An affinity-amplified immunoassay for juvenile hormone esterase.

A method is described for increasing the specificity of an immunoassay for catalytically active enzymes and is specifically illustrated with a sensitive assay for an important regulatory enzyme from insects. Trifluoromethyl ketone haptens, potent inhibitors of insect juvenile hormone esterase, were bound to proteins such as hemocyanin (keyhole limpet) and conalbumin (chicken embryo). Haptens containing a thiol group were conjugated using heterobifunctional coupling reagents, and haptens with a carboxylic acid moiety were conjugated by the mixed anhydride method. The trifluoromethyl ketone-protein conjugates, shown to retain their inhibitory activity against juvenile hormone esterase, were used as coating antigens in several solid-phase enzyme-linked immunosorbent assay formats along with specific antibodies raised in rabbits against purified juvenile hormone esterase. The previously unreported format, termed affinity-amplified immunoassay (AAIA), was successfully used for quantitative monitoring of low levels of the esterase in dilute hemolymph and egg homogenates from various lepidopteran insect species, as well as for detection of the native and mutant forms of the enzyme obtained in a recombinant baculovirus expression system. The AAIA format was more sensitive for the target esterase and detected only the catalytically active form of the enzyme.


Direct assay of membrane-associated protein kinase C activity in B lymphocytes in the presence of Brij 58.

This paper describes a simple and direct procedure for assaying Ca(2+)-dependent protein kinase C (PKC) activity in membrane fractions isolated from purified murine B lymphocytes (B cells) treated with phorbol 12-myristate 13-acetate (PMA). The results indicate that membrane-bound PKC in B cells, treated with PMA, can be measured directly in the presence of 0.5% Brij 58 by assaying the transfer of 32P from [gamma-32P]ATP to histone type III-S. This method obviates the need for partial purification of the protein kinase by ion-exchange chromatography prior to assaying PKC activity. The properties of membrane-associated PKC activity in B cells have been characterized, and the kinetics of PMA-induced translocation of PKC in cultured murine B cells, the rat glial tumor clone C6, and primary neonatal osteoblastic cells have been defined by this direct assay. The results obtained with B cells and the other cell lines indicate that this direct assay procedure could be useful for studies on the factors controlling PKC translocation in a variety of cultured mammalian cells.


Polymyxin B-horseradish peroxidase conjugates as tools in endotoxin research.

The peptide antibiotic Polymyxin B (PMB) binds to bacterial endotoxin (lipopolysaccharide, LPS). We prepared covalent conjugates of PMB and horseradish peroxidase (HRP) by periodation of HRP-linked oligosaccharides followed by direct condensation with PMB. In addition we prepared monoclonal antibodies (Mabs) to PMB. The PMB-HRP conjugates and anti-PMB Mabs were used to study in ELISA the binding of PMB to LPS from Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. In addition, PMB-HRP was used to quantify lipid A in ELISA, and to stain gram-negative bacteria histochemically. For the study of PMB-LPS interaction, PMB-HRP proved to be superior to the anti-PMB Mabs. PMB-HRP conjugates are useful general probes to detect or measure lipid A and LPS of various species using very simple methods and to stain bacteria, and they may obviate the need for many specific antisera. Thus, PMB-HRP conjugates are useful probes for endotoxin research.


Chromogenic redox assay for beta-lactamases yielding water-insoluble products. I. Kinetic behavior and redox chemistry.

We describe a chromogenic detection system for beta-lactamase which yields water-insoluble colored products. The assay is based on kinetic measurement of the appearance of color due to the beta-lactamase-initiated redox reaction. The substrates are C3' thiolate-substituted cephalosporins, which, after enzyme-catalyzed hydrolysis of the beta-lactam ring, undergo elimination of the thiolate ion. This thiolate, in a postenzymatic step, reduces the tetrazolium salts, which are water-soluble colorless compounds, to a colored water-insoluble precipitate of formazan. Our model in this study was a beta-lactamase Enterobacter cloacae P-99-catalyzed reaction of thiolacetate cephalosporin with several tetrazolium salts. We found that the reaction rate is dependent on the concentration of the electron carrier 5-methyl phenazinium methyl sulfate, the pKa of the C3' thiolate substituent of the cephalosporin substrate, and the reduction potential of the tetrazolium salts. A kinetic study of this system yielded a rate law for the reaction. We present a mechanism of the reaction and determination of the kinetic parameters for the process. The sensitivity of this kinetic assay is very high; we detect 3 x 10(-10) M beta-lactamase P-99, which is approximately 30 mIU. The assay times are very short, lasting from 2 to 5 min. The new assay system is particularly suitable for a rapid detection of beta-lactamases in bacterial colonies and in enzyme immunoassays where beta-lactamase may be used as the label.


Chromogenic redox assay for beta-lactamases yielding water-insoluble products. II. Heterogeneous sandwich assay for hCG.

A very sensitive and rapid heterogeneous sandwich enzyme immunoassay for human chorionic gonadotropin (hCG) is described. The assay is based on the application of the novel chromogenic redox substrate system for beta-lactamase which is used as label. The chromogen system consists of a thioacetylcephalosporin beta-lactamase substrate, which upon turnover by the enzyme label releases the thiolate with the concomitant reduction of the tetrazolium salt to a colored formazan. The concentration of the formazan is directly related to the amount of the hormone in the sample and is read spectrophotometrically. The enzyme-antibody conjugates, produced through use of heterobifunctional maleimide crosslinker, maintain 90% of the enzyme activity after 30 days at 25 degrees C. Concentrations of the hormone as low as 5 mIU/ml, equivalent to 25 fmol/ml, are detectable in 3 h.


A high-performance liquid chromatography-based radiometric assay for acyl-CoA:alcohol transacylase from jojoba.

Acyl-CoA:alcohol transacylase catalyzes the final step in the biosynthesis of storage liquid wax esters from acyl-CoA fatty acids and fatty alcohols in a limited number of microbes, algae, and Simmondsia chinensis Link (jojoba). An improved and automated method of enzyme assay for this catalyst from cotyledons of jojoba is described. The assay method uses reversed-phase C18 high performance liquid chromatography (HPLC) to separate the labeled C30:1 liquid wax product, [14C]-dodecanyl-octadecenoate, from the unreacted substrate, [14C]octadecenoyl-CoA (oleyl-CoA), and other components produced from enzymes present in the crude homogenate of jojoba cotyledons, including [14C]-octadecenoic acid (oleic acid) and [14C]octadecenol (oleyol). Methods are also described for microscale chemical synthesis in one vessel of 14C-radiolabeled substrates and products for the transacylase. These labeled reagents are required to confirm the HPLC separations of reaction products. The radioactive components are quantitated using an on-line flow-through scintillation detector enabling sensitive and precise analysis of the reaction products.


The use of streptavidin-biotin interaction for preparation of reagents for complement-dependent liposome immunoassay of proteins: detection of latrotoxin.

We have developed liposome sensitization by a protein, latrotoxin (LT), using immobilization of biotinylated LT via streptavidin with biotinylated phosphatidylethanolamine contained in liposomes. The use of such liposomes in the complement-dependent homogeneous liposome immune lysis assay (LILA) has allowed us to detect in the test sample as little as 2 micrograms/ml of polyclonal and 50-100 ng/ml of monoclonal IgG and IgM antibodies to LT. LT concentration in solution was determined by inhibition of immune lysis by free LT. The sensitivity of the LT assay varied from 1 x 10(-9) to 5-50 x 10(-9) M when antiserum (polyclonal antibodies) and monoclonal antibodies to LT were correspondingly used. The results show that a streptavidin-biotin spacer can be used to immobilize protein antigens on liposomes for a subsequent application in LILA. The suggested technique greatly simplifies the sensitization procedure and extends the applicability of the LILA.


Flow injection electrochemical enzyme immunoassay for theophylline using a protein A immunoreactor and p-aminophenyl phosphate-p-aminophenol as the detection system.

A competitive electrochemical enzyme immunoassay has been developed for the antiasthmatic drug theophylline, utilizing a controlled-pore glass-protein A immunoreactor and flow injection techniques. p-Aminophenyl phosphate, a substrate for alkaline phosphatase, has been used in this assay, and its hydrolysis product p-aminophenol was determined at +0.2 V versus the saturated calomel electrode. For each sample the antibody-protein A reaction takes place at near-neutral pH, and the complexes are eluted at acid pH. Serum theophylline has been determined by this method, and good relative standard deviations and percentage recoveries have been achieved.


High-performance liquid chromatographic determination of 3 alpha,5 beta-tetrahydroaldosterone in human urine with chemiluminescence detection.

A sensitive method for the determination of 3 alpha,5 beta-tetrahydroaldosterone (THALD) in human urine is described. The method uses high-performance liquid chromatography with chemiluminescence detection. Urinary THALD, released by enzyme hydrolysis, is isolated and concentrated using a Sephadex G-25M column and Bond-Elut C1 cartridges, and then oxidized by copper(II) acetate to form the corresponding glyoxal derivative. The glyoxal derivative is converted into the chemiluminescent quinoxaline by reaction with 4,5-diaminophthalhydrazide. The chemiluminescent quinoxaline is separated within 50 min on a reversed-phase column (TSKgel ODS-120T) with isocratic elution, followed by chemiluminescence detection; the chemiluminescence is produced by the reaction of the quinoxaline with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in alkaline solution. The detection limit for THALD is 0.6 pmol (220 pg) ml-1 in urine [1.5 fmol (0.53 pg) per 20 microliters injection] at a signal-to-noise ratio of 3. This method permits the sensitive and precise determination of THALD in human urine (50 microliters) from normal subjects and a patient with primary aldosteronism.


Determination of cadmium in biological samples by inductively coupled plasma atomic emission spectrometry after extraction with 1,5-bis(di-2-pyridylmethylene) thiocarbonohydrazide.

An inductively coupled plasma atomic emission spectrometric method for the determination of trace amounts of cadmium after extraction of the metal into isobutyl methyl ketone containing 1,5-bis(di-2-pyridylmethylene) thiocarbonohydrazide is described. The optimum extraction conditions were evaluated from a critical study of the effects of pH, concentration of extractant, shaking time and ionic strength. The detection limit for cadmium is 0.1 ng ml-1 and the calibration is linear from 0.2 to 140 ng ml-1. The relative standard deviation for ten replicate measurements is 2.9% for 2 ng ml-1 of cadmium. Results from the analysis of some certified biological reference materials are given.


Structural analysis of the non-dialysable urinary glucoconjugates of normal men.

Enzymic methods were employed to analyse the structure of the non-dialysable urinary glucoconjugates of ten healthy males. The excretion of the urinary glucoconjugates was determined from the glucosyl:galactosyl ratio after acid hydrolysis, and a mean value of 0.27 was obtained. The results of the specific actions of alpha- and beta-glucosidases showed that the non-dialysable urinary glucoconjugates contain a branched alpha-glucan fraction with 1,4- and 1,6-glucosidic bonds, and a beta-glucan fraction containing 1,4-glucosidic bonds.


A self-interpretive behavior analysis.

Although it rejects self-awareness as psychological bedrock, behavior-analytic theory can be stated self-inclusively, keeping the theorist within view. Its principles of discrimination and generalization have been elaborated to include concepts and higher order conditionalities, including those of logic and of awareness. Its violating a cultural bias that is called the "fundamental attribution error" may be a primary source of controversies. Its other disagreements with mainstream psychologies hinge more on contiguous versus remote causation than on mentalism versus antimentalism, which Skinner emphasized. The nonmediational, Skinnerian theorist is a participation in the world rather than an isolated self.


Radical behaviorism and scientific frameworks. From mechanistic to relational accounts.

A substantial portion of B. F. Skinner's scholarship was devoted to developing methods and terms for a scientific study of behavior. Three concepts central to scientific accounts--cause, explanation, and theory--are examined to illustrate the distinction between mechanistic and relational frameworks and radical behaviorism's relationship to those frameworks. Informed by a scientific tradition that explicitly rejects mechanistic interpretations, radical behaviorism provides a distinctive stance in contemporary psychology. The present analysis suggests that radical behaviorism makes closer contact with the "new world view" advocated by physicists and philosophers of science than does much of contemporary psychology.


From mechanistic to functional behaviorism.

A shift from mechanistic behaviorism to functional behaviorism is presented against the background of two historical traditions, one with an emphasis on form, the other with an emphasis on function. Skinner's work, which made more contributions to a functional behaviorism than to a mechanistic behaviorism, exemplifies this shift. The two traditions and an account of Skinner's development of functional relations are presented in order to show Skinner's contributions to aligning modern behavior analysis with the functional tradition.


Essentialism and selectionism in cognitive science and behavior analysis.

Contingencies of selection, be they phylogenetic or ontogenetic, merely set boundaries on units; they do not provide blueprints. Thus, variability is fundamental to all products of selection. Skinner, by characterizing the units of analysis in behavior as generic in nature, established his science squarely within the selectionist paradigm, thereby avoiding the tendency, common throughout psychology, to slip into essentialist analyses. The distinction between essentialism and selectionism is refined in this article, and prominent examples of essentialism in linguistics, theories of memory, theories of representation, associationism, and even in behavior analysis are identified. Recent trends in cognitive science--specifically, research on adaptive networks--is amenable to a selectionist interpretation, suggesting the possibility of future fruitful interactions with behavior analysis.


Models of proximate and ultimate causation in psychology.

B. F. Skinner saw behavior as a product of three levels of evolution. J. R. Kantor and Gregory Bateson noted similar relations. This article describes and applies basic evolutionary concepts to each level: (a) phylogenic, (b) ontogenic, and (c) cultural evolution. Each level is analyzed in terms of (a) units of selection, (b) variety of units required for the selection process, (c) selection pressures, (d) interactions among levels, and (e) implications for understanding and predicting behavior. Distinguishing between models of proximate and ultimate causation, as in biology, may help clarify research problems posed by, and facilitate better communication among, psychologists.


Teleological behaviorism.

A psychological science of efficient causes, using internal mechanisms to explain overt behavior, is distinguished from another psychological science, based on Aristotelian final causes, using external objects and goals to explain overt behavior. Efficient-cause psychology is designed to answer the question of how a particular act is emitted; final-cause psychology is designed to answer the question of why a particular act is emitted. Physiological psychology, modern cognitive psychology, and some parts of behaviorism including Skinnerian behaviorism are efficient-cause psychologies; final-cause psychology, a development of Skinnerian behaviorism, is here called teleological behaviorism. Each of these two conceptions of causality in psychology implies a different view of the mind, hence a different meaning of mental terms.


B. F. Skinner's legacy to human infant behavior and development.

B. F. Skinner's legacy to human behavioral research for the study of environment-infant interactions, and indeed for the conception of development itself, is described and exemplified. The legacy is largely the practicality, the efficiency, and the comparative advantage--relative to diverse other behavioral and nonbehavioral approaches--of using the operant-learning paradigm to organize and explain many of the sequential changes in behavior patterns conventionally thought to constitute infant development.


Case histories in the great power of steady misrepresentation.

The widespread misrepresentation of behaviorism in the scientific and popular literature has caused its contributions to the understanding of behavior to be systematically ignored or denied. This misrepresentation is manifested, in large part, as a form of academic folklore that codifies erroneous accounts of behaviorism's assumptions, findings, and goals. This article examines three representative "case histories" of the academic folklore about behaviorism: its alleged environmentalism, totalitarian aims, and intellectual intolerance. Because academic folklore has been highly resistant to the corrective efforts of behaviorists, explicit strategies are suggested for identifying and correcting folklore and for promoting more effective interdisciplinary communication.


Out of the laboratory and into the community. 26 years of applied behavior analysis at the Juniper Gardens Children's Project.

Application of Skinner's principles to socially significant human behavior had been well articulated by 1968 (Baer, Wolf, & Risley, 1968). Applications of these principles by Baer, Wolf, Risley, Hall, Hart, Christophersen, and their colleagues were in evidence as early as 1964 in the homes, schools, and clinics of inner-city Kansas City, Kansas, at the Juniper Gardens Housing Project. The work continues relatively uninterrupted, having contributed extensively to the literature of applied behavior analysis and the lives of community residents. This article describes the project and illustrates how applied behavioral research was initiated and extended, how the work addressed general concerns in psychology, and how it continues to address contemporary concerns within the community.


Breaking the structuralist barrier. Literacy and numeracy with fluency.

Behavior analysis is an example of a selection science, and behavioral programs that follow the tenets of selectionism, long advocated by B. F. Skinner, can have a large impact on social problems. This article describes the characteristics of selection sciences and their application in the Morningside Model of Generative Instruction, which addresses both adult literacy and children's learning and attention problems. School curricula are analyzed for their key component elements and underlying tool skills. Teaching procedures then establish and build these key components to fluency. New and complex repertoires then emerge with little or no instruction, producing curriculum leaps that allow students to make rapid academic advancement. Children typically gain more than two grade levels per school year, and adults advance two grades per month.


Is behavior analysis undergoing selection by consequences?

The legacy of B. F. Skinner's life is a natural science of behavior. The generality of its basic functional relations has led, by induction, to the unifying principle of selection by consequences, which accounts for morphological, behavioral, and cultural evolution. This principle both predicts and explains the observation that the science itself is becoming the object of differential selection in our culture. Public policy is increasingly being framed in terms of the effects of consequences on behavior, as illustrated by examples from education, economics, and politics. Survival of our culture may depend on our skill in managing this process.


B. F. Skinner, organism.

B. F. Skinner illustrated the power of behavior analysis by turning it upon his own behavior. This article considers parallels in the life and work of Charles Darwin and places Skinner's views on life and death in the context of his selectionist paradigm for psychology. The term organism plays a special role, and the account shows why B. F. Skinner might have regarded it as an appropriate title.


[Vertical transmission of HIV: descriptive epidemiology, risk factors and survival (II)].

Up until November 30, 1990, 415 children born to HIV-positive mothers were followed in order to study the vertical transmission rate and the survival of those infected, as well as the associated risk factors. According to CDC criteria, 264 children could be considered definitely classified, with 82 classified as infected (transmission rate: 31.1%) and 182 as sero-negative. The remaining 151 infants were younger than 15 months and classified as PO. Ninety percent of the studied mothers were infected through either the use of intravenous drugs or by sexual intercourse with HIV infected partner. The loss-of-antibody median age was one year. The median for the incubation period was 285 days, with a significant bimodal distribution regarding the child's age at diagnosis. Although the median survival time was longer than the study period, the fatality rate was estimated to be 22.0% and the percentage of children reaching age 4, 27%.


[Iga and IgG antireticuline in celiac disease. Their application as diagnostic markers of the disease].

We report the results of indirect immunofluorescent (IFI) detection of IgA and IgG antireticulin antibodies (IgA-ARA and IgG-ARA, respectively) in 283 serum samples from pediatric patients with coeliac disease (with and without gluten containing diets), patients with non-coeliac gastrointestinal disease, patients without gastrointestinal disease (control group) and patients with an increased risk for coeliac disease (diabetes mellitus, dermatitis herpetiformis or first grade relatives of coeliac patients). Our results indicate that IgA-ARA is a reproducible marker, with high positive (99-100%) and negative (100%) prediction values, when it is applied to children who have been on gluten containing diets for a long time (more than six months). The IgA-ARA measurement is not applicable in cases of selective IgA deficiency. Although IgG-ARA has a high predictive positive value, its low predictive negative value makes it a poor diagnostic tool. In the risk groups, our results suggest that these antibodies are useful in patient selection for intestinal biopsy.


[Comparative study of evoked otoacoustic emissions and auditory potentials of the brain stem in the neonatal period].

Evoked otoacoustic emissions (EOAE) are a recently discovered technique that is related to a good audiological status. It is a quick, simple and harmless test that can be used in neonatal screening. We report and compare here the results from a series of neonates in which we have performed both, this test and a brainstem response (ABR) test. The correlation between techniques was good, especially in normal positive neonates where the correlation was 100%. However, EOAE only explores the cochlear status and will not detect retrocochlear pathologies.


[Association between calcium content of drinking water and fractures in children].

The goal of this research was to determine the relationship between high levels of calcium in Majorca's water supplies and the prevalence of fractures in schoolchildren. The study was performed during two school terms and was done within the framework of the School Health System. Three towns of Majorca were selected because of the different content of calcium in their water supply. The fluorine content in the tap water was always under 0.5 mg/l. All of the towns have the same social and economic backgrounds. We surveyed 1,308 schoolchildren, between 11 and 14 years of age, about previous fractures. There is a statistically significant relationship between fractures and low calcium levels in the water supply. This association is stronger for children 11 years of age than for children 14 years old.


[Physiologic levels of bilirubin and behavior in healty infants during the neonatal period].

The purpose of this study was to evaluate behavioral differences in healthy newborns in relationship to bilirubin serum levels within the normal physiological range. Seventy-three healthy full-term newborns of adequate weight for their gestational age had bilirubin levels measured in a blood sample obtained by heel puncture on the third day of life. The Brazelton Neonatal Behavioral Assessment Scale (NBAS, 1984 version) was administered between the 60th and 80th hours of life. Results showed a mean bilirubin value of 123.12 mumol/L (7.2 mg/dL). There was an inverse correlation between serum bilirubin levels and behavior in regards to habituation, orientation, motor maturity and interaction of the NBAS. Although it can not be concluded that physiological bilirubin levels cause significant neurological disturbances, these results suggest that newborns with higher physiological bilirubin levels have some difficulties interacting with their caretakers.


[Nocturnal use of pirenzepine in young insulin dependent diabetics. Results of a 6-months treatment period].

Previous studies have demonstrated an elevation in GH in adult insulin-dependent diabetics which can be modified by administration of pirenzepina either IV or orally. In this study we have evaluated the mean nocturnal GH levels (MNGH) and HbA1-C levels in a group of young insulin-dependent diabetics, both before and after treatment with pirenzepina (Gastrozepin). The study population included 8 patients, 6 males and 2 females, between the ages of 12 and 17 years, with a mean of 15.6 years. Pirenzepina was administered during one month at a nightly oral dose of 0.6 mg/kg followed by 5 months of treatment with 1 mg/kg. The most important results obtained in the study are the following: 1) Nocturnal administration of pirenzepina did not significantly modify the MNGH in the study population (10.88 +/- 3.81 ng/ml vs 9.57 +/- 8.25 ng/ml, p > 0.05). 2) This pharmaceutical did not alter the plasma levels of HbA1-C (9.57 +/- 8.25 vs 10.01 +/- 2.30, p > 0.05). However, 5 out of 8 patients had a decrease in their nocturnal GH secretion after pirenzepina treatment. If only the 5 patients that responded to this treatment are considered, the differences in MNGH are significant (10.48 +/- 4.94 before treatment vs 4.35 +/- 2.53 following treatment, p < 0.05). Therefore, we conclude that oral pirenzepina treatment for 6 months, at the doses described, do not consistently decrease GH secretion in young diabetics nor does it decrease HbA1-C values in this group. However, further studies are necessary to establish the possible value of this treatment.


[Scrotal trauma in childhood].

Scrotal trauma has increase in frequency during recent years, largely as the result of sporting activities, physical aggressions and road accidents. Its importance lies in the likelihood of associated lesions of the testis and the spermatic cord. We report 41 cases of scrotal trauma. Thirty-seven cases were non-penetrating and four were penetrating (including a case of traumatic rupture of the testis). Nonsurgical treatment was applied in 30 cases (73.1%) and surgical treatment in 11 (26.8%). The diagnostic techniques employed included ultrasonography in 19 cases, gammagraphy in 10 cases, Doppler-effect ultrasonography in 2 cases and CAT in 2 cases. The decision to operate should be based on the clinical findings. Follow-up studies over 8-60 months were satisfactory in all cases, with no cases of testicular atrophy detected.


[Neonatal septicemia: incidence and risk factors].

The cases of 131 newborns with positive blood cultures have been reviewed. These children were born in our hospital between 1985 and 1990. We found that the incidence of sepsis was 4.3/1000 newborns. Early neonatal sepsis was the most frequent, representing 58% of the cases. Late sepsis in hospitalized infants represented 1.34% of the cases. Streptococcus B was the microorganism most frequently isolated (26.7%), followed by S. epidermidis (19.8%), E. coli (13.7%) and S. aureus (10.68%). In over 50% of the cases of early sepsis, maternal-fetal risk factors were present. Mortality was 7.6%, with half of these cases due to Streptococcus B.


[Incidence of IDDM in children (age 0-14 yrs) in Málaga 1982-1988].

The annual incidence of diabetes mellitus in children 0 to 14 years of age in the province of Malaga (Spain) between 1982 and 1988 was 11.4 cases per 100,000 inhabitants (range = 9.7-13.1). There was an inverse relationship with seasonal temperature; the onset of the disease occurred more frequently during the colder months. The highest incidence was found among children between 5 and 9 years of age, with 13.88 cases per 100,000. The male/female ratio was 1.3 in children between 0 and 5 years and in children between 5 and 9 years of age and 0.97 in children between 10-14 years. The onset of the disease as ketoacidosis remained constant at 25% and was independent of age. When compared with other European epidemiological studies, we found a higher incidence of diabetes mellitus than that in Central European or Mediterranean Countries.


[Main vascular access in situations of extreme urgency: intra-osseous infusion].

The intraosseous infusion is an easy technique to learn. The chosen places for infusion are: proximal tibia, distal tibia and distal femur. We have used this technique on thirteen occasions in emergency situations. In all cases, access was achieved in less than one minute. Saline, glucose and Ringer's solutions, sodium bicarbonate, blood, calcium chloride, epinephrine, atropine, succinylcholine, dopamine, dobutamine, isoproterenol, lidocaine and anticonvulsants may be infused. The doses are the same as those used in parenteral access and its efficacy is similar to that of a central venous line. Complications are rare. The most frequent is subcutaneous, and occasionally subperiosteal infiltration. Osteomyelitis appeared in 0.6% of the cases. The intraosseous infusion provides rapid, easy and safe vascular access in emergency situations. Therefore, we believe that this technique should be chosen in pediatric patients.


[Cholesterol and phospholipids in high density lipoproteins and their subfractions in a population in its second decade of life. The Burgos study].

Decreased levels of high-density lipoproteins (HDL) are related to a risk of ischaemic heart disease and are measured as HDL-cholesterol (HDLc), whereas little attention has been paid to HDL-phospholipids (HDLph). Regarding HDL subfractions (HDL2, HDL3) and risk of ischaemic heart disease, there are few studies in the literature and these are contradictory, especially those performed in subjects that were in their second decade of life. This study consisted of 322 healthy volunteers between 11 and 19 years of age. The HDL fractions and HDL3 subfractions were separated by precipitation with polyethylene glycol at defined pH and concentration. Concentrations of cholesterol and triglycerides were measured by enzymatic methods, except for HDL2c and HDL2ph, which were calculated by subtraction. Forty-two percent of the children with parental history of ischaemic heart disease, but only 26% of the children without parental history of heart disease, exhibited HDL3ph levels lower than 0.95 mmol/l (74 mg/dl). Levels of HDLc, HDL3c and HDL2c are relatively constant in girls after puberty and levels of HDLph and HDL3ph are increased; levels of HDLph are relatively constant in boys after puberty while levels of HDLc, HDL3c and HDL2c are decreased. Our results suggest that serum levels of HDL3ph are potential markers for a risk of ischaemic heart disease, together with other more classic risk factors.


[Vertebral density of hypercalciuric lithiasis. Its relation to calcium-protein intake and vitamin D metabolism].

Forty-two patients with calcium calculi were selected based on calciuria > 0.1 mmol/kg/d on an uncontrolled diet. To measure excretion of sodium, calcium, phosphates and hydroxyproline, a 24-hr urine sample was collected on the 4th day of a milk product-free diet, a fasting urine specimen was collected on the morning of the 5th day and another sample was taken 4 hr after the oral administration of calcium. On the 5th day, plasma levels of calcium, phosphates, intact parathyroid hormone (PTH), calcidiol and calcitriol were determined on an empty stomach and after administration of a calcium load. The results, compared to those of healthy subjects evaluated under the same conditions, enabled classification of the stone-formers as having dietary hypercalciuria (n = 18), when calciuria returned to normal on a low calcium diet, and idiopathic hypercalciuria (n = 24), when the urinary calcium level remained high. Patients with idiopathic hypercalciuria were then classified, according to Pak's criteria, as having absorptive hypercalciuria (n = 8), when the fasting calciuric levels was normal, renal hypercalciuria (n = 1), when fasting hypercalciuria with elevated circulating PTH was controlled by a calcium load, or undetermined hypercalciuria (n = 15) for those individuals with fasting hypercalciuria and normal plasma PTH levels. In addition, vertebral density was measured tomodensitometrically and expressed as a percentage of the normal as a function of sex and age based on a regression line calculated with the results of 239 normal subjects.(ABSTRACT TRUNCATED AT 250 WORDS)


[Drugs and aged subjects institutionalized in geriatric units].

Our one-year retrospective study evaluated drug use in a population of 136 patients over 60 years old hospitalized in a geriatric ward (moderate--and long-term institutionalization). After having classified the reason for hospitalization, the length of stay and the number of pathologies per patient, the number of prescribed medications was quantified by means of a drug-day unit (D-D). The mean number was 3.36 D-D, which was independent of sex, age and reason for admission. The most frequently prescribed medications treated central nervous system, cardiovascular and metabolic disorders. These results were compared to those of CREDOC 1981 and CREDES 1987. In addition, 14 iatrogenic episodes were identified in this study; aminoglycosides and cardiovascular drugs were responsible for the majority of them, despite the precautions taken in light of the therapeutic indications. The iatrogenesis thus identified was not due to biological abnormalities found in the elderly. In our opinion, this evaluation of drug use is a good predictive element for understanding iatrogenicity on a larger scale and thereby to attempt to avoid it.


[Temporal arteritis in young subjects. A trial of nosological classification apropos of 6 cases].

Six young-adult patients (19- to 32-years-old) are described: 3 men with temporally localized systemic vasculitis (thromboangiitis obliterans 2, Churg-Strauss angiitis 1) and 3 patients (2 men, 1 woman) with isolated temporal arteritis. Temporal arteritis in subjects under 40 years of age consists of either a temporal localization of systemic vasculitis (thromboangiitis obliterans or Buerger's disease, Churg-Strauss angiitis or polyarteritis nodosa) or a distinct entity of which only 12 biopsy-proven cases have been reported to date. The latter is differentiated from temporal (giant cell) arteritis of the older patient by a higher incidence in men, and the absence or rarity of general symptoms, ocular complications and an accelerated erythrocyte sedimentation rate. Two types of temporal arteritides in young adults seem to be distinguishable: an asymptomatic form with an isolated temporal nodule and a more symptomatic one. In some cases, temporal arteritis in young adults corresponds to a unique entity "juvenile temporal arteritis", which seems to be different from Takayasu's arteritis, localized forms of polyarteritis nodosa and Kimura's disease. Although its treatment remains difficult to define, therapy of the symptomatic form could include steroids, whereas the asymptomatic one seems to require only simple monitoring.


Stress proteins and reperfusion stress in the liver.

Blood reperfusion after temporary liver ischemia induces the expression of heat shock genes and the synthesis of heat shock proteins (hsps), in particular hsp 70. Induction requires a certain duration of ischemia, suggesting that cell damage before reperfusion is essential for activation of heat shock genes. The expression of the hsp 70 gene is preceded by activation of the cellular protooncogenes c-fos and c-jun. However, the product of these genes, which is transcription factor AP-1, seems unnecessary for activation of the hsp 70 gene, which does not require the integrity of protein synthesis. Hsp genes seem to behave as "early response genes," enabling the cell to respond to emergency situations.


Genotoxic-stress-response genes and growth-arrest genes. gadd, MyD, and other genes induced by treatments eliciting growth arrest.

As discussed throughout this paper, many mammalian DDI genes are associated with growth responses, including both positive responses to growth stimulation and negative responses involving transient growth arrest and terminal differentiation. It is interesting that several immediate-early genes encoding transcription factors, the jun genes, are DDI, are induced by terminal differentiation, and also are associated with positive growth responses. In negative growth-response genes, their control is complex and almost certainly involves multiple regulatory mechanisms. The role of growth-arrest genes after exposure to DNA-damaging agents is currently not known, but as growth arrest can have a protective effect on cells exposed to DNA-damaging agents in both bacteria and eukaryotes, some protective role(s) for the gadd genes may exist. Whatever the roles are for the individual gadd genes, the response of the gadd genes to DNA-damaging agents and other growth-arrest signals has been highly conserved during mammalian evolution, and it is likely that this stress response, as reflected by induction of one or more gadd genes, is present in most or perhaps all mammalian cells. Our findings that the gadd group overlaps with another group of growth-arrest genes, the MyD, indicate that these two groups combined define a new class of genes whose protein products are likely to play a role in cell growth cessation.


Oxidant carcinogenesis and antioxidant defense.

Growth promotion by oxidants is observed with cultured human and mouse fibroblasts as well as epidermal cells. It is expected to play a role in inflammation, fibrosis, and tumorigenesis. Indeed, oxidants trigger (patho)physiological reactions that resemble those induced by growth and differentiation factors. For example, active oxygen activates protein kinases, causes DNA breakage, and induces the growth competence-related protooncogenes c-fos and c-myc. The cellular antioxidant defenses affect the consequences of oxidant exposure. Transfectants of mouse epidermal cells that overproduce Cu,Zn-superoxide dismutase (SOD) were sensitized to the toxic effects of an extracellular burst of O2-. plus H2O2, whereas overproducers of catalase (CAT) were protected. Transfection of SOD overproducers with CAT corrected their hypersensitivity. Inducibility of the protooncogene c-fos by oxidants was diminished in SOD and CAT overproducers, albeit probably for different reasons. It is concluded that a fine balance of the multiple components of the antioxidant defense determines the growth response of cells to oxidative stress. In studies of the mechanism of the transcriptional induction of c-fos by oxidants, we identified the joint DSE-AP1 elements (dyad symmetry element, DSE) as major enhancer motifs in the 5'-upstream regulatory sequences of c-fos. Oxidants also increased the de novo synthesis of protein factors that bind to the fos-AP1 enhancer motif. Protein kinase and ADPR transferase inhibitors suppressed the transcriptional induction of c-fos as well as the increase in factor binding to fos-AP1. We conclude that protein phosphorylation and protein polyADP-ribosylation are required for the transcriptional induction of c-fos and the synthesis of protein factors that bind to fos-AP1. It is likely that the FOS and JUN proteins are among these factors and that they participate in the regulation of c-fos expression by oxidants.


Genomic heterogeneity of DNA repair. Role in aging?

The introduction and repair of DNA lesions are generally heterogeneous with respect to different genomic domains. In particular, the repair of helix-distorting damage, such as the cyclobutane pyrimidine dimers (CPD) induced by ultraviolet light occurs selectively in expressed genes. This is due in large part to the preferential repair of transcribed DNA strands, which is then reflected in a bias toward mutagenesis from persisting lesions in nontranscribed strands. Consequently, determination of overall genomic repair efficiencies may not be a good indicator of cellular sensitivity to agents that damage DNA. Although some studies suggest an age-related accumulation of altered nucleotides in DNA, we do not know the intragenomic distribution of those changes and whether they are relevant to the physiological aspects of aging. Subtle changes in the pattern of preferential repair during maturation could have profound effects on cell and tissue function. DNA repair has been analyzed in differentiating cell systems as possible models for aging. We have observed attenuated overall repair of CPD in differentiated rat myoblasts or PC12 neuron-like cells. In both model systems, several expressed genes have been shown to be repaired relatively efficiently but without strand specificity. In another model system of human HT1080 fibroblasts differentiating in the presence of dexamethasone, we demonstrated enhanced repair in the gene for plasminogen activator inhibitor I whose transcription is induced and, correspondingly, a reduced repair rate in the urokinase plasminogen activator gene whose transcription is suppressed. We conclude that any attempted correlation of the phenomena of aging with DNA repair should focus on the relevant genes in the tissue of interest.


Changes in calcium's role as a messenger during aging in neuronal and nonneuronal cells.

Alterations in calcium transport appear to be functionally significant. Treatment with drugs that promote calcium uptake partially reverse some of the age-related deficits in calcium-dependent processes. Thus, the relevance of decreased calcium coupled receptor binding is supported by the ability of 3,4-diaminopyridine to promote acetylcholine release by forebrain slices from aged mice. This drug also reduces the age-related depression in synaptosomal calcium uptake in aged rats and mice. 3,4-Diaminopyridine also reverses the age-related deficit in calcium transport, the age-related deficits in the tight rope test, and 8 arm maze performance. 3,4-Diaminopyridine is also effective in nonexcitable tissues, such as cultured skin fibroblasts; it increases the decreased cytosolic-free calcium. Depressed cell spreading of fibroblasts can be reversed by treatment of cells with the calcium ionophore A23187 which promotes calcium influx. 4-Aminopyridine, a similarly related compound, partially reverses short-term memory deficits in patients with Alzheimer's disease. Tetrahydroaminoacridine, an aminopyridine analog with anticholinesterase properties, produces clinical improvement in behavioral deficits due to Alzheimer's disease. Only recently has the aging brain become a subject of intense study. Evidently, the neurobiology of aging needs to develop its own theories to account for the unique aspects of brain aging as well as integrate them with the peripheral changes. An exciting but unexplored area of research in the aging brain concerns the coupling between calcium and the final end product, the induction of genes. Still unknown are the molecular events that set these processes in motion. In addition, whether conditions such as dietary restriction that increase longevity in certain rodents also retard age-related changes in calcium remains to be determined.


Apoptosis and T-cell repertoire selection in the thymus.

Thymic tolerance depends on induction of apoptosis (programmed cell death) in immature thymocytes by antigen/MHC complexes on dendritic cells (and possibly other bone marrow-derived APCs). Interactions with antigen/MHC complexes on thymic epithelial cells promote maturation of double-positive thymocytes to single-positive cells. However, the nature of the antigen/MHC complexes on thymic epithelial cells is unknown, and if the stromal cell interaction model as just outlined is correct, then presumably these complexes must be different from those presented on dendritic cells, otherwise all cells signaled for positive selection would be subject to negative selection by similar complexes on APCs. The nature of the signals provided by thymic epithelial cells versus dendritic cells is unknown and may provide a key to understanding the processes of positive and negative selection within the thymus. Clearly the intense selection of the T-cell repertoire within the thymus explains the high level of cell death observed within the immature thymocyte compartment. Such intensive selection shapes the T-cell repertoire in a way that provides an explanation for the genetic basis of immune responsiveness and for the susceptibility of certain individuals to autoimmunity.


Beta-endorphin concentrations in resting peripheral mononuclear cells and after treatment with PHA or serotoninergic drugs in human aging, Alzheimer's disease, and Down's syndrome.

We measured the concentrations of beta-endorphin in resting peripheral blood mononuclear cells obtained from normal subjects of different ages and from age-matched patients with Down's syndrome or Alzheimer's disease. We also measured beta-endorphin concentrations in peripheral blood mononuclear cells obtained from subjects of different ages after treatment with PHA or serotoninergic drugs. The results show that in normal subjects the concentrations of the peptide increase after 30 years of age and remain constant up to 99 years. After stimulation with PHA, the release of beta-endorphin in cells from subjects older than 30 years increases, leading to a decrease in contents, whereas it is unchanged in younger subjects. In patients with Down's syndrome or Alzheimer's disease, beta-endorphin concentrations in peripheral blood mononuclear cells behave similarly to those in age-matched normal subjects. Treatment in vivo with the serotoninergic agonist chlorimipramine induces an increase in beta-endorphin concentrations in peripheral blood mononuclear cells that is significantly greater in subjects over 30 years old than in younger subjects.


Biomarkers of aging in the neuroendocrine-immune domain. Time for a new theory of aging?

A common and generally accepted assumption is that with advancing age, the thymus undergoes progressive and irreversible involution. This is considered the main cause for the age-related deterioration of various immune functions and, ultimately, for the increased incidence of infectious, neoplastic, and automimmune diseases in old age. This assumption is no longer tenable because of several clear-cut demonstrations that age-related thymic involution is not an intrinsic and irreversible phenomenon. Various neuroendocrine or nutritional manipulations can to induce a regrowth of the thymus, even when applied in old age. This thymic reconstitution is followed by a consistent recovery of peripheral immune functions. These data strongly support the idea that thymic involution is a phenomenon secondary to age-related alterations in neuroendocrine-thymus interactions and that it is the disruption of such interactions in old age that is responsible for most of the age-associated dysfunctions. On the basis of this experimental and clinical evidence and as an alternative to purely immune or neuroendocrine theories of aging, a neuroendocrine-immune hypothesis is proposed. Further work is required to determine if the age-related disruption of neuroendocrine-immune interactions occurs because of progressive accumulation of stressor-dependent consequences at the level of one or the other system or if it may depend on a single common cause.


Protein synthesis, posttranslational modifications, and aging.

Posttranslational modifications of proteins are involved in determining their activities, stability, and specificity of interaction. More than 140 major and minor modifications of proteins have been reported. Of these, only a few have been studied in relation to the aging of cells, tissues, and organisms. These include phosphorylation, methylation, ADP-ribosylation, oxidation, glycation, and deamidation. Several of these modifications occur on proteins involved in crucial cellular processes, such as DNA synthesis, protein synthesis, protein degradation, signal transduction, cytoskeletal organization, and the components of extracellular matrix. Some of the modifications are the markers of abnormal and altered proteins for rapid degradation. Others make them less susceptible to degradation by normal proteolytic enzymes, and hence these accumulate during aging.


Relationship between antioxidants, prooxidants, and the aging process.

It is argued that reduced oxygen species may be one of the causal factors underlying the aging process. Experimental studies strongly support the view that the rate of metabolism is inversely associated with the rate of aging. It is pointed out that Pearl's rate of living theory is widely misunderstood, because of the mistaken belief that it advocates a fixed metabolic potential for different species or genotypes within a species. The in vivo level of oxidative stress tends to increase with age in insects and mammals as indicated by increased exhalation of alkanes. A search for the causes of this increase revealed that an age-associated decline in antioxidant defenses is neither widespread nor very impressive in magnitude. A comparison of antioxidant defenses (activities of SOD, catalase, and glutathione) in six different mammalian species did not suggest a clear association between these defenses and maximum life span potential of the species. In contrast, mitochondrial rates of O2- and H2O2 were found to increase with age in insects and mammals, and the MLSP of six mammals was found to be inversely correlated with liver mitochondrial rates of O2- and H2O2 generation. It seems that the age-related increase in oxidative stress is mainly due to the enhanced rate of O2- and H2O2 generation. It is hypothesized that variations in the rates of aging in different species, that are otherwise closely related phylogenetically, may be in part due to differences in rates of O2- and H2O2 production. Overall, the rates of oxidant generation are a better correlate of the rates of aging than are the levels of antioxidant defenses.


Oxidants are a major contributor to aging.

Very high level oxidative damage to DNA occurs during normal metabolism. In each rat cell the steady-state level of this damage is estimated to be about 10(6) oxidative adducts, and about 10(5) new adducts are formed daily. This endogenous DNA damage appears to be a major contributor to aging and to the degenerative diseases associated with aging such as cancer. The oxidative damage rate in mammalian species with a high metabolic rate, short life span, and high age-specific cancer rate such as rats is much higher than the rate in humans, long-lived mammals with a lower metabolic rate and a lower age-specific cancer rate. It is argued that deficiency of micronutrients that protect against oxidative DNA damage is a major contributor to human cancer. Epidemiological studies, a large body of experimental evidence, and theoretical work on the mechanisms of carcinogenesis point to mitogenesis as a major contributor to cancer. Dividing cells compared to nondividing cells are at an enormously increased risk for mutations in part due to the conversion of DNA adducts to mutations. Mitogenesis also increases the probability of gene amplification and loss of 5-methylcytosine. Dietary interventions that lower mitogenesis, such as calorie restriction, decrease the incidence of cancer.


Translational accuracy and the fitness of bacteria.

There are two aspects of the relationship between translational accuracy and the fitness of bacteria that I hope have been clarified in this review. One is that the impact of translational errors on the fitness of bacteria depends very much on nutritional conditions. It would seem that bacterial populations have the capacity to respond to different growth opportunities by the selection of suitable variants. It is particularly surprising how few mutations seem to be required to transform a slowly growing natural isolate with inefficient as well as inaccurate ribosomes into a growth-optimized laboratory strain. It would not be suprising if the selection of the slow, natural isolate phenotype under starvation conditions is equally facile. Another aspect of the accuracy-fitness relationship worth emphasizing is the strong impact of processivity errors and the weak impact of missense errors on the structures of proteins as well as on the growth of cells. What has been learned about translation mechanisms up to now is really only a preliminary to what remains to be discovered about the movements of tRNA, mRNA, and ribosomal subunits that support the processivity of translation. It would be very useful to have more direct methods at hand with which to study these movements. Likewise, the availability of methods to measure processivity errors in natural isolates would help to round out our view of the variability of the ribosomal mechanisms in nature.


Genetic analysis using the polymerase chain reaction.

The ability of PCR to amplify a specific DNA segment from a complex template in an automated reaction has made molecular analysis more accessible both to basic research and diagnostic laboratories. Modifications of the basic PCR method using generic primers now allow DNA amplification even in the absence of specific nucleotide sequence information. Although PCR has proved extremely valuable for detecting the presence or absence of a given sequence (e.g. diagnosis of infectious disease pathogens like HIV), it is uniquely suited to analyze genetic variation in the amplified DNA. The capacity to analyze DNA variation from limited samples like hair or cheek scrapings has made genetic diagnosis and population genetics studies much simpler and the amplification of specific sequences from individual cells (e.g. sperm) has opened new approaches to genetic mapping.


Genetics of retroviral integration.

Today the retroviral integration reaction is probably understood, both in terms of its genetics and chemistry, in as much detail as any eukaryotic recombination process. That understanding is in part due to its high efficiency (for it can be induced to occur synchronously in every cell of a culture); to its simplicity (for there is only one major protein player); to its accessibility (for the viral genome has provided all the cis- and trans-acting players); and to its willingness to perform well in vitro, ultimately with purified components. The process has thus made the classic transition from a phenomenon to be studied genetically to a reaction that can also be studied biochemically. The next advances in our understanding of the process of retroviral integration are likely to center on chemical issues. Some basic enzymological issues need to be addressed: we need to determine the oligomeric state of the native IN protein; its state when bound to linear viral DNA; the residues at the active site; the residues involved in sequence-specific recognition of DNA; and the points of contact between IN monomers. Much of this information will follow from detailed mutagenesis of expressed IN genes. A crucial step will be the determination of the structure of the IN protein at atomic resolution through X-ray diffraction analysis of protein crystals, a project underway in several laboratories. That structure may immediately suggest how the enzyme contacts and joins two DNA molecules, and will enormously facilitate the design and interpretation of mutational studies. It seems plausible that we can understand the IN protein as a machine as well as any nuclease or recombinase. A significant number of larger biological questions about integration remain unanswered and will require genetic approaches. What is the true structure of the preintegration complex in the cytoplasm? How does the complex enter the nucleus, and obtain access to the host DNA? Why, at least for most viruses in most cells, does integration depend on cell division? Why does efficient expression of the viral DNA to form progeny viral RNA and proteins depend on integration? How are target sites for integration on the host genome selected, and why are there "hot spots" for insertion? Are there host proteins that facilitate or participate in the integration reaction itself, and what are those proteins? Are any of those proteins involved in site selection?(ABSTRACT TRUNCATED AT 400 WORDS)


In vitro and in vivo activities of DQ-2556 and its mode of action.

DQ-2556 is a recently developed cephalosporin with a broad spectrum of antibacterial activity against both gram-positive and gram-negative bacteria. Its in vitro activity was roughly comparable to those of cefuzonam and cefpirome and greater than those of ceftazidime, cefepime, and cefclidin against gram-positive bacteria. Against gram-negative bacteria, DQ-2556 showed almost the same activity as those shown by cefpirome and cefepime. The activity was largely unaffected by culture medium pH or the addition of human serum. The protective effect of DQ-2556 in experimental gram-positive pathogen and Escherichia coli infections in mice was greater than those of ceftazidime, cefuzonam, cefepime, and cefclidin and was similar to that of cefpirome. Against Pseudomonas aeruginosa and Acinetobacter calcoaceticus infections, DQ-2556 was more active than cefuzonam and had activity similar to or less than those of ceftazidime, cefpirome, cefepime, and cefclidin. When cells of E. coli were exposed to various concentrations of DQ-2556, filamentous cells were observed at concentrations of 0.0008 micrograms/ml and greater, spheroplasts started to form at 0.025 micrograms/ml, and subsequent cell lysis was observed. The affinity of DQ-2556 to PBP 3 of E. coli, which participates in septum formation, as suggested by morphological observation, was two times greater than that of ceftazidime. DQ-2556 also had high affinities for PBPs 1B and 1A of E. coli. These results suggest that DQ-2556 is worthy for subsequent clinical trials.


Characterization of an azole-resistant Candida glabrata isolate.

A Candida (Torulopsis) glabrata strain (B57149) became resistant to fluconazole after a patient carrying the organism was treated with the drug at 400 mg once daily for 9 days. Growth of the pretreatment isolate (B57148) was inhibited by 50% with 0.67 microM ketoconazole, 1.0 microM itraconazole, and 43 microM fluconazole, whereas growth of B57149 was inhibited slightly by 10 microM ketoconazole but was unaffected by 10 microM itraconazole or 100 microM fluconazole. This indicates cross-resistance to all three azole antifungal agents. The cellular fluconazole content of B57149 was from 1.5- to 3-fold lower than that of B57148, suggesting a difference in drug uptake between the strains. However, this difference was smaller than the measured difference in susceptibility and, therefore, cannot fully explain the fluconazole resistance of B57149. Moreover, the intracellular contents of ketoconazole and itraconazole differed by less than twofold between the strains, so that uptake differences did not account for the azole cross-resistance of B57149. The microsomal cytochrome P-450 content of B57149 was about twice that of B57148, a difference quantitatively similar to the increased subcellular ergosterol synthesis from mevalonate or lanosterol. These results indicate that the level of P-450-dependent 14 alpha-demethylation of lanosterol is higher in B57149. Increased ergosterol synthesis was also seen in intact B57149 cells, and this coincided with a decreased susceptibility of B57149 toward all three azoles and amphotericin B. B57149 also had higher squalene epoxidase activity, and thus, more terbinafine was needed to inhibit the synthesis of 2,3-oxidosqualene from squalene. P-450 content and ergosterol synthesis both decreased when isolate B57149 was subcultured repeatedly on drug-free medium. This repeated subculture also fully restored the strain's itraconazole susceptibility, but only partly increased its susceptibility to fluconazole. The results suggest that both lower fluconazole uptake and increased P-450-dependent ergosterol synthesis are involved in the mechanism of fluconazole resistance but that only the increased ergosterol synthesis contributes to itraconazole cross-resistance.


In vitro susceptibilities of Actinobacillus actinomycetemcomitans to a number of antimicrobial combinations.

The in vitro susceptibilities of Actinobacillus actinomycetemcomitans to 14 antimicrobial combinations were studied by using the checkerboard titration technique. The results, expressed as the range of the fractional inhibitory concentration indices, were as follows: for metronidazole or its hydroxymetabolite combined with cefixime, 0.2 to 0.6; for moxalactam, 0.2 to 0.6; for penicillin G, 0.3 to 0.6; for tobramycin, 0.8 to 2.0; for erythromycin, 0.8 to 1.7; for ciprofloxacin, 0.2 to 0.6; for tetracycline, 0.8 to 1.2. Our observations indicated that the beta-lactam antibiotics as well as ciprofloxacin act synergistically with both metronidazole and its hydroxymetabolite against A. actinomycetemcomitans. Synergistic interactions were independent of the individual MICs of the antibiotics tested. Erythromycin, tobramycin, and tetracycline combined with either metronidazole or its hydroxymetabolite showed additive to indifferent effects against the five strains of A. actinomycetemcomitans, with the fractional inhibitory concentration indices ranging from 0.8 to 2.0. A. actinomycetemcomitans was found to be highly susceptible to ciprofloxacin (MIC of ciprofloxacin for 90% of strains tested, 0.010 micrograms/ml) and cefixime (MIC of cefixime for 90% of strains tested, 0.8 micrograms/ml). The results indicate that in patients who are allergic to penicillin, cefixime and ciprofloxacin may be useful alternative antibiotics in combination with metronidazole for the treatment of A. actinomycetemcomitans-associated periodontitis.


Prospective observational study of Klebsiella bacteremia in 230 patients: outcome for antibiotic combinations versus monotherapy.

Combination antimicrobial agent therapy has been advocated for treatment of gram-negative bacteremia, including that caused by Klebsiella spp. We performed a prospective, observational, 10-hospital collaborative study to evaluate the efficacy of antibiotic combination therapy versus that of monotherapy for 230 consecutive patients with Klebsiella bacteremia. The species involved were K. pneumoniae (82%), K. oxytoca (15%), and K. ozaenae (0.4%). Of the bacteremias, 26% were polymicrobial in nature. A total of 53% of cases were nosocomial infections. The most common portals were the urinary tract (28%), biliary tract (12%), lung (10%), and abdomen (9%). Some 49 and 51% of the patients had received monotherapy and antibiotic combination therapy (beta-lactam plus aminoglycoside), respectively; 14-day mortalities in the two groups were 20 and 18%, respectively. However, for the subgroup of patients who experienced hypotension within 72 h prior to or on the day of the positive blood culture, those patients who received combination therapy experienced significantly lower mortality (24%) than did those who received monotherapy (50%). We conclude that monotherapy with an antibiotic that is active in vitro against Klebsiella (beta-lactam or aminoglycoside) is sufficient therapy for less severely ill patients (immunocompetent, urinary tract portal, mentally alert, normal vital signs). On the other hand, for severely ill patients who experience hypotension, antibiotic combination therapy with a beta-lactam and an aminoglycoside agent is preferred.


Pharmacokinetics and transplacental passage of imipenem during pregnancy.

Imipenem pharmacokinetics were studied in early pregnancy (n = 7; length of gestation, 8.6 +/- 1.5 weeks, mean +/- standard deviation), in late pregnancy (n = 7; length of gestation, 38.7 +/- 1.4 weeks), and in the nonpregnant state (n = 6). A single dose of 500 mg of imipenem-cilastatin (1:1) was administered as a 20-min infusion. Multiple plasma and urine samples, as well as specimens of umbilical plasma and amniotic fluid from the pregnant subjects, were collected at frequent intervals for 8 h. Imipenem concentrations were assayed by specific microbiologic assay. The mean peak concentrations in plasma were 14.7 +/- 4.9, 14.9 +/- 5.2, and 43 +/- 28.3 micrograms/ml in early pregnancy, late pregnancy, and the nonpregnant state, respectively. The volumes of distribution were significantly larger during early pregnancy (0.98 +/- 0.45 liter/kg of body weight, P < 0.005) and late pregnancy (0.59 +/- 0.19 liter/kg, P < 0.05) than in the nonpregnant state (0.33 +/- 0.10 liter/kg), and total clearances from plasma were faster in early pregnancy (12.7 +/- 7.8 ml min-1 kg-1, P < 0.05) and late pregnancy (10.7 +/- 4.6 ml min-1 kg-1, P < 0.05) than in the nonpregnant state (5.77 +/- 1.19 ml min-1 kg-1). The mean concentrations in amniotic fluid were 0.07 +/- 0.01 and 0.72 +/- 0.85 micrograms/ml in early and late pregnancy. The mean umbilical venous and arterial drug concentrations were 1.72 +/- 1.22 and 1.64 +/- 1.22 micrograms/ml. The feto-maternal ratio at the time of cesarean section was 0.33 +/- 0.12. These results indicate that an adjustment of doses of imipenem may be required when treating pregnant women because of considerable changes in imipenem pharmacokinetics during pregnancy.


Efficacy and safety of amphotericin B colloidal dispersion compared with those of amphotericin B deoxycholate suspension for treatment of disseminated murine cryptococcosis.

The efficacy and safety of amphotericin B colloidal dispersion (ABCD) were compared with those of amphotericin B deoxycholate suspension (ABDS) (Fungizone) in a murine model of disseminated cryptococcosis. Mice were treated intravenously with either ABDS at 0.2, 0.8, or 3.2 mg/kg of body weight per dose or ABCD at 0.8, 3.2, 6.4, 12.8, or 19.2 mg/kg dose three times per week for 2 weeks. Excluding mice treated with ABDS at 3.2 mg/kg, which was acutely lethal in 100% of mice, and ABCD at 19.2 mg/kg, which also resulted in two early deaths, the survival of ABCD- and ABDS-treated groups was prolonged over survival of controls (P < or = 0.05). Survival of ABCD (3.2 mg/kg)-treated mice was improved over that of ABDS (0.2 mg/kg)-treated mice (P < 0.05); however, comparisons of mice given all other dosages of ABCD with mice given sublethal dosages of ABDS did not demonstrate differences in survival. Comparative fungal burdens in organs showed a decrease in liver (P < 0.05) and spleen (P < 0.05) burdens for ABCD with the 19.2-mg/kg therapy versus those with ABDS with the 0.8-mg/kg therapy and liver burdens for ABCD with the 12.8-mg/kg therapy versus ABDS with the 0.8-mg/kg therapy (P < 0.05). There was no difference in organ burdens between therapy with ABCD at 0.8 mg/kg and ABDS at 0.8 mg/kg. These data show that the efficacy of ABCD is equal to that of ABDS on a milligram-per-kilogram basis for murine disseminated cryptococcosis. Because of its decreased toxicity, greater efficacy with ABCD could be achieved through doses fourfold higher than the 100% lethal dose for ABDS. Thus, ABCD shows promise as an effective but less toxic alternative to ABDS for the treatment of disseminated cryptococcosis.


Pharmacokinetics of R 82913 in AIDS patients: a phase I dose-finding study of oral administration compared with intravenous infusion.

The pharmacokinetics of oral administration of R 82913, or tetrahydroimidazol [4,5,1-jk]-benzodiazepin-2(1H)-one or -thione (TIBO), was compared with those of intravenous administration in five AIDS patients. TIBO was administered as a single daily 1-h infusion of 100 mg for 29 days and orally as a single daily dose for 14 days with three consecutive regimens of 100, 200, and 100 mg with probenecid (1 g) daily. Each cycle was followed by a wash-out period. Oral bioavailability of TIBO appears to be low and is not improved by the adjunction of probenecid. Trough levels obtained with oral administration systematically remained far below the 90% inhibitory concentration of TIBO against human immunodeficiency virus type 1 (HIV-1). Tolerance of TIBO was excellent. No clinical efficacy could be demonstrated. p24 antigenemia decreased significantly in one patient under intravenous therapy. TIBO derivatives are promising anti-HIV-1 agents in vitro, but improvement of oral bioavailability is needed before implementation of long-term efficacy and tolerability studies. Moreover, rapid emergence of resistance, which has been recently documented, constitutes a major problem with most nonnucleoside reverse transcriptase inhibitors.


Acute and chronic effects of flucytosine on amphotericin B nephrotoxicity in rats.

The combination of intravenous flucytosine (FC) in 0.9% saline (NaCl) and amphotericin B (AmB) provides synergistic antifungal activity and is associated with a lower incidence of nephrotoxicity than with AmB treatment alone. This study was conducted to examine whether flucytosine can influence renal function and whether it can modify the acute and chronic renal responses to AmB in the rat. In the in situ perfused rat kidney, FC at a concentration of 10 mg/kg/min for 15 min had a vasodilator effect, increasing renal blood flow by 2.5 +/- 0.7 ml/min, an effect not observed with vehicle. After the infusion of FC was stopped for 15 min, AmB induced a decrease in renal blood flow similar to that with both FC and vehicle. In a second series of studies, AmB (5 mg/kg/day intraperitoneally) was administered to four groups of rats for 7 days. In addition, the following groups received the intravenous daily interventions indicated: group 1, 5% dextrose in water (15 ml/kg/12 h); group 2, FC (150 mg/kg/12 h) in 0.9% saline (15 ml/kg/12 h); group 3, 0.9% saline (15 ml/kg/12 h); and group 4, FC (150 mg/kg/12 h) in 5% dextrose in water. Group 1 sustained a 77% decrease in creatinine clearance over the 7 days and a threefold increase in serum creatinine concentration (P of < 0.05). Groups 2, 3, and 4 sustained significantly less nephrotoxicity, with no change in serum creatinine concentration and only 38, 41, and 53% decreases in creatinine clearance, respectively (P of < 0.05), compared with that for group 1. AmB levels in renal tissue varied inversely to creatinine clearance (r of 0.57, P of < or = 0.005). However, no significant differences were found in levels in tissue between groups (P of 0.06). The results of this study suggest that FC has a small but significant effect in reducing chronic AmB-induced nephrotoxicity. This amelioration of renal injury is independent of saline administration. There was evidence that the extent of renal uptake of AmB related to the efficiency of renal function at the end of the experiment.


Pharmacokinetics of cefepime dihydrochloride arginine in subjects with renal impairment.

In this study, the safety, tolerance, and pharmacokinetics of a single 1-g intravenous dose of cefepime (BMY-28142) were investigated. Twenty-three volunteers with various degrees of renal function were assigned to four trial groups according to glomerular filtration rates (GFR). Group IV consisted of five patients with end-stage renal disease undergoing treatment with hemodialysis. Cefepime concentrations in samples from plasma, urine, and infusion solutions were assayed with high-pressure liquid chromatography. The volume of distribution corresponded to the assumed extracellular fluid volume and did not differ significantly between the four groups. The area under the concentration-time curve increased as renal function decreased; in group II (GFR, 31 to 80 ml/[min x 1.73 m2]; n = 6), it was already three times higher than in group I (GFR, > or = 80 ml/[min x 1.73 m2]; n = 5). Mean residence time was 2.4, 6.8, 11.4, and 31.6 h for the four groups, respectively. Total clearance decreased (97.2, 34.6, 19.8, and 6.3 ml/[min x 1.73 m2]) with decreasing renal function, and a linear relationship between total plasma clearance and GFR was found with the regression equation y = 0.92x-2.0 (r = 0.991). Renal clearance was linearly correlated to GFR with the regression equation y = 0.87x-6.1 (r = 0.989), indicating that renal elimination is mainly by glomerular filtration. During hemodialysis, the extraction ratios were between 0.40 and 0.65. Dialysis clearance varied between 69.9 and 94.6 ml/(min x 1.73 m2).


Saperconazole therapy in a rabbit model of invasive aspergillosis.

The efficacy of orally and intravenously administered saperconazole against Aspergillus fumigatus was assessed in an immunosuppressed temporarily leukopenic rabbit model of invasive aspergillosis and compared with that of amphotericin B. Oral saperconazole at dosages of 5, 10, and 15 mg/kg of body weight per day improved survival compared with that of controls. In addition, saperconazole at 10 and 15 mg/kg/day reduced the tissue burden and reduced levels of circulating antigen, which correlated with increasing dosages of saperconazole. Intravenous saperconazole produced levels in serum more than 10-fold that of oral therapy. Intravenous saperconazole not only improved survival and reduced antigen levels but also significantly eradicated A. fumigatus from tissues compared with those of controls and was as effective as amphotericin B in these studies. Saperconazole was effective in the treatment of experimental invasive aspergillosis and demonstrates the potential of the newer azoles in therapy for invasive aspergillosis.


Spread of a newly found trimethoprim resistance gene, dhfrIX, among porcine isolates and human pathogens.

A plasmid-borne gene mediating trimethoprim resistance, dhfrIX, newly found among porcine strains of Escherichia coli, was observed at a frequency of 11% among trimethoprim-resistant veterinary isolates. This rather high frequency of dhfrIX could be due to the extensive use of trimethoprim in veterinary practice in Sweden. After searching several hundred clinical isolates, one human E. coli strain was also found to harbor the dhfrIX gene. Thus, the dhfrIX gene seems to have spread from porcine bacteria to human pathogens. Furthermore, the occurrence of other genes coding for resistant dihydrofolate reductase enzymes (dhfrI, dhfrII, dhfrV, dhfrVII, and dhfrVIII) among the porcine isolates was investigated. In addition, association of dhfr genes with the integraselike open reading frames of transposons Tn7 and Tn21 was studied. In colony hybridization experiments, both dhfrI and dhfrII were found associated with these integrase genes. The most common combination was dhfrI and int-Tn7, indicating a high prevalence of Tn7.


Activity of MS-8209, a nonester amphotericin B derivative, in treatment of experimental systemic mycoses.

The in vitro and in vivo toxicities and activities of MS-8209, a new hydrosoluble amphotericin B (deoxycholate-amphotericin B [D-AmB]; Fungizone) derivative, were studied. In vitro, MS-8209 was less toxic than AmB against renal tubular cells in primary culture and less active against Candida albicans and Cryptococcus neoformans. However, at 10-fold the AmB concentration, MS-8209 in vitro antifungal activity paralleled that of AmB. Fifty-percent lethal doses of MS-8209 and D-AmB in OF1 noninfected mice were 26 and 2.3 mg/kg, respectively. Therapeutic efficacy of MS-8209 was assessed in murine candidiasis, cryptococcosis, and aspergillosis. In each model of infection, we determined the maximum tolerated dosages of MS-8209 and D-AmB, i.e., the dosage inducing less than 15% mortality due to toxicity; the efficacies of MS-8209 and D-AmB at their respective maximum tolerated dosages were compared. In candidiasis, MS-8209 (15 mg/kg) significantly increased the survival time compared with D-AmB (0.5 mg/kg). Both compounds were equally effective at reducing CFU counts in the kidney. MS-8209 was the most effective agent for increasing the survival time in cryptococcal meningoencephalitis and for reducing CFU counts in spleen, brain, and lung during both cryptococcal pneumonia and meningoencephalitis. In aspergillosis, MS-8209 and D-AmB similarly prolonged the survival of treated mice compared with controls. These results show that when MS-8209 and D-AmB were used at the maximum tolerated dosage, MS-8209 was as effective as or more effective than D-AmB for the treatment of systemic mycoses. These findings warrant further experiments to study the pharmacokinetic properties and toxicity of MS-8209 under conditions of chronic administration.


Antimicrobial activities of clofazimine and B669 are mediated by lysophospholipids.

The susceptibilities of a range of gram-positive and gram-negative microbial pathogens to clofazimine and its analog B669 (0.1 to 32 micrograms/ml), as well as the effects of these agents on membrane phospholipid metabolism in Staphylococcus aureus and Escherichia coli, have been investigated in vitro. Gram-positive bacteria were found to be generally susceptible to these agents, whereas gram-negative organisms were uniformly resistant. Exposure of S. aureus to both agents (1 to 5 micrograms/ml), especially B669, caused dose-related enhancement of the activity of phospholipase A2, according to an increase in the release of 3H-radiolabeled arachidonate and lysophosphatidylethanolamine ([3H]LPE) from bacterial-membrane phospholipids. Treatment of E. coli with the riminophenazines also increased the release of [3H]arachidonate and [3H]LPE. Growth of gram-positive but not gram-negative bacteria was inhibited by LPE and lysophosphatidylcholine. Moreover, coincubation with alpha-tocopherol (vitamin E), a lysophospholipid complex-forming agent, or with lysophospholipase protected gram-positive bacteria against the riminophenazines as well as against lysophospholipids. The results from this study are consistent with a mechanism whereby lysophospholipids mediate the activities of the two drugs.


Cure of murine Trypanosoma brucei rhodesiense infections with an S-adenosylmethionine decarboxylase inhibitor.

The compound 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine (MDL73811), a potent inhibitor of S-adenosylmethionine decarboxylase, was effective in mice against six of eight clinical isolates of Trypanosoma brucei rhodesiense, the causative agent of East African sleeping sickness. In combination with the ornithine decarboxylase inhibitor DL-alpha-difluoromethylornithine (DFMO; Ornidyl), MDL73811 acted synergistically to cure seven of eight infections. MDL73811 was effective when given singly at 50 to 100 mg/kg of body weight per day for 7 days (osmotic pumps). In combination with subcurative DFMO levels (0.25 to 1.0% in drinking water for 7 days), the curative MDL73811 dose could be lowered to 25 or 50 mg/kg, depending on the isolate. Oral administration of the MDL73811-DFMO combination was also effective in an acute infection and in a long-term central nervous system model of Trypansoma brucei brucei infection. These data indicate that MDL73811 may be effective therapeutically in drug-refractory and late-stage East African trypanosomiasis.


Pharmacodynamics of once-daily amikacin in various combinations with cefepime, aztreonam, and ceftazidime against Pseudomonas aeruginosa in an in vitro infection model.

The pharmacodynamics of once-daily amikacin administered as monotherapy and in combination with aztreonam, ceftazidime, and cefepime against Pseudomonas aeruginosa ATCC 27853 and clinical isolate 16690 (moderately susceptible to ceftazidime) were investigated with an in vitro model of infection over a 24-h period. Monotherapy with aztreonam, ceftazidime, and cefepime and combinations of aztreonam with cefepime or ceftazidime were also studied. MICs and MBCs were determined for viable organisms at 24 h to test for the development of resistance. Once-daily amikacin demonstrated killing activity over the initial 8 h superior to those of all other drugs administered as monotherapy against both strains tested (P < 0.01). Regrowth by 24 h was greatest for the amikacin regimen (P < 0.01) but was apparent for all monotherapy regimens against both strains. No changes in susceptibilities were observed. All combination therapies containing once-daily amikacin achieved 99.9% reductions in log10 CFU/ml by 2.0 h against both strains, with no regrowth of organisms at 24 h. Aztreonam-cefepime and -ceftazidime combinations required approximately 5 to 6 h to achieve a 99.9% reduction in log10 CFU/ml. Although there was no difference in time to the 99.9% kill between the aztreonam-cefepime and -ceftazidime regimens against either strain, the killing activity of these combinations was significantly less than those of regimens containing once-daily amikacin (P < 0.01). Minor differences in the initial susceptibilities of beta-lactams and the monobactam aztreonam against P. aeruginosa may not be important for therapeutic outcomes when used in combination with single-daily aminoglycoside therapy.


Effect of sucralfate on pharmacokinetics of fleroxacin in healthy volunteers.

The effect of sucralfate on the pharmacokinetics of fleroxacin was assessed in 20 healthy male volunteers. The study was of a two-way crossover design in which subjects were randomized to one of the following two regimens at the time of entry: (i) a single 400-mg dose of fleroxacin alone or (ii) a 400-mg dose of fleroxacin given once and 1 g of sucralfate given every 6 h starting 24 h before fleroxacin treatment and continuing for 48 h after fleroxacin treatment. Blood samples were collected immediately before fleroxacin administration and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 16, 24, 36, and 48 h postdosing. Fleroxacin concentrations in plasma and urine were determined by high-performance liquid chromatography. While concurrent of fleroxacin and sucralfate resulted in a decrease in the area under the plasma concentration-time curve, a decrease in the maximum concentration, and an increase in the time to the maximum concentration (P < 0.05), these changes were modest compared with the interaction of other quinolones with sucralfate. The relative bioavailability of fleroxacin given with sucralfate, calculated from the area under the concentration-time curve, was 76% compared with that of fleroxacin alone. This is significantly better than the bioavailabilities of other quinolones (1.8 to 12.3%) when they are administered with sucralfate.


Fluoxetine hydrochloride enhances in vitro susceptibility to chloroquine in resistant Plasmodium falciparum.

The emergence of chloroquine resistance in Plasmodium falciparum has necessitated the development of alternate strategies for chemotherapy and chemoprophylaxis. One approach has been the identification of drugs that do not possess any intrinsic antimalarial activity when used alone but that potentiate the effect of currently available antimalarial drugs, such as chloroquine. We identified fluoxetine hydrochloride (Prozac), a commonly prescribed antidepressant, as another resistance modulator for drug-resistant P. falciparum. Studies with chloroquine-resistant clones and isolates from various geographical locations confirmed our initial observations with a chloroquine-resistant P. falciparum clone, W2. Fluoxetine concentrations of 500 nM were found to effectively modulate chloroquine resistance by 66% in clone W2. In comparison, verapamil at similar concentrations was observed to modulate chloroquine resistance in clone W2 by 61%. Neither fluoxetine nor verapamil was observed to possess any innate antimalarial activity. These data augment the current description of the chloroquine resistance phenotype and may provide additional insights into lead-directed synthesis of new antimalarial drugs.


Interleukin-1-induced nonspecific resistance to bacterial infection in mice is not mediated by glucocorticosteroids.

Preexposure to a low dose of interleukin-1 (IL-1; 3 to 30 micrograms/kg) 24 h before a lethal gram-negative bacterial infection prolonged survival in normal and granulocytopenic mice. To examine whether this protective effect is mediated by glucocorticosteroids, we first measured corticosterone concentrations in mice after administration of 80 and 800 ng of IL-1. IL-1 induced a dose-dependent increase in corticosterone levels in plasma. Next, the corticosterone peak induced by a protective dose of IL-1 (800 ng) was simulated by administration of synthetic human adrenocorticotropic hormone 1-24 (ACTH) in normal and neutropenic mice. Although corticosterone levels induced by pretreatment with IL-1 or ACTH were virtually identical, the ACTH-induced corticosterone peak was not associated with protection against Klebsiella pneumoniae infection in normal mice and Pseudomonas aeruginosa infection in neutropenic mice. This indicates that the protective effect of IL-1 pretreatment against gram-negative bacterial infection is not mediated by elevated levels of glucocorticosteroids. In addition, we found that plasma corticosterone concentrations during K. pneumoniae infection were significantly lower after pretreatment with IL-1 than after pretreatment with ACTH or vehicle, probably reflecting the better physical condition of IL-1-treated mice.


Pharmacokinetics of SCH-39304 in human immunodeficiency virus-infected patients following chronic oral dosing.

The pharmacokinetics of SCH-39304, an investigational, orally active, broad-spectrum antifungal agent, were evaluated in 17 adult, human immunodeficiency virus-positive males. Patients were studied on days 1 and 16 and were divided into the following three treatment groups: (i) patients with culture-proven oropharyngeal candidiasis who were not receiving concurrent zidovudine therapy and who were treated with 50 mg of SCH-39304 daily (n = 6); (ii) patients with culture-proven oropharyngeal candidiasis who were receiving concurrent zidovudine therapy and who were treated with 50 mg of SCH-39304 daily (n = 5); and (iii) patients with or without oropharyngeal candidiasis who were receiving concurrent zidovudine therapy and who were treated with 200 mg of SCH-39304 daily (n = 6). All patients received a single daily dose of the study medication for 16 days. Plasma samples for SCH-39304 concentration measurement were collected for 6 h following the initial dose and for 504 h following the day 16 dose. Urine was collected for 24 h following SCH-39304 administration on days 1 and 16. All samples were assayed for SCH-39304 by gas chromatography. Wide intersubject variations in SCH-39304 plasma concentration-versus-time profiles were observed on each study day. Absorption appeared to be slow, with mean day 1 peak plasma SCH-39304 concentrations of 1.2 micrograms/ml at 2.1 h (50 mg) and 3.9 micrograms/ml at 4.0 h (200 mg) after drug administration. Mean peak plasma SCH-39304 concentrations on day 16 were 7.6 micrograms/ml at 4.3 h (50 mg) and 17.2 micrograms/ml at 3.2 h (200 mg) after drug administration. Mean elimination half-lives on day 16 for the 50- and 200-mg daily dosages were 100 and 89 h, respectively. SCH-39304 was cleared primarily unchanged in the urine. Mean areas under the plasma concentration-versus-time curve (from 0 to 24 h) on day 16 reflect a lower than expected increase with the 200-mg/day regimen (314.5 microgram.h/ml) compared with that for the 50-mg/day regimen (139.9 microgram.h/ml), suggesting the potential for reduced bioavailability at higher dosages. No significant effect of concurrent zidovudine therapy on the kinetics of SCH-39304 was observed.


Pharmacokinetics of meropenem (ICI 194,660) and its metabolite (ICI 213,689) in healthy subjects and in patients with renal impairment.

The pharmacokinetics of meropenem (ICI 194,660) and its open-ring metabolite (ICI 213,689) were studied in 6 healthy volunteers and 16 patients with moderate to severe renal impairment after a single intravenous dose of 500 mg given as a 30-min infusion. Concentrations of unchanged meropenem in plasma and urine were measured by both microbiological and high-pressure liquid chromatographic (HPLC) assays. A good correlation was found between the two techniques. Pharmacokinetic parameters of unchanged meropenem were determined by using the HPLC data. The terminal half-life of unchanged meropenem increased in relation to the degree of renal impairment, being 1.2 h in subjects with normal renal function and 10 h in patients with end-stage renal failure. Total body clearance and renal clearance of unchanged meropenem are linearly related to creatinine clearance. The concentrations of the metabolite in plasma, which are very low in healthy subjects, significantly increased in uremic patients. The apparent half-life of ICI 213,689 increased in uremic patients and was about 35 h in patients with severe renal insufficiency. Meropenem and its metabolite are effectively removed by hemodialysis. The dialysis clearance of the unchanged drug was 81 +/- 22 ml/min. Dosage adjustments of meropenem will be necessary in patients with severe renal impairment.


In vitro antibiotic susceptibility of the newly recognized agent of ehrlichiosis in humans, Ehrlichia chaffeensis.

Ehrlichiosis in humans, a rickettsial disease recently discovered in the United States, is generally treated successfully with tetracyclines; however treatment with these agents is usually avoided with children and pregnant women. The in vitro susceptibility of Ehrlichia chaffeensis, the agent of human ehrlichiosis in the United States, was assessed by a quantitative evaluation of infected DH82 cells cultivated in 96-well microtiter plates in the presence of different concentrations of selected antibiotics. Extracellular MICs and MBCs were evaluated after 72 h of exposure to the antibiotics. Doxycycline and rifampin were found to exert rapidly bactericidal effects, with MBCs in the extracellular culture medium of less than 0.5 and 0.125 microgram/ml, respectively. E. chaffeensis was resistant to chloramphenicol, ciprofloxacin, erythromycin, co-trimoxazole, penicillin, and gentamicin, which had MICs greater than 16, 4, 8, 4, 40, and 32 micrograms/ml, respectively. These observations are consistent with the finding that human ehrlichiosis appears to respond to tetracycline therapy, which has been the therapy of first choice. Further clinical investigations are necessary to evaluate the role of rifampin in the treatment of human ehrlichiosis, especially in children.


Pharmacokinetics and tissue penetration of a single dose of ceftriaxone (1,000 milligrams intravenously) for antibiotic prophylaxis in thoracic surgery.

The pharmacokinetics and tissue penetration of ceftriaxone after a single intravenous injection of 1,000 mg to 17 patients for antibiotic prophylaxis in thoracic surgery were studied. The patients were scheduled for elective noncardiac thoracic surgery. Adequate levels in serum (higher than or equal to the MIC for 90% of isolates of Staphylococcus aureus, Streptococcus spp., Escherichia coli, Haemophilus influenzae, and Klebsiella pneumoniae) were found for all patients throughout the surgical procedures. Mean maximal (5-min) and final (24-h) ceftriaxone levels in serum were 157 +/- 42 and 8.6 +/- 4.5 mg/liter, respectively. The beta-phase elimination half-life was 8.6 +/- 3 h, the plasma clearance was 18.4 +/- 6.25 ml/min, and the apparent volume of distribution at steady state was 0.21 +/- 0.07 liters/kg. At the time of the thoracotomy, the ceftriaxone concentrations were 13.5 +/- 7.8 micrograms/g in thoracic wall fat and 27 +/- 9 micrograms/g in lung tissue. At the time of closure, the ceftriaxone concentration was 15 +/- 9 micrograms/g in thoracic wall fat. During the different steps of the surgical procedures, 100% of patients had adequate levels in tissue (higher than or equal to the MIC for 90% of isolates of Streptococcus spp., E. coli, H. influenzae, and K. pneumoniae). For S. aureus, 90 to 100% of patients had adequate tissue ceftriaxone levels.


Efficacies of liposome-encapsulated streptomycin and ciprofloxacin against Mycobacterium avium-M. intracellulare complex infections in human peripheral blood monocyte/macrophages.

Current treatments of disseminated infection caused by the Mycobacterium avium-M. intracellulare complex (MAC) are generally ineffective. Liposome-mediated delivery of antibiotics to MAC-infected tissues in vivo can enhance the efficacy of the drugs (N. Düzgüneş, V. K. Perumal, L. Kesavalu, J. A. Goldstein, R. J. Debs, and P. R. J. Gangadharam, Antimicrob. Agents Chemother. 32:1404-1411, 1988; N. Düzgüneş, D. A. Ashtekar, D. L. Flasher, N. Ghori, R. J. Debs, D. S. Friend, and P. R. J. Gangadharam, J. Infect. Dis. 164:143-151, 1991). We investigated the therapeutic efficacies of liposome-encapsulated streptomycin and ciprofloxacin against growth of the MAC inside human peripheral blood monocyte/macrophages. Treatment was initiated 24 h after infection of macrophages with the MAC and stopped after 20 h, and the cells were incubated for another 7 days. The antimycobacterial activity of streptomycin was enhanced when the drug was delivered to macrophages in liposome-encapsulated form, reducing the CFU about threefold more than the free drug did throughout the concentration range studied (10 to 50 micrograms/ml). With 50 micrograms of encapsulated streptomycin per ml, the CFU were reduced to 11% of the initial level of infection. Liposome-encapsulated ciprofloxacin was at least 50 times more effective against the intracellular bacteria than was the free drug: at a concentration of 0.1 microgram/ml, liposome-encapsulated ciprofloxacin had greater antimycobacterial activity than the free drug at 5 microgram/ml. With liposome-encapsulated ciprofloxacin at 5 micrograms/ml, the CFU were reduced by more than 1,000-fold at the end of the 7-day incubation period, compared with untreated controls. These results suggest that liposome-encapsulated ciprofloxacin or other fluoroquinolones may be effective against MAC infections in vivo.


Comparison of two antibiotic regimens (piperacillin plus amikacin versus ceftazidime plus amikacin) as empiric therapy for febrile neutropenic patients with cancer.

A total of 170 febrile episodes in neutropenic patients with cancer were randomly assigned to be treated with piperacillin-amikacin or ceftazidime-amikacin. The overall response rates were similar in both groups (68 and 65%, respectively). Response rates for clinically or microbiologically documented episodes were 54.5% for piperacillin-amikacin and 58.8% for ceftazidime-amikacin. Response rates for gram-negative bacillary infections were 65 and 73%, respectively. There was also no difference for gram-positive infections (31 and 50%, respectively). The toxicities were also comparable and consisted of skin rashes, hypokalemia, and diarrhea. Vancomycin was added if the fever persisted 72 h after the beginning of therapy; it increased the response rates to 94% when used with piperacillin-amikacin and 92% when used with ceftazidime plus amikacin. Our results suggest that the combinations show similar global efficacies in the treatment of febrile episodes in cancer patients.


Optimal dosing of trimethoprim-sulfamethoxazole when used with loperamide to treat traveler's diarrhea.

To explore the optimal dosing regimen for trimethoprim-sulfamethoxazole (TMP-SMX) when used in combination with loperamide to treat traveler's diarrhea, 190 U.S. adults with acute diarrhea were enrolled in a double-blind, randomized trial in Guadalajara, Mexico. All patients received loperamide (4-mg loading dose; 2 mg after each loose stool, not to exceed 16 mg/day for 3 days) and were randomized to receive a 3-day course of TMP-SMX (160:800 mg twice daily for six doses) (group A), a single large dose of TMP-SMX (320:1,600 mg) (group B), or a large loading dose (320:1,600 mg) followed by standard doses for 3 days (160:800 mg twice daily for five doses) (group C). Patients in group C responded best (P < 0.01), with 75% of subjects recovered from diarrhea in 12 h compared with 34 h (group A) and 33 h (group B). Similar differences in favor of group C were noted in the subset of patients who presented with moderate to severe diarrhea. On average, patients in group C took significantly (P < 0.05) less loperamide (1.2 mg) after the 4-mg loading dose compared with patients in group A (2.4 mg) or group B (2.0 mg). We conclude that the most efficacious treatment of traveler's diarrhea in the interior of Mexico is to take loperamide in usual doses to control symptoms in combination with a single large dose of TMP-SMX, which should then be continued for 3 days in standard doses.