RESUMO
Tuberculosis (TB) is a leading cause of death caused by Mycobacterium tuberculosis (M tb) and about one-third of the world's population is infected with TB. The household contacts of TB patients are at higher risk towards TB than general population. During the initial stages of infection, pro and anti-inflammatory cytokines are induced by innate immune cells, and the course of infection is influenced by general cytokine environment. These cytokines play an important role in the regulation of host immune responses against M tb. Therefore, it is necessary to understand the cytokines role in the immune mechanism to evaluate the correlation between the disease and the immune responses involved in TB. Our current study has focused on recombinant cytokines to understand their effects on cell proliferation and cytokine levels in culture supernatants. We observed that the mean proliferative responses to recombinant rhTNF-α were high and TNF-α levels were significantly low in APTB patients compared to their HHC and HC with p < 0.0375 and p < 0.0051 respectively. The mean proliferative responses to recombinant rhTGF-ß were significantly low in APTB when compared to HHC and HC with p < 0.0376, p < 0.0247 respectively, and TGF-ß levels were also significantly low in APTB and HHC compared to HC with p < 0.0468 and p < 0.0001 respectively. The lower cytokine secretions in culture supernatants might be due the autocrine signaling by recombinant cytokines towards the inflammatory response. Further, to validate these recombinant cytokines, a larger sample size could aid in identifying individuals at high risk for TB.
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Tuberculosis is caused by Mycobacterium tuberculosis (M tb), which is recognized by macrophages and produces inflammatory cytokines, and chemokines at the site of infection. The present study was proposed to understand the interaction of M tb antigens, cytokines, and chemokines. We have evaluated the chemokine MCP-1 levels and its expression in PBMCs stimulated with M tb antigens Ag85A, ESAT6 and recombinant cytokines rhTNF-α, rhIFN-γ, rhTGF-ß, and rhIL-10 in active pulmonary TB (APTB) patients, household contacts (HHC) at 0 months, 6 months and healthy controls (HC). We have observed low levels of MCP-1 with Ag85A, ESAT6, and rhTNF-α stimulations in APTB 0M compared to HHC and HC (p < 0.0067, p < 0.0001, p < 0.01, p < 0.005, p < 0.0065, p < 0.0001) and significantly increased after treatment with rhTNF-α. The MCP-1 levels with rhIFN-γ were high in APTB, HHC at 0 M and significant between APTB 0 M vs. 6 M, HHC vs. HC, and HHC 0M vs. 6M (p < 0.0352, p < 0.0252, p < 0.00062). The rhTGF-ß, rhIL-10 induced high MCP-1 levels in APTB, HHC compared to HC (p < 0.0414, p < 0.0312, p < 0.004, p < 0.0001) and significantly decreased after treatment with rhIL-10 (p < 0.0001). The MCP-1 expression was low with all the stimulations in APTB 0M when compared to HC and after treatment. Whereas, HHC shown low MCP-1 expression with rhTNF-α, rhIFN-γ and Ag85A and high with rhTGF-ß, rhIL-10 and ESAT6. In conclusion, the study determined the differential expression and production of MCP-1 with M tb antigens and recombinant cytokines. Further, cohort studies are required to study these interaction to identify the high risk individuals, which might help for TB control.
Assuntos
Antígenos de Bactérias , Quimiocina CCL2 , Citocinas , Mycobacterium tuberculosis , Proteínas Recombinantes , Humanos , Antígenos de Bactérias/imunologia , Quimiocina CCL2/imunologia , Quimiocina CCL2/metabolismo , Masculino , Mycobacterium tuberculosis/imunologia , Feminino , Proteínas Recombinantes/imunologia , Adulto , Citocinas/metabolismo , Proteínas de Bactérias/imunologia , Pessoa de Meia-Idade , Interferon gama/imunologia , Interferon gama/metabolismo , Tuberculose Pulmonar/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Interleucina-10/metabolismo , Interleucina-10/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Tuberculose/imunologia , Fator de Crescimento Transformador beta/imunologiaRESUMO
PURPOSE: The immune response induced by nucleotide-binding oligomerization domain-2(NOD2) is associated with the production of cytokines affected by the host's genetic background. The present study aimed to examine the effects of NOD2; 802C > T, 2105G > A polymorphisms associated with altered cytokine levels in patients with active pulmonary tuberculosis disease, Latent TB subjects (household contacts(HHC) and healthy controls(HC). METHODS: Genetic polymorphisms were analyzed by Restriction Fragment Length Polymorphism(RFLP) in 102-PTB patients, 102-HHC, and 132-HC. QuantiFERON-TB Gold In-Tube test was performed to identify latent TB infection in 60-HHC. Estimated their cytokine levels by ELISA in MDP (muramyl dipeptide) stimulated culture supernatants of all the groups. Further, we studied pre-mRNA structures by insilico analysis and relative gene expression by RT-PCR. RESULTS: Recessive genetic models of NOD2 802C > T SNP with TT genotype and AA genotype of NOD2 2105G > A SNP were significantly associated with increased TB risk in PTB patients and HHC compared with HC. In vitro stimulations were performed with NOD2 ligand MDP in PTB patients and latent TB subjects: QuantiFERON positive household contacts (QFT + ve HHC)and QuantiFERON negative household contacts(QFT-ve HHC). The results showed that reduced TNF-α and enhanced IL-12, IL-1ß indicate that these cytokines may play an essential role in the initial maintenance of cell-mediated immunity. Our study demonstrated the correlation between NOD2 polymorphism with IL-1ß, TNF-α, IL-12 levels. Insilico analysis represents the pre-mRNA secondary structures affected by NOD2 SNPs. We also observed the difference in m RNA levels in variant and wild genotypes. CONCLUSION: This finding may lead to the forthcoming development of immunotherapy and may be used as predictive markers to identify high-risk individuals for TB disease.
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Proteína Adaptadora de Sinalização NOD2/genética , Proteína Adaptadora de Sinalização NOD2/imunologia , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/imunologia , Acetilmuramil-Alanil-Isoglutamina/imunologia , Adulto , Citocinas , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Leucócitos Mononucleares/imunologia , Masculino , Polimorfismo de Nucleotídeo Único , Fatores de RiscoRESUMO
Forkhead Box Protein3 Transcription Factor (FOXP3) gene is an essential role player in the function and differentiation of regulatory T cells. Polymorphisms/mutations in FOXP3 gene cause Treg cell dysfunction, promote autoimmunity and inflammation. Based on this presumption, we screened 600 subjects from south India (equal number of diabetic (T2DM), diabetic nephropathy (T2DN) and healthy controls) for promoter and intronic (rs3761548C/A and rs2294021C/T) polymorphisms of FOXP3 gene. PCR-RFLP method used for genotyping, revealed an association of promoter SNP for both T2DM (OR = 2.41, 95% C.I = 1.67-3.49; p < 0.0001) and T2DN (OR = 2.16, 95% C.I = 1.45-3.24; p < 0.005). While intronic polymorphism with T2DN (OR = 1.91, 95% C.I = 1.28-2.84; p < 0.05). Further, in females rs3761548C/A showed 2.6 and 5.5-fold; rs2294021C/T showed 2.2- and 2.5-fold predisposition towards T2DM and T2DN respectively. Males exhibited a twofold risk (OR = 2.01, 95% C.I = 1.22-3.30; p < 0.05) towards T2DM with promoter and no association with intronic polymorphism. The combined genotypes in females with AA-CC; AA-TT predisposed and CA-CC; CA-CT protected heading towards T2DM and T2DN respectively, suggesting irrespective of type of allele at intronic locus AA and CA at promoter locus promote or protect the individual for diabetes and diabetic nephropathy, further confirmed by MLR. To our knowledge, the current study is the first of its kind that revealed an association of these polymorphisms of FOXP3 gene and gender influence on T2DM and T2DN among South Indians. Functional and cell-based studies on Treg cells are warranted to confirm our results that help to develop FOXP3/Treg based therapeutic interventions. Lack of data on Treg cells is the limitation of this study.
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Diabetes Mellitus Tipo 2/genética , Nefropatias Diabéticas/genética , Fatores de Transcrição Forkhead/genética , Predisposição Genética para Doença , Caracteres Sexuais , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Índia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo Genético , Polimorfismo de Fragmento de Restrição , Regiões Promotoras GenéticasRESUMO
The present study was designed to evaluate percentage of lymphocyte subsets in peripheral blood mononuclear cells of pulmonary tuberculosis patients with diabetes mellitus (TBDM) and household contacts (HHC) at the time of diagnosis and at different intervals of follow-up. T-lymphocyte subsets, monocytes and natural killer cells were evaluated using fluorescence associated cell sorting (FACS) in a total of 125 subjects including TBDM, pulmonary tuberculosis (PTB) patients, HHC, diabetes mellitus (DM) patients and healthy controls (HC), 25 in each category. CD4 proportion was significantly low in TBDM (p = 0.003), PTB (p = 0.0008) and HHC (p = 0.005) when compared to HCs and increased with treatment in PTB at 6 M and 12 M (p = 0.008). CD8 percentage was significantly low in DM (p = 0.01); Significantly high mean percentage was observed with respect to CD14 in TBDM (p = 0.008), PTB (p = 0.018), HHC (p = 0.008) and DM (p = 0.014); with CD16 in TBDM (p = 0.0001), PTB (p = 0.0001), HHC (p = 0.045); with CD56 in TBDM (p = 0.0003), PTB (p = 0.002) and HHC (p = 0.015) respectively when compared to HCs. These results indicate that TBDM patients have altered lymphocyte homeostasis and FACS analysis might have the potential of a non-invasive clinical indicator for the early detection and monitoring of TB in HHC.
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Diabetes Mellitus , Tuberculose Pulmonar , Seguimentos , Humanos , Leucócitos Mononucleares , Subpopulações de Linfócitos , Subpopulações de Linfócitos T , Tuberculose Pulmonar/complicações , Tuberculose Pulmonar/diagnósticoRESUMO
BACKGROUND & OBJECTIVES: High expression of arginase gene and its elevated level in serum and bronchial lavage reported in animal models indicated an association with the pathogenesis of asthma. This study was undertaken to assess the serum arginase activity in symptomatic asthma patients and healthy controls and to correlate it with cytokine levels [interleukin (IL)-4 and IL-13] and arginase I (ARG1) gene polymorphism. METHODS: Asthma was confirmed by lung function test according to the GINA guidelines in patients attending Allergy and Pulmonology Clinic, Bhagwan Mahavir Hospital and Research Centre, Hyderabad, India, a tertiary care centre, during 2013-2015. Serum arginase was analyzed using a biochemical assay, total IgE and cytokine levels by enzyme-linked immunosorbent assay and genotyping of ARG1 for single-nucleotide polymorphisms (SNPs) rs2781666 and rs60389358 using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: There was a significant two-fold elevation in the arginase activity in asthmatics as compared to healthy controls which correlated with disease severity. Non-atopic asthmatics showed elevated activity of arginase compared to atopics, indicating its possible role in intrinsic asthma. Levels of serum IL-13 and IL-4 were significantly high in asthma group which correlated with disease severity that was assessed by spirometry. A positive correlation was observed between arginase activity and IL-13 concentration. Genetic analysis of ARG1 SNPs revealed that rs2781666 G/T genotype, T allele and C-T haplotype (rs60389358 and rs2781666) were associated with susceptibility to asthma. INTERPRETATION & CONCLUSIONS: This study indicated that high arginase activity and IL-13 concentration in the serum and ARG1 rs2781666 G/T genotype might increase the risk of asthma in susceptible population. Further studies need to be done with a large sample to confirm these findings.
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Arginase/genética , Asma/genética , Predisposição Genética para Doença , Interleucina-13/genética , Adolescente , Adulto , Arginase/sangue , Asma/sangue , Asma/fisiopatologia , Feminino , Estudos de Associação Genética , Genótipo , Haplótipos , Humanos , Interleucina-13/sangue , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Fatores de Risco , Adulto JovemRESUMO
Live attenuated vaccines are generally highly efficacious and often superior to inactivated vaccines, yet the underlying mechanisms of this remain largely unclear. Here we identify recognition of microbial viability as a potent stimulus for follicular helper T cell (TFH cell) differentiation and vaccine responses. Antigen-presenting cells (APCs) distinguished viable bacteria from dead bacteria through Toll-like receptor 8 (TLR8)-dependent detection of bacterial RNA. In contrast to dead bacteria and other TLR ligands, live bacteria, bacterial RNA and synthetic TLR8 agonists induced a specific cytokine profile in human and porcine APCs, thereby promoting TFH cell differentiation. In domestic pigs, immunization with a live bacterial vaccine induced robust TFH cell and antibody responses, but immunization with its heat-killed counterpart did not. Finally, a hypermorphic TLR8 polymorphism was associated with protective immunity elicited by vaccination with bacillus Calmette-Guérin (BCG) in a human cohort. We have thus identified TLR8 as an important driver of TFH cell differentiation and a promising target for TFH cell-skewing vaccine adjuvants.
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Ativação Linfocitária/imunologia , Viabilidade Microbiana/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Receptor 8 Toll-Like/imunologia , Vacinas Atenuadas/imunologia , Adulto , Animais , Formação de Anticorpos/imunologia , Diferenciação Celular/imunologia , Feminino , Humanos , Masculino , SuínosRESUMO
Single nucleotide polymorphisms (SNPs) of cytokine genes have been found to be involved in the clinical outcome of Tuberculosis. The present study was aimed to identify the high risk genotypes in Tuberculosis patients and their household contacts. A total of 490 subjects were studied which includes 150 active pulmonary tuberculosis patients (APTB), 190 household contacts (HHC) and 150 healthy controls (HC). The SNPs of TNF-α (-308A/G), IL-10(-1082G/A) and IL-6(-174G/C) were performed by ARMs PCR. The IL-10 GA genotype showed significant association in APTB and HHC and was 2.3 times higher risk in APTB and 3.7 times in HHC compared to HCs. The A allele was found to be significantly associated with the risk of disease. The CC genotype of IL-6 was found to be significantly associated in APTB and an insignificant positive association in HHCs. The multifactor dimensionality reduction (MDR) analysis indicated that the genotypes of IL-6 were showing high risk with GA genotype of IL-10. In conclusion the gene interaction may be useful for identification of genotypes as biomarkers to distinguish high risk individuals.
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Interleucina-10/genética , Interleucina-6/genética , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Tuberculose Pulmonar/genética , Fator de Necrose Tumoral alfa/genética , Adolescente , Adulto , Fatores Etários , Estudos de Casos e Controles , Características da Família , Marcadores Genéticos/genética , Predisposição Genética para Doença/genética , Técnicas de Genotipagem , Humanos , Tuberculose Latente/genética , Tuberculose Pulmonar/imunologia , Adulto JovemRESUMO
PURPOSE: Association of cytokine genes reflects their susceptibility towards infection and disease in household contacts (HHC) of pulmonary tuberculosis (PTB) patients. Hyperglycemia, a common factor in diabetics might influence their risk towards mycobacterium tuberculosis infection and disease development. This study determines the association of IL-6 and IL-18 cytokine gene variants of TB patients with diabetes mellitus (TBDM) and their HHC in Hyderabad. METHODS: Single nucleotide polymorphisms of IL-6 (-174 G>C and -572 G>C) and IL-18 (-137 G>C and -607 C>A) cytokine genes were genotyped by Amplification Refractory Mutation System and Restriction Fragment Length polymerase chain reaction in total of 705 subjects comprising of TBDM, their HHC, PTB, DM and Healthy controls (HC). RESULTS: At IL-6 -174G>C variant, GG genotype, G allele in TBDM and TBDM HHC, at -572G>C variant, C allele in TBDM and GG haplotype in TBDM HHC were showing positive association, however DM have not shown any association at IL-6 polymorphic sites. With respect to the IL-18 gene polymorphisms, at -137 G>C variant, GG genotype was positively associated in PTB while at -607 C>A variant positive association was shown with AC genotype in TBDM, their HHC and DM; GACC diplotype in TBDM and GCGC in PTB. CONCLUSION: Our findings suggest that susceptible combination of IL-6 and IL-18 cytokine genes associated with disease in the HHCs highlight their risk of inclination towards the disease.
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Diabetes Mellitus Tipo 2/genética , Interleucina-18/genética , Interleucina-6/genética , Polimorfismo de Nucleotídeo Único , Tuberculose Pulmonar/genética , Adulto , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/epidemiologia , Características da Família , Feminino , Genótipo , Humanos , Índia , Masculino , Pessoa de Meia-Idade , Tuberculose Pulmonar/complicações , Tuberculose Pulmonar/epidemiologiaRESUMO
Prolonged treatment of tuberculosis (TB) often leads to poor compliance, default and relapse, converting primary TB patients into category II TB (Cat IITB) cases, many of whom may convert to multi-drug resistant TB (MDR-TB). We have evaluated the immunotherapeutic potential of Mycobacterium indicus pranii (MIP) as an adjunct to Anti-Tubercular Treatment (ATT) in Cat II pulmonary TB (PTB) patients in a prospective, randomized, double blind, placebo controlled, multicentric clinical trial. 890 sputum smear positive Cat II PTB patients were randomized to receive either six intra-dermal injections (2 + 4) of heat-killed MIP at a dose of 5 × 108 bacilli or placebo once in 2 weeks for 2 months. Sputum smear and culture examinations were performed at different time points. MIP was safe with no adverse effects. While sputum smear conversion did not show any statistically significant difference, significantly higher number of patients (67.1%) in the MIP group achieved sputum culture conversion at fourth week compared to the placebo (57%) group (p = 0.0002), suggesting a role of MIP in clearance of the bacilli. Since live bacteria are the major contributors for sustained incidence of TB, the potential of MIP in clearance of the bacilli has far reaching implications in controlling the spread of the disease.
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Vacinas contra a Tuberculose/efeitos adversos , Tuberculose Pulmonar/terapia , Vacinação/métodos , Vacinas de Produtos Inativados/efeitos adversos , Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium/imunologia , Vacinas contra a Tuberculose/uso terapêutico , Vacinação/efeitos adversos , Vacinas de Produtos Inativados/uso terapêuticoRESUMO
Asthma is a chronic inflammatory disease of the lower airways characterised by intermittent airway narrowing and airflow obstruction. The aim of this study was to examine the association of IL-13 Arg 130 Gln (A/G) and -1112C/T cytokine gene polymorphisms and to know the secretion of IL-13 cytokine levels and the interactions between the IL-13 130A/G and IL-13Rα1/IL-4Rα complex cytokine genes. The study population comprised of atopic and non-atopic asthma patients and healthy controls (HC) (N = 120). Single nucleotide polymorphisms (SNPs) were determined by restriction fragment length polymorphism (RFLP). IL-13 cytokine serum levels were measured by enzyme-linked immunosorbent assay (ELISA), and homology modelling of IL-13 A/G cytokine gene was performed through in silico analysis. In IL-13 130A/G cytokine gene AG, GG genotypes (p < 0.0042, OR = 2.87, CI 1.46-5.65; OR = 1.92, CI 1.06-3.48) were found to be significant in atopic asthma patients vs HC. The mean IL-13 serum cytokine levels were found to be significantly high in atopic (38.48 ± 36.54) and non-atopic (36.05 ± 34.54) asthma patients whereas total serum IgE levels were significantly high at p < 0.0001 in atopic and low in non-atopic asthma patients at p < 0.003 compared to HC. In silico analysis indicated that residue IL-13 130 with charge modifying variants was crucial in ligand-receptor interactions. IL-13 cytokine serum levels were significantly high in atopic and non-atopic asthma patients compared to HC. The GG genotype of IL-13 130A/G cytokine gene might be involved in the induced production of total IgE and IL-13 cytokine serum levels suggesting IL-13 may be important in the signalling of asthma.
Assuntos
Asma/genética , Interleucina-13/genética , Polimorfismo de Nucleotídeo Único/genética , Adulto , Asma/metabolismo , Estudos de Casos e Controles , Feminino , Genótipo , Humanos , Hipersensibilidade Imediata/sangue , Hipersensibilidade Imediata/genética , Imunoglobulina E/sangue , Interleucina-13/sangue , Interleucina-13/fisiologia , Masculino , Transdução de SinaisRESUMO
Conventionally, facultative intracellular pathogen, Mycobacterium tuberculosis, the tuberculosis (TB) causing bacilli in human is cleared by cell-mediated immunity (CMI) with CD4(+) T cells playing instrumental role in protective immunity, while antibody-mediated immunity (AMI) is considered non-protective. This longstanding convention has been challenged with recent evidences of increased susceptibility of hosts with compromised AMI and monoclonal antibodies conferring passive protection against TB and other intracellular pathogens. Therefore, novel approaches toward vaccine development include strategies aiming at induction of humoral response along with CMI. This necessitates the identification of mycobacterial proteins with properties of immunomodulation and strong immunogenicity. In this study, we determined the immunogenic potential of M. tuberculosis Zinc metalloprotease-1 (Zmp1), a secretory protein essential for intracellular survival and pathogenesis of M. tuberculosis. We observed that Zmp1 was secreted by in vitro grown M. tuberculosis under granuloma-like stress conditions (acidic, oxidative, iron deficiency, and nutrient deprivation) and generated Th2 cytokine microenvironment upon exogenous treatment of peripheral blood mononulear cells PBMCs with recombinant Zmp1 (rZmp1). This was supported by recording specific and robust humoral response in TB patients in a cohort of 295. The anti-Zmp1 titers were significantly higher in TB patients (n = 121) as against healthy control (n = 62), household contacts (n = 89) and non-specific infection controls (n = 23). A significant observation of the study is the presence of equally high titers of anti-Zmp1 antibodies in a range of patients with high bacilli load (sputum bacilli load of 300+ per mL) to paucibacillary smear-negative pulmonary tuberculosis (PTB) cases. This clearly indicated the potential of Zmp1 to evoke an effective humoral response independent of mycobacterial load. Such mycobacterial proteins can be explored as antigen candidates for prime-boost vaccination strategies or extrapolated as markers for disease detection and progression.
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Several cytokine gene variants have shown to be associated with host susceptibility to infectious diseases including tuberculosis (TB). High rates of transmission were identified within household members of TB patients. In this study, we examined whether single nucleotide polymorphisms of IFN-γ +874A/T and IL-12 +1188A/C affect susceptibility to TB. Genomic DNA from patients with active disease, their household contacts HHC and healthy controls HC was genotyped for IFN-γ +874A/T and IL-12 +1188A/C SNPs by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). IFN-γ +874 AA and AT genotypes were significantly with different frequencies in patients and total HHC as compared to HC (p<0.0001). In patients IL-12 +1188 AC and CC genotypes were associated with TB (p<0.003, p<0.008). In total HHC AC, CC genotypes and both alleles (A&C) were significantly different as compared to HC (p<0.004, p<0.001, p<0.034) and the same result was obtained when HHC were stratified into related (p<0.02, p<0.001) and unrelated (p<0.009, p<0.017) individuals. Allelic frequencies, however, were significant only in related contacts (p<0.021). Generalized multifactor dimensionality reduction method (GMDR) testing revealed high risk combinations of several genotypes in IFN-γ & IL-12 genes. Our findings suggest an important role of genetic variations of IFN-γ and IL-12 for susceptibility to TB.
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Interferon gama/genética , Interleucina-12/genética , Tuberculose Pulmonar/genética , Adolescente , Adulto , Análise Mutacional de DNA , Características da Família , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Humanos , Índia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Adulto JovemRESUMO
Household contacts of tuberculosis patients are at high risk of infection and development of active disease. In this study we evaluated the cytokine production and mRNA expression of IFN-γ, TNF-α, IL-10&IL-6 stimulated with r32kDa M. bovis BCGAg in active pulmonary tuberculosis patients (APTB), household contacts (HHC) and healthy controls (HC). The results showed the stimulated levels of IFN-γ and TNF-α were low while IL-10 levels were high in APTB and HHC compared to HC. IL-6 has not shown any significant difference. The mRNA expression of TNF- α was 8 fold high in HCs compared to APTB and HHC. The IL-6 expression was 2.2 fold &1 fold less in APTB and HHC compared to HCs. Multinomial logistic regression analysis indicated that the stimulated levels of IFN-γ & IL-6 and sex significantly predicted the HHC group from HCs at p<0.05.In conclusion further follow up studies with r32kd antigen might help to identify the high risk individuals.
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Busca de Comunicante , Citocinas/metabolismo , Habitação , Leucócitos Mononucleares/metabolismo , Mycobacterium tuberculosis/imunologia , RNA Mensageiro/metabolismo , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/transmissão , Adolescente , Adulto , Fatores Etários , Antígenos de Bactérias/imunologia , Estudos de Casos e Controles , Proliferação de Células , Células Cultivadas , Citocinas/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Interações Hospedeiro-Patógeno , Humanos , Testes de Liberação de Interferon-gama , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/microbiologia , Modelos Logísticos , Masculino , Análise Multivariada , Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/patogenicidade , Valor Preditivo dos Testes , RNA Mensageiro/genética , Fatores de Risco , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/metabolismo , Tuberculose Pulmonar/microbiologia , Adulto JovemRESUMO
BACKGROUND: Household contacts of diagnostically established tuberculosis (TB) patients are highly susceptible to disease development. It is surmised that cytokines perhaps play a synergistic and a prognostic role in the activation of the otherwise latent infection in these house hold contacts. Evaluation of the cytokines and any of their inherent polymorphisms might provide a useful diagnostic tool in evaluating the immune regulation and the progression of the disease. The cytokines thus released in a paracrine manner in serum may also provide an indirect measure of the cytokine function. OBJECTIVE: The present study was aimed to evaluate the levels of TNF-α, IL-10 & IL-6 cytokines and their correlation with genotype variants amongst tuberculosis patients and their household contacts. METHODS: The cytokine levels were estimated in serum by enzyme-linked immunosorbent assay (ELISA) and their polymorphisms were studied by amplification refractory mutation system polymerase chain reaction (ARMs PCR) in active pulmonary tuberculosis patients (APTB = 150), household contacts (HHC = 190), and healthy controls (HC = 150). RESULTS: The median values of TNF-α cytokine were significantly high among APTB and HHC compared to HCs (P< 0.0001 and 0.0001). IL-6 levels also were elevated among APTB compared to HHC and HC, and a significant difference was observed between APTB and HHC at P<0.0001; APTB & HC at P< 0.04; HHC & HC at P< 0.01. The IL-10 levels were low in APTB compared to HHC and HCs and no significant difference was observed. TNF-α/IL-10 ratio was significant and indicated Th1 predominance in APTB and HHC. IL-6/IL-10 showed pronounced Th1 expression in APTB and Th2 in HHC and HC. The ROC analysis indicated that both IL-10 and IL-6 can be used to decide the risk of exposed individual to a disease. The results of multivariate analysis indicate that IL-10 (-1082) GA genotype was significantly associated with p<0.028 in APTB. No significant association was observed between genotypes, other serum cytokine levels and clinical characteristics between APTB, HHC and HCs. CONCLUSION: Large sample size with follow-up at different time points may further illuminate the role of IL-10 and IL-6 cytokines as a prognostic marker in house hold contacts.
Assuntos
Citocinas/genética , Citocinas/metabolismo , Família , Variação Genética , Genótipo , Tuberculose/genética , Tuberculose/metabolismo , Adolescente , Adulto , Alelos , Análise de Variância , Estudos de Casos e Controles , Citocinas/sangue , Feminino , Humanos , Mediadores da Inflamação , Interleucina-10/sangue , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-6/sangue , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Polimorfismo de Nucleotídeo Único , Curva ROC , Fatores de Risco , Tuberculose/sangue , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Adulto JovemRESUMO
Multi-drug-resistant (MDR) tuberculosis is a major public health problem worldwide. Drug resistance arises due to non-compliance of antibiotic therapy. Herein, we explored the therapeutic options available ranging from conservative treatment approaches to alternate adjunct therapies such as mesenchymal stromal cell (MSC) therapy interventions. It is attractive to understand the scientific rationale of using cells as drugs, in particular mesenchymal stem/stromal cells. The review dwells and attempts to analyze the mechanistic approaches of the current treatment modalities to modern therapies. MSCs have demonstrated profound capacity to regenerate and repair. They appear to modulate that the activities of dendritic cells regulate T cells, both in vivo and in vitro. While there seems to be some benefit of such therapies, its use warrants further research. The merits and de-merits of autologous therapy/allogeneic therapy are ill understood. The challenges of requirement of large number of cells for infusion, the route of administration, choice of timing are complex issues that need to be addressed. Furthermore, the host immune responses, environmental factors and epigenetic mechanisms compound the problem. Although, clinical studies are being performed using autologous MSCs in different inflammatory models, it is important that such an intervention should be based on sound scientific rationale. The current review examines the immunomodulatory properties of MSCs, its interactions with other cell types, in assessing the basis for autologous/allogeneic cell-based therapies in the treatment of XDR/MDR tuberculosis.
Assuntos
Células Dendríticas/imunologia , Imunoterapia/métodos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/imunologia , Células Estromais/imunologia , Linfócitos T/imunologia , Tuberculose Resistente a Múltiplos Medicamentos/terapia , Animais , Ensaios Clínicos como Assunto , Epigênese Genética , Humanos , Imunidade Inata , Regeneração , Células Estromais/transplante , Tuberculose Resistente a Múltiplos Medicamentos/imunologiaRESUMO
Applied tissue engineering in regenerative medicine warrants our enhanced understanding of the biomaterials and its function. The aim of this study was to evaluate the proliferation and differentiation potential of human adipose-derived stem cells (hADSCs) grown on chitosan hydrogel. The stability of this hydrogel is pH-dependent and its swelling property is pivotal in providing a favorable matrix for cell growth. The study utilized an economical method of cross linking the chitosan with 0.5% glutaraldehyde. Following the isolation of hADSCs from omentum tissue, these cells were cultured and characterized on chitosan hydrogel. Subsequent assays that were performed included JC-1 staining for the mitochondrial integrity as a surrogate marker for viability, cell proliferation and growth kinetics by MTT assay, lineage specific differentiation under two-dimensional culture conditions. Confocal imaging, scanning electron microscopy (SEM), and flow cytometry were used to evaluate these assays. The study revealed that chitosan hydrogel promotes cell proliferation coupled with > 90% cell viability. Cytotoxicity assays demonstrated safety profile. Furthermore, glutaraldehyde cross linked chitosan showed < 5% cytotoxicity, thus serving as a scaffold and facilitating the expansion and differentiation of hADSCs across endoderm, ectoderm and mesoderm lineages. Additional functionalities can be added to this hydrogel, particularly those that regulate stem cell fate.
Assuntos
Tecido Adiposo/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Quitosana/química , Hidrogéis/química , Células-Tronco/metabolismo , Tecido Adiposo/citologia , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Células-Tronco/citologiaRESUMO
BACKGROUND: Concurrent occurrence of HIV and Tuberculosis (TB) infections influence the cellular environment of the host for synergistic existence. An elementary approach to understand such coalition at the molecular level is to understand the interactions of the host and the viral factors that subsequently effect viral replication. Long terminal repeats (LTR) of HIV genome serve as a template for binding trans-acting viral and cellular factors that regulate its transcriptional activity, thereby, deciding the fate of HIV pathogenesis, making it an ideal system to explore the interplay between HIV and the host. METHODOLOGY/PRINCIPAL FINDINGS: In this study, using biotinylated full length HIV-1 LTR sequence as bait followed by MALDI analyses, we identified and further characterized human-Zinc-finger-protein-134 (hZNF-134) as a novel positive regulator of HIV-1 that promoted LTR-driven transcription and viral production. Over-expression of hZNF-134 promoted LTR driven luciferase activity and viral transcripts, resulting in increased virus production while siRNA mediated knockdown reduced both the viral transcripts and the viral titers, establishing hZNF-134 as a positive effector of HIV-1. HIV, Mycobacteria and HIV-TB co-infections increased hZNF-134 expressions in PBMCs, the impact being highest by mycobacteria. Corroborating these observations, primary TB patients (nâ=â22) recorded extraordinarily high transcript levels of hZNF-134 as compared to healthy controls (nâ=â16). CONCLUSIONS/SIGNIFICANCE: With these observations, it was concluded that hZNF-134, which promoted HIV-1 LTR activity acted as a positive regulator of HIV propagation in human host. High titers of hZNF-134 transcripts in TB patients suggest that up-regulation of such positive effectors of HIV-1 upon mycobacterial infection can be yet another mechanism by which mycobacteria assists HIV-1 propagation during HIV-TB co-infections. hZNF-134, an uncharacterized host protein, thus assumes a novel regulatory role during HIV-host interactions. Our study provides new insights into the emerging role of zinc finger proteins in HIV-1 pathogenesis.
Assuntos
Infecções por HIV/fisiopatologia , Repetição Terminal Longa de HIV/genética , Mycobacterium/fisiologia , Tuberculose/fisiopatologia , Células Cultivadas , Células HEK293 , Infecções por HIV/genética , Humanos , Microscopia Confocal , Mycobacterium/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
BACKGROUND: Cytokines are the hallmark of immune response to different pathogens and often dictate the disease outcome. HIV infection and tuberculosis (TB) are more destructive when confronted together than either alone. Clinical data related to the immune status of HIV-TB patients before the initiation of any drug therapy is not well documented. This study aimed to collect the baseline information pertaining to the immune status of HIV-TB co-infected patients and correlate the same with CD4+T cell levels and viral loads at the time of diagnosis prior to any drug therapy. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the cytokines, CD4+T cell levels and viral loads to determine the immune environment in HIV-TB co-infection. The study involved four categories namely, Healthy controls (n=57), TB infected (n=57), HIV infected (n=59) and HIV-TB co-infected (n=57) patients. The multi-partite comparison and correlation between cytokines, CD4+T-cell levels and viral loads prior to drug therapy, showed an altered TH1 and TH2 response, as indicated by the cytokine profiles and skewed IFN-γ/IL-10 ratio. Inadequate CD4+T cell counts in HIV-TB patients did not correlate with high viral loads and vice-versa. When compared to HIV category, 34% of HIV-TB patients had concurrent high plasma levels of IL-4 and TNF-α at the time of diagnosis. TB relapse was observed in 5 of these HIV-TB co-infected patients who also displayed high IFN-γ/IL-10 ratio. CONCLUSION/SIGNIFICANCE: With these studies, we infer (i) CD4+T-cell levels as baseline criteria to report the disease progression in terms of viral load in HIV-TB co-infected patients can be misleading and (ii) co-occurrence of high TNF-α and IL-4 levels along with a high ratio of IFN-γ/IL-10, prior to drug therapy, may increase the susceptibility of HIV-TB co-infected patients to hyper-inflammation and TB relapse.
Assuntos
Linfócitos T CD4-Positivos/citologia , Coinfecção/sangue , Infecções por HIV/sangue , Interleucina-4/sangue , Tuberculose/sangue , Fator de Necrose Tumoral alfa/sangue , Carga Viral , Adulto , Fármacos Anti-HIV/uso terapêutico , Coinfecção/diagnóstico , Coinfecção/tratamento farmacológico , Coinfecção/imunologia , Infecções por HIV/diagnóstico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Humanos , Pessoa de Meia-Idade , Tuberculose/diagnóstico , Tuberculose/tratamento farmacológico , Tuberculose/imunologiaRESUMO
Interleukin (IL-10), an anti-inflammatory cytokine, is known to have dual effect on the host immune system. One of these roles is that it provides an effective autoregulatory mechanism which protects the host from excessive inflammation and tissue damage which is in part initiated by the Th1 driven pro-inflammatory immune responses during infections (such as TB, HIV and malaria). However, though beneficial, this autoregulatory mechanism is at times exploited by pathogens which evade elimination by Th1 driven immune response leading to chronic infections. The main aim of this study therefore was to study the influence of IL-10 polymorphism in relation to its levels with respect to HIV-TB co-infection. A total of 452 participants were categorized into HIV (121), active tuberculosis (TB) (118), HIV-TB (HT) (106) groups and healthy control group (107). Polymorphism for IL-10 gene (positions -1082, -819, -592) was studied using ARMS-PCR, RFLP. IL-10 and IFN-γ levels in antigen stimulated cultures were measured using ELISA. Statistical analysis was performed using Chi-Square (χ(2)) test, One-way ANOVA and t-tests. IL-10 (-1082) GG genotype was positively associated with HIV-TB, whereas AG with HIV and AA with TB. The cohort with GG genotype also had significantly high stimulated levels of IL-10 compared to AG and AA. AC genotype was significantly frequent in HIV-TB group at IL-10 (-592) position when compared with controls. HIV positive individuals with GG genotype at IL-10 (-1082) position and high IL-10 levels may have a high risk of developing TB co-infection.