgA1 protease activity despite the fact that different culture media were used. By direct immunofluorescence testing, binding of IgA to Haemophilus ducreyi organisms could be demonstrated.


Monoclonal antibody based capture ELISA/ELIFA for the detection of Chlamydia psittaci in veterinary clinical specimens.

A capture ELISA/ELIFA system based on monoclonal capture and biotinylated monoclonal detection antibody with specificity for an epitope on chlamydial lipopolysaccharide (LPS) is described. The assay is fast, specific and detects a minimum dose of approximately 2000 chlamydial particles (C. psittaci). Unlike other commercially available test kits, it comprises a proteinase K treatment to exclude non-specific binding of antibodies to Fc receptors. In contrast to the sophisticated and cumbersome isolation procedures, even non-specialized laboratories may use this assay system for the investigation of clinical samples of different origin for the presence of C. psittaci within a short period of time. Due to the genus specificity of the monoclonal antibody used C. trachomatis and C. pneumoniae can be detected as well.


Detection of Mycoplasma contamination in primary calf kidney cell cultures.

A total of 31 primary cell culture preparations of calf kidney including their processing stages (kidney, washing fluid, cell suspension, cell culture monolayer) were investigated for mycoplasmas by cultural methods. Mycoplasma (M.) arginini, was isolated from 8 out of 27 investigated samples of calf kidney from 3 out of 26 washing fluids, 5 out of 20 cell suspensions, and from 9 out of 21 cell culture monolayers. Furthermore, M. arginini was repeatedly found in throat swabs of the cell laboratory technicians. The results give indications as to the source and route of mycoplasma infections of primary cell cultures.


Pertussis whole cell vaccine: relation between intracerebral protection in mice and antibody response to pertussis toxin, filamentous hemagglutinin and adenylate cyclase.

N:NIH mice were vaccinated according to the WHO recommendations for the potency test with the Second International Standard for Pertussis Vaccine (ISPV). Blood for serological investigation was taken from the animals on day 14 post immunization before intracerebral challenge with Bordetella pertussis 18323 was done. The relationship between anti-pertussis toxin, anti-filamentous hemagglutinin and anti-adenylate cyclase antibody levels as measured by ELISA and protection from intracerebral challenge was studied. The proportion of surviving mice increased in correlation with increasing anti-PT titres; a protective level of 4 ELISA units/ml was found. Such relationship between protection against intracerebral challenge and antibody titres was not found for anti-FHA nor for anti-AC antibodies, thus suggesting that these antibodies do not play an important role in protection in this model. The excellent correlation between anti-PT antibody titres and protection suggests that the measure of anti-PT response could be a useful tool for estimating the potency of whole-cell vaccines. The development of an alternative method for testing the potency of pertussis whole-cell vaccines based on the anti-PT response should be considered.


Urinary tract infection due to a mucoid (M) form of Salmonella. A "new" transformation from M form into T1 form.

An eighty-year-old patient suffering from prostatic hypertrophy developed cystitis associated with fever, macrohematuria and significant bacteriuria. In urine cultures, growth of a mucoid (M) form of Salmonella was seen which changed into a T1 form after having been stored at room temperature or passed through U tubes. While the M form did not agglutinate in Salmonella O and H antisera, H antigens of the T1 form could be identified as l,v and 1.7. The isolate was therefore designated S.I M----T1:l,v:1.7. To date, no such M-T1 variation has been described. For diagnostic and epidemiologic purposes Salmonella M forms should be transformed into the N form or a T form (as in our case), because it is possible to demonstrate O or T and H antigens in these forms. The method of transformation and the pathogenesis of urinary excretion of salmonellae are briefly described.


A simplified agarose gel electrophoresis for rotavirus detection.

The use of agarose gel electrophoresis for rotavirus detection is a suitable routine method if simplifying modifications are introduced. Incorporation of ethidium bromide into the agarose gel permits its use for several times in the course of one week without further staining of the RNA segments. The electrophoresis chamber with a UV-light-transparent bottom is put directly onto the transilluminator and the RNA bands of the rotavirus can be visualized without manipulating the gel tray. During the winter of 1990/91, we found a good sensitivity (98%) of this method in comparison to a latex agglutination test (Slidex Rota-Kit 2, bio Mérieux).


A 41.7 kDa serine protease from Clostridium perfringens type A: degradation of purified human serum proteins.

Two clinical isolates of Clostridium perfringens type A produced a novel caseinolytic serine protease. Both enzymes had a molecular weight of 41.7 kilodaltons and an isoelectric point of 9.1. The two enzymes were immunogenic for rabbits and closely related serologically. Both enzymes partially degraded the heavy chains of human immunoglobulins (Ig) G and IgM, but not IgA. Purified human complement (C) components C3, C5, C8, and C9 were attacked; C1q was refractory. Both enzymes were active against human transferrin, alpha 1-antitrypsin, alpha 2-macroglobulin, haptoglobin, type III fibrinogen, and fibronectin. C-reactive protein was refractory.


Optimization of culture conditions for toxin production of type G Clostridium botulinum.

Culture conditions were optimized for toxin production of Clostridium botulinum type G, the last toxigenic serotype described. Six factorial experiments were performed to assess the effect of medium composition (nutrients, metal ions, sterile soil, pH), incubation conditions (time, temperature and anaerobiosis) and associated microorganisms (Bacillus subtilis, Lactobacillus plantarum) on the toxinogenesis of C. botulinum type G. A significant (4 to 10 fold) improvement of toxin production was obtained by using an optimized medium (3% proteose-peptone, 0.5% trypticase, 1.1% glucose, 0.5% yeast extract, adjusted to pH 8.0) and incubation of the culture for 12 days at 26 degrees C in a nitrogen atmosphere.


A sensitive method for the detection of enterotoxigenic Escherichia coli by the polymerase chain reaction using multiple primer pairs.

In this study, a polymerase chain reaction (PCR) method has been developed for the detection of enterotoxigenic Escherichia coli (ETEC). Three different sets of oligonucleotide primers synthesized were used to amplify the enterotoxin genes of heat-labile (LTh) and heat-stable (STIa and STIb) enterotoxins of ETEC. These primers amplified a 627, 240, or 169 base pair (bp) DNA fragment from LTh, STIa and STIb gene, respectively, of the reference ETEC strains. The addition of RNase A (10 micrograms/ml) to the PCR reaction solution diminished nonspecific amplification of DNA fragments other than the enterotoxin genes. Five types of ETEC strains corresponding to the LTh, STIa, STIb, LTh-STIa, or LTh-STIb genotypes were distinguished by a single procedure of PCR using the mixture of the three sets of primers. PCR, hybridization, and conventional methods were subjected to one hundred stool specimens from diarrheal patients. It was found that PCR was the most sensitive method among them. These results suggested that PCR with triple primer pairs would be useful for the laboratory diagnosis of ETEC in the stool specimens.


Subtyping of Legionella pneumophila serogroup 1 strains isolated in Germany using monoclonal antibodies.

Legionella pneumophila serogroup 1 (Lp1) strains isolated from patients and hot water supplies in different locations of Germany were subtyped using seven monoclonal antibodies (mabs) in the indirect immunofluorescence test (IFA) and in part, using a dot blot assay. Four of these mabs were produced in Dresden. Three mabs (mab 33G3, mab 32A12 and mab 144c2) were kindly supplied by J. Joly, Quebec, Canada. Altogether, seven antigenic variants were found among Lp1 strains isolated in Germany. Patient strains belonged to the Philadelphia, Benidorm, Knoxville, France, Olda-Heysham and Bellingham subgroups, whereas environmental isolates reacted like the Bellingham, Oxford, Philadelphia, Knoxville and France strains. The majority of patient strains (15 out of 26, 58%) reacted with our mab 3/1 (corresponding to mab 2 of the standard panel), but only 26 out of 118 environmental strains (22%) isolated from 4 of 15 hot water supplies did so (p less than 0.05). The majority of water-borne Lp1 strains reacted with a mab specific of the Bellingham subgroup. Three water systems under study were associated with human legionellosis. Two of them contained Bellingham-like strains, one Philadelphia-like legionellae.


Indirect haemagglutination test for the serodiagnosis of brucellosis using stabilised sheep red blood cells.

Sheep red blood cells (SRBCs) stabilised with formaldehyde, glutaraldehyde, pyruvic aldehyde and double aldehyde were evaluated, in the indirect haemagglutination (IHA) test, for their suitability for the serodiagnosis of brucellosis in bovines. Serum samples from 23 standard tube agglutination test (STAT)-positive and 12 clinically suspected but STAT-negative cows were titrated by the IHA test. Double aldehyde and glutaraldehyde-stabilised SRBCs were found to be more sensitive in the IHA than the formaldehyde or pyruvic aldehyde-stabilised SRBCs. Double aldehyde and glutaraldehyde treatments enabled the IHA to clearly differentiate between the normal and the diseased cows. Double aldehyde-stabilised cells have an edge over the glutaraldehyde-stabilised SRBCs because the former treatment makes the SRBCs readily available for antigen coating without tannic acid activation.


Is Escherichia coli invading tubuloepithelial cells?

The adhesion of Escherichia coli to host epithelium cells is the very first step of urinary tract infections followed by the internalization of the bacteria into these cells. These steps are influenced by several surface antigens or products of the pathogen, e.g. fimbriae or adhesins, K antigen, and hemolysin. The bacterial adherence and the internalization of several isogenic E. coli O18 strains differing in the expression of K5 antigen, hemolysin, and type of fimbriae were measured by using a permanent line of porcine tubuloepithelial cells (LLC-PK1). Strains with K5 antigen were reduced in their adherence and internalizability as compared to the K-negative strains. The expression of hemolysin by these strains lead to an increase of adherence and internalization. The internalization of bacteria is influenced mainly by their adherence to the epithelial cells. Thus, the engorgement of attached bacteria is rather a kind of endocytosis than an invasion of bacteria.


In vitro and in vivo activities of KRM-1648, a newly synthesized benzoxazinorifamycin, against Mycobacterium marinum.

The in vitro and in vivo activities of KRM-1648, one of the newly synthesized benzoxazinorifamycins, against M. marinum were compared with those of rifampin. The MIC values of KRM-1648 determined by the agar dilution method on 7H11 medium against 10 strains of M. marinum were 32-128 times and even more below those of rifampin. In an in vivo experiment, KRM-1648 was markedly effective in terms of the incidence of gross skin lesions and the number of CFUs in the lungs and spleen. Its efficacy was much greater than that of rifampin.


The clinical significance of Acinetobacter baumannii in blood cultures.

Acinetobacter baumannii was isolated from blood cultures in 31 patients in 4 intensive care units at a large teaching hospital over a period of 6 months. In 23 of these patients, Acinetobacter was also isolated from tracheal aspirates. Catheter-related infection could be identified as the cause of bacteremia in 14 cases, whereas pneumonia was present in only 6 cases. All patients were seriously ill and 7 (24%) died of their infection.


Heat stability of parvovirus B19: kinetics of inactivation.

Heat inactivation of parvovirus B19 (B19) was studied in a culture of hematopoietic progenitor cells generated in vitro from peripheral human blood. After inoculating cell cultures with identical volumes of plasma (MII) containing B19 (B19-MII) heat-treated (60 degrees C) for various periods of time, a time-dependent inactivation of the input virus was determined by a decrease of viral DNA replication. No B19 DNA was detected after infection with B19-MII heat-treated for 20 min or more by Southern blot. Viral B19 protein production decreased time-dependently and was not detected after infection with samples treated for 12 min at 60 degrees C or more determined by the enzyme immunoassay. This study indicates that infectivity of B19 virus in plasma can be reduced in vitro by heat-treatment (60 degrees C). However, this does not mean that the heat treatment completely inactivated B19 virus.


Epidemiology and ecology of tick-borne encephalitis in the eastern part of Germany between 1960 and 1990 and studies on the dynamics of a natural focus of tick-borne encephalitis.

From 1960 to 1990, attempts to isolate TBE virus from ticks and small mammals were made and investigations to detect TBE antibody in small mammals, game and humans were performed in the five new federal Länder of Germany. The confirmed TBE cases for which the site of exposure could be determined were also registered. As a result of these epidemiological and ecological investigations, a map is presented showing the natural foci of TBE which are primarily located in the subatlantic and mountainous climatic regions. TBE was endemic in the area of investigation from 1960 to 1990 showing a morbidity of up to 0.7 per 100,000 inhabitants which decreased in recent years to 0.02 per 100,000 inhabitants. The natural foci of TBE virus in eastern Germany showed a high activity between 1960 and 1970. Since that time, there have hardly been any cases of human disease and TBE virus could no longer be detected, neither in ticks nor in small mammals. Taking the natural focus on the Island of Usedom as an example, attempts have been made to elucidate whether such foci have become extinct or whether they have persisted. From 1983 to 1989, a surveillance programme was performed to detect antibodies to TBE virus in small mammals and game and to attempt to culture virus from ticks and small mammals. The attempts to isolate virus from a total of 8200 ticks were negative. Attempts to isolate virus from the brains of small mammals were also negative. Antibody prevalence in 446 small mammals and 500 animals of game was ca. 1%. At one site, the sero-positive reactions converted from 0% (1983-1988) to 4.5% (1989) among small mammals population. The investigations performed on the Island of Usedom have shown that this natural focus has not disappeared but is in a state of endemic latency. Moreover, the seroconversion observed in the small mammals population shows that further surveillance of such foci is necessary. This becomes obvious by the sporadic occurrence of single TBE cases as well as by a low antibody prevalence of 1% in small mammals and game. The epidemiological situation in eastern Germany is thus completely different from that in western Germany where an average of 70-120 TBE cases per year are registered, occurring mainly in Bavaria and Baden-Württemberg.


Studies on glycolytic enzymes of Plasmodium knowlesi, a simian malaria parasite, and infected host erythrocytes.

Almost all of the important enzymes of glycolytic sequence were studied in isolated schizonts of Plasmodium knowlesi as well as in normal and P. knowlesi parasitized rhesus red blood cells. Significant activities of all the glycolytic enzymes assayed were observed in cell-free schizonts and the levels of all were found to be considerably elevated in infected host erythrocytes, confirming that the consumption of glucose is dramatically increased in host red cells, consequent to malaria infection.


Phage susceptibility, enterotoxigenicity and antibiograms of Staphylococcus aureus strains isolated from human wounds and diarrhoea.

The phage types, enterotoxigenicity and antibiograms of Staphylococcus aureus strains isolated from human diarrhoea and skin wounds in Nigeria were determined. Of 194 strains tested, 140 (72.2%) were typable using a combination of phages in the International Phage Set (IPS) for human strains of S. aureus and the bovine phage set. IPS phages lysed 118 (60.8%) strains while 96 (49.5%) were susceptible to bovine phages. S. aureus strains from adult diarrhoea were significantly (P less than or equal to 0.01; x2) more sensitive to bovine phages (52.8%) than to IPS human phages (16.7%). Strains isolated from wounds were however significantly (P less than or equal to 0.001; x2) more susceptible to IPS human phages (72.9%) than to bovine phages (41.5%). Phage group III strains were predominant amongst diarrhoeal isolates while wound strains were most susceptible to group II phages. Phage 119, a bovine phage, lysed all 19 strains of diarrhoeal origin sensitive to group M phages but all wound strains were resistant. Overall, 132 (68.0%) strains were enterotoxigenic producing staphylococcal enterotoxins A (SEA), B (SEB), C (SEC) or a combination of these. A majority of diarrhoeal strains elaborated SEC while SEB production was predominant amongst wound strains. Of the eight antimicrobial agents used, S. aureus strains were most resistant to penicillin (71.6%) and least to sulfamethoxazole/trimethoprim (1.0%). Occurrence of resistance to one or more antibiotics was higher amongst wound strains (97.5%) than amongst strains isolated from diarrhoea (52.6%). It was concluded that bovine phage 119 may be a useful epidemiologic marker for S. aureus strains of bovine origin associated with human diarrhoea in Nigeria. It is however difficult to ascribe any aetiological significance to these strains because other enteropathogens not assayed for may have been present.


Serotyping and further characterization of group B streptococcal isolates from Indonesia.

Serotyping of 103 streptococci of serological group B isolated from human clinical specimens in Indonesia revealed mainly type pattern Ia/c and type patterns III and V, either alone or in combination with protein antigens c or R. Few cultures had serotype II and IV, none of the cultures had type antigen Ib or X. Extracts of one group B streptococcal culture reacted with antibodies specific to type antigens II, IV and R and one culture with antibodies specific to candidate type 7271. Protein antigen c was present as c alpha, c alpha, beta but not as c beta component. Group B streptococci with type antigen R grew generally as a granular sediment with clear supernatant, formed compact colonies in soft agar and reacted positively in the salt aggregation test. Group B streptococci without surface antigen R grew mainly with a uniform turbidity of the growth media, formed diffuse colonies in soft agar and were negative in the salt aggregation test.


Postoperative wound infections complicating adult spine surgery.

A review of 19 consecutive patients treated for deep wound infections after spinal surgery was performed. Patients were analyzed for preoperative risk factors, original diagnosis requiring surgery, onset of infection, presentation, treatment, and outcome. These patients (10 men, nine women), with a mean age of 44 years (range 18-74) were treated for 13 deep and six subcutaneous infections: 16 spine infections and nine graft site infections (six with both). All 19 presented with draining wounds on an average of 17 days after surgery (range 4-86). Staphylococcus aureus was cultured in 14. Patients were treated with i.v. antibiotics for an average of 3 weeks (range 0-14) followed by oral antibiotics for an average of 6 weeks (range 2-12). Surgical treatment included an average of 1.8 debridements (range 0-5), primary closure in two, delayed primary closure in seven, and healing by secondary intent in 10. All but three patients were noted to be malnourished at the onset of their infection, with a total lymphocyte count of less than 2,000. Three patients required removal of their hardware. Eighteen of 19 healed or their infections were stabilized, 17 of 18 arthrodeses fused, and no patient had neurological deterioration secondary to the infection. The treatment outlined produced satisfactory results in all but one patient. We conclude that postoperative spine infections are frequently associated with poor nutrition. Although definitive diagnosis is determined by the culture, clinical exam is helpful in establishing a presumptive diagnosis and, thus, earlier institution of surgical and antibiotic treatment.


Tuberculosis of the spine: a review of 236 operated cases in an underdeveloped region from 1954 to 1964.

Antituberculous medication for treatment of tuberculosis has been used for nearly a half century. However, tuberculosis of the spine still shows up from time to time in the developed countries and it is still a common scourge in half of the world today. The author reviews his treatment of tuberculosis of the spine over 30 years' full-time work in developing countries. He gives an analytical breakdown of his first 10 years of operating on 236 cases, both paralytic and nonparalytic. Changes in treatment developed with experience. Posterior spinal fusion gave good results. Radical evacuation of the abscess alone gave poor results. Best results were obtained in the combination of the two. This surgery provided a stable, pain-free spine and shortened period of time that antituberculous medication was required. A true lateral approach for spinal decompression was developed and is described. This procedure can be performed by ordinary orthopedic teams in developing countries. It is safer than the transthoracic approach and gives better exposure.


Immediate postoperative stability of the atlantoaxial articulation: a biomechanical study comparing simple midline wiring, and the Gallie and Brooks procedures.

A biomechanical study was undertaken to quantitate the immediate postoperative stability of the atlantoaxial articulation after stabilization by simple midline wiring, as well as the Gallie and Brooks procedures. Fresh adult cadaveric cervical spines were mounted for testing such that only the C1-2 motion segment and the load deflection curves generated in flexion, extension, and torsion moved. Spines were tested after the creation of a fracture at the base of the odontoid process with transection of the transverse ligament and after stabilization by each of the surgical techniques. Mean apparent stiffness values evaluated the biomechanical properties of these three commonly performed procedures. Although all tested procedures restored supraphysiologic mechanical properties in all modes tested (p less than 0.001), the Brooks technique was stiffer than the other two methods (p less than 0.001). Although many factors must be considered when selecting a particular surgical technique, the biomechanical data obtained in this study may be of assistance to the surgeon in choosing the type of construct for upper cervical stabilization and arthrodesis.


The thermoplastic Minerva body jacket: a clinical comparison with other cervical spine splinting techniques.

A retrospective analysis of the efficacy of a variety of external stabilization techniques used in 155 cases of unstable cervical spine injuries is presented. The movement at each intervertebral level was evaluated during thermoplastic Minerva body jacket stabilization in 18 additional patients. Many currently available approaches to external stabilization of the cervical spine were, thus, assessed. Thermoplastic Minerva body jacket stabilization offered superior segmental immobilization compared with published data for the halo. It is concluded that some unstable injuries to the high cervical spine might best be treated with a halo device, whereas mid to low cervical injuries and the remaining upper cervical spine injuries appear to be optimally treated with a Minerva jacket. Lesser injuries may be treated with a variety of available orthoses. The thermoplastic Minerva body jacket offers a superior limitation of intervertebral movement compared with other commonly used braces, including the halo jacket, for most cervical spine injuries. The technique of application of the thermoplastic Minerva body jacket is reviewed.


Noncontiguous injuries of the spine.

A total of 372 consecutive spinal injury patients were evaluated at the Regional Spinal Cord Injury Center of Delaware Valley. Of these, 39 patients (10.5%) were found to have noncontiguous spinal column injuries. Fewer than half of the patients in our series could be classified into previous classification systems (Calenoff, Gupta) of noncontiguous spinal fractures. Fifteen fractures in 12 patients were missed on presentation on admission and 25% of these patients had a progressive neurologic deficit as a result of improper initial immobilization. The location of missed fractures were found to be primarily at the extremes or junctures of the spine (i.e., cervicothoracic, thoracolumbar). Complete spinal roentgenographic evaluation is recommended in the workup of suspected spinal column injury patients, and additional imaging modalities (i.e., tomograms, computed tomography scans, and magnetic resonance imaging) may be necessary in those areas of the spine difficult to visualize.


Anterior transpedicular instrumentation of the lumbar spine: an anatomical study.

Lumbar burst fractures or massive tumorous destruction of the lumbar spine generally require a combined anterior-posterior surgical approach for adequate decompression and stabilization. Anatomical studies in 10 adult cadavers with 100 pedicle screw placements from a single strictly anterior approach showed that this new method represents a safe and promising way for stable lumbar spine instrumentation, avoiding the risks of a bilateral approach. The technique provides a stability comparable with bilateral instrumentation. Exact knowledge of the geometry of the corresponding vertebral bodies and the spinal canal by computed tomography scanning is a prerequisite for this surgical technique. First clinical applications using this technique reconfirm the experiences of the anatomical study.


Unstable thoracolumbar and lumbar burst fractures treated with the AO fixateur interne.

Twenty-five consecutive patients with unstable thoracolumbar and lumbar burst fractures were surgically treated with the AO Fixateur Interne (Synthes USA, 1690 Russell Rd., Paoli, PA). Indications for surgery included a progressive neurologic deficit, spinal canal compromise greater than 50%, vertebral body collapse greater than 50%, or sagittal angulation greater than 20 degrees. Twenty males and five females ranging in age from 16 to 60 years (average 31) were treated surgically and prospectively followed. Twenty-one fractures occurred at the thoracolumbar junction (T10-L2) and 4 in the lumbar spine (L3-5). Twenty-four patients were followed for a minimum of 12 months, ranging from 12 to 38 months (average 22); one patient was lost to follow-up after 1 month postoperatively. Preoperatively, 12 patients had partial neurologic deficits; postoperatively, 11 improved at least 1 Frankel grade. Preoperatively, the 12 patients with partial neurologic deficits averaged 45 points (range 24-49) on the lower extremity motor index scale. After surgery, these patients improved an average of five points (range 1-23) on the motor index scale. Both patients with complete spinal cord injuries remained unchanged neurologically postoperatively; no patients deteriorated neurologically after surgery. The average preoperative sagittal kyphosis at the fracture site was +16 degrees (range +10 degrees to +31 degrees); the immediate postoperative sagittal angular correction averaged -4 degrees (lordosis) and ranged from +12 degrees (kyphosis) to -26 degrees (lordosis). At last follow-up, the sagittal angular correction remained unchanged in three patients and decreased in 21 patients to an average of +5 degrees (range +37 degrees to -14 degrees).(ABSTRACT TRUNCATED AT 250 WORDS)


Cervical spondylolysis.

The term cervical spondylolysis describes a long-standing, perhaps congenital defect of the pars interarticularis of a cervical vertebra. We report 10 new cases of cervical spondylolysis and review the literature. All patients in this report were treated nonoperatively with subsequent symptomatic improvement. Cervical spondylolysis must be differentiated from its traumatic counterparts radiographically. Characteristic radiographic findings include well-corticated margins at the defect, a characteristic "bow tie" deformity, and ipsilateral dysplastic facets. Compensatory hypertrophic changes of the adjacent articular processes, spina bifida, and spondylolisthesis are frequently, but not always, seen in conjunction with cervical spondylolysis. The vast majority of patients with radiographically proven cervical spondylolysis can be treated confidently with conservative measures. Surgical intervention should be reserved for those who fail nonoperative management or who exhibit neurologic compromise referable to an unstable spondylolytic defect.


Esophageal perforation after anterior cervical plate fixation: a report of two cases.

We report two cases of esophageal complications due to migration of screws after anterior plate fixation of the cervical spine. Close observation for screw back out is needed, particularly in those patients with associated neurologic deficits or osteoporosis.


Central retinal artery occlusion: a complication of the knee-chest position.

A rare complication of the knee-chest position used for lumbar disk surgery is reported. Central retinal artery occlusion resulted in permanent loss of vision secondary to direct ocular pressure. Prevention is the only rational treatment for this unfortunate condition.


Extraosseous epidural tumor of immunoglobulin D myeloma.

We report a rare case of an immunoglobulin D (IgD) myeloma mass that occupied the lumbar epidural space. The patient presented with paraparesis combined with an extremely high concentration of serum IgD, and azotemia. A complete remission was obtained after combination chemotherapy, but the patient died from an epidural tumor in the high thoracic region 19 months after the onset of symptoms.


Torulopsis glabrata vertebral osteomyelitis.

A case of Torulopsis glabrata osteomyelitis of the thoracic spine producing spinal cord compression and myelopathy is reported. The patient displayed several of the predisposing factors to T. glabrata infection, including diabetes mellitus, a history of abdominal surgery, and intravenous catheterization with hyperalimentation. The patient was successfully managed with surgical decompression, debridement, and curettage of the affected bone, postoperative immobilization, and systemic amphotericin B therapy. Her pain was relieved, spinal cord function recovered, and the vertebrae healed uneventfully. T. glabrata osteomyelitis is a rare and unpredictably progressive infection. This case demonstrates that it requires prompt therapy and close observation.


Male-mediated teratogenesis and other reproductive effects: biologic and epidemiologic findings and a plea for clinical research.

This paper reviews biologic and epidemiologic evidence that prefertilization and perifertilization exposures to fathers influence a variety of reproductive outcomes, including fertilization, miscarriage, low birth weight, congenital anomalies, cancer, and neurodevelopmental and other childhood health problems. Males and females bring an equal number of chromosomes to their progeny, but their genomes may affect different aspects of reproduction. While the key male role principally ends at fertilization, there is growing experimental and human evidence that factors relating both to prefertilization and perifertilization exposure also play a role post fertilization. Some negative human epidemiologic findings reflect the fact that routinely gathered information usually generates detailed descriptions of maternal exposures and does not collect records regarding prefertilization paternal exposures. The absence of extensive human evidence should be interpreted as a deficiency in research rather than an absence of male-mediated adverse reproductive outcomes. More than 60 different compounds or industrial processes have been identified as increasing defects in human sperm and possibly increasing the risk to offspring from male-mediated exposures. Further research needs to include better characterizations of both maternal and paternal prefertilization and perifertilization exposures, in order to assess more accurately their relative effects. Pediatricians confronted with adverse pregnancy and antenatal health outcomes should obtain detailed information on relevant prefertilization exposures of both parents.


Does early exposure to maternal smoking affect future fertility in adult males?

Animal data suggest that prenatal exposure to certain tobacco smoke components such as nicotine may affect the development of the male gonadal axis, which may in turn affect future adult fertility. There are no previous epidemiologic studies on the potential effects of early (prenatal and childhood) exposure to maternal smoking on the reproductive system in adult male offspring. To investigate this question, we used data from a follow-up study of reproductive function and fertility among young adult sons of mothers who had participated in a randomized clinical trial of diethylstilbestrol use during pregnancy. We observed no significant effects of early exposure to maternal smoking on conventional semen characteristics, hormone levels (follicle stimulating hormone [FSH], luteinizing hormone [LH] and testosterone), urogenital abnormalities and diseases, or perceived infertility problems. Current active smoking by the men was, however, associated with a significant decrease in the percentage of sperm with normal morphology.


Caffeine effects on meiotic maturation in hamster oocytes in vitro.

The effect of caffeine on meiotic maturation in cultured hamster oocytes was investigated. Meiotic status was scored from chromatin spreads of oocytes previously exposed to caffeine (0, 0.00017, 0.0017, 0.017, 0.17, 1.7, 2.4, 5.1, and 10.2 mM) for up to 20 h. While concentrations of caffeine less than 0.017 mM failed to affect significantly the onset of meiotic resumption, 0.0017 mM caffeine significantly decreased the proportion of oocytes progressing normally to telophase I-metaphase II, and concomitantly increased the proportion of both diploid MII and aneuploid oocytes. In addition, 0.17 to 10.2 mM caffeine induced a dose-dependent increase in the proportion of meiotically arrested oocytes, with less than 5% oocytes progressing normally through to the final stages of meiotic maturation at 10.2 mM caffeine. Taken together, these data show that caffeine at concentrations as low as 0.0017 to 0.017 mM interfere with progression of meiotic maturation, and that concentrations higher than 0.017 mM delay initiation of this process. Since caffeine peaks at 0.017 mM in the plasma of women following a cup of brewed coffee, we conclude that caffeine-induced perturbations of oocyte meiotic maturation may be responsible, at least in part, for the recently revealed correlation between caffeine intake and reduced fertility in women.


Disruption of female rat vaginal cyclicity by daily treatment with imipramine.

Intact, adult female rats were injected for 21 days with 10 mg/kg of the tricyclic antidepressant imipramine. During this time, there was almost total suppression of reproductive cyclicity as indicated by the absence of vaginal proestrus and by the absence of sexual receptivity. However, there was no evidence for a permanent disturbance in the female reproductive system. When treatment with imipramine was terminated, vaginal cyclicity resumed within the next 2 to 8 days. The generality of the current findings to other antidepressants or to the human female is currently unknown. However, since human females are the primary consumers of antidepressants, caution should be exercised when females are given antidepressant therapy.


Effects of oocyte exposure to local anesthetics on in vitro fertilization and embryo development in the mouse.

The effect on fertilization and development of local anesthetics routinely used during ultrasound-guided oocyte retrieval in women undergoing in vitro fertilization was examined in a mouse in vitro fertilization system. Mouse oocytes were exposed in vitro to lidocaine, chloroprocaine, and bupivacaine at concentrations of 0 (control), 0.01, 0.1, 1.0, 10.0, 100.0 micrograms/mL for 30 min, washed, and then inseminated. In vitro oocyte fertilization at 24 and 48 h and embryo development at 72 h were determined. Bupivacaine adversely affected mouse in vitro fertilization and embryo development only at the highest exposure concentration, 100 micrograms/mL, while lidocaine and chloroprocaine produced adverse effects at concentrations as low as 1.0 and 0.1 microgram/mL, respectively. Furthermore, an adverse dose-related effect on fertilization and embryo development was shown for lidocaine and chloroprocaine, but not for bupivacaine. These data demonstrate that the local anesthetics, lidocaine (L), chloroprocaine (C), and bupivacaine (B), adversely affect mouse in vitro fertilization and embryo development in the order of C greater than L greater than B.


Genotoxic and embryotoxic effects of gonadotropin-hyperstimulated ovulation of murine oocytes, preimplantation embryos, and term fetuses.

Compared to spontaneous ovulation, gonadotropin-hyperstimulated ovulation (superovulation) in mice resulted in a fourfold increase in the number of preimplantation embryos 3 days post coitum, 50% of which died before term. Both in vitro development of embryos during the preimplantation period and transfer of morulae from superovulated females to pseudopregnant untreated foster mothers indicate that the prenatal loss occurring shortly before implantation up to term is due to maternal factors rather than to direct hormonal effects on oocytes or early embryos. Indeed, no genotoxic events could be observed in 4-cell to blastocyst stage embryos from superovulated female mice as revealed by the chromosomal aberration test and the sister chromatid exchange assay. Chromosome analysis of the pronuclei from mouse zygotes showed an increased rate of aberrations in oocyte-derived nuclei after superovulation in comparison to spontaneous ovulation. The present data suggest that aberrant murine oocytes may be fertilized, but they do not survive the first cleavage stages. The result is discussed with respect to the high incidence of chromosomal abnormalities found in human oocytes after gonadotropin-hyperstimulated ovulation.


Alterations in hypothalamic and pituitary hormone levels induced by neonatal treatment of female mice with diethylstilbestrol.

Neonatal female mice of the NMRI strain were treated with the synthetic estrogen diethylstilbestrol (DES), 5 micrograms daily for the first 5 days after birth, or with vehicle only. Levels of LH and FSH (pituitary and serum) and LHRH (hypothalamus) were measured by radioimmunoassay (RIA) in 6- to 56-day-old females with 6- to 7-day intervals. Compared to controls, DES-treated females had low levels of LH on days 6, 12, and 21 in the pituitary, and on days 6 and 12 in serum; increased LH levels were seen in both the pituitary and serum on day 42. The FSH levels of DES-treated females were decreased on days 6 and 12 in the pituitary and on day 6 in serum; an increased FSH content occurred on day 21 in the pituitary. In the preoptic area and basal hypothalamus of DES-treated females, levels of LHRH were increased on day 21 and decreased on day 42. On day 56, the serum levels of FSH and LH and the hypothalamic content of LHRH were similar in controls and DES females. A second study including both synthetic and natural estrogen was performed in 12-day-old females. Treatment with 10(-2) micrograms DES or lower doses or 5 micrograms estradiol-17 beta (E2) on days 1 to 5 after birth had no depressive effect on serum LH. The hypothalamic-pituitary-ovarian feedback system reacted similarly to ovariectomy and E2 challenge in 15-day-old control and DES-treated females. DES-treated 56-day-old females had a reduced LH response to ovariectomy but increased response to exogenous E2 compared to controls. These results show that neonatal treatment with DES has pronounced effects on the hypothalamic-pituitary system in the developing female mouse, which may be of importance for the altered ovarian function in these females as adults.


Reproductive toxicity of theobromine and cocoa extract in male rats.

The toxicities of theobromine and cocoa extract on the reproductive tract of male rats were compared in the present study. A cocoa powder extract containing 117 mg theobromine/g extract was prepared using 85% boiling methanol. Sprague-Dawley rats were weighed and dosed daily for 31 days with vehicle, 250 mg/kg theobromine, 2.14 g/kg cocoa extract (117 mg theobromine/g extract), or 0.43 g/kg cocoa extract by oral gavage. The animals were sacrificed on day 32. One testis and epididymis were removed and weighed. The epididymis was saved for the determination of epididymal sperm reserves. The remaining testis was fixed by whole body glutaraldehyde perfusion and processed for morphologic examination. A decrease in body weight gain and epididymal weights were observed in theobromine and high-dose cocoa-extract-treated groups. Theobromine and high-dose cocoa extract caused vacuolation within the Sertoli cell, abnormally shaped spermatids, and failed release of late spermatids in treated animals. Most of the vacuolations were found in the earlier and middle stage seminiferous tubules (stages I to VIII). However, the frequency of some parameters of testis alterations were significantly lower in the high-dose cocoa-extract-treated group compared to the theobromine-treated group. These data demonstrate the ability of a cocoa extract containing theobromine to alter testis structure in a similar pattern but with reduced intensity compared to that observed after oral exposure to pure theobromine.


Testicular toxicity of methylmercury: analysis of cellular distribution pattern at different stages of the seminiferous epithelium.

Stage-specific distribution of methylmercury (MM) and spermatogenic changes were analyzed in rats administered 5 or 10 micrograms MM/kg, ip, daily for 15, 30, 60, and 90 days. MM deposition, as grain number/cm2 was noted in basal portions at later stages on day 15, which increased gradually by day 90. MM deposition was in the order of stages IV, VII, XIV, IX, being higher in adluminal portions on days 30 and 60. MM-enriched cytoplasmic masses leaked out through disintegrated tubular membrane on days 60 and 90. Epithelial damage, at stages late XIV through IV, V through VI, VII through VIII, XIII through mid-XIV, and IX through XII, accorded with the gradual deposition of MM. As profound cell death occurred between zygotenes to pachytenes and dividing spermatocytes to step 1 spermatids, the spermatids were conspicuously decreased at later times. It is possible that MM distorts the barrier system at stages IX through XII, gets distributed within the tubule, and hence may pose a direct or Sertoli cell mediated effect at stages XII through early XIV in a dose-duration-MM burden related manner.


Quinone reductase activity in the first trimester placenta: effect of cigarette smoking and polycyclic aromatic hydrocarbons.

Quinone reductase (QR) is considered a major protective enzyme against cancer in the organism. In this study, the activity of QR was measured in first trimester placental tissue using colorimetric techniques. There were no significant differences between the mean enzyme activity of women who smoked more than 20 cigarettes per day during pregnancy and of nonsmokers (0.50 +/- 0.09 compared with 0.51 +/- 0.15 mumol/mg protein/10 min, respectively). Among the polycyclic aromatic hydrocarbons (PAH) studied, dimethyl benzanthracene (DMBA) increased QR activity in a dose-dependent manner in the first trimester placental explants at the 10- to 100-microM range after 6 h of incubation (440% increase) with the highest concentration. The effect of other PAH of different potency added at 50-microM concentrations showed that benz(a)anthracene (BA), dibenzo(a,h)anthracene (DBHA), dibenzo(a,c)anthracene (DBCA), or chrysene (CH) caused a significant 2- to 3-fold increase in the enzymatic activity after 6 h of incubation. At 24 h 50-microM DBCA effect was also stimulatory, while the 10-microM DMBA effect almost reached statistical significance. However, no differences were encountered in the response of placental tissues to PAH between cigarette smokers and nonsmokers at 6 and 24 h. The present data indicate that placental QR activity is increased by exposure to PAH in vitro, but it does not appear to be affected by in vivo exposure to cigarette smoking. Thus, the early placenta appears to have a significant potential to inactivate carcinogens/mutagens locally, thereby limiting their transfer to the embryo.


Embryotoxicity of 25 psychotropic drugs: a study using CHEST.

Twenty five psychotropic drugs were ranked according to the embryotoxicity dose ranges estimated by the Chick Embryotoxicity Screening Test (CHEST). The chick results were compared with some data for common laboratory mammals. In 17 psychotropic drugs a deleterious dose-dependent effect upon the embryonic cardiovascular system was disclosed, terminating in immediate cardiac arrest.


[The role of genetic factors in the development of ischemic heart disease in patients with chronic nonspecific lung diseases].

The investigations of the clinico-genetic factors in chronic bronchitis sufferers with phenotype A revealed atherogenic changes in lipoprotein spectrum and suppression of plasma fibrinolytic activity. It is suggested that chronic staphylococcal infection may contribute to high coagulating blood properties in relevant patients.


[Clinico-morphological characteristics of intrahepatic cholestasis in hepatitis A and delta infection].

Clinical and morphological characteristics were studied for a course of acute viral hepatitis A and delta with long-term cholestatic syndrome in asymptomatic carriers of HBsAg. 149 patients with moderate disease were examined, 53 of them had long-standing cholestasis syndrome. 96 viral hepatitis patients free of cholestasis served control. It is believed that the emergence of long-term intrahepatic cholestasis may be due to the following mechanisms of sinusoid lumina as a result of a drastic enlargement of multinuclear hepatocytes, hyperplasia and hypertrophy of Kupffer's cells. impaired binding of bilirubin in zone III of hepatic acinus. In cholestatic form of hepatitis A there occurs more pronounced reduction of sinusoid volume compared to other variants of viral hepatitis run.


[Vertebrogenic lesions of the peripheral nervous system in pregnant women and puerperae].

Neurologic appearance of spinal osteochondrosis was assessed in 112 pregnant women and puerperae . In 75 females osteochondrosis involved the lumbosacral and in 37 ones cervical-thoracic regions. In the majority of the patients (79%) vertebrogenic lesions manifested with reflex syndromes. Provoking factors and the clinical pattern of the disease are considered with special emphasis on the role of pregnancy and labour as promoters of pain recurrences and the onset of new symptoms in women with neurological picture of osteochondrosis.


[Effect of psychopathological disorders in patients with unstable stenocardia on serum lipids and the indicators of lipid peroxidation].

A total of 193 males suffering from unstable angina pectoris (UAP) were examined against 20 control subjects with stable angina pectoris (SAP) for the serum levels of lipids and activity of lipid peroxidation. The parameters studied varied between the groups. 152 UAP patients exhibited psychopathological disturbances, mainly agitated melancholia. These patients (78.8%) were noticed to have atherogenic shifts in serum lipids and lipid peroxidation products levels increased as compared to those without psychoemotional problems. It was found that in UAP psychopathologic disturbances contribute to the progress of coronary insufficiency this justifying the use of psychotherapy in combination with drugs in UAP patients.


Increased cotinine elimination and cotinine-N-oxide formation by phenobarbital induction in rat and mouse.

The metabolic fate of cotinine, the major metabolite of nicotine, was studied in phenobarbital-induced and non-induced isolated perfused rat lung and liver and in isolated hepatocytes of rats and mice. The non-induced lung tissue showed low cotinine metabolizing capacity while the perfused liver was approximately four times more active. After phenobarbital pretreatment the metabolism of cotinine was increased eight-fold in the intact liver. A substantial increase in cotinine metabolism was also found in isolated hepatocytes from PB-induced rats and in cultured mouse hepatocytes grown in a medium supplemented with PB. This was paralleled by an increased formation of cotinine-N-oxide which could be inhibited by 100 microM metyrapone. In contrast, the pulmonary elimination of cotinine was not affected by PB. A dominant role of primary N-oxidation of nicotine compared to C-oxidation was apparent in non-induced rat liver. After PB treatment the rate of nicotine-N'-oxide formation dropped markedly while the cotinine related pathways were increased causing an inversion of the N- to C-oxidation ratio. In the lung, cotinine formation was the preferred metabolic pathway of nicotine already in non-induced organs. The pattern of nicotine metabolites was not altered by PB induction. In conscious PB-induced rats receiving nicotine orally or intravenously, 3'-hydroxycotinine was found as the main urinary metabolite of nicotine while only a small fraction was excreted as cotinine-N-oxide. This discrepancy between the profile of nicotine metabolites in perfused liver and lung and in the urine in vivo indicates that extrahepatic organs other than the lung may be important sites of cotinine metabolism.


Effects of nicotine receptor agonists on acetylcholine release from the isolated motor nerve, small intestine and trachea of rats and guinea-pigs.

The effects of nicotine receptor agonists on the release of [3H]acetylcholine from the phrenic nerve, the small intestine and the trachea were investigated to characterize neuronal nicotine receptors within the peripheral nervous system. Contraction of the indirectly-stimulated hemidiaphragm was recorded to investigate desensitization of the postsynaptic muscular nicotine receptors. Nicotine, cytisine, 1,1-dimethyl-4-phenylpiperazinium and 2-(4-aminophenyl)-ethyl-trimethyl-ammoniumiodide caused a concentration-dependent (0.1-30 microM) increase in evoked [3H]acetylcholine release from the phrenic nerve, whereby bell-shaped concentration-response curves were obtained. The rank order of decreasing potency was: nicotine greater than cytisine greater than 1,1-dimethyl-4-phenylpiperazinium greater than 2-(4-aminophenyl)-ethyl-trimethyl-ammoniumiodide. The presynaptic effects of nicotine depended strongly on the exposure time: facilitation occurred after a short 20 s exposure and inhibition after a 3 min exposure, whereas nicotine no longer affected evoked [3H]acetylcholine release after a 15 min exposure. Pre-exposure (40 min) of the phrenic nerve to 0.3 microM nicotine prevented any subsequent modulatory effect of a high nicotine concentration. In contrast, the contraction of the indirectly-stimulated hemidiaphragm remained unaffected in the presence of 0.3-30 microM nicotine, but a concentration of 1 mM nicotine abolished skeletal muscle contraction. Nicotine (10 microM) produced a substantial release of [3H]acetylcholine in the small intestine but not in the isolated trachea. The present experiments show presynaptic nicotine receptors at the phrenic nerve, which, under appropriate conditions, can mediate facilitation of evoked transmitter release. These neuronal receptors appear more sensitive to desensitizing conditions than the postsynaptic muscular nicotine receptors.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of cigarette smoking or ingestion of nicotine on platelet 5-hydroxytryptamine (5-HT) levels in smokers and non-smokers.

Platelets of healthy smokers and non-smokers were prepared and their content of 5-hydroxytryptamine was determined by HPLC with electrochemical detection. Platelet 5-HT levels in smokers (728 +/- 156 pmol per 10(8) platelets, mean +/- SEM, n = 9) were significantly higher than those in non-smokers (353 +/- 156 pmol per 10(8) platelets, n = 11). Smoking of a single cigarette caused a transient increase in platelet 5-HT levels by about 350% in non-smokers, but had no additional effect in smokers. Similarly, chewing of nicotine gum (4-8 mg nicotine) resulted in a transient increase in platelet 5-HT by about 100% in non-smokers, but not in smokers. In conclusion, smoking of cigarettes can cause an increase in platelet 5-HT, most likely via an enhanced supply of 5-HT from enterochromaffin cells which can be stimulated via nicotine receptors.


Lack of effect of topically applied nicotine on pial arteriole diameter and blood-brain barrier integrity in the cat.

In the present study, the vasomotor effects of nicotine, its interaction with local chemical factors and norepinephrine, and its effects on the permeability of the blood-brain barrier (BBB) were investigated. Using perivascular microapplication, 10(-6) M nicotine was found not to exert a vasomotor effect by itself or to modify the vasodilating effect of an increase in perivascular H+, K+ and adenosine concentration. The constrictor effect of a decrease in H+, K+ or an increase in norepinephrine concentration in the perivascular space was also not altered by 10(-6) M nicotine, indicating a lack of interaction between nicotine and the compounds tested. Using cortical superfusion and intravital fluorescence microscopy nicotine (10(-7) to 10(-3) M) was also found not to affect the diameter of pial arteries during superfusion periods of 30 min each. The integrity of BBB could be demonstrated in time-matched solvent controls over 3 h using intravenously-infused FITC-labelled dextran (MW 70,000) as tracer. During cortical superfusion with 10(-7) to 10(-5) M nicotine the permeability of the BBB was not increased compared with the time-matched controls. However, during superfusion with 10(-4) and 10(-3) M nicotine, tracer extravasation could be quantified by computer-aided image analysis. The extravasation index (EI) increased by up to eight times. These data indicate that only toxic concentrations of nicotine increase BBB permeability to FITC-dextran 70,000.


Effect of nicotine on mRNA levels encoding opioid peptides, vasopressin and alpha 3 nicotinic receptor subunit in the rat.

The effect of acute and chronic nicotine treatment of rats on the mRNA levels coding for the three opioid peptide precursors, for provasopressin and for the alpha 3 subunit of nicotinic receptors in brain, pituitary and/or adrenal medulla of rats was investigated. Nicotine was found to increase the levels of proenkephalin mRNA in the adrenal medulla, but did not affect the levels of PENK mRNA in striatum, hypothalamus and hippocampus. The mRNA levels of prodynorphin were increased together with that of provasopressin in the hypothalamus after nicotine, whereas the prodynorphin mRNA levels in the hippocampus and the striatum remained unchanged. Nicotine treatment resulted in an increase in the pro-opiomelanocortin mRNA levels in the anterior pituitary and in a decrease in the intermediate pituitary, but did not change the levels of pro-opiomelanocortin mRNA in the hypothalamus. The levels of mRNA coding for the alpha 3 subunit of nicotinic receptors in the hypothalamus and the adrenal medulla remained unchanged. The increase in the prodynorphin and provasopressin mRNA levels in the hypothalamus was most pronounced 1 day after s.c. application of two doses of 0.4 mg/kg nicotine (about 100% above control). A smaller increase in mRNA concentrations (about 30%) was found after tonic infusion of the drug for 4 days (4 mg/kg per day), whereas no change was observed after tonic infusion of nicotine for 7 and 14 days indicating the development of complete tolerance.(ABSTRACT TRUNCATED AT 250 WORDS)


Differential effects of acute and chronic nicotine treatment on MPTP-(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) induced degeneration of nigrostriatal dopamine neurons in the black mouse.

Evidence exists for a negative correlation between Parkinson's disease and smoking. The present and previous studies indicate that nicotine treatment can markedly alter the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in the black mouse based on biochemical determinations of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels in neostriatum and substantia nigra 2 weeks after MPTP injection. Acute intermittent treatment with (-)nicotine starting 10 min before the MPTP injection partly protected against MPTP-induced neurotoxicity in the neostriatum and substantia nigra. Also, a partial protection was observed in the substantia nigra when (-)nicotine was given together with MPTP in an acute intermittent treatment schedule. Conversely, chronic infusion of (-)nicotine via minipumps produced a dose-related enhancement of MPTP-induced DA neurotoxicity in the neostriatum. It is suggested that the protective activity of nicotine in the MPTP model is related to a blockade of MPP+ uptake into the DA cells via increased DA release. Conversely, the nicotine enhancement of MPTP-induced DA toxicity is suggested to be caused by a failure of the nicotinic cholinoceptors to desensitize to the chronic (-)nicotine exposure, leading to increased chronic influx of Na+ and Ca2+ ions via the ion channels of the nicotinic cholinoceptors located on the DA neurons with associated increased Ca ion toxicity and increased energy demands.


A new role for nicotine: selective inhibition of thromboxane formation by direct interaction with thromboxane synthase in human promyelocytic leukaemia cells differentiating into macrophages.

Thromboxane, one of the major oxygenated arachidonic acid metabolites of human macrophages, is the most potent vasoconstricting and proaggregatory molecule known. In addition, thromboxane has been shown to be related to host defence mechanisms. We studied the effects of nicotine and its major metabolites on thromboxane formation using cultured macrophage-like cells (HL-60), microsomal assays and purified thromboxane synthase. In intact cells, nicotine, cotinine and methylnicotine at submicromolar concentrations inhibited the rate of conversion of both arachidonic acid and the unstable endoperoxide prostaglandin H2 into thromboxane but not into other eicosanoids. This indicates that nicotine selectively inhibits thromboxane synthase at concentrations that are readily observed in the circulation of smokers. Microsomal assays revealed that nicotine decreased the maximal velocity of thromboxane synthase without affecting the apparent affinity of the enzyme for its substrate. In contrast, no effect of nicotine on kinetic parameters of prostaglandin H synthase or prostacyclin synthase could be observed. Difference spectra, using purified thromboxane synthase, revealed that nicotine directly interacts with the enzyme, presumably by binding the nitrogen of the nicotine ring structure to the iron of the cytochrome P-450 component of thromboxane synthase.


Nicotine increases ciliary beat frequency by a direct effect on respiratory cilia.

We studied the direct effects of nicotine on the ciliary beat frequency (CBF) undisturbed by interference from mucus secretion by using epithelial strips from ferret tracheae which contain no goblet cells and, because the glands were left behind in the submucosa, no gland tissue either. Strips were studied in a chamber perfused with medium M-199 at 37 degrees C at a perfusion rate of 1.6 ml min-1 using a perfusion pump (Braun, Melsungen). CBF was determined photometrically, the signal being subjected to a fast Fourier transformation. We measured CBF continuously for 10 min (5 min with and 5 min without perfusion). Under baseline conditions without nicotine, it decreased from 23.4 +/- 0.8 to 22.2 +/- 0.5 Hz during perfusion and increased from 22.0 +/- 0.8 to 23.3 +/- 0.8 Hz during the subsequent period without perfusion. Nicotine increased CBF transiently, i.e., the effect was demonstrable only during perfusion, being strongest during the first 3 min of perfusion. At 10(-5) M, the increase in CBF was significant only during the 3rd minute, but at 10(-4) and 10(-3) M, CBF was elevated significantly throughout most of the perfusion period compared with control tissues perfused with medium M-199 only. Thus, at 2 min, CBF was 22.8 +/- 0.6 (mean +/- SE) in tissues perfused with medium M-199 only but was 24.3 +/- 0.8 Hz (NS, Student's unpaired t-test), 26.6 +/- 0.5 Hz (P = 0.001), and 26.8 +/- 1.2 Hz (P = 0.01) in tissues perfused with 10(-5), 10(-4), and 10(-3) M nicotine (dissolved in medium M-199), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


Smoking influences the atherogenic potential of low-density lipoprotein.

The possible influence of smoking on the low-density lipoprotein (LDL) and its biological activity was investigated. Plasma LDL was prepared from healthy male smokers and nonsmokers, and oxidized with Cu (II) as prooxidant. Oxidized LDL from smokers generated significantly more lipid peroxidation products, so-called thiobarbituric acid reactive substances (TBARS), when compared to oxidized nonsmoker LDL. Analysis of vitamin E levels in LDL obtained from both smokers and nonsmokers revealed that the vitamin E content of smoker LDL was significantly less than that of nonsmoker LDL. The amounts of cholesteryl esters formed in cultured P388. D.1 macrophages were greater in the presence of smoker LDL than with nonsmoker LDL. The data suggest that some of the proatherogenic effects of smoking may be related to oxidative modification of LDL and alteration of its biological activity.


Alpha 1-proteinase inhibitor and mucus proteinase inhibitor in human lung emphysema.

The role of the antiproteases alpha 1-proteinase inhibitor (alpha 1PI) and mucus proteinase inhibitor (MPI) in human lung emphysema was investigated by measuring their amount and functional activity against trypsin, leukocyte elastase, and pancreatic elastase in the bronchoalveolar lavage fluid (BALF). In addition, leukocyte elastase was quantified in the lavage samples by measuring the concentration of the elastase-alpha 1PI-complex. The study population consisted of 38 patients (5 nonsmokers, 8 former smokers, 25 smokers) with acquired emphysema (i.e., emphysema which is not caused by alpha 1PI deficiency), and 44 individuals (16 nonsmokers, 8 former smokers, 20 smokers) without emphysema. No differences were found between patients with and without emphysema in the activities of alpha 1PI and MPI, or in the concentration of alpha 1PI. The concentration of MPI was significantly higher in the BALF of patients with emphysema than in that of patients without emphysema (p = 0.025). A significantly higher concentration of elastase-alpha 1PI complex was found in patients with emphysema than in those without emphysema (p = 0.041). This finding could reflect the higher proteinase burden to which patients with emphysema are exposed. The increase of MPI in lavage fluid of patients with emphysema seems to be the result of increased production in emphysematous lungs. However, it remains unclear why patients develop emphysema while showing an increased content of MPI.


Volatile N-nitrosamines in mainstream cigarette smoke: occurrence and formation.

Analytical data is presented for the occurrence of the three major volatile N-nitrosamines in cigarette tobacco and mainstream smoke, namely N-nitrosodimethylamine (NDMA),- N-nitrosoethylmethylamine (NEMA) and N-nitrosopyrrolidine (NPYR) as well as for the occurrence of the corresponding precursor amines and non-volatile N-nitrosamino acids in tobacco. Experimental studies using the C20 reference cigarette show that NPYR present in mainstream smoke results from direct transfer of preformed NPYR (ca. 1.5%), decarboxylation of N-nitrosoproline in tobacco (ca. 10%), pyrolytic nitrosation of pyrrolidine in tobacco (ca. 37%) and concerted decarboxylation/nitrosation of proline (ca. 52%) during tobacco pyrolysis.


Tobacco-specific nitrosamines--metabolism and biological monitoring of exposure to tobacco products.

Tobacco-specific nitrosamines are derived from nicotine and related tobacco alkaloids and can be detected in tobacco products as well as in mainstream and sidestream smoke. Two of them, N-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, are strong carcinogens in laboratory animals. Because of its organospecificity for the lung, the latter is considered to be a causative factor in tobacco-related human lung cancer. Upon metabolic activation both nitrosamines give rise to a common reactive intermediate binding to macromolecules such as DNA and haemoglobin and hydrolysing to 4-hydroxy-1-(3-pyridyl)-1-butanone. Because of easy access to large quantities of haemoglobin from blood samples, it is most suitable for biomonitoring human exposure to tobacco-specific nitrosamines. A highly sensitive analytical method for determination of femtogram amounts of 4-hydroxy-1-(3-pyridyl)-1-butanone provides an approach to assess individual exposure to active and passive smoking.


Determinants of a genotoxic effect of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in human diploid fibroblasts.

The induction of micronuclei was studied in human diploid fibroblasts incubated in the presence of the tobacco-specific nitrosamine NNK. We used four fibroblast strains having a high capacity of O6-alkylguanine DNA alkyltransferase (13.0-23.3 pmol O6-methylguanine repaired per 8 x 10(6) cells) and four strains that showed no detectable repair capacity. Incubation with NNK doubled the frequency of micronuclei in repair-deficient cells but failed to evoke any effect in the proficient cell strains. Control experiments were performed with the direct methylating agent MNNG and in the presence of inhibitors of either metabolic activation or alkyltransferase. The results showed that the genotoxicity of NNK is dependent on the relationship between its metabolic activation and the constitutive DNA repair. This supports earlier findings that low constitutive levels of O6-alkylguanine DNA alkyltransferase may increase susceptibility to lung cancer after exposure to DNA methylating agents.


Systemic genotoxic effects of tobacco-related nitrosamines following oral and inhalational administration to Sprague-Dawley rats.

An ex vivo model to detect nonspecific DNA damage in different rat tissues has been developed and employed to study systemic properties of tobacco-specific N-nitrosamines. One hour after treatment of rats with the carcinogens, primary, intact cells were isolated from various organs. Viability of the cells was monitored by trypan blue exclusion. Genotoxicity was determined by alkaline elution, in situ nick translation or microgel electrophoresis. We found that oral application of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces genotoxic effects in the liver (3.125-50 mg/kg), whereas N-nitrosonornicotine (NNN) is only moderately active (50-100 mg/kg). Furthermore, oral administration of NNK, NNN, and of N-nitrosodimethylamine (NDMA), induces DNA damage in the nasal cavity. In peripheral blood lymphocytes genotoxicity of NDMA (less than 2 mg/kg), but not of NNK (50 mg/kg), was observed. NDMA and NNK are just as genotoxic in the liver when administered by inhalation as orally (effective doses: 0.1-1 and 50 mg/kg, respectively). For human cancer, these results indicate that in addition to the susceptibilities in local organs (oral cavity after snuff dipping and lung after tobacco smoke inhalation), these nitrosamines also pose a risk systemically for more remote organs.


Tobacco and alcohol and the risk of head and neck cancer.

We carried out two case-control studies on the relative risk of head and neck cancer in association with tobacco and alcohol consumption. The first study carried out at the ENT Department of the University hospitals of Heidelberg and Giessen (FRG) comprised 200 male patients with squamous cell cancer of the head and neck and 800 control subjects matched for sex, age, and residential area (1:4 matching design). Of the tumour patients, 4.5% had never smoked, in contrast to 29.5% of the control group. The average tobacco and alcohol consumption of the patients was approximately twice as high as in the control subjects. The highest alcohol and tobacco consumption was observed in patients suffering from oropharyngeal cancer. Tobacco and alcohol increased the risk of head and neck cancer in a dose-dependent fashion and acted as independent risk factors. In heavy smokers (greater than 60 pack-years) a relative risk of 23.4 (alcohol adjusted) was calculated. Combined alcohol and tobacco consumption showed a synergistic effect. The risk ratio increased more in a multiplicative than in an additive manner. Oral and laryngeal cancer were associated with the highest tobacco-associated risk values. The highest ethanol-associated risk values were associated with oropharyngeal and laryngeal cancer. The second study was carried out at the ENT Department of the University of Heidelberg on 164 males with squamous cell carcinoma of the larynx and 656 control subjects matched for sex, age and residential area (1:4 matching design). Of the cases, 4.2% had never smoked, compared with 28.5% of the control subjects.(ABSTRACT TRUNCATED AT 250 WORDS)