Mohan Natrajan

Mycobacterium indicus pranii as stand-alone or adjunct immunotherapeutic in treatment of experimental animal tuberculosis.

Background & objectives: Mycobacterium w (M.w) is a saprophytic cultivable mycobacterium and shares several antigens with M. tuberculosis. It has shown good immunomodulation in leprosy patients. Hence in the present study, the efficacy of M.w immunotherapy, alone or in combination with multi drug chemotherapeutic regimens was investigated against drug sensitive M. tuberculosis H37Rv and three clinical isolates with variable degree of drug resistance in mice. Methods: BALB/c mice were infected with M. tuberculosis H37Rv (susceptible to all first and second line drugs) and three clinical isolates taken from the epository of the Institute. The dose of 200 bacilli was used for infection via respiratory route in an aerosol chamber. Chemotherapy (5 days/wk) was given one month after infection and the vaccinated group was given a dose of 1×107 bacilli by subcutaneous route. Bacterial load was measured at 4 and 6 wk after initiation of chemotherapy. Results: M.w when given along with chemotherapy (4 and 6 wk) led to a greater reduction in the bacterial load in lungs and other organs of TB infected animals compared to. However, the reduction was significantly (P<0.05) more in terms of colony forming units (cfu) in both organs (lungs and spleen). Conclusion: M.w (as immunomodulator) has beneficial therapeutic effect as an adjunct to chemotherapy.

Expression of CXCL10 (IP-10) and CXCL11 (I-TAC) chemokines during Mycobacterium tuberculosis infection and immunoprophylaxis with Mycobacterium indicus pranii (Mw) in guinea pig.

Mycobacterium indicus pranii (earlier known as Mycobacterium w) has been used as an immunmodulatory agent in leprosy and tuberculosis by mediating the release of various cytokines and chemokines. CXCL10 (IP-10) and CXCL11 (I-TAC) chemokines are involved in T-cell migration and stimulation of natural killer cells in Mycobacterium tuberculosis infection. In this study, the effect of heat killed M. indicus pranii (alone and in conjunction with chemotherapy) on disease progression was determined by colony forming units (CFUs) in guinea pig lung following their aerosol infection and the expression levels of CXCL10 and CXCL11 were studied by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) and in situ RT-PCR. Four groups of animals included; infection only (Rv), immunoprophylaxis (RvMw), chemotherapy (RvCh) and combination of immunoprophylaxis with chemotherapy (RvChMw). In the group where immunoprophylaxis was given in combination with chemotherapy, the CFU counts reduced significantly at 4th week post-infection as compared to animals that received immunoprophylaxis or chemotherapy alone. At the same time, all groups of animals had elevated expression of CXCL 10 which was significantly high only in animals that received Mw with or without chemotherapy. Unlike to CXCL 10, study demonstrated suppressed expression CXCL 11 in both immunoprophylaxis as well as chemotherapy groups that became up-regulated in synergistic response of immunoprophylaxis and chemotherapy. Taken together, data indicates that the expression of CXCL10 and CXCL11 positively correlates with anti-tubercular treatment (at least with combination of immunoprophylaxis and chemotherapy). Therefore, prior immunization with Mw appears to be a good immunomodulator for release of chemokines and augments the effect of chemotherapy.

In situ hybridization in the histological diagnosis of early and clinically suspect leprosy.

The present study tests the utility of the in situ hybridization procedure for M. leprae rRNA in the histological diagnosis of early leprosy and clinically suspect leprosy, both diagnostically demanding situations. The histological confirmation obtained with routine histopathology (Haematoxylin-Eosin staining for studying morphologic alterations and Fite-Faraco staining for demonstration of acid-fast bacilli) were 32% for early leprosy and 25% for clinically suspect leprosy. With performance of the in situ hybridization on the histologically unconfirmed cases, the positivity rates obtained were 58.8% and 55%, respectively. The results of the study confirm the utility of the procedure in the diagnostically difficult situations of early and suspect leprosy, and it is proposed that the procedure be employed in situations of clinical doubt.

High Resolution Structural Changes of Schwann Cell and Endothelial Cells in Peripheral Nerves Across Leprosy Spectrum

ABSTRACT A systematic ultrastructure of peripheral nerves across the spectrum of leprosy was studied with an aim to better understanding the pathogenesis of nerve involvement in leprosy using light and electron microscope. The pathogenesis of nerve destruction varies in leprosy considerably along the spectrum. The study has begun to shed new light on some aspects of the infection of Mycobacterium leprae (M. lepare) and phenomenon has opened new avenue of research and possible mechanism of pathogenesis in TT/BT/BL/LL leprosy. In tuberculoid type (TT) and borderline tuberculoid (BT) leprosy, the degenerative changes of Schwann cells (SCs) and presence of perineural and perivascular cuffing by mononuclear cells. The endoneurial blood vessel (EBV) showed thickening of basement membrane with hypertrophy of EC leading to narrowing or complete occlusion of lumen and causing ischemia. However, borderline lepromatous (BL) and lepromatous leprosy (LL) foamy macrophages and vacuolated SC contain numerous small dense materials, irregular in shape and size was prominent and, considered to be degenerated and fragmented M. Leprae. The dense materials were also found in the cytoplasm of vascular EC. It was revealed that besides SC, the EC of EBV frequently harbor M. leprae in LL. The lumen of the EBV was wide open with enlarged nucleus. In the present study, the ultrastructural characteristics suggest that hypersensitivity mechanisms are possibly responsible for nerve damage in TT/BT leprosy. However, the study indicates that the mechanisms of nerve damage in BL/LL are basically different wherein hypersensitivity appears to play a very limited role.

Genital tuberculosis in females

The morbidity and mortality due to tuberculosis (TB) is high worldwide, and the burden of disease among women is significant, especially in developing countries. Mycobacterium tuberculosis bacilli reach the genital tract primarily by haematogenous spread and dissemination from foci outside the genitalia with lungs as the common primary focus. Genital TB in females is a chronic disease with low-grade symptoms. The fallopian tubes are affected in almost all cases of genital TB, and along with endometrial involvement, it causes infertility in patients. Many women present with atypical symptoms which mimic other gynaecological conditions. A combination of investigations is needed to establish the diagnosis of female genital TB (FGTB). Multidrug anti-TB treatment is the mainstay of management and surgery may be required in advanced cases. Conception rates are low among infertile women with genital TB even after multidrug therapy for TB, and the risk of complications such as ectopic pregnancy and miscarriage is high. More research is needed on the changing trends in the prevalence and on the appropriate methods for diagnosis of FGTB.

Expression and Analysis of CXCL8 and CXCL10 Chemokines in Human SkinLesions Infected with M.leprae

This study is focused to analyze the expression profile of CXCL8 and CXCL10 chemokines genes and to address the contribution of these chemokines in late phase of M.leprae infection in human skin lesion samples. In this study skin biopsy samples from leprosy patients (n=35) were collected including BL-LL=12, BB=14, BT=8 and healthy volunteers (n=3). All the biopsy samples were collected in RNALater for mRNA expression study as well as 10% buffered formalin (BF) for histopathologic analysis. Total RNA was isolated from collected samples and cDNA was prepared. Level of mRNA expression of CXCL8 and CXCL10 chemokine genes was measured via q-PCR and insitu RT-PCR to locate the presence of CXCL8 and CXCL10 chemokines inside M. leprae infected tissue. The mRNA expression of CXCL8 was found relatively elevated in lepromatous (BL-LL) skin samples significantly as compared to BB category of leprosy; whereas in BT cases down-regulation was recorded. CXCL10 mRNA expression was found elevated in lepromatous (BL-LL) cases than other cases (BT, BB) of leprosy samples. In histopathological examination, 59.38% maximum infiltration was observed in BL-LL cases of leprosy and in-situ RT-PCR confirmed the presence of chemokines genes in tissue sections. Interestingly, chemokines CXCL-10 and CXCL-8 showed elevated expression in BL-LL category. The study advocated that CXCL10 and CXCL-8 possibly may have a role in lepromatous (BL-LL) form of leprosy.

Potential of adjunctive Mycobacterium w (MIP) immunotherapy in reducing the duration of standard chemotherapy against tuberculosis

INTRODUCTION The need to shorten the treatment duration in tuberculosis has always been felt. Immunotherapy in combination with chemotherapy has been considered a promising approach for this purpose into tuberculosis. We studied the adjuvant immunotherapeutic activity of Mycobacterium indicus pranii (MIP or Mw) in combination with conventional chemotherapy using guinea pig of pulmonary tuberculosis infected with Mycobacterium tuberculosis H37Rv via aerosol. METHODS Experimental animals treated with standard chemotherapy and immunotherapy (MIP) separately and in combination of both. Guinea pig lungs evaluated following infection and subsequent therapy at predefine time point. Various cytokine mRNA expressions levels were quantified by quantitative reverse transcriptase PCR at the 4th, 8th and 12th week post-infection of M. tuberculosis. RESULTS We determined the time required for bacterial clearance from guinea pig lungs. Standard chemotherapy (RvCh) compared to the animals where chemotherapy plus Mw immunotherpay (RvChMwT) was given. It took 12 weeks to achieve bacterial clearance in the RvCh group while this was achieved in 8 weeks in RvChMwT group. Pro-inflammatory cytokines (IFN-γ, IL-2, IL-12p35 and TNF-α) level were higher in RvCh, RvChMwT and RvMwT group, while the IL-10 and TGF-β were suppressed. CONCLUSION Cytokine expression level showed that Mw in conjunction with chemotherapy enhances the effect of pro-inflammatory cytokines (such as, IFN-γ, IL-2, IL-12 and TNF-α) and reduces the production and effect of anti-inflammatory cytokines (like IL-10 and TGF-β) thereby restoring the pro-inflammatory / anti-inflammatory cytokines balance. Thus, the present study indicates that subject to rigorous testing by other parameters, Mw (MIP) as adjunct immunotherapy has potential for reducing treatment duration.

Expression profile of CXCL12 chemokine during M. tuberculosis infection with different therapeutic interventions in guinea pig

Mycobacterium indicus pranii (MIP) already established as an immune-modulator in mycobacterial infections generates immune response by acting on CXC chemokines. In the present study, the immunomodulatory effect of MIP in conjunction with chemotherapy against M.tb infection was evaluated by colony forming units (CFUs) following aerosol infection to guinea pig and by measuring CXCL12 chemokine expression using q-PCR and in situ RT-PCR. Different experimental groups included, infection (Rv), immunoprophylaxis (RvMw), chemotherapy (RvCh) and combination of immunoprophylaxis+chemotherapy (RvChMw) group and normal healthy (NH) group. In the combination of immunoprophylaxis+chemotherapy (RvChMw) group, the CFU counts reduced significantly (p<0.001) at 4th week of infection as compared to other treated groups (RvMw and RvCh group). The expression of CXCL12 was recorded in all the treated groups of animals. The study demonstrated suppressed expression of CXCL 12 in both immunoprophylaxis as well as chemotherapy groups (6th and 8th week) that become elevated in immunoprophylaxis plus chemotherapy group (10th week), at which time point no CFUs were detected in RvCh and RvChMw group. The findings indicate that the expression of CXCL12 is associated with good response to anti - tubercular treatment. Thus, prior immunization with MIP appears to show good immunomodulatory effect to release CXCL12 chemokine during infection and also correlates with enhanced effect to chemotherapy.

Immunoprophylaxis in Guinea Pigs with Mycobacterium indicus pranii (Mw) in Combination with Standard Chemotherapy of M. tuberculosis Infection ImprovesLung Pathology

Tuberculosis continues to be a major global health problem. BCG (Bacillus Calmette–Guerin) has been used as a vaccine to control tuberculosis, however, the protective value of the vaccine has been reported be highly variable across different populations. During the last three decades, several workers have investigated the potential of protective efficacy of Mycobacterium indicus pranii (MIP or Mw) against mycobacterial infections (leprosy and tuberculosis) which has been used as an immunoprophylactic tool and also as an adjunct to chemotherapy. The present study, reports the beneficial effect of prior immunization with MIP in terms of improvement in histopathological findings and reduction of bacterial burden, as augmenter to the effects of chemotherapy in experimental tuberculosis.