Cell-Mediated Immune Responses of Healthy Laboratory Volunteers to Sonicate Antigens Prepared from the Most Prevalent Strains of Mycobacterium tuberculosis from South India Harboring a Single Copy of IS6110

Our restriction fragment length polymorphism (RFLP) studies have shown that the most prevalent (40%) strains of Mycobacterium tuberculosis from South India contain a single copy of the IS6110 insertion sequence and are of importance in studying virulence and immunity. Sonicate antigens from seven such strains were used to study in vitro T-cell proliferation and gamma interferon (IFN-�) and interleukin-12 (IL-12) secretion as markers of protective immunity in 25 healthy subjects positive for purified protein derivative (PPD). The standard PPD and heat-killed H37Rv antigens induced the maximum levels of T-cell proliferation and IFN-� secretion but low levels of IL-12. All sonicate antigens induced T-cell proliferation and IFN-� secretion with strong positive correlation. Our results suggest that sonicate antigens from the most prevalent and recent strains of M. tuberculosis from clinical isolates have the potential to induce T-cell activation and may allow newer and specific antigens to be further characterized for diagnosis and vaccine development

Humoral immune responses of normals and tuberculosis patients to multiple sonicate antigens prepared from the most prevalent strains of Mycobacterium tuberculosis harbouring single copy of IS6110 from South India

The search for newer species-specific antigens from prevalent strains of Mycobacterium tuberculosis (Mtb) associated with active disease will add substantially to improve the presently available diagnostic tools. We studied the protein profiles of 13 sonicate antigens (S1– S13) prepared from the prevalent strains. The humoral immune response was also studied in 30 normal and 30 tuberculosis (TB) patients. The sonicate antigens S10 and S12 showed maximum differential protein bands in low molecular mass region of 10–30 kDa on SDS–PAGE. Our ELISA results showed significant increase in Mtb-specific IgG antibodies in TB plasma for H37Rv, followed by PPD, S1 and S10 antigens. Immunoblot analysis of S10 antigen showed specific recognition pattern at low molecular mass region by TB plasma alone with 12–77% positivity of protein bands. Thus the sonicate antigen S10 was found to be discriminatory by ELISA and Western blot and hence a good candidate for further purification of its individual proteins to be evaluated for diagnosis

Humoral Immune Response in Tuberculous Pleuritis

Tuberculous pleuritis is a good human model to understand the local and protective immune response against tuberculosis, due to the self-limitedness of the disease. Although the cellular immune response has been well characterised in tuberculous pleurisy, much less is known about the humoral immune response operating at the site of infection. To understand the humoral immune response, B cells were enumerated in peripheral blood mononuclear cells (PBMC) and pleural fluid mononuclear cells (PFMC) of tuberculous (TP) and non-tuberculous pleuritis patients (NTP). The levels of IgG, IgA and IgM antibodies for PPD, culture filtrate (CF) and sonicate antigens (Son Ag) were assessed in plasma (BL) and pleural fluid (PF) and a western blot was carried out with the CF antigen. The percentage of CD19+B-cells was similar in PBMC and PFMC of TP patients but was significantly lower in PFMCs of NTP patients. The IgG levels for PPD and CF antigens were higher in PF of TP than NTP patients. The antigen recognition patterns did not differ in plasma and pleural fluid of the same patient in both groups pointing out the passive diffusion of the plasma to the pleura. The antigens 25, 31, 33, 70, 110, 124 and 132 kDa were recognized exclusively by the TP patients. Thus our study showed that the local humoral response in TP did not differ from the systemic response. However, the humoral response differed in TP patients when compared to NTP patients

Role of TNF-a in host immune response in tuberculous pleuritis

Tumour Necrosis Factor (TNF)-a, a pro-inflammatory cytokine has a dual role in host immunity and immunopathology of tuberculosis and is considered to be pivotal for determining the clinical course of the disease, either beneficial or detrimental. The assessment of TNF-a in pleural tuberculosis will help us to understand its role in host defence mechanism against Mycobacterium tuberculosis (MTB) infection. In this study, TNF-a and IFN-g levels were measured in plasma and pleural fluid of both tuberculosis (TB) and non- TB patients and in the supernatants of blood and pleural fluid mononuclear cells (PBMCs and PFMCs) stimulated in vitro with PPD, culture filtrate and heatkilled (MTB). In addition, apoptosis induced by PPD and MTB was also studied. TNF-a and IFN-g were significantly elevated in pleural fluid than in plasma of pleural tuberculosis patients, suggesting the compartmentalization of Th1 cytokine-secreting cells at the site of disease. In vitro stimulation of PFMCs with PPD and MTB showed a significant increase in these cytokine levels and also enhanced apoptosis of these cells. This increase in TNF-a levels may contribute to the containment of infection by synergizing with IFN- g to activate infected macrophages or by the regulation of T-cell apoptosis

Comparison of localized versus systemic levels of Matrix metalloproteinases (MMPs), its tissue inhibitors (TIMPs) and cytokines in tuberculous and non-tuberculous pleuritis patients

The interaction of Matrix metalloproteinases (MMPs), its tissue inhibitors (TIMPs) and pro-inflammatory cytokines in response to Mycobacterium tuberculosis (MTB) infection is important to understand the immune response at the site of infection. We compared the levels of MMPs, TIMPs and cytokines in plasma (BL) and pleural fluid (PF) of tuberculosis (TB) and non tuberculosis (NTB) patients. Comparison between BL and PF showed significantly higher levels of MMP-1, TIMP-1 and -3 in TB PF; of MMP-7, -8, -9 in BL of both groups. Also, levels of MMP-1,-8,-9 and TIMP-3 were significantly higher in TB PF compared to NTB. Cytokines INF-γ, TNF-α, and IL-6 significantly increased in PF of both groups. A positive correlation of MMPs with TIMPs in TB, MMP-1 and -9 with IL-6 in TB PF and MMP-9 with IFN-γ in NTB PF was observed. This study implicates the possible usage of MMPs as bio-markers aiding diagnosis in TB pleuritis

Proteomics Analysis of Three Different Strains of Mycobacterium tuberculosis under In vitro Hypoxia and Evaluation of Hypoxia Associated Antigen’s Specific Memory T Cells in Healthy Household Contacts

In vitro mimicking conditions are thought to reflect the environment experienced by M. tuberculosis inside the host granuloma. The majority of the in vitro dormancy experimental models used laboratory adapted strain H37Rv or Erdman strain over the prevalent clinical strains involved during disease outbreaks. Thus, we included the most prevalent clinical strains (S7 and S10) of M. tuberculosis from south India in addition to H37Rv for our in vitro oxygen depletion (hypoxia) experimental model. Cytosolic proteins were prepared from the hypoxic cultures, resolved by two-dimensional electrophoresis (2-DE) and protein spots were characterized by mass spectrometry. Totally 49 spots were characterized as over-expressed or newly appeared between the 3 strains. Two antigens (ESAT-6, Lpd) out of the 49 characterized spots were readily available in recombinant form in our lab. Hence, these 2 genes were overexpressed, purified and used for in vitro stimulation of whole blood collected from healthy household contacts (HHC) and active pulmonary tuberculosis patients (PTB). Multicolour flow cytometry analysis showed high levels of antigen specific CD4+ central memory T cells in circulation of HHC when compared to PTB (p<0.005 for ESAT-6 and p<0.0005 for Lpd). This shows proteins that are predicted to be upregulated during in vitro hypoxia in most prevalent clinical strains would bring the possible potential immunogens. In vitro hypoxia experiments with most prevalent clinical strains would also bring the probable true representative antigens that involved during adaption mechanism

Leptin response in patients with tuberculous pleuritis

Background & objectives: Tuberculous pleuritis is used as a model to understand the protective immune response in tuberculosis. It is predominated by Th1 response at the site of infection, where a possible role for the leptin, a known enhancer of Th1 response, could be speculated. Hence, we investigated leptin levels in pleural effusions in patients with both tuberculous (TP) and non-tuberculous (NTP) pleural effusion. Methods: Leptin and cytokine levels were assessed in serum and pleural fluid of TP and NTP patients (N = 20 each) by ELISA. Multivariate regression analysis were performed to find the possible determinants of leptin taking leptin as the dependent and body mass index (BMI), gender, source of leptin [i.e., serum or pleural fluid (PF)], age and disease status as independent variables. Results: PF leptin levels were significantly higher than serum leptin levels in both the groups however the PF leptin levels were significantly lower in TP subjects compared to NTP. The results showed that the leptin was found to be dependent on BMI but not on the other parameters. However, regression analysis based on the source of leptin showed males to be a better predictor of leptin. No correlation was observed between leptin and measured immune parameters. Interpretation & conclusions: Our findings demonstrated that the decreased leptin levels were associated with reduction in BMI but not with the disease status in tuberculous pleuritis