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

A Correlation between Phagocytosis and Apoptosis in THP-1 Cells Infected with Prevalent Strains fo Mycobacterium tuberculosis

The innate ability of infected macrophages to undergo programmed cell dealth (apoptosis) and curtail the infection is crucial for the host defense. Although phagocytosis and intracellular killing mechanisms leading to apoptosis in macrophages are highly effective in eliminating the infecting tuberculous bacilli, some Mycobacterium tuberculosis (Mtb) straIns have evolved strategies to inhibit this microbicidal function and make use of macrophage for its successful and prolonged survival. Two clinical strains of Mtb (S7 and S10) found to be prevalent and primitive, based on molecular epidemiological studies, were used to study the magnitude in induction of apoptosis in THP-1 cells at various time points of infection and to correlate it with phagocytosis. The percentage of phagocytosis did not show any strain-specific association with differentiated THP-1 cells. But in the phagocytic index, the clinical strains showed a low dose of infection in the 1-10 bacilli category thereby exerting less burden on the cells. The induction of apoptosis was strain dependent. The THP-1 cells infected with H37Ra and S10 showed an increase in apoptosis at all time points while the S7 strain induced minimum apoptosis. A negative correlation between apoptosis and phagocytic index was observed in the 1-10 category and a positive correlation in the > 20 category of the phagocytic index. This novel observation indicates that the magnitude of THP-1 cell apoptosis is a function of the number of internalized mycobacteria. These results indicated a differential mode of infection by clinical strains and their adaptation to different survival strategies that may lead to immune suppression and pathogenesis of the disease

Th2-type immune response observed in healthy individuals to sonicate antigen prepared from the most prevalent Mycobacterium tuberculosis strain with single copy of IS6110

Different Mycobacterium tuberculosis strains operate different immune evasion strategies for their survival in the host. This mainly depends on the virulence of the strain and the host immune responses. The most virulent strains are actively involved in the transmission, widely spread in the community and induce differential immune responses. We evaluated the immune response of a sonicate antigen prepared from one predominant strain (S7) from M. tuberculosis harbouring a single copy of IS6110. Significant lymphoproliferative response against purified protein derivative from tubercle bacillus (PPD) and H37Rv antigens was observed in PPD positive normal individuals and tuberculosis patients. Interferon-gamma (IFN-c) levels against these antigens were significantly increased in normal individuals but not in tuberculosis patients. The antigen S7 showed marginal T-cell proliferation but did not induce IFN-c secretion in both groups. Conversely, it induced significantly high levels of cytokine interleukin 4 (IL-4) in normal individuals. The macrophage cytokines, IL-12 and tumour necrosis factor alpha (TNF-a), did not show S7 antigen specific stimulation. The intracellular cytokine further confirmed an increase in IL-4+/CD4+ T-cells and a decrease in IFN-c+/CD4+ T-cells upon stimulation. The antibody response showed an increase in IgG and IgA levels against this antigen in normal individuals. These observations suggest that antigen S7 modulates the immune response towards T helper cell type 2 by suppressing T helper cell type 1 protective immune response in PPD positive normal individuals. We speculate that some components of this sonicate antigen are associated with immunosuppressive response

Phenotypic modulation in Mycobacterium tuberculosis infected neutrophil during tuberculosis

Background & objectives: Polymorphonuclear leucocytes (PMN) or neutrophils infiltrate to the inflammatory sites and phagocytose mycobacteria thereby inhibiting the bacillary spread initially until the accumulated macrophages get activated. The present study was carried out to highlight the interaction of neutrophils with the two clinical isolates (S7 and S10) of Mycobacterium tuberculosis and the subsequent morphological changes. Methods: Dextran purified neutrophils from normal and TB patients infected with M. tuberculosis\ud isolates were cultured for 3 and 18 h time points. At the end of termination, the cell surface expression of CD16, CD69, CXCR2 and induction of apoptosis were analyzed using flow cytometry. Cytokines and chemokines were estimated in supernatants by ELISA. Results: All infected PMN showed decrease in CD16 at both time points in normals while at 18 h in TB group. Interestingly, CD69 expression was significantly high at early time point in TB-PMN compared to normals. The high expression of CXCR2 was sustained in infected TB-PMN at both the time points. S7 and S10 infected neutrophils showed high phagocytic indices compared to H37Rv in both the groups. A significant increase in apoptosis was observed at both the time points in infected TB-PMN but only at 18 h in normals. Increased pro-inflammatory cytokine (TNF-α) and chemokine (IL-8) response was observed in infected neutrophils at 3 h in both the groups. Interpretation & conclusions: This study demonstrates the varying degree of modulation of neutrophil functions in both the groups. TB-PMN was more competent in amplifying the innate immune response and conferring protection at the early phase of infection. However, the response was not strain specific in either of these groups