Mohideen S. Jawahar

Regulatory role of vitamin D receptor gene variants of Bsm I, Apa I, Taq I, and Fok I polymorphisms on macrophage phagocytosis and lymphoproliferative response to mycobacterium tuberculosis antigen in pulmonary tuberculosis.

The regulatory role of vitamin D receptor (VDR) gene variants of BsmI, ApaI, TaqI, and FokI polymorphisms on vitamin D3-modulated macrophage phagocytosis with live Mycobacterium tuberculosis and lymphoproliferative response to M. tuberculosis culture filtrate antigen (CFA) was studied in patients with pulmonary tuberculosis (n = 46) and in normal healthy subjects (NHS) (n = 64). Vitamin D3 at a concentration of 1 × 10−7 M enhanced the phagocytic potential of normal subjects who had a phagocytic index of less than 20%. This increase was seen in subjects with the genotypes BB (p = 0.017), AA (p = 0.016), tt (p = 0.034), and FF (p = 0.013) and the extended genotype BBAAtt (p = 0.034). Normal subjects with BBAAtt performed better phagocytosis than individuals with bbaaTT genotype (p = 0.034). Vitamin D3 at 10−9, 10−8, and 10−7 M concentrations suppressed the lymphoproliferative response to CFA antigen in normal subjects. This decreased lymphocyte response was observed in normal individuals with the genotypes BB (p = 0.0009), tt (p = 0.016), and FF (p = 0.008) and the extended genotype BBAAtt (p = 0.02). Addition of vitamin D3 had no significant effect on macrophage phagocytosis and lymphoproliferative response to CFA in pulmonary TB patients. This may be due to the unresponsive nature of the cells to the action of vitamin D3 or the downregulated VDR expression by virtue of the disease, which renders them inactive. The genotypes BB, tt, and the extended genotype BBAAtt may be associated with increased expression of VDR which in turn regulate the action of vitamin D3 and modulate the immune functions to M. tuberculosis in NHS.

Impact of HIV Infection on the Recurrence of Tuberculosis in South India

BACKGROUND There is limited information on the relative proportion of reactivation and reinfection at the time of recurrence among human immunodeficiency virus (HIV)-infected and HIV-uninfected patients who are successfully treated for tuberculosis infection in India. METHODS HIV-infected and HIV-uninfected patients with sputum culture-positive pulmonary tuberculosis were treated with short-course regimens and followed up for 36 months at the Tuberculosis Research Centre, South India. Bacteriologic recurrences were documented, and typing of strains was performed using 3 different genotypic techniques: restriction fragment length polymorphism (RFLP) by IS6110, spoligotyping, and mycobacterial interspersed repeat unit (MIRU)-variable number tandem repeat (VNTR). DNA fingerprints of paired Mycobacterium tuberculosis isolates (baseline and recurrence) were compared. RESULTS Among 44 HIV-infected and 30 HIV-uninfected patients with recurrent tuberculosis during the period July 1999 to October 2005, 25 and 23 paired isolates, respectively, were typed using all 3 methods. Recurrence was due to exogenous reinfection in 88% of HIV-infected and 9% of HIV-uninfected patients (P<.05). Among recurrent isolates, the HIV-infected patients showed more clustering, as well as a higher proportion of drug resistance, including multidrug resistance. CONCLUSIONS In India, a tuberculosis-endemic country, most recurrences after successful treatment of tuberculosis are due to exogenous reinfection in HIV-infected persons and endogenous reactivation in HIV-uninfected persons. Strategies for prevention and treatment of tuberculosis infection must take these findings into consideration.

Effect of vitamin D3 on phagocytic potential of macrophages with live Mycobacterium tuberculosis and lymphoproliferative response in pulmonary tuberculosis.

Immune responses are elicited through antigen presentation and recognition by macrophages and T-lymphocytes, respectively. The immunomodulatory effect of vitamin D3 on macrophage phagocytic potential with live Mycobacterium tuberculosis, spontaneous and M. tuberculosis culture filtrate antigen induced lymphocyte responses were studied in pulmonary tuberculosis patients (PTBPs) (n = 31) and normal healthy subjects (NHSs) (n = 43). Vitamin D3 at a concentration of 10−7 M significantly enhanced the macrophage phagocytosis of live M. tuberculosis in normal subjects with low phagocytic potential (less than 10%) (p = 0.015). No such increase was observed in PTBPs. Vitamin D3 significantly decreased the spontaneous lymphoproliferative response (p = 0.022) and increased the apoptosis of peripheral blood mononuclear cells in PTBPs (p = 0.024). In normals, vitamin D3 increased the spontaneous lymphoproliferative response. An inverse correlation between macrophage phagocytosis and spontaneous response was observed in NHSs, whereas a direct correlation was seen between vitamin D3-treated cells in normal subjects under in vitro condition. Vitamin D3 decreased the M. tuberculosis culture filtrate antigen induced lymphocyte response significantly in normal subjects (p = 0.0003), while it had no influence on the lymphocyte response in PTBPs. The present study suggests that exposure to vitamin D3 increases the phagocytic potential and spontaneous lymphoproliferative response but brings down the antigen-induced response in normals. In tuberculosis, addition of vitamin D3 has no significant effect on antigen-induced lymphoproliferative response. This may be due to the unresponsive nature of the cells to the action of vitamin D3 by virtue of the disease, which renders them inactive.

Expansion of Pathogen-Specific T-Helper 1 and T-Helper 17 Cells in Pulmonary Tuberculosis With Coincident Type 2 Diabetes Mellitus

BACKGROUND Type 2 diabetes mellitus (DM) is a major risk factor for the development of active pulmonary tuberculosis, although the immunological mechanisms underlying this interaction remain unexplored. The influence of poorly controlled diabetes on pathogen-specific T-helper 1 (Th1) and T-helper 17 (Th17) responses have not been examined. METHODS To identify the role of Th1 and Th17 cells in tuberculosis with coincident DM, we examined mycobacteria-specific immune responses in the whole blood of individuals who had tuberculosis with DM and compared them to those in individuals who had tuberculosis without DM. RESULTS Tuberculosis coincident with DM is characterized by elevated frequencies of monofunctional and dual-functional CD4(+) Th1 cells following Mycobacterium tuberculosis antigen stimulation and elevated frequencies of Th17 subsets at both baseline and following antigen stimulation. This was associated with increased systemic (plasma) levels of both Th1 and Th17 cytokines and decreased baseline frequencies of natural regulatory T cells but not interleukin 10 or transforming growth factor β. CONCLUSIONS Therefore, our data reveal that tuberculosis in persons with DM is characterized by elevated frequencies of Th1 and Th17 cells, indicating that DM is associated with an alteration in the immune response to tuberculosis, leading to a biased induction of Th1- and Th17-mediated cellular responses and likely contributing to increased immune pathology in M. tuberculosis infection.

Randomized Clinical Trial of Thrice-Weekly 4-Month Moxifloxacin or Gatifloxacin Containing Regimens in the Treatment of New Sputum Positive Pulmonary Tuberculosis Patients

Background Shortening tuberculosis (TB) treatment duration is a research priority. This paper presents data from a prematurely terminated randomized clinical trial, of 4-month moxifloxacin or gatifloxacin regimens, in South India. Methods Newly diagnosed, sputum-positive HIV-negative pulmonary TB patients were randomly allocated to receive gatifloxacin or moxifloxacin, along with isoniazid and rifampicin for 4 months with pyrazinamide for first 2 months (G or M) or isoniazid and rifampicin for 6 months with ethambutol and pyrazinamide for first 2 months (C). All regimens were administered thrice-weekly. Clinical and bacteriological assessments were done monthly during treatment and for 24 months post-treatment. The Data and Safety Monitoring Board recommended termination of the trial due to high TB recurrence rates in the G and M regimens. Results Of 416 patients in intent-to-treat analysis, 6 (5%) of 124, 2 (2%) of 110 and 2 (2%) of 137 patients with drug-susceptible TB in the G, M and C arms respectively had unfavorable response at the end of treatment; during the next 24 months, 17 (15%) of 115, 11 (11%) of 104 and 8 (6%) of 132 patients respectively, had TB recurrence. Of 38 drug-resistant patients 1 of 8 and 3 of 26 in the G and C arms respectively had unfavourable response at the end of treatment; and TB recurrence occurred in 2 of 7 and 2 of 23 patients, respectively. The differences in TB recurrence rates between the G and C arms was statistically significant (p = 0.02). Gastro-intestinal symptoms occurred in 23%, 22% and 9% of patients in the G, M and C arms respectively, but most reactions were mild and manageable with symptomatic measures; 1% required regimen modification. Conclusions 4-month thrice-weekly regimens of gatifloxacin or moxifloxacin with isoniazid, rifampicin and pyrazinamide, were inferior to standard 6-month treatment, in patients with newly diagnosed sputum positive pulmonary TB. Trial Registration Clinical Trials Registry of India CTRI/2012/10/003060

Type 2 Diabetes Mellitus Coincident with Pulmonary Tuberculosis Is Associated with Heightened Systemic Type 1, Type 17, and Other Proinflammatory Cytokines

RATIONALE Type 2 diabetes mellitus is a major risk factor for the development of active tuberculosis, although the biological basis underlying this susceptibility remains poorly characterized. OBJECTIVES AND METHODS To identify the influence of coincident diabetes mellitus on cytokine levels in pulmonary tuberculosis, we examined circulating levels of a panel of cytokines and chemokines in the plasma of individuals with tuberculosis with diabetes and compared them with those of individuals without diabetes. MEASUREMENTS AND MAIN RESULTS Tuberculosis with diabetes is characterized by elevated circulating levels of type 1 (IFN-γ, tumor necrosis factor-α, and IL-2), type 2 (IL-5), and type 17 (IL-17A) cytokines but decreased circulating levels of IL-22. This was associated with increased systemic levels of other proinflammatory cytokines (IL-1β, IL-6, and IL-18) and an antiinflammatory cytokine (IL-10) but not type 1 IFNs. Moreover, tuberculosis antigen-stimulated whole blood also showed increased levels of proinflammatory cytokines. Finally, type 1 and type 17 cytokines in plasma exhibit a significant positive correlation with hemoglobin A1C levels, indicating that impaired control of diabetes is associated with this proinflammatory milieu. Multivariate analysis revealed that the association of proinflammatory cytokines with diabetes mellitus was not influenced by age, sex, or other metabolic parameters. CONCLUSIONS Our data reveal that tuberculosis with diabetes is characterized by heightened cytokine responsiveness, indicating that chronic inflammation underlying type 2 diabetes potentially contributes to increased immune pathology and poor control in tuberculosis infection.

Plasma Heme Oxygenase-1 Levels Distinguish Latent or Successfully Treated Human Tuberculosis from Active Disease

Background Tuberculosis (TB) is associated with oxidative stress and the induction of host anti-oxidants to counteract this response. Heme oxygenase-1 (HO-1) is a critical promoter of cytoprotection in diverse disease models including mycobacterial infection. Nevertheless, the pattern of expression of HO-1 in human tuberculosis has not been studied. Here, we examine expression of HO-1 in M. tuberculosis-exposed and -infected individuals and test its ability to distinguish active from latent and successfully treated TB cases. In addition, we assess correlations between plasma levels of HO-1 and cytokines closely associated with the immunopathogenesis of TB. Methods Cross-sectional and longitudinal analyses of levels of HO-1, acute phase proteins and pro-inflammatory cytokines were performed in plasma samples from individuals with active pulmonary, extra-pulmonary or latent TB infection and healthy controls as part of a prospective cohort study in South India. Results Systemic levels of HO-1 were dramatically increased in individuals with active pulmonary and extra-pulmonary tuberculosis and particularly those with bilateral lung lesions and elevated bacillary loads in sputum. HO-1 levels effectively discriminated active from latent tuberculosis with higher predictive values than either C-reactive protein or serum amyloid protein. Moreover, there was a marked reduction in HO-1 levels in active TB cases following anti-tuberculous therapy but not in those who failed treatment. Pulmonary TB patients displaying the highest concentrations of HO-1 in plasma exhibited significantly elevated plasma levels of interleukin (IL)-10, interferon (IFN)-γ and IL-17 and diminished levels of tumor necrosis factor (TNF)-α. Conclusion These findings establish HO-1 levels as a potentially useful parameter for distinguishing active from latent or treated pulmonary tuberculosis, that is superior in this respect to the measurement of other acute inflammatory proteins.

Effect of vitamin D3 on chemokine expression in pulmonary tuberculosis.

1,25 Dihydroxy vitamin D(3) (vitamin D(3)) is an immunomodulator and its deficiency has been associated with susceptibility to tuberculosis. We have studied the immunoregulatory role of vitamin D(3) on various chemokine expression in pulmonary tuberculosis. Peripheral blood mononuclear cells obtained from 21 pulmonary tuberculosis (PTB) patients and 24 healthy controls (HCs) were cultured for 48 h with culture filtrate antigen (CFA) of Mycobacterium tuberculosis with or without vitamin D(3) at a concentration 1 × 10(-7)M. The relative mRNA expression of monocyte chemoattractant protein-1 (MCP-1, CCL2), macrophage inflammatory protein-1α (MIP-1α, CCL3), macrophage inflammatory protein-1β (MIP-1β, CCL4), and regulated upon-activation, normal T cell-expressed and secreted (RANTES, CCL5) and IFN-γ inducible protein-10 (IP-10, CXCL10) chemokines were estimated from 48 h old macrophages using real-time polymerase chain reaction (RT-PCR). The culture supernatants were used to estimate the various chemokines including monokine induced by IFN-γ (MIG, CXCL9) levels using cytometric bead array. In HCs, vitamin D(3) significantly suppressed the MCP-1 mRNA expression of CFA stimulated cells (p=0.0027), while no such effect was observed in PTB patients. Vitamin D(3) showed no significant effect on MIP-1α, MIP-1β and RANTES in both the study groups. The CFA induced IP-10 mRNA and protein expression was significantly suppressed by vitamin D(3) in both the study groups (p<0.05). A similar suppressive effect of vitamin D(3) was observed with MIG protein in healthy controls (p=0.0029) and a trend towards a suppression was observed in PTB patients. The suppressive effect of vitamin D(3) is more prominent in CXC chemokines rather than CC chemokines. This suggests that vitamin D(3) may down regulate the recruitment and activation of T-cells through CXC chemokines at the site of infection and may act as a potential anti-inflammatory agent.

IL-10 Dependent Suppression of Type 1, Type 2 and Type 17 Cytokines in Active Pulmonary Tuberculosis

Background Although Type 1 cytokine responses are considered protective in pulmonary tuberculosis (PTB), their role as well as those of Type 2, 17 and immunoregulatory cytokines in tuberculous lymphadenitis (TBL) and latent tuberculosis (LTB) have not been well studied. Aim and Methods To identify cytokine responses associated with pulmonary tuberculosis (TB), TB lymphadenitits and latent TB, we examined mycobacterial antigen-specific immune responses of PTB, TBL and LTB individuals. More specifically, we examined ESAT-6 and CFP-10 induced Type 1, Type 2 and Type 17 cytokine production and their regulation using multiplex ELISA. Results PTB individuals exhibited a significantly lower baseline as well as antigen-specific production of Type 1 (IFNγ, TNFα and IL-2); Type 2 (IL-4) and Type 17 (IL-17A and IL-17F) cytokines in comparison to both TBL and LTB individuals. TBL individuals exhibited significantly lower antigen-specific IFNγ responses alone in comparison to LTB individuals. Although, IL-10 levels were not significantly higher, neutralization of IL-10 during antigen stimulation resulted in significantly enhanced production of IFNγ, IL-4 and IL-17A in PTB individuals, indicating that IL-10 mediates (at least partially) the suppression of cytokine responses in PTB. Conclusion Pulmonary TB is characterized by an IL-10 dependent antigen-specific suppression of Type 1, Type 2 and Type 17 cytokines, reflecting an important association of these cytokines in the pathogenesis of active TB.

Heightened plasma levels of heme oxygenase-1 and tissue inhibitor of metalloproteinase-4 as well as elevated peripheral neutrophil counts are associated with TB-diabetes comorbidity.

BACKGROUND The increased prevalence of type 2 diabetes mellitus (T2DM) in countries endemic for TB poses a serious complication in the clinical management of this major infectious disease. Understanding the impact of T2DM on TB and the determinants of comorbidity is critical in responding to this growing public health problem with better therapeutic approaches. Here, we performed an exploratory study assessing a series of biologic parameters that could serve as markers of pathogenesis in TB with T2DM. METHODS Cross-sectional analyses of levels of heme oxygenase-1 (HO-1), acute phase proteins, tissue metalloproteinases, and tissue inhibitors of metalloproteinase (TIMPs) as well as cytokines and chemokines were performed in plasma samples from individuals with active pulmonary TB or with coincident TB and T2DM from South India. RESULTS Compared with patients with TB without diabetes, those with coincident T2DM exhibited increased Mycobacterium tuberculosis bacillary loads in sputum. Plasma levels of HO-1 but not of other acute phase proteins were higher in patients with TB and T2DM than in patients without diabetes, independent of bacillary sputum loads. HO-1 concentrations also positively correlated with random plasma glucose, circulating glycosylated hemoglobin, and low-density lipoprotein levels. Moreover, patients with coincident TB and T2DM exhibited increased plasma levels of TIMP-4 and elevated peripheral blood neutrophil counts, which, when considered together with HO-1, resulted in increased power to discriminate individuals with active TB with and without T2DM. CONCLUSIONS Elevated plasma levels of HO-1 and TIMP-4 and peripheral blood neutrophil counts are potential single and combined markers of pathogenesis in TB and T2DM.