Utpal Sengupta

Risk behaviour, sexually transmitted infections and HIV among long-distance truck drivers: a cross-sectional survey along national highways in India

Objective:To report HIV and sexually transmitted infection (STI) prevalence and sexual behaviour of long-distance truckers on four national highway routes from a large, cross-sectional, national-level trucker survey in India. Methods:Seven trans-shipment locations covering the bulk of Indias transport volume along four routes, north-west (NW), north-south (NS), north-east (NE) and south-east (SE) were identified as survey sites. A total of 2066 long-distance truckers were selected using a two-stage, time–location cluster sampling approach and, after consent, interviewed about their sexual behaviour. Urine and blood sample were tested for selected STIs. Results:Overall, HIV prevalence among truckers was found to be 4.6%, with prevalence highest on the SE route (6.8%) and lowest on the NS (2.4%). Positive HSV-2 serology, which was tested in a 10% subsample, was low along three routes, 10.0%, 12.8% and 6.7% for the NE, NS and NW, respectively, but 38.7% in the SE. The truckers from the SE were found to be more likely to have sex with paid partners than the NE route. Moreover, truckers who owned their trucks were more likely than those who did not use condoms consistently with paid partners, and truckers who drive trucks owned by their relatives/friends are more likely than others to have any STI. Conclusions:Low self-risk perception for HIV (9.9%), low consistent condom use with non-paid partners (18.6%) and wives (3%), low reported exposure to any interventions (25.6%) and low levels of ever having taken an HIV test (16.5%) make truckers an important bridge population requiring strengthened interventions.

Serodiagnostic Efficacy of Mycobacterium tuberculosis 30/32-kDa Mycolyl Transferase Complex, ESAT-6, and CFP-10 in Patients with Active Tuberculosis

Elimination of tuberculosis (TB) largely depends upon definitive rapid diagnosis and treatment. Widely used diagnostic tests do not qualify for use in a developing country due to lack of either desired accuracy or their cost. In the present study an enzyme-linked immunosorbent assay was used to evaluate the diagnostic potential of an immuno-dominant 30/32-kDa mycolyl transferase complex (Ag85 complex) and Mycobacterium tuberculosis-specific proteins (ESAT-6 and CFP-10) of the RD1 region. Higher sensitivity (84.1%) with Ag85 complex was observed compared with ESAT-6 (64.9%) and CFP-10 (66%), with almost similar specificity (Ag85: 85.2%, ESAT-6: 88.9%, CFP-10: 85.2%), whereas the individual components of Ag85 complex, i.e. Ag85A, Ag85B, and Ag85C, showed sensitivities of 44.6, 34, and 80.9% and specificities of 55.6, 74.1, and 40.7% respectively. A cocktail of Ag85 complex, ESAT-6, CFP-10, Ag85A, Ag85B, and Ag85C antigens also could not help in increasing either sensitivity (51.1%) or specificity (85.2%). Furthermore, immunoblot analysis using clinical isolates as well as a standard strain (H37Rv) of M. tuberculosis also showed strong reactivity of sera from TB patients to Ag85 complex and, to a lesser extent, also to ESAT-6. To conclude, use of Ag85 complex along with ESAT-6 and CFP-10 seems to be promising in minimizing the heterogeneous sero-responses of adult TB cases.

Reciprocity between Regulatory T Cells and Th17 Cells: Relevance to Polarized Immunity in Leprosy

T cell defect is a common feature in lepromatous or borderline lepromatous leprosy (LL/BL) patients in contrast to tuberculoid or borderline tuberculoid type (TT/BT) patients. Tuberculoid leprosy is characterized by strong Th1-type cell response with localized lesions whereas lepromatous leprosy is hallmarked by its selective Mycobacterium leprae specific T cell anergy leading to disseminated and progressive disease. FoxP3+ Regulatory T cells (Treg) which are essential for maintaining peripheral tolerance, preventing autoimmune diseases and limiting chronic inflammatory diseases also dampen proinflammatory T cells that include T helper 17 (Th17) cells. This study is aimed at evaluating the role of Treg cells in influencing other effector T cells and its relationship with the cytokine polarized state in leprosy patients. Peripheral blood mononuclear cells from of BT/TT (n = 15) and BL/LL (n = 15) patients were stimulated with M. leprae antigen (WCL) in presence of golgi transport inhibitor monensin for FACS based intracellular cytokine estimation. The frequency of Treg cells showed >5-fold increase in BL/LL in comparison to BT/TT and healthy contacts. These cells produced suppressive cytokine, IL-10 in BL/LL as opposed to BT/TT (p = 0.0200) indicating their suppressive function. The frequency of Th17 cells (CD4, CD45RO, IL-17) was, however, higher in BT/TT. Significant negative correlation (r = -0.68, P = 0.03) was also found between IL-10 of Treg cells and IL-17+ T cells in BL/LL. Blocking IL-10/TGF-β restored the IL-17+ T cells in BL/LL patients. Simultaneously, presence of Th17 related cytokines (TGF-β, IL-6, IL-17 and IL-23) decreased the number of FoxP3+ Treg cells concomitantly increasing IL-17 producing CD4+ cells in lepromatous leprosy. Higher frequency of Programmed Death-1/PD-1+ Treg cells and its ligand, PDL-1 in antigen presenting cells (APCs) was found in BL/LL patients. Inhibition of this pathway led to rescue of IFN-γ and IL-17 producing T cells. Results indicate that Treg cells are largely responsible for the kind of immunosuppression observed in BL/LL patients. This study also proves that Treg cells are profoundly affected by the cytokine milieu and this property may be utilized for benefit of the host.

Diagnostic Potential of Ag85C in Comparison to Various Secretory Antigens for Childhood Tuberculosis

Childhood tuberculosis is difficult to diagnose. A rapid, simple and relatively inexpensive diagnostic test will be crucial to future control efforts. Therefore, efficacy and diagnostic potential of different secretory antigens of Mycobacterium tuberculosis (CFP‐10, Ag85complex, Ag85 A, B, C) and their combinations along with ESAT‐6 in the detection of antibody profiles of childhood tuberculosis cases were evaluated using ELISA technique and reactivity was compared with the gold standards (smear, culture and IS6110 targeted PCR). In the present study, 88 fresh, untreated childhood tuberculosis (TB) cases, 17 children undergoing anti‐tubercular therapy, 17 children having disease other than TB and 25 healthy children were included. ROC curve analysis was used to calculate the sensitivity and specificity of each antigen for antibody detection. Ag85C was found to be showing highest sensitivity of 89.77% and specificity of 92% among all the antigens used (P < 0.0001). Positivity with antigen was 95% in smear and culture negative patients. Antibody reactivity was noted in 92.62% of patients who were positive for IS6110 by PCR. Cocktail of all the antigens showed 67.1% sensitivity and 80% of specificity. Sensitivity of 29.55%, 57.95%, 64.77% and specificity of 80%, 72%, 64% was observed using CFP‐10, Ag85complex and Ag85B. Low reactivity of 31.82% in patients and least specificity of 24% was noted with Ag85A. Our finding demonstrates the potential of Ag85C in the detection of antibody in childhood TB cases and this antigen showed good concordance with PCR positivity.

Mycobacterial antigen(s) induce anergy by altering TCR- and TCR/CD28-induced signalling events: insights into T-cell unresponsiveness in leprosy.

Present study investigates the role of Mycobacterium leprae (M. leprae) antigens on TCR- and TCR/CD28-induced signalling leading to T-cell activation and further correlates these early biochemical events with T-cell anergy, as prevailed in advanced stages of leprosy. We observed that both whole cell lystae (WCL) and soluble fraction of M. leprae sonicate (MLSA) not only inhibited TCR, thapsigargin and ionomycin induced calcium fluxes by diminishing the opening of calcium channels, but also TCR- or TCR/CD28-induced proximal signalling events like phosphorylation of Zap-70 and protein kinase-C (PKC) activity. Study of TCR- and TCR/CD28-induced downstream signals revealed that M. leprae antigens curtail phosphorylation of both Erk1/2 and p38MAPK, consequently altering terminal signalling events like reduced binding of NFAT on IL-2 promoter and transcription of IL-2 gene, diminished expression of activation markers (CD25 and CD69). Furthermore, M. leprae fractions significantly inhibited IL-2 secretion and T-cell blastogenesis in healthy individuals. Altogether, results suggest that M. leprae interferes with TCR/CD28-induced upstream as well as downstream signalling events resulting in reduced IL-2 production and thus inhibition in T-cell proliferation, which might be responsible for T-cell unresponsiveness leading to stage of immunosuppression and consequently, for the progression of disease.

Proteomic analysis of outer membrane proteins of Edwardsiella tarda

Aims:  The purpose of this study was to identify outer membrane proteins (OMPs) of Edwardsiella tarda.

Molecular mimicry between HSP 65 of Mycobacterium leprae and cytokeratin 10 of the host keratin; Role in pathogenesis of leprosy

Mycobacteria are known to induce autoimmune response in the host. Anti-host keratrin antibodies (AkAbs) might be responsible for the autoimmune phenomena in leprosy patients as majority of leprosy lesions are manifested in the skin and occurrence of keratosis is not an uncommon feature. The aim of this study was to find out the level of AkAbs in leprosy patients across the spectrum and to explore its correlation with the clinical manifestation of the disease. Further, mimicking epitopes of keratin and Mycobacterium leprae components were characterized. We screened 140 leprosy patients (27 BT, 28 BL, 41 LL, 25 T1R, 19 ENL), 74 healthy controls (HC) and 3 psoriasis patients as positive control. Highest AkAbs level was observed in the psoriasis patients followed by T1R, LL, BL, ENL, TT/BT. AkAbs level was significantly (p<0.05) higher in all the groups of leprosy patients except TT/BT in comparison to HC. Significant positive correlation was found between number of lesions and level of AkAbs in leprosy patients. Highest lympho-proliferation for keratin protein was observed in T1R, followed by BL/LL, TT/BT, ENL. Lympho-proliferation was significantly (p<0.05) higher in all groups of leprosy patients except ENL in comparison to HC. Interestingly, it was noted that hyperimmunization of inbred strains of female BALB/c mice and rabbit with M. leprae soluble antigen (MLSA) induce higher level of AkAbs. The percentage of FoxP3(+) expressing Treg cells (total CD4(+)CD25(+)FoxP3(+) andCD4(+)CD25(+hi)FoxP3(+)) in splenocytes and lymph nodes of hyperimmunized mice were declined in comparison to control mice. Further, it was found that this autoimmune response can be adoptively transferred in naïve mice by splenocytes and lymph node cells as well as T cells. Comparative molecular characterization between keratin and MLSA noted a cross-reactivity/similarity between these two antigens. The cross-reactive protein of keratin was found to be in molecular weight range ≈74-51kDa and at pI 4.5 while the cross-reactive protein of MLSA was found to be in molecular weight ≈65kDa and at pI 4-4.5. Cross-reactive protein of keratin and MLSA was identified and characterized by MALDI-TOF/TOF analysis and Mascot software. It was found that the keratin (host protein) which reacted with anti-M. leprae sera is cytokeratin-10 and MLSA which reacted with anti-keratin sera is heat shock protein 65 (HSP 65). Seven B-cell epitopes of cytokeratin-10 and HSP 65 was found to be similar by multiple sequence alignment using ClustalW server and out of which 6 B-cell epitopes were found to be on the surface of HSP 65. In conclusion, our study provides evidence for the existence of molecular mimicry between cytokeratin-10 of keratin (host protein) and 65kDa HSP (groEL2) of M. leprae. Presence of heightened CMI response of leprosy patients to keratin and positive correlation of AkAbs level with number of lesions of leprosy patients showed the clinical evidence for its role in the pathogenesis in leprosy.

Production of monoclonal antibodies specific to major outer membrane protein of Edwardsiella tarda

Edwardsiella tarda is an important cause for hemorrhagic septicemia in fish and gastro and extra-intestinal infections in humans. Monoclonal antibodies (MAbs) were produced against outer membrane proteins (OMPs) of E. tarda ET-7, isolated from diseased snakehead (Ophiocephalus punctatus). Two stable hybridoma clones, designated as 3F10 and 2C3 MAbs were found to be potentially specific for E. tarda by indirect enzyme linked immunosorbent assay (ELISA). These MAbs recognized major immunogenic OMP band at 44kDa in Western blotting. Both MAbs belonged to the IgG1 isotype and recognized different epitopes of OMP as seen by competitive ELISA. These MAbs strongly reacted with all 17 isolates of E. tarda used in our study by indirect ELISA and Western blotting. Interestingly, no reaction was observed with the reference strain of E. tarda (MTCC 2400). The sensitivity of 3F10 MAb to detect whole cells of E. tarda was up to a level of 1x10(4)CFU/ml in indirect ELISA. No cross-reactivity of MAbs were seen with Escherichia coli, Salmonella arizonae, Pseudomonas fluorescens, Aeromonas hydrophila, Vibrio cholerae, Flavobacterium ferrugineum and Mycobacterium tuberculosis. These MAbs could be used for specific detection of E. tarda infection in fish by immunoassays.

Phenolic-glycolipid-1 and lipoarabinomannan preferentially modulate TCR- and CD28-triggered proximal biochemical events, leading to T-cell unresponsiveness in mycobacterial diseases

BackgroundAdvanced stages of leprosy show T cell unresponsiveness and lipids of mycobacterial origin are speculated to modulate immune responses in these patients. Present study elucidates the role of phenolicglycolipid (PGL-1) and Mannose-capped lipoarabinomannan (Man-LAM) on TCR- and TCR/CD28- mediated signalling.ResultsWe observed that lipid antigens significantly inhibit proximal early signalling events like Zap-70 phosphorylation and calcium mobilization. Interestingly, these antigens preferentially curtailed TCR-triggered early downstream signalling events like p38 phosphorylation whereas potentiated that of Erk1/2. Further, at later stages inhibition of NFAT binding, IL-2 message, CD25 expression and T-cell blastogenesis by PGL-1 and Man-LAM was noted.ConclusionAltogether, we report that Man-LAM and PGL-1 preferentially interfere with TCR/CD28-triggered upstream cell signalling events, leading to reduced IL-2 secretion and T-cell blastogenesis which potentially could lead to immunosupression and thus, disease exacerbation, as noted in disease spectrum.

Induction of lepromin reactivity in cured lepromatous leprosy patients: impaired chemokine response dissociates protective immunity from delayed type hypersensitivity

Delayed Type Hypersensitivity (DTH) and protective immunity are thought to be tightly linked. Remarkable similarity exists between their cellular and immune mechanisms. However, their dissociation is also well known. Here we investigate the immunological mechanisms relevant for their dissociation in a group of non-relapsing cured lepromatous leprosy (CLL) patients. In these patients, using lepromin reaction as a model system of DTH we report critical role of tissue chemokine response in synchronous manifestation of these linked phenomena. Results indicate elevation of the threshold of tissue chemokine induction thus dissociating DTH from protective immunity in lepromin -ive CLL patients. We also show that the DTH anergy in these subjects is not an absolute one but depends on the strength of the stimulus. Our data provide insights into the intricate relationship between DTH and immunity and highlight the persistent presence of effector immune mechanisms involving these two pathways in apparently unresponsive lepromatous leprosy patients.