Exploring the Role of C-C Motif Chemokine Ligand-2 Single Nucleotide Polymorphism in Pulmonary Tuberculosis: A Genetic Association Study from North India.

The C-C motif chemokine ligand-2 (CCL2) was evidenced to be associated with tuberculosis susceptibility in some ethnic groups. In the present study, effort was made to find out the association of CCL2-2518 A>G and -362 G>C variants with susceptibility to TB in a population from North India. The genotyping was carried out in 373 participants with pulmonary TB (PTB) and 248 healthy controls (HCs) for CCL2-2518 A>G and -362 G>C polymorphisms by PCR-RFLP and by melting curve analysis using fluorescence-labeled hybridization fluorescent resonance energy transfer (FRET) probes, respectively, followed by DNA sequencing in a few representative samples. Genotype and allele frequencies were compared by the chi-squared test and crude and Mantel-Haenszel (M-H) odds ratio (OR). OR was calculated using STATA/MP16.1 software. Further, CCL2, IL-12p70, IFN-γ, TNF-α, and TGF-ß levels were measured in serum samples of these participants using commercially available kits. Our analysis indicated that the homozygous mutant in both -2518 GG (OR = 2.07, p = 0.02) and -362 CC (OR = 1.92, p = 0.03) genotypes was associated with susceptibility to pulmonary TB. Further, heterozygous genotypes -2518AG (OR = 0.60, p = 0.003) and -362GC (OR = 0.64, p = 0.013) provide resistance from PTB disease. Haplotype analysis revealed AC haplotype (p = 0.006) to be a risk factor associated with PTB susceptibility. The serum CCL2 level was significantly elevated among participants with -2518 AA genotype compared to -2518 GG genotype. CCL2 level was observed to be positively correlated with IL12p70, IFN-γ and TNF-α, thus suggesting the immunological regulatory role of CCL2 against pulmonary tuberculosis. CCL2-2518 GG and -362 CC genotypes were found to be associated with susceptibility to pulmonary tuberculosis and CCL2-2518AG and CCL2-362GC with resistance from PTB. AC haplotype was found to be a risk factor for PTB in the present study. It may be hypothesized from the findings that -2518G allele could be responsible for lower production of CCL2 which leads to defective Th1 response and makes a host susceptible for pulmonary tuberculosis.

Leprosy-specific B-cells within cellular infiltrates in active leprosy lesions.

Leprosy is a spectral disease with polar lepromatous and tuberculoid forms correlating with enhanced humoral and cell-mediated immunity, respectively, against Mycobacterium leprae and the borderline forms, borderline lepromatous, midborderline, and borderline tuberculoid showing in-between clinical and immunological characteristics. Histopathologically, the cellular infiltrates of leprosy lesions show predominantly the presence of interacting T-cells and antigen presenting cells like macrophages, whereas the presence of B-cells has only been sporadically reported. The present study demonstrates by immunohistochemical techniques the presence of B-cells, including plasma cells, in active lesions from lepromatous leprosy, skin smear negative borderline lepromatous, and paucibacillary borderline tuberculoid leprosy. Furthermore, the study demonstrates the in situ production of M leprae-specific antibodies from BT lesions using an organotypic skin explant culture model. Finally, analysis of the cytokine release profile in supernatants of lesional organotypic skin cultures showed a microenvironment conducive to the differentiation and maturation of B-cells. The results demonstrate the presence of different functionally active B-cell stages within lesions of patients with leprosy, including borderline tuberculoid patients, which could secrete anti-M leprae-specific antibodies. However, their role in leprosy pathology remains to be elucidated.

Increased level of urinary nitric oxide metabolites in leprosy patients during type 2 reactions and decreased after antireactional therapy.

OBJECTIVES: To assess the urinary nitric oxide metabolites in lepromatous patients in ENL (type 2 reactions) and to compare these metabolites after subsidence of reactions following antireactional therapy. Further to compare the levels in a group of lepromatous leprosy patients without reactions. DESIGN: The initial urine samples were collected from lepromatous leprosy patients when they came with ENL before commencing antireactional therapy and repeat samples were taken after resolution of ENL. Morning urine samples were collected from LL patients without reactions. Nitrites and nitrates in urine were measured using commercially available kit. Mean levels of nitric oxide metabolites of LL patients with ENL and without ENL were compared by student's 't' test. The level during ENL and after resolution was compared by paired 't' test. RESULTS: The nitric oxide metabolites were analyzed in 14 LL patients with ENL and after resolution of ENL and in 5 LL patients without reaction. The level of urinary nitric oxide metabolite is higher in LL patients in ENL reaction compared to LL patients without reaction (P < 0.04). These levels were reduced significantly with resolution of reaction following antireactional therapy (P < 0.004). CONCLUSION: The findings of this study suggested that the NO/NOM excretion is increased in leprosy patients during ENL episodes. With antireactional therapy (steroids) and clinical improvement the levels are reduced.

Leprosy reactions: humoral and cellular immune responses to M. leprae, 65kDa, 28kDa, and 18 kDa antigens.

This study examines the immune responses against some stress proteins of Mycobacterium leprae in leprosy patients with and without leprosy reactions. Leprosy patients showed a higher level of antibodies to all antigens compared to healthy controls. The antibody response to 18kDa antigen was significantly higher in patients with Type 1 reaction compared to those of TT or borderline patients without Type 1 reaction, or those with Type 2 reaction. Borderline (BT/BL), lepromatous (LL) and patients with reactions (Type 1 and Type 2) had higher levels of antibodies to M. leprae soluble extract (MLSE) and 65kDa than those of the tuberculoid (TT) group. LL, borderline patients, and patients with Type 1 reaction had a higher level of antibody to 28kDa than those of healthy controls. However, no significant differences could be observed in antibody response to these antigens (MLSE, 65kDa, and 28kDa) between patients with reaction and without reaction. A significant proportion of TT/BT patients showed positive lymphoproliferative response to MLSE compared to BL/LL patients. In addition, the lymphoproliferative response to MLSE was significantly greater in patients with Type 1 reaction compared to patients without reaction. No difference in proliferative response to 65kDa could be observed in any of these groups. The finding of high levels of antibodies against stress proteins in patients with Type 1 reactions, especially to 18 kDa antigen, along with a heightened lymphoproliferative response to MLSE is suggestive of a coexistence of cell mediated and humoral immunity in leprosy patients during Type 1 reactions. On the other hand, in Type 2 reactions no significant role of stress proteins could be demonstrated except a heightened lymphoproliferative response to the 28 kDa antigen.

Immunological profile of treated lepromatous leprosy patients

The immune responses of 19 treated lepromatous patients who had remained smear negative for a long period were assessed for specific cell-mediated immunity (CMI), anti-Mycobacterium leprae antibodies and cytokine release in response to challenge with M. leprae soluble antigen (MLSA). All of these patients remained anergic to Mitsuda lepromin. Lymphoproliferation in response to M. leprae antigen was noted in only two patients. Significant reduction in the phenolic glycolipid I (PGL-I) antibody response in treated patients with no difference in the M. leprae 35-kDa antibody response was observed when these responses were compared with those of active lepromatous patients. More treated patients produced interleukin-2 (IL-2) and interferon gamma (IFN-gamma) than did active patients. On the other hand, fewer treated patients produced IL-10 than did active patients. These limited findings suggest that the host immune response makes an attempt toward upregulation of CMI in some treated LL/BL patients.

Determination of circulating IgG subclasses against lipoarabinomannan in the leprosy spectrum and reactions

IgG subclasses against lipoarabinomannan of mycobacteria were analyzed in the sera of leprosy patients. Patients with active leprosy [tuberculoid and lepromatous, patients undergoing erythema nodosum leprosum (ENL) and reversal reactions] and inactive cases (tuberculoid and lepromatous who were cured after chemotherapy) were included in this study. Active lepromatous patients had higher levels of IgG subclasses, except IgG4, compared to active tuberculoid patients. Some of the inactive cases (lepromatous patients cured after chemotherapy) were positive for the IgG1, IgG2 and IgG3 subclasses. However, their levels are lower than active lepromatous cases. On the other hand, no difference in the subclass levels between the active and inactive tuberculoid groups could be observed. While a significant fall in the level of IgG3 in ENL was observed as compared to lepromatous leprosy without ENL, higher levels of IgG1 and IgG2 were found in patients with reversal reactions compared to their active counterparts without reactions.