PubMed Health⌕ Search

Biomedical subjects

Bhupinder Singh

Publications and source records attributed to Bhupinder Singh.

At least 19 recordsLinked to original sources

D-galactonate metabolism in enteric bacteria: a molecular and physiological perspective.

D-galactonate, a widely prevalent sugar acid, was first reported as a nutrient source for enteric bacteria in the 1970s. Since then, decades of research enabled a description of the modified Entner-Doudoroff pathway involved in its degradation and reported the structural and biochemical features of its metabolic enzymes, primarily in Escherichia coli K-12. However, only in the last few years, the D-galactonate transporter has been characterized, and the regulation of the dgo operon, encoding the structural genes for the transporter and enzymes of D-galactonate metabolism, has been detailed. Notably, in recent years, multiple evolutionary studies have identified the dgo operon as a dominant target for adaptation of E. coli in the mammalian gut. Despite considerable research on dgo operon, numerous fundamental questions remain to be addressed. The emerging relevance of the dgo operon in host-bacterial interactions further necessitates the study of D-galactonate metabolism in other enterobacterial strains.

Operon↗

Fluoride in drinking water and human urine in Southern Haryana, India.

The objective of this study was to determine the fluoride content in drinking water and urine samples of adolescent males aged 11-16 years living in Southern Haryana, India. A total of 30 drinking water sources in the studied habitations were assessed for fluoride contamination. Fluoride was estimated in the urine of 400 male children randomly selected from these habitations. The fluoride concentration in drinking water and urine samples was determined using USEPA fluoride ion selective electrode method. The mean fluoride concentration in drinking water samples of Pataudi, Haily Mandi and Harsaru villages was 1.68+/-0.35, 3.22+/-1.18 and 1.78+/-0.12 mg/l, respectively. The mean urinary fluoride concentration was 2.26+/-0.024 mg/l at Pataudi, 2.48+/-0.77 mg/l at Haily Mandi and 2.43+/-0.84 mg/l at Harsaru village. The higher fluoride levels in the urine of children may be associated to higher fluoride levels in drinking water. The accuracy of measurements was assessed with known addition method in water and urine. Mean fluoride recovery was 98.0 and 99.1% in water and urine. The levels obtained were reproducible with in +/-3% error limit.

Adolescent↗

Studies on dissolution enhancement and mathematical modeling of drug release of a poorly water-soluble drug using water-soluble carriers.

Role of various water-soluble carriers was studied for dissolution enhancement of a poorly soluble model drug, rofecoxib, using solid dispersion approach. Diverse carriers viz. polyethylene glycols (PEG 4000 and 6000), polyglycolized fatty acid ester (Gelucire 44/14), polyvinylpyrollidone K25 (PVP), poloxamers (Lutrol F127 and F68), polyols (mannitol, sorbitol), organic acid (citric acid) and hydrotropes (urea, nicotinamide) were investigated for the purpose. Phase-solubility studies revealed AL type of curves for each carrier, indicating linear increase in drug solubility with carrier concentration. The sign and magnitude of the thermodynamic parameter, Gibbs free energy of transfer, indicated spontaneity of solubilization process. All the solid dispersions showed dissolution improvement vis-à-vis pure drug to varying degrees, with citric acid, PVP and poloxamers as the most promising carriers. Mathematical modeling of in vitro dissolution data indicated the best fitting with Korsemeyer-Peppas model and the drug release kinetics primarily as Fickian diffusion. Solid state characterization of the drug-poloxamer binary system using XRD, FTIR, DSC and SEM techniques revealed distinct loss of drug crystallinity in the formulation, ostensibly accounting for enhancement in dissolution rate.

Calorimetry, Differential Scanning↗

Sp1 is up-regulated in cellular and transgenic models of Huntington disease, and its reduction is neuroprotective.

Interactions between mutant huntingtin (Htt) and a variety of transcription factors including specificity proteins (Sp) have been suggested as a central mechanism in Huntington disease (HD). However, the transcriptional activity induced by Htt in neurons that triggers neuronal death has yet to be fully elucidated. In the current study, we characterized the relationship of Sp1 to Htt protein aggregation and neuronal cell death. We found increased levels of Sp1 in neuronal-like PC12 cells expressing mutant Htt, primary striatal neurons, and brain tissue of HD transgenic mice. Sp1 levels were also elevated when 3-nitropropionate (3-NP) was used to induce cell death in PC12 cells. To assess the effects of knocking down Sp1 in HD pathology, we used Sp1 siRNA, a heterozygous Sp1 knock-out mouse, and mithramycin A, a DNA-intercalating agent that inhibits Sp1 function. The three approaches consistently yielded reduced levels of Sp1 which ameliorated toxicity caused by either mutant Htt or 3-NP. In addition, when HD mice were crossed with Sp1 heterozygous knock-out mice, the resulting offspring did not experience the loss of dopamine D2 receptor mRNA characteristic of HD mice, and survived longer than their HD counterparts. Our data suggest that enhancement of transcription factor Sp1 contributes to the pathology of HD and demonstrates that its suppression is beneficial.

Animals↗

Formulation and optimization of controlled release mucoadhesive tablets of atenolol using response surface methodology.

The aim of the current study was to design oral controlled release mucoadhesive compressed hydrophilic matrices of atenolol and to optimize the drug release profile and bioadhesion using response surface methodology. Tablets were prepared by direct compression and evaluated for bioadhesive strength and in vitro dissolution parameters. A central composite design for 2 factors at 3 levels each was employed to systematically optimize drug release profile and bioadhesive strength. Carbopol 934P and sodium carboxymethylcellulose were taken as the independent variables. Response surface plots and contour plots were drawn, and optimum formulations were selected by feasibility and grid searches. Compressed matrices exhibited non-Fickian drug release kinetics approaching zero-order, as the value of release rate exponent (n) varied between 0.6672 and 0.8646, resulting in regulated and complete release until 24 hours. Both the polymers had significant effect on the bioadhesive strength of the tablets, measured as force of detachment against porcine gastric mucosa (P < .001). Polynomial mathematical models, generated for various response variables using multiple linear regression analysis, were found to be statistically significant (P < .01). Validation of optimization study, performed using 8 confirmatory runs, indicated very high degree of prognostic ability of response surface methodology, with mean percentage error (+/- SD) as -0.0072 +/- 1.087. Besides unraveling the effect of the 2 factors on the various response variables, the study helped in finding the optimum formulation with excellent bioadhesive strength and controlled release.

Adrenergic beta-Antagonists↗

Bioavailability of immediate- and extended-release formulations of glipizide in healthy male volunteers.

BACKGROUND AND OBJECTIVE: An extended-release glipizide formulation using a hydrophilic matrix system containing hydrophilic polymers has been developed for use in diabetes mellitus. This study compared the pharmacokinetic parameters of immediate- and extended-release formulations of glipizide 5mg in healthy male volunteers. METHODS: In a single-dose, four-period, four-treatment, Latin-square crossover study, the bioavailability of immediate-release glipizide 5mg (Glynase) [GL], extended-release glipizide 5mg (Glynase) XL [GLXL], Glucotrol XL [GTXL], and the new formulation developed in our laboratory [GLPF]) was compared. Plasma glipizide levels of the four formulations were determined at different time intervals, and pharmacokinetic parameters were analysed using a two-compartment body model. RESULTS: The mean peak plasma concentration (C(max)) of the immediate-release formulation (523+/-60 ng/mL) was significantly higher (p<0.05) than those of the three extended-release formulations (403+/-24, 349+/-37 and 426+/-55 ng/mL for GLXL, GTXL and GLPF, respectively). Mean time to reach C(max) was 1.83+/-0.3 hours for GL, 4.41+/-1.2 hours for GLXL, 3.21+/-0.8 hours for GTXL and 3.24+/-0.4 hours for GLPF. The order of magnitude of area under the plasma concentration-time curve was GTXL (5591 ng . h/mL)>GLXL (4,771 ng . h/mL)>GLPF (4,537 ng . h/mL)>GL (1,897 ng . h/mL). The mean residence time was 3.14+/-0.59 hours for GL, 8.26+/-0.81 hours for GLXL, 9.70+/-2.70 hours for GTXL and 7.87+/-1.93 hours for GLPF. Extended-release glipizide formulations maintained effective plasma drug concentrations for approximately 24 hours. Plasma levels of glipizide fluctuated less with GTXL than with the other two extended-release formulations. CONCLUSION: The newly developed formulation (GLPF) maintained effective levels of glipizide for a period of more than 20 hours, with quicker onset of action than the other two formulations. This formulation may be more economical than glipizide GITS.

Adolescent↗

Excessive daytime sleepiness in a trainee military pilot.

A 21-yr-old army trainee pilot was noted by his colleagues to be frequently falling asleep in his class and was reported to medical authorities. Despite a number of investigations and review by two sleep specialists, a formal diagnosis could not be made. During his medical review board three questions were posed: does the trainee have excessive daytime sleepiness (EDS), or is his sleepiness a variation of normal; if he is excessively sleepy, what is the most likely diagnosis; and should the trainee be allowed to continue to undertake pilot training based on most likely diagnosis and/or his history. The most likely diagnosis was considered to be idiopathic hypersomnia, a relatively uncommon condition which is a diagnosis of exclusion. The condition was considered incompatible with flight duties in a pilot under training due to the potential for compromise of performance, mission completion, and flight safety. Stimulant medications may control symptoms, but are incompatible with flying duties in the Australian Defense Force. This case illustrates the difficulties in determining aeromedical disposition in borderline cases of EDS where a clear diagnosis cannot be made.

Adult↗

Emergence of ciprofloxacin resistant Neisseria gonorrhoeae in north India.

BACKGROUND AND OBJECTIVES: Though quinolones have been recommended as a single dose treatment for uncomplicated gonorrhoea, there have been reports of treatment failure with fluoroquinolones. In this study we determined the antimicrobial susceptibility levels of consecutive isolates of Neisseria gonorrhoeae to examine the emergence of ciprofloxacin resistance N. gonorrhoeae. METHODS: Minimum inhibitory concentration (MIC) of ciprofloxacin and other drugs (penicillin, tetracycline, ciprofloxacin and ceftriaxone) was determined by agar dilution method. MIC was interpreted according to the NCCLS guidelines. beta lactamase production was detected by iodometric method and chromogenic cephalosporin method using nitrocefin disc. RESULTS: A total of 45 consecutive isolates of N. gonorrhoeae were obtained from patients with suspected acute gonococcal uretheritis. Of the 45 isolates, 35 (77.7%) were resistant to ciprofloxacin, 16 (35.5%) showed MIC value greater than 8 microg/ml. All isolates were sensitive to ceftriaxone while 21 isolates (46.6%) were resistant to penicillin and 23 (51%) to tetracycline. Ten isolates (22%) were found to be beta-lactamase producers. INTERPRETATION AND CONCLUSION: Ciprofloxacin resistant N. gonorrhoeae is on the rise in and around Chandigarh (north India). Thus, periodic surveillance of susceptibility levels of N. gonorrhoeae is essential to prevent the dissemination of drug resistant strains in the community.

Anti-Bacterial Agents↗

Optimizing drug delivery systems using systematic "design of experiments." Part I: fundamental aspects.

Design of an impeccable drug delivery product normally encompasses multiple objectives. For decades, this task has been attempted through trial and error, supplemented with the previous experience, knowledge, and wisdom of the formulator. Optimization of a pharmaceutical formulation or process using this traditional approach involves changing one variable at a time. Using this methodology, the solution of a specific problematic formulation characteristic can certainly be achieved, but attainment of the true optimal composition is never guaranteed. And for improvement in one characteristic, one has to trade off for degeneration in another. This customary approach of developing a drug product or process has been proved to be not only uneconomical in terms of time, money, and effort, but also unfavorable to fix errors, unpredictable, and at times even unsuccessful. On the other hand, the modern formulation optimization approaches, employing systematic Design of Experiments (DoE), are extensively practiced in the development of diverse kinds of drug delivery devices to improve such irregularities. Such systematic approaches are far more advantageous, because they require fewer experiments to achieve an optimum formulation, make problem tracing and rectification quite easier, reveal drug/polymer interactions, simulate the product performance, and comprehend the process to assist in better formulation development and subsequent scale-up. Optimization techniques using DoE represent effective and cost-effective analytical tools to yield the "best solution" to a particular "problem." Through quantification of drug delivery systems, these approaches provide a depth of understanding as well as an ability to explore and defend ranges for formulation factors, where experimentation is completed before optimization is attempted. The key elements of a DoE optimization methodology encompass planning the study objectives, screening of influential variables, experimental designs, postulation of mathematical models for various chosen response characteristics, fitting experimental data into these model(s), mapping and generating graphic outcomes, and design validation using model-based response surface methodology. The broad topic of DoE optimization methodology is covered in two parts. Part I of the review attempts to provide thought-through and thorough information on diverse DoE aspects organized in a seven-step sequence. Besides dealing with basic DoE terminology for the novice, the article covers the niceties of several important experimental designs, mathematical models, and optimum search techniques using numeric and graphical methods, with special emphasis on computer-based approaches, artificial neural networks, and judicious selection of designs and models.

Drug Delivery Systems↗

Optimizing drug delivery systems using systematic "design of experiments." Part II: retrospect and prospects.

The first study on application of Design of Experiments (DoE) in optimizing drug formulation appeared in 1967. Since then the number of literature reports on the use of DoE optimization in development of drug delivery technologies has been piling up steadily. Such systematic techniques find their use in every type of conventional dosage form and modern drug delivery system. The drug delivery devices investigated for optimization using response surface methodology include controlled release compressed matrices, microparticulates, macroparticulates, vesicular systems, floating systems, bioadhesive systems, semisolids, transdermals, and inhalations. The optimized processes mainly include extrusion-spheronization, pelletization, microencapsulation, coating, granulation, and tableting. Part I of this article [Crit Rev Ther Drug Carrier Syst 2005; 22(1):27-106] dealt with the salient steps involved in the DoE optimization methodology using diverse experimental designs. Part II deals with various retrospective literature findings as well as the prospective application of such DoE procedures while optimizing varied drug delivery technologies. A vast account of various DoE reports on optimization of diverse drug delivery system and processes along with the critical graphical analysis of various designs, input, and response variables have been presented here in a categorical form. Such an explicit and updated review on drug delivery optimization has not been published anywhere else in the recent past.

Biological Availability↗

Design, development and optimization of nimesulide-loaded liposomal systems for topical application.

Nimesulide, a non-steroidal anti-inflammatory drug, was incorporated into multilamellar liposomes to improve its performance on topical administration. The drug was loaded onto liposomes employing thin film hydration technique. Various process and formulation variables were investigated to obtain the liposomal products of desired quality. Liposomes were monitored for percent drug entrapment, after separating the unentrapped drug by mini column centrifugation, for vesicular properties (such as size distribution profile, morphological attributes and agglomeration tendency), drug diffused through synthetic semipermeable membrane, and drug leakage. Systematic optimization studies were carried out using 3(2) factorial design to select the optimized liposomal composition with reference to percent drug entrapment, drug diffusion and leakage. The optimized batch of liposomes was subjected to drug permeation and drug retention studies employing rat skin and human cadaver skin. In comparison to methanolic solution of pure nimesulide, liposomal formulations were found to retain higher amounts of nimesulide in the skin. Anti-inflammatory studies, using carragenan-induced rat paw edema model, indicated significantly better performance of liposomally entrapped nimesulide in comparison to the marketed gel formulation and the Carbopol gel containing nimesulide.

Administration, Topical↗

Effect of textile effluents on growth performance of wheat cultivars.

Laboratory experiments were conducted to study the effect of different concentrations in the range of 0-100% textile effluents (untreated and treated) on seed germination (%), delay index (DI), plant shoot length and root length, plant biomass, chlorophyll content and carotenoid of three different cultivars of wheat. The textile effluent did not show any inhibitory effect on seed germination at low concentration (6.25%). The other reported plant parameters also followed the similar trend. Seeds germinated in undiluted effluents did not survive for longer period. Based on the tolerance to textile effluent, the wheat cultivars have been arranged in the following order: PBW-343 < PBW-373 < WH-147. It has also been concluded that effect of the textile effluent is cultivar specific and due care should be taken before using the textile effluent for irrigation purpose.

Biomass↗

Cystamine increases L-cysteine levels in Huntington's disease transgenic mouse brain and in a PC12 model of polyglutamine aggregation.

Cystamine, a small disulfide-containing chemical, is neuroprotective in a transgenic mouse and a Drosophila model of Huntington's disease (HD) and decreases huntingtin aggregates in an in vitro model of HD. The mechanism of action of cystamine in these models is widely thought to involve inhibition of transglutaminase mediated cross-linking of mutant huntingtin in the process of aggregate formation/stabilization. In this study we show that cystamine, both in vitro and in a transgenic mouse model of HD (R6/2), increases levels of the cellular antioxidant L-cysteine. Several oxidative stress markers increase in HD brain. We provide further evidence of oxidative stress in mouse HD by demonstrating compensatory responses in R6/2 HD brains. We found age-dependent increases in forebrain glutathione (GSH), and increased levels of transcripts coding for proteins involved in GSH synthesis and detoxification pathways, as revealed by quantitative PCR analysis. Given the general importance of oxidative stress as a mediator of neurodegeneration we propose that an increase in brain L-cysteine levels could be protective in HD. Furthermore, cystamine was dramatically protective against 3-nitropropionic acid-induced striatal injury in mice. We suggest that cystamine's neuroprotective effect in HD transgenic mice results from pleiotropic effects that include transglutaminase inhibition and antioxidant activity.

Animals↗

Potential therapeutic applications of some antinutritional plant secondary metabolites.

Plant-based formulations have been used since ancient times as remedial measures against various human and animal ailments. Over the past 20 years interest in traditional medicines has increased considerably in many parts of the world. Whereas modifications in lifestyles, including diet, have had a profound effect on the increased risks of various diseases, there is considerable scientific evidence, both epidemiological and experimental, regarding vegetables and fruits as key features of diets associated with reduced risks of diseases such as cancers and infections. This has led to the use of a number of phytometabolites as anticarcinogenic and cardioprotective agents, promoting a dramatic increase in their consumption as dietary supplements. There are changing perceptions regarding the therapeutic potential of various plant secondary metabolites (PSMs), some of which have also been known to possess certain antinutritional qualities. The knowledge gained at the cellular and molecular levels, and biological activities of PSMs including tannin-polyphenols, saponins, mimosine, flavonoids, terpenoids, and phytates, would be useful in planning for future epidemiological studies and human cancer prevention trials, especially when a large pure dosage is not the option to deliver the active compounds to many tissues. It is well observed that alteration of cell cycle regulatory gene expression is frequently found in tumor tissues or cancer cell lines, and studies have suggested that the herbal-based or plant-originated cell cycle regulators might represent a new set of potential targets for anticancer drugs. The recent upsurge of interest in this area of research and advances made therein indicate that the impact of a number of diseases affecting humans and animals may be lessened, if not prevented, by simple dietary intake of PSMs with putative therapeutic properties.

Anti-Infective Agents↗

Morphological and biochemical effects of immunosuppressive drugs in a capillary tube assay for endothelial dysfunction.

Immunosuppressive drugs common in clinical transplantation are known to have untoward effects on the vascular system. The effects of some drugs, notably cyclosporin A (CyA), have been studied on the vascular system, while those of others have not. In the vascular system, endothelial cells are the predominant cell type exposed to intravascular concentrations of immunosuppressive drugs. We therefore studied the effects of drugs common in clinical transplantation on endothelial cells in a capillary tube assay. The endothelial cells in the capillary tubes are morphologically more similar to those in the microvasculature than endothelial cells in monolayers. We studied the kinetics and extent of capillary tube formation and prostacyclin (PGI2) and endothelin-1 (ET-1) release from the in vitro capillaries to determine the morphological and biochemical effects of five immunosuppressive agents on endothelial function. We found a significant difference in the morphological and biochemical effects of the two common calcineurin inhibitors, CyA and tacrolimus (FK506) on capillary morphology in vitro. The former had a pronounced injurious effect on the morphology of the in vitro capillaries, while the latter did not. CyA also significantly increased ET-1 release by the capillaries, but FK506 did not. Mycophenolate mofetil (MMF) was the only other agent that had a moderately injurious effect on the morphology of the in vitro capillaries. Sirolimus (rapamycin) and dexamethasone, similar to FK506, had no effect on the capillary morphology. All these agents, except dexamethasone, increased PGI2 release. Our data suggest that CyA adversely affects the morphology of the microvasculature and that this is mediated, at least partly, by an increased ET-1 release by endothelial cells exposed to CyA. These findings describe a novel effect of CyA and MMF on endothelial cells that could be relevant to understanding the mechanisms of immunosuppressive drug-mediated endothelial injury in clinical transplantation.

Cell Survival↗

Rofecoxib: an update on physicochemical, pharmaceutical, pharmacodynamic and pharmacokinetic aspects.

Rofecoxib (MK-966) is a new generation non-steroidal anti-inflammatory agent (NSAID) that exhibits promising anti-inflammatory, analgesic and antipyretic activity. It selectively inhibits cyclooxygenase (COX)-2 isoenzyme in a dose-dependent manner in man. No significant inhibition of COX-1 is observed with rofecoxib up to doses of 1000 mg. The pharmacokinetics of rofecoxib has been found to be complex and variable. Mean oral bioavailability after single dose of rofecoxib (12.5, 25 or 50 mg) is 93% with t(max) varying widely between 2 and 9 h. It is highly plasma-protein bound and is metabolized primarily by cytosolic reductases to inactive metabolites. Rofecoxib is eliminated predominantly by hepatic metabolism with a terminal half-life of approximately 17 h during steady state. Various experimental models and clinical studies have demonstrated rofecoxib to be superior, or at least equivalent, in anti-inflammatory, analgesic and antipyretic efficacy to comparator nonselective NSAIDs in osteoarthritis, rheumatoid arthritis and other pain models. Emerging evidence suggests that rofecoxib may also find potential use as supportive therapy in various pathophysiologic conditions like Alzheimer's disease, and in various malignant tumours and polyps, where COX-2 is overly expressed. Rofecoxib is generally well-tolerated. Analysis of data pooled from several trials suggests that rofecoxib is associated with fewer incidences of clinically symptomatic gastrointestinal ulcers and ulcer complications vis-à-vis conventional NSAIDs. However, this gastropreserving effect may be negated by concurrent use of low-dose aspirin for cardiovascular risk reduction. Rofecoxib tends to show similar tolerability for renal and cardiothrombotic events as compared with nonnaproxen nonselective NSAIDs. No clinically significant drug interaction has been reported for rofecoxib except with diuretics, where it reverses their salt-wasting effect and thus can be clinically exploited in electrolyte-wasting disorders. There is only modest information about the physicochemical and pharmaceutical aspects of rofecoxib. Being poorly water soluble, its drug delivery has been improved using varied formulation approaches. Although it is stable in solid state, rofecoxib is photosensitive and base-sensitive in solution form with its degradation mechanistics elucidated. Analytical determinations of rofecoxib and its metabolites in biological fluids employing HPLC with varied types of detectors have been reported. Isolated studies have also been published on the chromatographic and spectrophotometric assay of rofecoxib and its degradants in bulk samples and pharmaceutical dosage forms. The current article provides an updated overview on the physicochemical, pharmaceutical, pharmacokinetic and pharmacodynamic vistas of rofecoxib.

Anti-Inflammatory Agents, Non-Steroidal↗

Effect of hypobaric hypoxia on auditory sensitivity.

INTRODUCTION: Previous studies on the effect of hypobaric hypoxia on auditory sensitivity are not readily interpretable, in most cases because the potential effect of ambient pressure on stimulus level was not considered. In this study, auditory sensitivity to 1, 8, 12, and 16 kHz tones was compared between conditions of hypoxia and normoxia at the same simulated altitude (3700 m). METHOD: In the hypoxic condition, the partial pressure of oxygen in the inspired air was allowed to decrease with increasing altitude. In the normoxic condition, the partial pressure of oxygen was maintained at a level equivalent to that experienced at mean sea level (MSL). This comparison also controlled for any effect resulting from physiological consequences of hypobaria other than hypoxia (such as a change in middle-ear impedance). RESULTS: A small (2.57 dB) reduction in sensitivity across the frequency range tested was observed. CONCLUSION: A reduction in sensitivity of this magnitude would not be expected to have a large impact on the effectiveness of information transfer via the auditory modality.

Adult↗

A new in vitro model to study endothelial injury.

BACKGROUND: Endothelial dysfunction or "endothelialitis" is a prominent feature in several disease states ranging from atherosclerosis to transplant rejection. This dysfunction is also caused by drugs such as cyclosporin A (CyA) and leads to allograft vasculopathy and eventual graft loss. Despite the frequency and importance of this injury, there is no model to study the morphological effects of endothelial injury and dysfunction in vitro. METHODS: We utilized a model in which mouse endothelial cells (SVEC 4-10) can be induced to form capillary tubes by culturing on a laminin-rich matrix (Matrigel). In this morphological model of endothelial cell function, we studied the effect of varying doses of CyA on two parameters of tube formation: initiation of tube formation and disruption of mature capillary tubes. As a positive control we used IFN-gamma, which inhibited capillary tube formation. We developed this assay in 96-well culture plates to test several samples simultaneously. RESULTS: The assay could be adapted to a 96-well format by optimizing the cell density. Endothelial dysfunction was seen when the endothelial cells were incubated with cyclosporin A, which affected both morphological parameters of tube formation. At higher doses (2-20 microg/ml) CyA both inhibited capillary tube formation and disrupted mature capillary tubes. At lower doses CyA only inhibited the initiation of tube formation; it did not disrupt mature capillary tubes. IL-2 (400-1000 pg/ml) and IFN-gamma (10-400 pg/ml) inhibited initiation of tube formation but did not disrupt mature capillary tubes. None of these agents, including high doses of CyA, impaired endothelial cell viability. CONCLUSION: CyA-induced endothelial dysfunction can be modeled in vitro by this novel morphological assay of capillary tube formation. This assay can discern mild and severe degrees of endothelial dysfunction. The different effects of low and high levels of CyA on capillary tube formation imply that similar dysfunction in vivo may be responsible for allograft vasculopathy caused by CyA. This novel model can also be utilized to study other forms of vasculitis.

Animals↗