Regulatory role of 1, 25-dihydroxyvitamin D3 and vitamin D receptor gene variants on intracellular granzyme A expression in pulmonary tuberculosis.

Vitamin D receptor (VDR) genotypes have been shown to be associated with differential susceptibility or resistance to tuberculosis. The influence of FokI, BsmI, ApaI and TaqI variants of VDR gene on 1, 25(OH)(2) D(3) modulated granzyme A expression of cytotoxic lymphocytes induced by culture filtrate antigen (CFA) of Mycobacterium tuberculosis was studied in 40 pulmonary tuberculosis (PTB) patients and 49 normal healthy subjects (NHS) by flow cytometry. In both the study groups, addition of 1, 25(OH)(2) D(3) (10(-7)M) significantly reduced the percentage of granzyme A positive cells in both unstimulated (NHS, p<0.0001; PTB, p=0.02) and stimulated culture conditions (CFA, NHS, p<0.0001; PTB, p=0.0001) which correlated positively with the IFN-gamma levels (unstimulated, p=0.01; CFA stimulated, p=0.004) in NHS. The ApaI aa genotype and bbaaTT extended genotype were associated with a significantly decreased percentage of granzyme A positive cells in NHS (p<0.05). Our results suggest that 1, 25(OH)(2) D(3) suppresses granzyme A probably by down-regulating Th1 cytokine response. Moreover, the VDR gene variants might regulate cytotoxic T-cell response via 1, 25(OH)(2) D(3) mediated suppression of granzyme A expression in tuberculosis.

Cytokine gene polymorphisms and cytokine levels in pulmonary tuberculosis.

Polymorphisms in the cytokine genes are known to influence cytokine levels and may be associated with outcome of infections. We investigated the polymorphisms in the cytokine genes namely IFN-gamma (+874 and +5644), IL-2 (-330 and +160), IL-4 (VNTR), IL-6 (-174), IL-10 (-1082 and -819) and IL-12B (+1188) in 188 normal healthy subjects (NHS) and 166 pulmonary tuberculosis patients (PTB) using polymerase chain reaction-based methods. To study the influence of cytokine gene polymorphisms on cytokine levels, phytohaemagglutinin and culture filtrate antigen of Mycobacterium tuberculosis-induced cytokine levels were measured by ELISA from 72-h-old peripheral blood mononuclear cell culture supernatants. Significantly decreased frequency of TT genotype of IL-2 -330 polymorphism (p=0.024, odds ratio (OR) 0.53, 95% CI 0.31-0.92) was observed in patients compared to NHS. The genotype frequencies of other polymorphisms were not different between patients and NHS. IL-12p40 levels were significantly decreased among NHS with AA genotype of IL-12B gene polymorphism compared to NHS with AC genotype (p<0.05). Increased levels of IL-12p40 were observed among patients with CC genotype of IL-12B gene compared to patients with other genotypes (p<0.01). The present study suggests that the TT genotype of IL-2 -330 polymorphism may be associated with the protection to PTB in south India. Further, +1188 polymorphism of IL-12B gene either alone or in combination with closely linked genes may regulate IL-12p40 production and may play a major role on acquired immunity to tuberculosis.

Regulatory region polymorphisms of vitamin D receptor gene in pulmonary tuberculosis patients and normal healthy subjects of south India.

Vitamin D receptor (VDR) gene variants are associated with differential susceptibility or resistance to tuberculosis in different ethnic groups. We investigated the polymorphisms in the 5' regulatory region of VDR gene in 206 normal healthy subjects and 166 patients with pulmonary tuberculosis from south India. Cdx-2 polymorphism was studied by polymerase chain reaction (PCR) with allele-specific primers, while genotyping of A1012G was done by PCR-based restriction fragment length polymorphism. A significantly decreased frequency of Cdx-2 G allele (P = 0.016) and G/G genotype (P = 0.010) and an increased frequency of A-A haplotype (A allele of Cdx-2 and A allele of A1012G) (P = 0.015) were observed in patients compared to controls. The study suggests that Cdx-2 G/G genotype may be associated with protection and A-A haplotype with susceptibility to tuberculosis.

Regulatory role of promoter and 3' UTR variants of vitamin D receptor gene on cytokine response in pulmonary tuberculosis.

Vitamin D receptor (VDR) gene variants are shown to regulate immune response in tuberculosis. We studied the influence of VDR promoter (Cdx-2 and A1012G), 3' untranslated region (Apa I, Bsm I, and Taq I) and start codon (Fok I) polymorphisms on 1,25(OH)(2)D(3)-modulated IL-12p40, IFN-gamma, IL-10, and IL-5 response to live Mycobacterium tuberculosis and its culture filtrate antigen (CFA) in 60 normal healthy subjects and 51 pulmonary tuberculosis patients. In peripheral blood mononuclear cell cultures with CFA and 1,25(OH)(2)D(3), IL-12p40, and IFN-gamma levels were significantly decreased (p < 0.05) and IL-10 levels were significantly increased (p < 0.05) in patients with GG genotype. The extended genotype bbaaTT (baT haplotype) was associated with decreased IL-12p40 and IFN-gamma levels and significantly increased IL-10 levels (p < 0.05). The Cdx-2 GG genotype and baT haplotype are associated with a suppressed Th1 and increased IL-10 response, which suggests that 1,25(OH)(2)D(3) probably through the VDR polymorphic variants augments the anti-inflammatory response at the site of M. tuberculosis infection.

1, 25 Dihydroxyvitamin D3 modulated cytokine response in pulmonary tuberculosis.

1, 25 Dihydroxyvitamin D(3) (1, 25(OH)(2) D(3)) has gained significant importance in tuberculosis with regard to its immunoregulatory activities. Our aim was to evaluate the effect of 1, 25(OH)(2) D(3) on cytokine response to Mycobacterium tuberculosis antigens in pulmonary tuberculosis. Peripheral blood mononuclear cells obtained from 60 healthy controls and 52 pulmonary tuberculosis patients were cultured with culture filtrate antigen (CFA) of M. tuberculosis and live M. tuberculosis with and without 1, 25(OH)(2) D(3) (10(-9), 10(-8)and 10(-7)M concentrations). The culture supernatants were used to estimate IL-8, IL-6, TGF-beta, IL-10, IFN-gamma, IL-12p40, IL-2, IL-4 and IL-5 levels by ELISA. 1, 25 Dihydroxyvitamin D(3) significantly suppressed IL-12p40 and IFN-gamma production in response to CFA and live M. tuberculosis with a maximum suppression at 10(-7)M concentration (p<0.0001). In CFA stimulated cultures, addition of 1, 25(OH)(2) D(3) significantly suppressed IL-8, IL-6 and IL-10 whereas the IL-2 levels were significantly increased in controls. It variably influenced the Th2 cytokines, showing an increased trend for IL-4 and suppressed IL-5 levels. We report that 1, 25(OH)(2) D(3) differentially modulates production of cytokines in response to M. tuberculosis antigens by predominantly suppressing IL-12p40 and IFN-gamma production in a dose dependent manner. Our results suggest a role for vitamin D in restricting acquired immune response against tuberculosis by regulating cytokine production.

Interferon gamma (IFNgamma) & interleukin-4 (IL-4) gene variants & cytokine levels in pulmonary tuberculosis.

BACKGROUND & OBJECTIVES: Cytokine gene polymorphisms may alter Th1/Th2 balance with major implications in tuberculosis. The aim of our study was to find out whether Interferon gamma +874A and IL-4 -590T polymorphisms were associated with susceptibility to pulmonary tuberculosis as well as the level of IFNgamma and IL-4 in south Indian population. METHODS: Interferon gamma +874A and IL-4 -590T promoter polymorphisms were studied in 129 pulmonary tuberculosis (PTB) patients and 127 normal healthy subjects (NHS) and were associated with culture filtrate and live Mycobacterium tuberculosis induced IFNgamma and IL-4 production in peripheral blood mononuclear cells (PBMCs). IL-4 gene variants were also associated with IgG antibody levels against M. tuberculosis culture filtrate antigen. RESULTS: The variant IFNgamma genotypes and IFNgamma levels between genotypes did not differ significantly in patients and controls. Significantly increased frequency of variant IL-4 'CT' genotype in PTB patients (P<0.05) and 'CC' genotype in control group (P<0.01) was observed. IL-4 levels were detectable in very few subjects and the IgG levels did not differ between the three IL-4 genotypes. INTERPRETATION & CONCLUSION: The study suggests a lack of functional association of Interferon gamma +874A polymorphism in tuberculosis in south Indian population. The higher frequency of IL-4 'CT' genotype in PTB suggests a possible association of IL-4 -590T promoter polymorphism with susceptibility to tuberculosis, and the 'CC' genotype may be associated with protection.

Elevated percentage of perforin positive cells in active pulmonary tuberculosis.

BACKGROUND & OBJECTIVES: Perforin is one of the major effector molecules of cytotoxic cells associated with killing of cells harbouring intracellular bacterial infection. The precise role of perforin positive cells in tuberculosis still remains controversial. The present study was done to determine the number of circulating CD4(+) and CD8(+) perforin positive cells to assess the level of cytotoxic response against Mycobacterium tuberculosis in patients with pulmonary tuberculosis. METHODS: Intracellular perforin and surface CD4 and CD8 staining of peripheral blood lymphocytes was done using specific monoclonal antibodies and enumerated using flowcytometry. RESULTS: A significantly decreased total lymphocytes (P<0.01), CD4 (P<0.001) and CD8 (P<0.01) lymphocyte counts in PTB patients was observed compared to normal healthy individuals (NHS). Intracellular perforin staining showed significantly elevated percentages of total (P<0.05) and CD8 (P<0.01) perforin positive cells in PTB patients compared to NHS. However, the absolute counts of total, CD4 and CD8 cells positive for perforin were similar in patients and NHS. INTERPRETATION & CONCLUSION: Our results suggest that during active stage of pulmonary tuberculosis there was an increased percentage of CD8 cells positive for perforin, irrespective of their absolute counts. Further, CD8(+) perforin positive cells may have increased cytolytic activity against M. tuberculosis in active pulmonary tuberculosis.