1. Molecular Biology and Molecular Epidemiology of Mycobacterium tuberculosis
Principal Investigator: Dr. Sujatha Narayanan
2. Regulatory role of HLA-DR2 on immune functions in pulmonary tuberculosis:
Principal Investigator: Dr. P.Selvaraj 3. Immune Response in tuberculous pleuritis (TP) - Th1/ Th2 paradigm:
Principal Investigator: Dr. Sulochana Das
4. Identification of immunoreactive T-cell antigens of M. tuberculosis through proteomic techniques:
Principal Investigator: Dr. Alamelu Raja
5. Genotypic Testing For Drug Resistant Mutations in HIV Strains in South India
Principal Investigator: Dr. Soumya Swaminathan 6. Influence of Active Tuberculosis on Chemokine and Chemokine Receptor Expression in HIV-infected Persons
Principal Investigator: Dr. Sudha Subramanyan
7. Study of interaction between HIV and TB with special reference to immune activation markers
Principal Investigator: Dr. Elizabeth L. Hanna
-----------------------------------------------------------------------------------------------------------------------------------
1. Molecular Biology and Molecular Epidemiology of Mycobacterium tuberculosis
Principal Investigator: Dr. Sujatha Narayanan
We work on three broad objectives, namely Functional Genomics of M.tuberculosis, Molecular Epidemiology of M.tuberculosis and Host Signal transduction in response to M. tuberculosis.
a. Functional Genomics
Collaboration with AECOM under AIRTP programme
(i) Serine Threonine kinases of M.tuberculosis
By using a combination of biochemical and molecular biology techniques we have cloned and characterized two serine/threonine protein kinases (STPK's), PknE & I, from M.tuberculosis. Knock out mutants for these kinases have been constructed and research is in progress to determine the functions of these molecules in the physiology of the organism and the pathology of the disease. Future work will involve identifying the interacting substrates for these kinases using a functional genomic approach including microarrays to probe physiological functions.
(ii) Development of Vaccine candidates
Funding: ICMR
Our present goal is to develop a recombinant BCG based epitope vaccine for tuberculosis. In this regard, we plan to graft epitopes from immudominant antigens of M.tuberculosis and express them in BCG and evaluate the immunogenicity and protective efficacy of the constructs.
(iii) Gene Regulation of Mycobacterium tuberculosis
Gene regulation of the first inducible gene of mycobacteria namely acetamidase gene of M.smegmatis and gro ES gene of M.tuberculosis has been extensively analysed. The gene regulation of GMP synthetase gene of M.tuberculosis has also been studied.
b. Molecular Epidemiology of M.tuberculosis
Funding: Model Dots Project
By using various techniques to fingerprint (Restriction Fragment Length Polymorphism, Spoligotyping or spacer oligotyping, MIRU Mycobacterial Intersperced Repeat Sequence Deletion microarray etc.) we have understood the transmission dynamics of the mycobacterial strains prevalent in South India. We have also deciphered the genotype of the M.tuberculosis of S.India especially Model DOTS area to be of ancient lineage (East African Indian/Indo Oceanic group). We have found that in South India the infection is mostly endogenous reactivation in “ non HIV TB patients” and exogenous reinfection in “HIV TB patients”.
c. Host Signal Transduction
Active research is underway to 1) investigate the role of MAPK in mycobacterial survival (using THP-1 cell line as the study model 2) look into the correlation between the pathogenicity of mycobacterial species and the ability to induce the activation of MAPK cascades.
top
2. Regulatory role of HLA-DR2 on immune functions in pulmonary tuberculosis:
Principal Investigator: Dr. P.Selvaraj
Our earlier studies revealed the association of HLA-DR2 with susceptibility to pulmonary tuberculosis. The regulatory role of HLA-DR2 on immune functions such as macrophage phagocytosis, perforin positive cells and Th1 and Th2 cytokine production are being studied in pulmonary tuberculosis.
(ii) Regulatory role of variant genotypes of Vitamin-D receptor gene on Cytokine response in pulmonary tuberculosis
Our earlier studies revealed the immunoregulatory role of vitamin D receptor gene variants on vitamin D 3 modulated phagocytosis and lymphoproliferative responses. The study has been further extended to understand the influence of vitamin D 3 and vitamin D receptor gene variants on Mycobacterium tuberculosis antigen induced cytokine response, intracellular cytokine positive CD4+ and CD8+ cells and Granzyme A positive cells in pulmonary tuberculosis.
(iii) Cytokine gene polymorphisms and cytokine levels in pulmonary tuberculosis
Th 1 and Th2 cytokines play an important role in the immune response against tuberculosis and alteration in their levels contribute to the out come of the infection. Single nucleotide polymorphisms in the cytokine genes may influence the cytokine levels which may be associated with susceptibility or resistance to tuberculosis. These aspects are being studied.
(iv) Human Leucocyte Antigen (HLA) and non-HLA gene polymorphism studies in HIV and HIV-TB patients
Funding: ICMR Task Force
The importance of host genetic factors (HLA and non-HLA) on susceptibility or resistance to HIV infection and the variability in disease progression towards AIDS has been emphasized by many studies. The association of HLA -A and –B (class I) and -DR, -DQ and –DP (class II) polymorphisms as well as non-HLA gene polymorphisms (Mannose binding lectin, Vitamin D receptor, Chemokine receptor and Chemokine genes) with susceptibility or resistance to HIV infection and HIV with active tuberculosis are being studied.
top
3. Immune Response in tuberculous pleuritis (TP) - Th1/ Th2 paradigm:
Principal Investigator: Dr. Sulochana Das
Tuberculous pleuritis provides one of the best human models to understand the mechanism of immunity to tuberculosis at the site of infection. Our group focuses to understand the correlates of protective immune response in tuberculosis, using tuberculous pleuritis as a model. The cellular and the soluble components of pleural fluid are studied to understand the in vivo immune response in TP and also the in vitro correlates of protective immune response to tuberculosis are studied. The various components of this study include determination of cellular architecture and the cytokine profile of pleural fluid, testing cell proliferation and apoptosis, Dual-signal phenomenon in tuberculous pleuritis using mycobacterial antigens etc.
(ii) Role of chemokines in tuberculous Immunity
In this study, we focus to address the role of mesothelial cells and secreted chemokines help in inducting the transmigration of immune cells from the systemic circulation to the site of infection. The consequence of the present study would shed light on the lining of the lung, the pleura and the mesothelial cells.
(iii) Role of Dendritic cells in tuberculous Immunity
The degree of maturation induced by different clinical strains of M.tb and the signaling mechanism involved in inhibition of DC maturation by M.tb is currently addressed with the objective to study the degree of virulence of M.tb related to inhibition of maturation of DCs and to evaluate the impact of DC activation by M.tuberculosis on T cells.
(iv) Human monocyte and macrophage apoptosis induced by Mycobacterium tuberculosis strains and its implication on cell mediated immune response
In this study we selected some of the single-copy clinical strains (IS6110) to study how the immune responses are modulated in response to these strains and to characterize the antigenic components unique to these strains.
top
4. Identification of immunoreactive T-cell antigens of M. tuberculosis through proteomic techniques:
Principal Investigator: Dr. Alamelu Raja
The main objective of this project is to find the fractions which induce higher IFN-γ
in protected population and not in the TB patients. The cut-off was ascertained using mean of IFN-γ
level + 2 SD of TB patients. Any fraction inducing IFN-γ level above the cut-off value was classified as positive. B ased on the positivity, the fractions were separated into major groups: (i) 10 HHC & 0 TB positive (ii) 9 HHC & 0 TB positive (iii) 8 HHC & 0 TB positive etc. Tandem mass spectrometry (ESI MS/MS) was carried out in all these fractions, in Pro. John Belisle's laboratory, Fort Collins. With the help of mass data and SDS-PAGE, the active antigen in each fraction will be identified by bio-informatic approaches.
(ii) Interferon gamma assay for latent TB in HIV infection
Funding: R03 Grant of NIAID, NIH
The aim of this study is to assess the role of Interferon Gamma (IFN-γ), Interleukin-4 and Interleukin-4 d 2 in the diagnosis of latent tuberculosis in the following subjects – (i) Healthy controls (HHC), with high risk for LTBI, (ii) Healthy controls (HC), with low risk for LTBI, (iii) HIV +ve, (iv) TB +ve and (v) HIV-TB
(iii) Cytotoxic cell response in M.tuberculosis infection:
The aim of this project is to analyse the cytotoxic cell response in tuberculosis and study the mechanisms of cytotoxicity. Response to whole M.tuberculosis H37Rv, purified antigens ESAT-6 and CFP-10 and overlapping 20-mer peptides of the 2 antigens are being studied, with lymphoproliferation of CD4 and CD8 cells and IFN-γ secretion, as read out. The study subjects are the two groups as described above, "susceptible" and "protected".
Cytolytic molecule response was also studied in these two groups to M.tuberculosis. An increase in Granzyme A and B levels was observed in patients than HHC. Cytotoxic T (CD8) cells were found to produce more Granzyme A and B when compared to helper cells (CD4)
(iv) Innate immunity in HIV infection:
Another project to demonstrate natural killer (NK) and NKT cell mediated innate immune response in HIV-TB has been recently started. NK cells are innate effector cells of the immune system, which exert a prompt cytolytic activity against infected and tumor cells without prior stimulation. The study subjects are as follows: (i) HIV seropositive pulmonary TB patients (HIV-TB), (ii) HIV seronegative TB patients (TB), (iii) HIV seropositive patients without TB (HIV), (iv) Normal healthy subjects (NHS)
top
5. Genotypic Testing For Drug Resistant Mutations in HIV Strains in South India
Principal Investigator: Dr. Soumya Swaminathan
Antiretroviral combination therapy is a major advance in the treatment of HIV infection. Four classes of antiretroviral drugs (ARV) are now available to treat HIV-1 infection Nucleoside Reverse Transcriptase Inhibitors (NRTI), Non Nucleoside Reverse Transcriptase Inhibitors (NNRTI), Protease inhibitors (PI) and Fusion inhibitors (FI). The emergence of resistance to these ARVs is an important cause of treatment failure. The viral mutants exhibit genotypic mutations in the reverse transcriptase (RT), protease (prot) & envelope (env) genes. The mutations have been well characterized in subtype B strains, because this subtype is predominant in the western world where ARV has been extensively used. In developing countries like India, where subtype C is predominant, ARV has only been recently introduced. In India, the National AIDS Control Organization (NACO) instituted antiretroviral therapy in government hospitals in 2004. The standard drug regimen consists of Stavudine, Lamuvidine and Nevirapine. Further, in the PMTCT program single dose Nevirapine for the mother at the time of labour and the newborn, has been introduced all over the country, following antenatal screening for HIV. Hence more data are needed on resistance of subtype C to ARV.
HIV Drug resistance testing will be performed in pediatric population (ARV failures) as well as ART-naïve; patients enrolled in ongoing clinical trials and recently infected pregnant women enrolled in the PMTCT program.
(ii) Evaluation of cellular immune response to infection with HIV-1 subtype C in South Indians, with special reference to gag epitope mapping
We hypothesize that the viral epitopes recognized by Indian HIV+ individuals may be different, as the HLA background of the individuals as well as the viral subtypes common in the region would determine the pattern of the immune reactivity to natural HIV-1 infection and presumably vaccines. This study will examine CTL responses to HIV-1 peptides in the South Indian population.
top
6. Influence of Active Tuberculosis on Chemokine and Chemokine Receptor Expression in HIV-infected Persons
Principal Investigator: Dr. Sudha Subramanyan
Tuberculosis is the major opportunistic infection of HIV-1-infected patients in developing countries. Concurrent infection with TB results in immune cells having enhanced susceptibility to HIV-1 infection, which facilitates entry and replication of the virus. Cumulative data from earlier studies indicate that TB provides a milieu of continuous cellular activation and irregularities in cytokine and chemokine circuits that favor viral replication and disease progression. To better understand the interaction of the host with HIV-1 during active tuberculosis, we investigated in vivo expression of the HIV-1 coreceptors, CCR5 and CXCR4, and circulating levels of the inhibitory beta-chemokines, macrophage inflammatory protein-1-alpha (MIP-1alpha), macrophage inflammatory protein-1-beta (MIP-1beta), and regulated upon activation T cell expressed and secreted (RANTES), in HIV-positive individuals with and without active pulmonary tuberculosis. We found a significant decrease from normal in the fraction of CD4+ T cells expressing CCR5 and CXCR4 in individuals infected with HIV. However, CCR5 and CXCR4 expression did not differ significantly between HIV patients with and without tuberculosis. Higher amounts of MIP-1alpha, MIP-1beta, and RANTES were detected in plasma of HIV-1-positive individuals, particularly those with dual infection, although the increase was not found to be statistically significant.
top
7. Study of interaction between HIV and TB with special reference to immune activation markers
Principal Investigator: Dr. Elizabeth L. Hanna
In persons infected with HIV, immune mechanism triggered by Mycobacterium tuberculosis may lead to increased HIV replication and disease progression. We studied the impact of active tuberculosis and its treatment on changes in three plasma markers of systemic immune activation. Plasma samples were obtained from 42 HIV+TB+ and 38 HIV-TB+ patients, at the time of diagnosis of active TB and at the end of 6 months of anti tuberculosis treatment (ATT). Thirty-seven HIV+TB- and 62 HIV-TB- individuals served as controls. Markers of immune activation, including neopterin, Beta 2 -Microglobulin ( b 2 -M) and soluble tumor necrosis factor receptor type I (sTNF-RI) were measured using commercial ELISA kits. Plasma levels of neopterin, b 2 -M and sTNF-RI were elevated in all TB patients, with the highest levels in HIV+TB+ patients with advanced disease. Neopterin and sTNF-RI levels decreased significantly in both HIV+TB+ positive and HIV-TB+ patients at the end of 6 months of ATT but remained significantly higher than control values. HIV+TB+ patients with CD4 counts <200 cells/µl had the highest levels at baseline with a steep fall in neopterin and sTNF-R1 during treatment. b 2 -M levels did not decline significantly following ATT in either of the groups. Both HIV and TB increase plasma levels of immune activation markers including neopterin, b 2 -M and sTNF-RI. Anti-TB Treatment reduces activation marker levels in TB patients with and without HIV co-infection. However, a high proportion of HIV+TB+ patients continue to have levels well above the normal range, indicating that underlying immune activation persists despite TB treatment.
top |
