Kalyan Goswami

T-Cell Assays for Tuberculosis Infection: Deriving Cut-Offs for Conversions Using Reproducibility Data

Background Although interferon-gamma release assays (IGRA) are promising alternatives to the tuberculin skin test, interpretation of repeated testing results is hampered by lack of evidence on optimal cut-offs for conversions and reversions. A logical start is to determine the within-person variability of T-cell responses during serial testing. Methodology/Principal Findings We performed a pilot study in India, to evaluate the short-term reproducibility of QuantiFERON-TB Gold In Tube assay (QFT) among 14 healthcare workers (HCWs) who underwent 4 serial QFT tests on day 0, 3, 9 and 12. QFT ELISA was repeated twice on the same sets of specimens. We assessed two types of reproducibility: 1) test-retest reproducibility (between-test variability), and 2) within-person reproducibility over time. Test-retest reproducibility: with dichotomous test results, extremely high concordance was noticed between two tests performed on the same sets of specimens: of the 56 samples, the test and re-test results agreed for all but 2 individuals (κ = 0.94). Discordance was noted in subjects who had IFN-γ values around the cut-off point, with both increases and decreases noted. With continuous IFN-γ results, re-test results tended to produce higher estimates of IFN-γ than the original test. Within-person reproducibility: when continuous IFN-γ data were analyzed, the within-person reproducibility was moderate to high. While persons with negative QFT results generally stayed negative, positive results tended to vary over time. Our data showed that increases of more than 16% in the IFN-γ levels are statistically improbable in the short-term. Conclusions Conservatively assuming that long-term variability might be at least twice higher than short-term, we hypothesize that a QFT conversion requires two conditions to be met: 1) change from negative to positive result, and 2) at least 30% increase in the baseline IFN-γ response. Larger studies are needed to confirm our preliminary findings, and determine the conversion thresholds for IGRAs.

Novel microfilaricidal activity of nanosilver

Purpose The currently available drug repertoire against lymphatic filariasis, a major health hazard in the developing world, is inadequate and is fraught with serious limitations. Thus, the development of an effective antifilarial strategy has become a global research thrust mandated by the World Health Organization. Nanoparticles of silver endowed with antibacterial potency are known to induce apoptosis in eukaryotic cells. The present study was designed to investigate the possible microfilaricidal efficacy of silver nanoparticles and to establish the validity of apoptotic rationale in antifilarial drug designing. Methods This report analyzed the effect of nanoparticles of silver as well as gold (size range: 10–15 nm) on the microfilariae of Brugia malayi obtained from the lavage of peritoneal cavities of infected jirds (Meriones unguiculatus). The study included a microfilarial motility assay, a trypan blue exclusion test, a poly(adenosine diphosphate-ribose) polymerase activity study, ethidium bromide/acridine orange differential staining, and transmission, as well as scanning electron microscopic evaluation of ultrastructural changes in microfilariae. Results The study demonstrates that nanoparticles of silver, but not of gold, elicited significant loss in microfilarial motility. Differential staining of parasites with ethidium bromide and acridine orange, poly(adenosine diphosphate-ribose) polymerase activity in microfilarial lysate, and electron microscopic findings underscored apoptotic death of parasites attributable to nanosilver. In a trypan blue exclusion test, the 50% lethal dose of nanosilver was measured to be 101.2 μM, which was higher than the recorded complete inhibitory concentration value (50.6 μM), thus supporting nanosilver as a potential drug candidate against lymphatic filariasis. Conclusion The present report provides the first ever conclusive proof in support of apoptosis as a novel stratagem in antifilarial drug designing and nanoscale silver as a valid lead in research on antifilarial therapeutics. The main embargo about the current drug diethylcarbamazine citrate is its empirical use without rationale. Effective microfilaricidal activity of nanosilver at relatively low concentrations as reported in this study, with evidence of the induction of apoptosis in microfilariae, projects nanosilver as a potential drug adjuvant against lymphatic filariasis. The much higher 50% lethal dose value of nanosilver compared to the complete inhibitory concentration value reported in this study argues in favor of a safe therapeutic window of this agent in its antifilarial efficacy.

Exploration of 2, 4-diaminopyrimidine and 2, 4-diamino-s-triazine derivatives as potential antifilarial agents.

In view of the mandate from the World Health Organization (WHO) for developing novel drug candidates against human lymphatic filariasis, dihydrofolate reductase (DHFR) inhibitors are explored as potential antifilarial agents. The in vitro biological evaluation of an in-house library of 12 diverse antifolate compounds with 2,4-diaminopyrimidine and 2,4-diamino-s-triazine structural features against Brugia malayi is reported. To confirm the DHFR inhibitory potential of these compounds, reversal studies using folic acid and folinic acid were undertaken. Inhibition of DHFR can induce apoptosis; in this light, preliminary evidence of apoptosis by test compounds was detected using ethidium bromide-acridine orange staining and the poly(adenosine diphosphate-ribose) polymerase (PARP) inhibition assay. Among the evaluated compounds, 3 showed significant activity against both microfilariae and adult worms. The effects of 2 of these compounds were mostly reversed by folic acid, validating DHFR inhibitory activity. Partial reversal of the effect of 2 compounds by folinic acid and non-reversal of the effect of the third compound both by folic and folinic acids are discussed. This study opens new avenues for the discovery of lead molecules by exploiting the folate pathway against one of the major neglected tropical diseases, filariasis.

In vitro biological evaluation of biguanides and dihydrotriazines against Brugia malayi and folate reversal studies.

Dihydrofolate reductase (DHFR) is a well-known target for antibacterial and anticancer therapy. DHFR inhibitors are useful for protozoan parasites, but are yet to be explored against metazoan species; hence the present work was designed to evaluate the efficacy of DHFR inhibitors against filariasis, one of the major neglected tropical diseases. Molecules from our in-house library of synthetic antifolate agents (biguanide and dihydrotriazine derivatives) were evaluated along with the antimalarial drug pyrimethamine and the antibacterial drug trimethoprim in an in vitro model against Brugia malayi microfilariae (Mf). Three biguanides and two dihydrotriazines were more potent than trimethoprim and pyrimethamine against B. malayi Mf. Trimethoprim, pyrimethamine and four of the five compounds active against Mf were also active against adult worms. To probe the mechanism of action of the compounds, reversal of activity of active compounds by folic acid and folinic acid was studied. In conclusion, DHFR inhibitors could be used as leads for new antifilarial drugs.

Brugia malayi cystatin therapeutically ameliorates dextran sulfate sodium-induced colitis in mice.

Helminth immunomodulation in the host has been shown to have therapeutic implications in inflammatory bowel diseases. In this study we aimed to evaluate the therapeutic effect of Brugia malayi recombinant cystatin (rBmCys) in a dose‐dependent manner on dextran sulfate sodium (DSS)‐induced colitis in mice.

Possible implication of oxidative stress in anti filarial effect of certain traditionally used medicinal plants in vitro against Brugia malayi microfilariae.

Introduction: Tropical disease research scheme of World Health Organization has duly recognized traditional medicine as alternative for antifilarial drug development. Polyphenolic compounds present in traditionally used herbal medicines are natural antioxidants; however, paradoxically they may exert pro-oxidant effect. Popular drug diethyl carbamazine citrate harnesses the innate inflammatory response and the consequent oxidative assault to combat invading microbes. Methods: With this perspective, extracts of Vitex negundo L. (roots), Butea monosperma L. (leaves), Aegle marmelos Corr. (leaves), and Ricinus communis L. (leaves) were selected to explore the possible role of oxidative rationale in the antifilarial effect in vitro. Results: Apart from the last, other three plant extracts were reported to have polyphenolic compounds. Dose-dependent increase was found in the levels of lipid peroxidation and protein carbonylation for all the three plant extracts except Ricinus communis L. (leaves). Such increase in oxidative parameters also showed some degree of plant-specific predilection in terms of relatively higher level of particular oxidative parameter. High degree of correlation was observed between the antifilarial effect and the levels of corresponding oxidative stress parameters for these three plants. However, extracts of Ricinus communis L. (leaves) which is relatively deficient in polyphenolic ingredients recorded maximum 30% loss of motility and also did not show any significant difference in various stress parameters from corresponding control levels. Conclusion: These results reveal that targeted oxidative stress might be crucial in the pharmacodynamics.

Brugia malayi soluble and excretory-secretory proteins attenuate development of streptozotocin-induced type 1 diabetes in mice

Understanding the modulation of the host‐immune system by pathogens‐like filarial parasites offers an alternate approach to prevent autoimmune diseases. In this study, we have shown that treatment with filarial proteins prior to or after the clinical onset of streptozotocin‐induced type‐1 diabetes (T1D) can ameliorate the severity of disease in BALB/c mice. Pre‐treatment with Brugia malayi adult soluble (Bm A S) or microfilarial excretory‐secretory (Bm mf ES) or microfilarial soluble (Bm mf S) antigens followed by induction of diabetes led to lowering of fasting blood glucose levels with as many as 57·5–62·5% of mice remaining nondiabetic. These proteins were more effective when they were used to treat the mice with established T1D as 62·5–71·5% of the mice turned to be nondiabetic. Histopathological examination of pancreas of treated mice showed minor inflammatory changes in pancreatic islet cell architecture. The therapeutic effect was found to be associated with the decreased production of cytokines TNF‐α & IFN‐γ and increased production of IL‐10 in the culture supernatants of splenocytes of treated mice. A switch in the production of anti‐insulin antibodies from IgG2a to IgG1 isotype was also seen. Together these results provide a proof towards utilizing the filarial derived proteins as novel anti‐diabetic therapeutics.

Metabolic Syndrome in the Rural Population of Wardha, Central India: An Exploratory Factor Analysis

Background and Objectives: Metabolic syndrome – a plausible precondition for type II diabetes and cardiovascular diseases is also on rise. To understand the mechanistic complexity of metabolic syndrome it is imperative to study the specific contribution of the determinants of metabolic syndrome. Such study can help to identify the most significant factor which may be of use in early detection as well as prevention efforts. Such information is scarcely available from India and especially from rural India. Hence, the present study was undertaken to explore for such factor which might be considered crucial for development of such pathogenesis particularly in rural population of Wardha. Methods: A cross-sectional study comprising of 300 subjects was carried out in rural area of Primary Health Center, attached to medical college with approximate 31,000 populations. The anthropometric parameters such as height, weight, waist circumference were measured. Overnight fasting samples were collected for lipid profile (total cholesterol, triglyceride, high density lipoproteins, low density lipoproteins, very low density lipoproteins) and fasting blood glucose levels. The National Cholesterol Education Programme Adult Treatment Panel, ATP-III guidelines were used to categorize the study subjects. As many of the variables are highly intercorrelated, exploratory factor analysis was carried out to reduce the data to a smaller number of independent factors that accounts for the most of the variances in the data. Principal component analysis was used as a method of extraction. Results: For both sexes, three factors were extracted accounting for about 71% variance in the measured variables. An adiposity factor which accounted for highest explained variance (28%), was the initial factor extracted. It was loaded positively by waist circumference, triglyceride, and very low density lipoprotein and negatively loaded by high density lipoprotein. Second factor extracted was a cholesterol factor which explained about 20% variance. It was positively loaded by total cholesterol and low density lipoprotein. Blood pressure factor was third to be extracted which again explained about 20% variance. It was positively loaded by systolic and diastolic blood pressure. Interpretation and Conclusion: The results clearly indicate the significance of visceral adiposity over the obesity in general or simple abdominal obesity measured anthropometrically as a pathogenic determinant of the metabolic syndrome. The most consistent factor has been found to be dyslipidemia which explained major share of the observed variance and the most significant load of this factor being rested on triglyceride and the VLDL level. Hence, we conclude measurement of triglyceride might be a rewarding screening parameter for assessment of cardio-metabolic risk in general populace and warrants a large scale study focusing into this issue.

SXP–RAL Family Filarial Protein, rWbL2, Prevents Development of DSS-Induced Acute Ulcerative Colitis

Helminthic infections lead to the release of various molecules which play an important role in modulation of the host immune system. Such filarial proteins with immunomodulatory potential can be used for therapeutic purpose in inflammatory and immune mediated diseases. In the present study, we have explored the prophylactic effect of filarial SXP–RAL family protein of Wuchereria bancrofti i.e. rWbL2 protein in DSS induced inflammatory ulcerative colitis in a mouse model. Prior treatment of rWbL2, followed by induction of colitis, showed significantly reduced disease severity as indicated by the decreased disease manifestations and improved macroscopic and microscopic inflammation. This preventive effect was found to be associated with increased release of anti-inflammatory cytokine IL-10 and decreased release of proinflammatory cytokines IFN-γ, TNF-α, IL-6 and IL-17 by the splenocytes of treated mice. From this study, it can be envisaged that pretreatment with filarial protein, rWbL2, can prevent the establishment of ulcerative colitis in BALB/c mice. The underlying immunological mechanism may involve the up-regulation of Th2 immune response with down-regulation of Th1 response.

Therapeutic potential of the immunomodulatory proteins Wuchereria bancrofti L2 and Brugia malayi abundant larval transcript 2 against streptozotocin-induced type 1 diabetes in mice.

Epidemiological and experimental evidence has supported the concept of using helminths as alternative bio-therapeutic agents in the treatment of type 1 diabetes (T1D). In the current study, two filarial proteins, recombinant Wuchereria bancrofti L2 (rWbL2) and Brugia malayi abundant larval transcript 2 (rBmALT-2) have been investigated, individually and in combination, for their therapeutic potential in streptozotocin (STZ)-induced T1D. The rWbL2 and rBmALT-2 proteins, when administered individually or in combination, have resulted in lowering of the blood glucose levels and reducing the incidence of T1D in mice. In addition, these proteins have led to reduced lymphocytic infiltration and decreased islet damage and inflammation. The curative effect was found to be associated with the suppression of release of tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), and increased production of interleukin (IL)-4, IL-5 and IL-10 cytokines by the splenocytes of the diabetic mice. Insulin-specific IgG1 and antigen-specific IgE antibodies were found to be elevated in the sera of mice treated with rWbL2 and rBmALT-2 proteins. From the findings in this study, it can be envisaged that both of these filarial immunomodulatory proteins have the potential to ameliorate T1D by altering the regulatory immune responses.