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.

Immunological depiction of synthetic B-cell epitopes of Mycobacterium tuberculosis.

Background: To combat the tuberculosis (TB) epidemic, the development of a better and faster diagnosis or more effective vaccine is essential. Pulmonary TB (PTB) is one of the major causes of morbidity and mortality. Early diagnosis of TB is difficult. Serological assays have been performed with several antigens of laboratory strains such as Mycobacterium tuberculosis H37Rv which have not been found to be highly sensitive. In the present study, various peptides were synthesized which were predicted on the basis of immunoreactivity and differential expression in clinical isolates of M. tuberculosis compared to their expression in a laboratory strain of M. tuberculosis. Therefore, the aim of this study was to compare the antibody levels in PTB and healthy controls against these peptides. Methods: An effort was made to evaluate antibody response to peptides derived from proteins Rv2588c, Rv0512, Rv0148, Rv0896, and Rv0635 of M. tuberculosis in PTB patients and healthy individuals through enzyme-linked immunosorbent assay. Five milliliters of venous blood samples was collected from each participant, and serum was separated and stored until use. Results: Antibody levels against these peptides, Rv2588c, Rv0512, Rv0148, Rv0896. and Rv0635 in 139 PTB patients and 52 healthy controls were evaluated. Higher immune response was observed in PTB patients when compared with healthy individuals. Strong immunoglobulin G responses with high percentage, considerable difference among patients and healthy controls was observed with P < 0.0001. Conclusion: In our study, we found significant statistical differences in antibody levels in PTB patients and healthy individuals against these peptides. These peptides are suggestive of being a potential new candidate (s) for early diagnosis of TB.

SWATH-MS analysis of plasma proteins among Indian HIV-1 infected patients.

The identification and characterization of plasma proteins in drug resistant and drug sensitive in HIV-1 infected/AIDS patients were carried out using the SWATH-MS protocol. In total, 204 proteins were identified and quantified, 57 proteins were differentially expressed, out of which 25 proteins were down regulated and 32 proteins were up regulated in drug resistant patients. Six proteins such as complement C4-A, immunoglobulin heavy variable 1-2, carboxylic ester hydrolase, fibulin-1, immunoglobulin lambda constant7, secreted phosphoprotein 24 were differentially expressed in individuals with drug resistant HIV as compared to individuals with drug sensitive HIV. Gene ontology of 57 differentially expressed proteins was analysed and documented.

Toll-like receptor 2 (-196 to -174) del and TLR1 743 A > G gene polymorphism-a possible association with drug-resistant tuberculosis in the north Indian population.

Objectives: The objective of this study is to analyze the association between TLR2 deletion (-196 to -174) and TLR1 743 A > G gene polymorphism with drug resistant tuberculosis (PTB, MDR-TB, and XDR-TB) in a population from Agra, Uttar Pradesh. Methods: The present case-control study included 101 pulmonary TB patients, 104 multidrug-resistant TB patients, 48 extremely drug-resistant TB patients, and 130 healthy and unrelated controls residing in the same locality. The genotyping method for TLR2 deletion (-196 to -174) was carried out by allele-specific polymerase chain reaction (PCR), and TLR1 743 A > G gene polymorphism was performed by hybridization probe chemistry in Roche Real-Time PCR. Genotype and allele frequencies were analyzed by the chi-square test. Cytokine levels were measured by ELISA and compared using Mann-Whitney and Kruskal-Wallis tests. Results: The frequency of heterozygous (Ins/del) genotypes for TLR2 (-196 to -174) polymorphism was predominant in XDR-TB patients (0.57), whereas heterozygous A/G genotype for TLR1 743 A > G single nucleotide polymorphism (SNP) was predominant in healthy controls (0.57) for TLR1 743 A > G gene polymorphism. The heterozygous genotype of TLR2 deletion polymorphism was found to be significantly higher in XDR-TB (p = 0.0001). TLR1 743 A > G SNP, AG genotypes were found to be significantly associated with healthy controls than PTB (p = 0.047). The level of serum cytokines (IL-6, TNF-α, and IFN-γ) was also found to be significantly different among TB patients and healthy controls. Conclusion: The findings suggested that in the present population, the heterozygous (Ins/Del) genotype and deletion allele of TLR2 deletion (-196 to -174) polymorphism are associated with the risk for the development of drug-resistant TB. Furthermore, for TLR1 743 A > G gene polymorphism, A/G genotype, and G allele are found associated with healthy controls, suggesting the protective role against TB.

Systematic review and meta-analysis of human Toll-like receptors genetic polymorphisms for susceptibility to tuberculosis infection.

Epidemiological data from the world health organization (WHO) show that Globally an estimated 10 million (range, 8.9-11.0 million) people around the world were infected with TB in 2019. M.tuberculosis (M.tb) is the major cause of tuberculosis. Infection with M.tb has varied host immune responses because of the host genetic factor and its response to the infection. Genetic polymorphism in TLRs imparts susceptibility or resistance to the host against several diseases. In the present study, a systematic review and meta-analysis were performed to describe the relationship among various TLRs and SNPs involved in M.tb infection and their association with susceptibility to pulmonary tuberculosis in various populations of the world. PubMed and Scihub databases from 2008 to 2019 were searched and 58 articles were shortlisted for the present study to explore the association between TLRs gene polymorphisms and susceptibility to tuberculosis infection. The combined analysis showed that the polymorphisms TLR1 (rs5743618), TLR1 (rs4833095), TLR2 (-196 to -174) del, TLR2 (rs3804099), TLR4 (rs4986790), TLR4 (rs4986791), TLR4 (rs7873784), TLR6 (rs5743810), TLR8 (rs3764880), TLR9 (rs5743836), TLR9 (rs352139) were significantly associated with TB disease in certain ethnic population. In our meta-analysis study, we have also found variations between studies in some polymorphism, for example. The TLR1 (rs 5743618), TLR2 (rs5743708), TLR4 Asp299Gly, TLR4 Thr399Ile, TLR4 (rs7873784), TLR6 (rs5743810), TLR9 (rs5743836) was associated with the protection against TB. Meta-analysis was performed between polymorphisms and pulmonary tuberculosis to define increase or decrease in susceptibility to tuberculosis in various populations, which indicated that a relationship exists between SNPs/host genetic factors and susceptibility or resistance in patients suffering from pulmonary tuberculosis our finding concludes that this gene polymorphism may be associated with susceptibility to TB. The present study adds value to the various researches and studies going on various populations of the world in better understanding the role of TLR polymorphism in TB.

Genomic signatures of protease and reverse transcriptase genes from HIV-1 subtype C isolated from first-line ART patients in India.

Genomic signatures of the protease and reverse transcriptase gene of HIV-1 from HIV infected North Indian patients who were under ART from 1 to ≤ 7 years were analyzed. The DNA from plasma samples of 9 patients and RNA from 57 patients were isolated and subjected to amplification for the protease and reverse transcriptase gene of HIV-1 subtype C. Then sequencing was carried out following the WHO dried blood spot protocol. The drug resistance mutation patterns were analyzed using the HIV Drug Resistance Database, Stanford University, USA. Lamivudine-associated drug-resistance mutations such as M184V/M184I, nevirapine-associated drug resistance mutations Y181C and H221Y, and efavirenz-associated drug resistance mutations M230I were observed in reverse transcriptase gene of archived DNA of two HIV-1 infected patients. No mutation was observed in the remaining 7 patients. Various computational tools and websites like viral epidemiological signature pattern analysis (VESPA), hyper mutation, SNAP version 2.1.1, and entropy were utilized for the analysis of the signature pattern of amino acids, hyper mutation, selection pressure, and Shannon entropy in the protease and reverse transcriptase gene sequences of the 9 archived DNA, 56 protease gene and 51 reverse transcriptase gene from the HIV-1 DNA amplified sequences of RNA. The HIV-1 Subtype-C (Gene bank accession number: AB023804) and first isolate HXB2 (Gene bank accession number: K03455.1) was taken as reference sequence. The signature amino acid sequences were identified in the protease and reverse transcriptase gene, no hyper mutation, highest entropy was marked in the amino acid positions and synonymous to non-synonymous nucleotide ratio was calculated in the protease and reverse transcriptase gene of 9 archived DNA sequences, 56 protease and 51 reverse transcriptase gene sequences of HIV-1 Subtype C isolates.

Detection of Drug Resistance Mutations in the Reverse Transcriptase Gene of HIV-1-Infected North Indian Population Failing First-Line Antiretroviral Therapy "A Follow-Up Cohort Study".

We aim to characterize the drug resistance mutations in reverse transcriptase gene of HIV-1 subtype C-infected North Indian population in those who are failing first-line antiretroviral therapy (ART) and if these mutations are associated with mortality. We also attempted the assessment of switch over to second-line antiretroviral therapy in these patients. Based on the immunological marker CD4 count (<350 cubic/mm), 192 HIV/AIDS patients were selected and viral load was estimated in those who were enrolled from December 2009 to November 2016. Based on viral load, genotyping was carried out in 57 HIV-1 isolates (VL ≥1,000 copies/mL) by sequencing and drug resistance mutations were examined through the Stanford HIV Drug Resistance Database, USA. Among them, 21 (36.84%) first-line ART failure patients were shifted to second-line ART. These patients were followed for a period wide ranging from 10 months to 11 years. Drug resistance mutation M184V (ATG to GTA) (63.15%) associated with lamivudine and abacavir and K103N (AAG or AAA to AAU) (36.84%) associated with efavirenz and nevirapine were predominantly identified in first-line ART failure patients. During follow-up, it was observed that 3 out of 21 who were in second-line ART died, whereas 9 out of 36 died who were in the first-line ART. No mutation could be associated with mortality although TAM-2 mutations were absent in patients who died. This study indorses the need for a facility for viral load estimation and resistance monitoring in each treatment failure patient and availability of appropriate antiretroviral therapies.

Identification and differential expression of serotransferrin and apolipoprotein A-I in the plasma of HIV-1 patients treated with first-line antiretroviral therapy.

BACKGROUND: Plasma proteins are known to interfere the drug metabolism during therapy. As limited information is available regarding the role of plasma proteins in HIV drug resistance during ART in HIV/AIDS patients, the present study aimed to identify and characterize the differentially expressed plasma proteins in the drug resistant and drug respondent groups of HIV-1 infected patients with > 6 years of first line ART. METHODS: Four-drug resistant (treatment failure) and four-drug respondent (treatment responder) patients were selected for plasma proteomic analysis based on viral load and drug resistance associated mutations from a cohort study designed on the first line ART patients who were enrolled in the antiretroviral therapy center, Sarojini Naidu Medical College, Agra, India from December 2009 to November 2016. After depleting high abundant proteins, plasma proteins were resolved using two-dimensional gel electrophoresis on IPG strips, pH range of 3-10. Spots were selected in the gel based on the density of staining which was common in the drug resistant and drug respondent groups separately. The fold change of each spot was calculated using image-J. Each protein spot was identified using the matrix assisted laser desorption/ionization-time of flight/time of flight (MALDI-TOF/TOF) after tryptic digestion. Peptide peaks were identified through flex analysis version 3.3, and a search against a protein data base using the internal Mascot. Gene ontology study was completed through STRING v.11 and Panther15.0. RESULTS: Out of eight spots from 2D gel samples analyzed by MALDITOF/TOF, two proteins were found to have significant score (> 56) after Flex analysis. These two proteins were identified to be apolipoprotein A1 and serotransferrin. The fold change expression of these two proteins were analyzed in drug resistant and drug respondent group. Apolipoprotein-A1 and serotransferrin were observed to be expressed 1.76 and 1.13-fold more respectively in drug respondent group compared to drug resistant group. The gene ontology analysis revealed the involvement of these two proteins in various important physiological processes. CONCLUSION: Apolipoprotein A-I and serotransferrin were found to be expressed more in drug respondent group compared to drug resistant group.

Autoimmunity to Tropomyosin-Specific Peptides Induced by Mycobacterium leprae in Leprosy Patients: Identification of Mimicking Proteins.

Background: It has been shown earlier that there is a rise in the levels of autoantibodies and T cell response to cytoskeletal proteins in leprosy. Our group recently demonstrated a rise in both T and B cell responses to keratin and myelin basic protein in all types of leprosy patients and their associations in type 1 reaction (T1R) group of leprosy. Objectives: In this study, we investigated the association of levels of autoantibodies and lymphoproliferation against myosin in leprosy patients across the spectrum and tried to find out the mimicking proteins or epitopes between host protein and protein/s of Mycobacterium leprae. Methodology: One hundred and sixty-nine leprosy patients and 55 healthy controls (HC) were enrolled in the present study. Levels of anti-myosin antibodies and T-cell responses against myosin were measured by ELISA and lymphoproliferation assay, respectively. Using 2-D gel electrophoresis, western blot and MALDI-TOF/TOF antibody-reactive spots were identified. Three-dimensional structure of mimicking proteins was modeled by online server. B cell epitopes of the proteins were predicted by BCPREDS server 1.0 followed by identification of mimicking epitopes. Mice of inbred BALB/c strain were hyperimmunized with M. leprae soluble antigen (MLSA) and splenocytes and lymph node cells of these animals were adoptively transferred to naïve mice. Results: Highest level of anti-myosin antibodies was noted in sera of T1R leprosy patients. We observed significantly higher levels of lymphoproliferative response (p < 0.05) with myosin in all types of leprosy patients compared to HC. Further, hyperimmunization of inbred BALB/c strain of female mice and rabbit with MLSA revealed that both hyperimmunized rabbit and mice evoked heightened levels of antibodies against myosin and this autoimmune response could be adoptively transferred from hyperimmunized to naïve mice. Tropomyosin was found to be mimicking with ATP-dependent Clp protease ATP-binding subunit of M. leprae. We found four mimicking epitopes between these sequences. Conclusion: These data suggest that these mimicking proteins tropomyosin and ATP-dependent Clp protease ATP-binding subunit of M. leprae or more precisely mimicking epitopes (four B cell epitopes) might be responsible for extensive tissue damage during type1 reaction in leprosy.

Association of Toll like receptor 2 and 9 gene variants with pulmonary tuberculosis: exploration in a northern Indian population.

Tuberculosis (TB) is a disease of global importance. There is an increasing recognition of the role of Toll like receptors, important pattern recognition receptors of host immune system, in determining the susceptibility or resistance to TB in various populations. In an attempt to examine the importance of Toll like receptors in immune response to Mycobacterium tuberculosis infection, we explored two variants each of TLR2 and TLR9 in a population residing in Uttar Pradesh, India. Genotyping was performed to detect -196 to -174 del polymorphism and G2258A SNP (Arg753Gln, rs5743708) in TLR2 gene and -T1237C (rs5743836) and G2848A (rs352140) SNP in TLR9 gene in patients with pulmonary TB and healthy controls. The A allele of G2848A SNP in TLR9 gene was found with a marginally higher frequency among TB patients as compared to healthy controls, suggesting that A allele at position 2848 of TLR9 gene may be associated with susceptibility to TB in North Indian population [p = 0.05, Mantel-Haenszel OR = 1.34, 95% CI (1.0-1.82)].

Association of Nitric Oxide Synthase2 gene polymorphisms with leprosy reactions in northern Indian population.

The pathogen Mycobacterium leprae causes leprosy that affects mainly skin and nerves. Polymorphisms of certain genes are substantiated to be associated with the susceptibility/resistance to leprosy. The present investigation addressed the association of Nitric Oxide Synthase2 gene polymorphisms and leprosy in a population from northern part of India. A total of 323 leprosy cases and 288 healthy controls were genotyped for four NOS2 promoter variants (rs1800482, rs2779249, rs8078340 and rs2301369) using FRET technology in Real Time PCR. None of these SNPs in promoter sites was associated with susceptibility/resistance to leprosy. NOS2 rs1800482 was found to be monomorphic with GG genotype. However, NOS2-1026T allele was observed to be in higher frequency with leprosy cases (BL and LL) who were not suffering from any reactional episodes compared to cases with ENL reaction {OR=0.30, 95% CI (0.10-0.86), p=0.024}. NOS2-1026GT genotype was more prevalent in cases without reaction (BT, BB and BL) compared to RR reactional patients {OR=0.38, 95% CI (0.17-0.86), p=0.02}. Although haplotype analysis revealed that no haplotype was associated with leprosy susceptibility/resistance with statistical significance, GTG haplotype was noted to be more frequent in healthy controls. These SNPs are observed to be in linkage disequilibrium. Although, these SNPs are not likely to influence leprosy vulnerability, -1026G>T SNP was indicated to have noteworthy role in leprosy reactions.

Molecular mimicry between Mycobacterium leprae proteins (50S ribosomal protein L2 and Lysyl-tRNA synthetase) and myelin basic protein: a possible mechanism of nerve damage in leprosy.

Autoantibodies against various components of host are known to occur in leprosy. Nerve damage is the primary cause of disability associated with leprosy. The aim of this study was to detect the level of autoantibodies and lympho-proliferative response against myelin basic protein (MBP) in leprosy patients (LPs) and their correlation with clinical phenotypes of LPs. Further, probable role of molecular mimicry in nerve damage of LPs was investigated. We observed significantly high level of anti-MBP antibodies in LPs across the spectrum and a positive significant correlation between the level of anti-MBP antibodies and the number of nerves involved in LPs. We report here that 4 B cell epitopes of myelin A1 and Mycobacterium leprae proteins, 50S ribosomal L2 and lysyl tRNA synthetase are cross-reactive. Further, M. leprae sonicated antigen hyperimmunization was responsible for induction of autoantibody response in mice which could be adoptively transferred to naive mice. For the first time our findings suggest the role of molecular mimicry in nerve damage in leprosy.

Toll-like Receptor 1 743 A>G, 1805 T>G & Toll-like Receptor 6 745 C>T gene polymorphism and tuberculosis: a case control study of north Indian population from Agra (India).

Infection with Mycobacteriumtuberculosis possibly depends on host genetic factors and is thought to be the major cause of differential susceptibility to the disease. In the present study, 205 pulmonary tuberculosis cases and 127 healthy controls were studied for the association of Toll-like Receptor (TLR) variants (TLR1 variants 743A>G and 1805T>G, and TLR6 variant 745 C>T) in north Indian population. The frequency of heterozygous genotypes (AG) in TB cases (0.47) and HCs (0.61), differed significantly (p value = 0.02). The association of AG genotypes in HCs was adjusted for gender as gender was observed to be a confounder and M-H OR was found to be 0.62 (p = 0.044). On categorizing the cases basing on AFB smear positivity, the heterozygous genotypes (AG) was found to be associated with low bacillary load (scanty and 1+) (P = 0.002). No association was observed for either TLR1 1805 T>G or TLR6 745 C>T polymorphism. Level of serum IL6 was found to be significantly higher among healthy controls with TLR1 GG genotype compared to healthy controls with AA (p = 0.035) and AG (p = 0.005) genotypes. Thus, it may be suggested that the heterozygous condition for TLR1 743 A>G provide resistance from the disease. However, in depth study is required to understand the mechanism for possible protective responses.

Molecular mimicry between HSP 65 of Mycobacterium leprae and cytokeratin 10 of the host keratin; role in pathogenesis of leprosy.

Mycobacteria are known to induce autoimmune response in the host. Anti-host keratrin antibodies (AkAbs) might be responsible for the autoimmune phenomena in leprosy patients as majority of leprosy lesions are manifested in the skin and occurrence of keratosis is not an uncommon feature. The aim of this study was to find out the level of AkAbs in leprosy patients across the spectrum and to explore its correlation with the clinical manifestation of the disease. Further, mimicking epitopes of keratin and Mycobacterium leprae components were characterized. We screened 140 leprosy patients (27 BT, 28 BL, 41 LL, 25 T1R, 19 ENL), 74 healthy controls (HC) and 3 psoriasis patients as positive control. Highest AkAbs level was observed in the psoriasis patients followed by T1R, LL, BL, ENL, TT/BT. AkAbs level was significantly (p<0.05) higher in all the groups of leprosy patients except TT/BT in comparison to HC. Significant positive correlation was found between number of lesions and level of AkAbs in leprosy patients. Highest lympho-proliferation for keratin protein was observed in T1R, followed by BL/LL, TT/BT, ENL. Lympho-proliferation was significantly (p<0.05) higher in all groups of leprosy patients except ENL in comparison to HC. Interestingly, it was noted that hyperimmunization of inbred strains of female BALB/c mice and rabbit with M. leprae soluble antigen (MLSA) induce higher level of AkAbs. The percentage of FoxP3(+) expressing Treg cells (total CD4(+)CD25(+)FoxP3(+) andCD4(+)CD25(+hi)FoxP3(+)) in splenocytes and lymph nodes of hyperimmunized mice were declined in comparison to control mice. Further, it was found that this autoimmune response can be adoptively transferred in naïve mice by splenocytes and lymph node cells as well as T cells. Comparative molecular characterization between keratin and MLSA noted a cross-reactivity/similarity between these two antigens. The cross-reactive protein of keratin was found to be in molecular weight range ≈74-51kDa and at pI 4.5 while the cross-reactive protein of MLSA was found to be in molecular weight ≈65kDa and at pI 4-4.5. Cross-reactive protein of keratin and MLSA was identified and characterized by MALDI-TOF/TOF analysis and Mascot software. It was found that the keratin (host protein) which reacted with anti-M. leprae sera is cytokeratin-10 and MLSA which reacted with anti-keratin sera is heat shock protein 65 (HSP 65). Seven B-cell epitopes of cytokeratin-10 and HSP 65 was found to be similar by multiple sequence alignment using ClustalW server and out of which 6 B-cell epitopes were found to be on the surface of HSP 65. In conclusion, our study provides evidence for the existence of molecular mimicry between cytokeratin-10 of keratin (host protein) and 65kDa HSP (groEL2) of M. leprae. Presence of heightened CMI response of leprosy patients to keratin and positive correlation of AkAbs level with number of lesions of leprosy patients showed the clinical evidence for its role in the pathogenesis in leprosy.