Association of CC-chemokine ligand-2 gene polymorphisms with leprosy reactions.

BACKGROUND: C-C motif chemokine ligand 2, a gene that codes for a protein involved in inflammation. Certain SNPs in the CCL2 gene have been studied for their potential associations with susceptibility to various diseases. These SNPs may affect the production and function of the CCL2 protein, which is involved in the recruitment of immune cells to the site of inflammation. Variations in CCL2 may influence the immune response to Mycobacterium leprae infection. OBJECTIVE: To investigate the association of the C-C motif chemokine ligand-2 single nucleotide polymorphisms with leprosy. METHODS: CCL2 single nucleotide polymorphisms were analyzed in a total of 975 leprosy patients and 357 healthy controls. Of those, 577 leprosy and 288 healthy controls were analyzed by PCR-RFLP for CCL2 -2518 A>G, 535 leprosy and 290 controls for CCL2 -362 G>C, 295 leprosy and 240 controls for CCL2 -2134 T>G, 325 leprosy and 288 controls for CCL2 -1549 A>T SNPs by melting curve analysis using hybridization probe chemistry and detection by fluorescence resonance energy transfer (FRET) technique in Realtime PCR. The levels of CCL2, IL-12p70, IFN-γ, TNF-α, and TGF-ß were estimated in sera samples and correlated with CCL2 genotypes. RESULTS: The frequency of the GCT (-2518 A>G, -362 G>C, -2134 T>G) haplotype is observed to be higher in leprosy patients compared to healthy controls (P = 0.04). There was no significant difference observed in genotypic frequencies between leprosy patients and healthy controls {(-2518A>G, p = 0.53), (-362 G>C, p = 0.01), (-2134 T>G, p = 0.10)}. G allele at the -2134 site is predominant in leprosy (borderline) without any reaction (8 %) compared to borderline patients with RR reactions (2.1 %) (P = 0.03). GG genotype (p = 0.008) and G allele at -2518 (p = 0.030) of the CCL 2 gene were found to be associated with patients with ENL reaction. An elevated level of serum CCL2 was observed in leprosy patients with the -2518 AA and AG genotypes (p = 0.0001). CONCLUSIONS: G allele and GG genotype at the CCL2 -2518 site are associated with a risk of ENL reactions.

Notch signaling induces lymphoproliferation, T helper cell activation and Th1/Th2 differentiation in leprosy.

The present study evaluates role of Notch1 signaling in the regulation of T cell immunity in leprosy. Peripheral blood mononuclear cells from leprosy patients and healthy controls were activated with Mycobacterium leprae antigens along with activation of Notch1 signaling pathway and then lymphoproliferation was analyzed by lymphocytes transformation test and the expression of Notch1 and its ligands DLL1, Jagged1 and Jagged 2, T cell activation marker and Th1-Th2 cytokines on Th cells in PBMCs of study subjects were analyzed by flow cytometry. Further, these parameters were also analyzed after inhibition of Notch1 signaling pathway. Higher percentage of Notch1expressing Th cells were noted in TT/BT cases and higher percentage of DLL1 expressing Th cells in TT/BT and BL/LL cases. M. leprae antigens were found to induce the expression of Jagged1 on Th cells. Interestingly activation of Notch1 signaling pathway induced lymphoproliferation in BL/LL cases in response of PGL-1. Activation of Notch1 signaling was also found to induce the expression of T cell activation markers CD25, CD69 and Th1 cytokine IFN-γ in response to M. leprae antigens. Immunomodulation through Notch1 signaling seen in our study could be helpful in augmenting Th1 response in leprosy.

Transcription factors STAT-4, STAT-6 and CREB regulate Th1/Th2 response in leprosy patients: effect of M. leprae antigens.

BACKGROUND: Leprosy is an ideal human disease to study T cell regulation as patients show correlation between cytokine skewed Th1-Th2 responses and clinical forms of the disease. The Role of transcription factors on the modulation of Th1 and Th2 responses by M. leprae antigens has not been adequately studied. In the present study, we studied the effect of M. leprae antigens on transcription factors STAT-4, STAT-6 and CREB and their correlation with Th1/Th2 cell mediated immune responses in leprosy. METHODS: Leprosy patients of both categories of tuberculoid leprosy (BT/TT) and lepromatous leprosy (BL/LL) were selected from the OPD of NJ1L & OMD, (ICMR), Agra and healthy individuals (H) were chosen from the staff and students working in the institute. Peripheral blood mononuclear cells (PBMCs) of the study subjects were stimulated with M. leprae antigens (WCL, MLSA, and PGL-1). Sandwich ELISA was done in the culture supernatants of healthy and leprosy patients to detect IL-4, IL-10 and IFN-γ. Further, expression of IFN-γ and IL-4 and activation of STAT4, STAT6 and CREB transcription factors in CD4+ T cell with or without stimulation of M. leprae antigens was investigated by flow cytometry. RESULTS: Lepromatous leprosy patients showed significantly lower IFN-γ and higher IL-4 levels in culture supernatant and significantly low expression of IFN-γ and higher expression of IL-4 by CD4+ T cells than healthy individuals with or without antigenic stimulation. Antigenic stimulation significantly increased IL-10 in BL/LL patients but not in BT/TT patients or healthy individuals. PGL-1 stimulation led to significantly higher activation of STAT-6 in BT/TT and BL/LL patients in comparison to healthy individuals. All the three antigens led to activation of CREB in healthy and BT/TT patients but not in BL/LL patients. CONCLUSION: Our findings show that M. leprae antigens differentially modulate activation of T cell transcription factors STAT-4/STAT-6 and CREB. These transcription factors are well known to regulate Th1 and Th2 mediated immune response which in turn could play vital role in the clinical manifestations of leprosy. These observations may help to determine how these T cell transcription factors affect the development of immune dysfunction and whether these new pathways have a role in immunomodulation in intracellular diseases like leprosy and TB.

Asociación de Mycobacterium leprae viable con leprorreacciones / No disponible

La hipótesis de trabajo es que el Mycobacterium leprae (M. leprae) viable desempeña un papel crucial en la precipitación de la reacción tipo 1 (T1R) en lepra. Materiales y métodos: Se estudiaron un total de 165 nuevos pacientes multibacilares. Para demostrar la presencia de M. leprae se emplearon tres técnicas en las lesiones en reacción T1R (+), como son el crecimiento en la almohadilla plantar (MFP), detección inmunohistoquímica de la proteína secretora M. leprae Ag85, y 16s rRNA - empleando RT-PCR in situ. Como controles, se emplearon biopsias de BEC y lesiones sin reacción T1R (-). Hallazgos: Un número significativamente mayor de homogeneizados de biopsia de lesión obtenidos al inicio, de casos T1R (+) han presentado crecimiento en MFP, demostrando la presencia de bacterias viables comparado con T1R (-) (P = 0·005). En contraste, pocas biopsias BEC resultaron positivas tanto en T1R (+) como en T1R (-). Con respecto a Ag85, mientras que la positividad total fue más elevada en T1R (+) (74%), la positividad de la tinción (Grado ≥ 2+) era significativamente mayor en el grupo T1R (+) BT-BB 11/20 (55%). En las muestras de biopsias en casos de T1R recurrentes, tanto el Ag 85 como el 16s rRNA, la positividad (62% y 100%) fue mayor en el grupo T1R (+). Se propone que las bacterias viables son componentes esenciales en las T1R y la diferencia en la calidad de los bacilos y no la cantidad o la ratio de inactivos/viables desempeña un papel en la precipitación de T1R. En conclusión, los hallazgos demuestran que los bacilos «metabólicamente activos» de M. leprae son un componente/requisito fundamental de la T1R y la proteína secretora Ag 85, quizás sea el precipitador de T1R (AU)

The working hypothesis is that, viable Mycobacterium leprae (M. leprae) play a crucial role in the precipitation of Type 1 reaction (T1R) in leprosy. Material and Methods: A total of 165 new multibacillary patients were studied. To demonstrate presence of viable M. leprae in reactional lesion (T1R+), three tests were used concurrently viz. growth in the mouse foot pad (MFP), immunohistochemical detection of M. leprae secretory protein Ag85, and 16s rRNA - using in situ RT- PCR. Mirror biopsies and non reactional lesions served as controls T1R (-). Findings: A significantly higher proportion of lesion biopsy homogenates obtained at onset, from T1R (+) cases have shown unequivocal growth in MFP, proving the presence of viable bacteria, as compared to T1R (-) (P , 0·005). In contrast, few Mirror biopsies were positive in both T1R (+) and T1R (-). With respect to Ag85, while the overall positivity was higher in T1R (+) (74%), however the intensity of staining (Grade ≥ 2+) was disproportionately higher in T1R (+) BT-BB lesions 11/20 (55%). In the rebiopsies obtained during a repeat episode of T1R, Ag 85 as well as 16s rRNA, positivity (62% & 100%) was higher in T1R (+). It is inferred therefore «viable» bacteria are an essential component in T1R and difference in the quality of bacilli, not the quantity or the ratio of dead to viable play a role in the precipitation of T1R. In conclusion, the findings show that «metabolically active» M. leprae is a component/prerequisite and the secretory protein Ag 85, might be the trigger for precipitation of T1R (AU)

Association of viable Mycobacterium leprae with Type 1 reaction in leprosy.

UNLABELLED: The working hypothesis is that, viable Mycobacterium leprae (M. leprae) play a crucial role in the precipitation of Type 1 reaction (T1R) in leprosy. MATERIAL AND METHODS: A total of 165 new multibacillary patients were studied. To demonstrate presence of viable M. leprae in reactional lesion (T1R+), three tests were used concurrently viz. growth in the mouse foot pad (MFP), immunohistochemical detection of M. leprae secretory protein Ag85, and 16s rRNA--using in situ RT-PCR. Mirror biopsies and non reactional lesions served as controls (T1R-). FINDINGS: A significantly higher proportion of lesion biopsy homogenates obtained at onset, from T1R(+) cases have shown unequivocal growth in MFP, proving the presence of viable bacteria, as compared to T1R(-) (P < 0.005). In contrast, few Mirror biopsies were positive in both T1R(+) and T1R(-). With respect to Ag85, while the overall positivity was higher in T1R(+) (74%), however the intensity of staining (Grade 2+) was disproportionately higher in T1R(+) BT-BB lesions 11/20 (55%). In the rebiopsies obtained during a repeat episode of T1R, Ag 85 as well as 16s rRNA, positivity (62% & 100%) was higher in T1R(+). It is inferred therefore 'viable' bacteria are an essential component in T1R and difference in the quality of bacilli, not the quantity or the ratio of dead to viable play a role in the precipitation of T1R. In conclusion, the findings show that 'metabolically active' M. leprae is a component/prerequisite and the secretory protein Ag 85, might be the trigger for precipitation of T1R.

High resolution structural changes of Schwann cell and endothelial cells in peripheral nerves across leprosy spectrum.

A systematic ultrastructure of peripheral nerves across the spectrum of leprosy was studied with an aim to better understanding the pathogenesis of nerve involvement in leprosy using light and electron microscope. The pathogenesis of nerve destruction varies in leprosy considerably along the spectrum. The study has begun to shed new light on some aspects of the infection of Mycobacterium leprae (M. lepare) and phenomenon has opened new avenue of research and possible mechanism of pathogenesis in TT/BT/BL/LL leprosy. In tuberculoid type (TT) and borderline tuberculoid (BT) leprosy, the degenerative changes of Schwann cells (SCs) and presence of perineural and perivascular cuffing by mononuclear cells. The endoneurial blood vessel (EBV) showed thickening of basement membrane with hypertrophy of EC leading to narrowing or complete occlusion of lumen and causing ischemia. However, borderline lepromatous (BL) and lepromatous leprosy (LL) foamy macrophages and vacuolated SC contain numerous small dense materials, irregular in shape and size was prominent and, considered to be degenerated and fragmented M. Leprae. The dense materials were also found in the cytoplasm of vascular EC. It was revealed that besides SC, the EC of EBV frequently harbor M. leprae in LL. The lumen of the EBV was wide open with enlarged nucleus. In the present study, the ultrastructural characteristics suggest that hypersensitivity mechanisms are possibly responsible for nerve damage in TT/BT leprosy. However, the study indicates that the mechanisms of nerve damage in BL/LL are basically different wherein hypersensitivity appears to play a very limited role.

Mycobacterium indicus pranii as stand-alone or adjunct immunotherapeutic in treatment of experimental animal tuberculosis.

BACKGROUND & OBJECTIVES: Mycobacterium w (M.w) is a saprophytic cultivable mycobacterium and shares several antigens with M. tuberculosis. It has shown good immunomodulation in leprosy patients. Hence in the present study, the efficacy of M.w immunotherapy, alone or in combination with multi drug chemotherapeutic regimens was investigated against drug sensitive M. tuberculosis H37Rv and three clinical isolates with variable degree of drug resistance in mice. METHODS: BALB/c mice were infected with M. tuberculosis H37Rv (susceptible to all first and second line drugs) and three clinical isolates taken from the epository of the Institute. The dose of 200 bacilli was used for infection via respiratory route in an aerosol chamber. Chemotherapy (5 days/wk) was given one month after infection and the vaccinated group was given a dose of 1x107 bacilli by subcutaneous route. Bacterial load was measured at 4 and 6 wk after initiation of chemotherapy. RESULTS: M.w when given along with chemotherapy (4 and 6 wk) led to a greater reduction in the bacterial load in lungs and other organs of TB infected animals compared to. However, the reduction was significantly (P<0.05) more in terms of colony forming units (cfu) in both organs (lungs and spleen). CONCLUSION: M.w (as immunomodulator) has beneficial therapeutic effect as an adjunct to chemotherapy.

In situ hybridization in the histological diagnosis of early and clinically suspect leprosy.

The present study tests the utility of the in situ hybridization procedure for M. leprae rRNA in the histological diagnosis of early leprosy and clinically suspect leprosy, both diagnostically demanding situations. The histological confirmation obtained with routine histopathology (Haematoxylin-Eosin staining for studying morphologic alterations and Fite-Faraco staining for demonstration of acid-fast bacilli) were 32% for early leprosy and 25% for clinically suspect leprosy. With performance of the in situ hybridization on the histologically unconfirmed cases, the positivity rates obtained were 58.8% and 55%, respectively. The results of the study confirm the utility of the procedure in the diagnostically difficult situations of early and suspect leprosy, and it is proposed that the procedure be employed in situations of clinical doubt.

10-12 years follow-up of highly bacillated BL/LL leprosy patients on combined chemotherapy and immunotherapy.

This study reports the follow-up results of 36 highly bacillated untreated BL/LL cases who were serially allocated to three treatment groups. Group I patients received a modified WHO regimen (Rifampicin 600 mg once a month supervised, 50 mg of Clofazimine and 100 mg of Dapsone daily unsupervised) and BCG 0.1 mg per dose 6 monthly; group II patients received the same multi-drug treatment (MDT) and Mw (2 x 10(8) killed bacilli per dose) 6 monthly: group III patients received the same MDT with 0.1 ml of distilled water 6 monthly and acted as a control. Treatment was continued till smear negativity. All these three groups were comparable by their initial clinical score, bacteriological index (BI), viable bacilli as assessed by the mouse foot pad (MFP), bacillary adenosine triphosphate (ATP) content and also histologically at the time of starting treatment. All these parameters were evaluated every 6 months. The vaccines were well tolerated. All the patients in group I became smear negative by 3.5 years, in group II in 3 years whereas those in group III took 5 years. The incidence of reactions was the same in all the groups during the first 2 years, however, patients of group III (MDT + placebo) continued to have reactions up to 3 years. No viable bacilli could be detected in the local and distal sites as estimated by MFP and bacillary ATP after 12 months in both the immunotherapy groups. These could be detected in patients on MDT alone up to 24 months of therapy. Histologically patients in both the immunotherapy groups (groups I and II) showed accelerated granuloma clearance, histological upgrading and non-specific healing without granuloma formation both at the local and distal sites and this was achieved much earlier compared to the MDT + placebo group. Thus, by the addition of immunotherapy the effective treatment period of achieving bacteriological negativity could be reduced by about 40%, time period of reactions reduced by 33% and there were no reactions and/or relapses in the 10-12 years post-treatment follow-up.

Relapses in paucibacillary patients after treatment with three short term regimens containing rifampin

Three multidrug regimens all containing rifampin and dapsone have been tried for the treatment of 278 cases of paucibacillary leprosy. Regimen I was the one recommended by the WHO Study Group. Regimen II was the same as Regimen I with depsone alone continued for a further 6 months. Regimen III was the same as Regimen II but rifampin was given daily for the first 7 days. The patients were comparable with regard to disease classification, lepromin status, bacteriological status, and number of lesions. As reported earlier, the disease inactivity rates by 1 year of treatment were much greater with Regimens II and III than with Regimen I (94% and 97% vs 76%). Early reaction was seen in 6% of those in Regimen III and in none in Regimens I and II. Late reaction was observed in 9% of those in Regimen I and none in Regimens II and III. During 3 1/2 years of follow up, 13% of the cases in Regimen I, 1% in Regimen II, and 2% in Regimen III relapsed. Since the patients in the three regimens were otherwise comparable, it is concluded that the high inactivity rate, low relapse rate (1%-2%), and no early or late reaction as observed in Regimen II patients were because of adequate treatment