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BACKGROUND: Genetic elements are known to influence susceptibility to tuberculosis (TB). P2X7R is a candidate gene with multiple single-nucleotide polymorphisms (SNPs) that has the potential to influence an individual's ability to kill the intracellular pathogen Mycobacterium tuberculosis. OBJECTIVE: To explore the role of five functional polymorphisms of P2X7R in susceptibility or resistance to TB in a North Indian Punjabi population. DESIGN: A case-control study was conducted among 245 TB patients (145 pulmonary TB [PTB] and 100 extra-pulmonary TB [EPTB]) and 247 healthy controls. DNA extracted from samples of peripheral blood was analysed for five SNPs of P2X7R using amplification refractory mutation system-polymerase chain reaction (PCR) [-762(T/C), +1513(A/C), DNA sequencing +1729(T/A)] and PCR-restriction fragment length polymorphism [+489(G/A), +946(G/A)] methods. RESULTS: Of the three loss-in-function polymorphisms, +1513(A/C) showed a statistically significant association with TB susceptibility, while the other two (+946 and +1729) sites were found to be monomorphic in our population. The only gain-in-function polymorphism (+489), and -762 promoter polymorphisms failed to reveal differences in genotypic or allelic distributions. CONCLUSION: The C allele at the +1513 site was identified as a risk factor for TB in this North Indian Punjabi population; the +1729 site was found to be monomorphic, unlike its polymorphic distribution in a South Indian TB patient population.

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BACKGROUND: The renin-angiotensin system (RAS) is an important facet of blood pressure regulation physiology. Treatment of essential hypertension targets the RAS using Angiotensin Converting Enzyme Inhibitors (ACEIs). However, ACEIs are not uniformly effective and show inter-individual pharmacodynamic variations. AIM: To assess the correlation between genetic polymorphisms in the genes coding for RAS components (angiotensin converting enzyme (ACE I/D), α-adducin (ADD1) and ß1 -adrenoreceptor (ß1-ADR)) and response to Ramipril. MATERIALS AND METHODS: We recruited 120 patients with essential hypertension who were administered Ramipril monotherapy initially, followed by combination therapy, if needed, based on their responses. Relationship between genotypes of the three candidate genes and decrease in the blood pressure (BP) was analyzed. RESULTS: One hundred and six patients were evaluable at the end of the study period and 21 different genotypes were observed among them. Seven of them were classified as responders after 8 weeks and at the end of 12 weeks, an additional 77 (72.64%) were deemed responders. 19/22 non-responders were treated with combination therapy and 7/19 (36.84%) showed a response to the same. There was a significant difference between the proportions of responders and non-responders among the genotypes of the ADD1 and ß1-ADR genes (P=0.005 and 0.003, respectively). The best predictors of response to Ramipril 5 mg daily were the II/GG/SS, II/TG/SS, II/GG/SG, ID/GG/SS, ID/GG/SG and ID/TT/SS and DD/GG/SS; II/GG/GG, II/TT/SG, ID/TG/SG, ID/TT/SG, DD/GG/SG and DD/GG/GG were moderately predictive and II/TT/SS, II/TG/GG, ID/TG/GG, DD/TG/SG and DD/TG/GG were poorly predictive of response. DISCUSSION: Variable responses to Ramipril may be the result of genetic factors. CONCLUSION: Pre-prescription genotyping may help individualize treatment.

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Although India accounts for the highest tuberculosis (TB) burden in the world, the diversity in prevalent Mycobacterium tuberculosis strains is very poorly documented. Tuberculosis specific deletion 1 (TbD1) is a marker that has been used to differentiate ancient from modern strains. We report for the first time TbD1-based diversity in clinical M. tuberculosis isolates circulating in the North Indian states of Himachal Pradesh and Punjab. The present study documents a very high prevalence of modern strains in North India, which is in contrast to earlier studies that emphasised the predominance of ancestral strains for the majority of TB cases in India.

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BACKGROUND: Development of tuberculosis (TB) disease is an outcome of complex host-pathogen interactions. The purinergic P2X(7) receptors are adenosine triphosphate gated molecules shown to induce killing of intracellular Mycobacterium tuberculosis, followed by apoptosis of the infected macrophage. A single nucleotide polymorphism in exon 13 of the P2X(7) receptors gene at +1513 position has been shown to abolish the function of this receptor and to be associated with increased susceptibility to TB in some ethnic groups. OBJECTIVE: To explore the association of +1513 (A-->C) polymorphism in TB patients in Punjab, North India. DESIGN: A case-control study was conducted by studying peripheral blood samples from 204 TB patients (181 pulmonary, 23 extra-pulmonary) and 177 controls with no history of TB. P2X(7) +1513 (A-->C) polymorphism was studied using amplification refractory mutation system analysis. RESULT: The distribution of +1513 A/C genotypes in the TB patient and the control groups revealed a statistically significant association with TB (P = 0.002). CONCLUSION: The +1513C allele is a risk factor for the development of TB in the North Indian Punjabi population.

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Extra pulmonary tuberculosis (EPTB) constitutes around 20% of all tuberculosis cases in India. Conventional methods are of limited use in diagnosing this form of the disease. Polymerase chain reaction (PCR) has emerged as a sensitive and specific tool for documenting the presence of Mycobacterium tuberculosis in clinical samples but lacks quantitative ability. The present study evaluates peripheral blood as an alternative clinical specimen for diagnosing EPTB. Peripheral blood samples from 38 EPTB and 89 non tuberculous subjects were analyzed for the presence of tubercle bacilli by MPB 64 gene based PCR method. The assay gave an overall sensitivity of 60.53% with negative predictive value of 76.92% which is superior to present gold standard of mycobacterial culture (10.53 and 72.36%). Additionally, 43.82% of non tuberculous subjects gave positive results with the PCR, thus mitigating the clinical utility of this test. An in-house Competitive PCR (C-PCR) assay was used to determine the mycobacterial load in peripheral blood from culture positive, culture negative EPTB patients and non tuberculous controls which ranged from 7498-12498, 602-4797 and 101-800 genome equivalent (ge)/mL, respectively. The data clearly demonstrated that C-PCR assay can furnish insightful information in diagnosing extra pulmonary disease.

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BACKGROUND: Tumour necrosis factor (TNF)-alpha is a pleiotropic, pro-inflammatory cytokine of 17 kDa, whose gene is localized on the short arm of chromosome 6. It has a G-308A polymorphism in the promoter region, which is known to be associated with its differential production; the A allele being the high producer. The circulating level of TNF-alpha is under genetic control and implicated in the pathophysiology of asthma and tuberculosis. Since raised levels of TNF-alpha have been found in asthma and tuberculosis, we looked for the association of TNF-alpha G-308A polymorphism in patients with these diseases. METHODS: A total of 300 blood samples from patients (155 with asthma, 145 tuberculosis) and 211 normal healthy controls were collected. The G-308A polymorphism was studied using amplification refractory mutation system analysis. RESULTS: The distribution of G/A alleles in the two patient groups when compared with normal controls revealed a statistically significant association with asthma (p = 0.016) but not with tuberculosis (p = 0.178). CONCLUSION: The data support the common variant common disease hypothesis, which emphasizes that common genetic variations may participate as critical players in inciting common diseases.

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SETTING: Two hospitals, Sri Guru Ram Das Institute of Medical Sciences and Research and the TB and Chest Hospital, Amritsar, Punjab. OBJECTIVE: To explore genetic diversity among the clinical Mycobacterium tuberculosis isolates prevalent in Punjab. DESIGN: Fifty-six random clinical isolates of M. tuberculosis were cultured from the sputum specimens of pulmonary tuberculosis patients. DNA was extracted from cultured biomass and analysed using the mycobacterial interspersed repetitive units (MIRU) typing method. RESULTS: MIRU typing of 51 isolates revealed 45 different patterns, with a combined Hunter-Gaston discriminatory index (HGDI) of 0.990. Five clinical isolates failed to amplify for one or more MIRUs and were excluded from the analysis. The remaining isolates were categorised in three groups based on the allelic heterogeneity of individual MIRUs. MIRU 10, 16, 26 and 31 were highly discriminant, with an HGDI value >0.6; MIRU 4, 23, 24, 39 and 40 were designated as moderately discriminant (HGDI value 0.6-0.3) and MIRU 2, 20 and 27 were poorly discriminant (HGDI value <0.3). CONCLUSION: MIRU typing and the HGDI values revealed that M. tuberculosis strains from Punjab are genetically quite heterogeneous.

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PURPOSE: Different stages of hepatitis B virus (HBV) infection can be defined by serum HBV DNA levels. This study attempts to (1) investigate serum HBV DNA levels in inactive carriers and patients with chronic HBV (CHB) infection and (2) define cut-off value between inactive carriers and HBeAg (precore antigen of HBV) negative CHB patients in Indian population. METHODS: One hundred and forty samples encompassing 42 inactive HBsAg carriers and 98 CHB patients (53 HBeAg-positive and 45 HBeAg-negative) were analysed. Serum HBV DNA levels were determined employing an in-house competitive polymerase chain reaction (cPCR) assay. RESULTS: The HBeAg-positive patients were found to have the maximum median HBV DNA load, which was significantly higher than the HBeAg-negative ones (median; 1.25 x 10(8) vs. 2.30 x 10(5) copies/mL ; P<0.05). Interestingly, the latter group has significantly higher HBV DNA levels than the inactive carriers (median; 2.30 x 10(5) vs. 4.28 x 10(3) copies/mL; P<0.05). The 2.5 x 10(4) copies/ml HBV DNA levels were optimal for discriminating CHB patients (HBeAg-negative) from inactive carriers with 75.6 and 78.6% sensitivity and specificity, respectively. CONCLUSIONS: Despite the extensive overlapping of HBV DNA levels in inactive carriers and HBeAg negative CHB patients, 2.5 x 10(4) copies/mL is the most favourable cut-off value to classify these individuals and would be imperative in the better management of this dreadful disease.

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Hepatitis B virus (HBV) infection is a major public health problem and a leading cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma. Worldwide, there are about 350 million carriers of this pathogen and India bears the second highest carrier pool in the world. Early diagnosis and measurement of viral load in hepatitis B patients is very helpful for the better management of this disease. The existing methods for viral quantification are either cumbersome or expensive. Since viral replication correlate well with HBV DNA levels a new sensitive, reliable and cost effective competitive PCR assay has been developed for quantifying the viral load in the serum of hepatitis B patients. The S gene based cPCR assay was able to detect as low as 100 genome equivalent/ml of HBV DNA from human serum and was applied to determine viral load among inactive and chronic hepatitis B carriers demonstrating the usefulness of the developed test.

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The molecular quantification of Mycobacterium tuberculosis (TB) from clinical samples can improve the management of TB. Competitive polymerase chain reaction (C-PCR) is an accepted technique often used for this purpose, and IS6110 is the most popular target in such studies. As the number of these elements varies from 0 to 16 in clinical isolates, it is prone to give inconsistent results. A simple PCR-based approach is described in this study to generate a novel competitor for a single copy 38 kDa gene for the development of C-PCR for the quantification of the M. tuberculosis genome.

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Bronchial asthma is an inflammatory disorder in which genetic and environmental factors play an important role. Several susceptible genes have been identified using whole genome scan and candidate gene approaches. Tumor necrosis factor alpha, a pro-inflammatory cytokine, is one such gene that figures prominently in such investigations. The secreted levels of this cytokine are under genetic control and attributed to the presence of single nucleotide polymorphism G-308 A in the promoter region of its gene. However, the association of this polymorphism varies from population to population. As there are no data available on North Indians, the present study aims to fill this void. For this, 366 subjects (155 asthmatic and 211 normal control subject) were genotyped using Amplification Refractory Mutation System Analysis (ARMS-PCR) and agarose gel electrophoresis. The distribution of G/A alleles between the two groups revealed statistically significant differences (p = 0.016). Furthermore, the asthma patients categorized on the basis of pattern (Seasonal versus Perennial) and age of onset of disease (Childhood versus Late Onset) revealed significant association with only seasonal (p = 0.021) and late onset asthmatic groups (p = 0.039). The data from the present study strongly suggest an association between TNF-alpha and asthma in the North Indian population.

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Nitric oxide (*NO) is a short-lived free radical with many functions including vasoregulation, synaptic plasticity, and immune modulation and has recently been associated with AIDS pathology. Various pathophysiological conditions, such as viral infection, trigger inducible nitric oxide synthase (iNOS) to synthesize NO in the cell. NO-derived species can react with thiols of proteins and form nitrosothiol adducts. HIV-1 protease (HIV-PR) contains two cysteine residues, Cys67 and Cys95, which are believed to serve a regulatory function. We have found that HIV-PR is inactivated by nitric oxide produced in vitro by NO donors and by iNOS. Sodium nitroprusside inhibited HIV-PR by 70%, and S-nitroso-N-acetylpenicillamine completely inhibited the enzyme. Furthermore, iNOS generated sufficient NO to inhibit HIV-PR activity by almost 90%. This inactivation was reversed by the addition of reducing agents. Treatment of HIV-PR with NO donors and ritonavir (a competitive peptide inhibitor) indicates that NO exerts its effect through a site independent of the active site of HIV-PR. Using electrospray ionization mass spectrometry, we found that NO forms S-nitrosothiols on Cys67 and Cys95 of HIV-PR which directly correlate with a loss of activity. These data indicate that NO may suppress HIV-1 replication by directly inhibiting HIV-PR.

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Nitric oxide (NO) is a naturally occurring free radical with many functions. The oxidized form of NO, the nitrosonium ion, reacts with the thiol group of cysteine residues resulting in their modification to S-nitrosothiols. The human immunodeficiency virus type 1 (HIV-1) protease (HIV-PR) has two cysteine residues that are conserved amongst different viral isolates found in patients with acquired immunodeficiency syndrome (AIDS). In an active dimer, these residues are located near the surface of the protease. We have found that treatment of HIV-PR with different NO congeners results in loss of its proteolytic activity and simultaneous formation of S-nitrosothiols. Sodium nitroprusside inhibited HIV-PR up to 70% and S-nitroso-N-acetylpenicillamine completely inhibited the protease within 5 min of treatment. The pattern of inhibition by NO donors is comparable to its inhibition by N-acetyl pepstatin. Using electrospray ionization-mass spectrometry, we identified the modification of HIV-PR by NO as that of S-nitrosation. Our findings point towards a possible role of NO in mediating resistance to HIV-1 infection.

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In searching for a candidate mechanism for the immunosuppressive as well as fibrogenic consequences of cyclosporine usage, we have explored the hypothesis that cyclosporine stimulates transcription of transforming growth factor-beta 1 (TGF-beta 1), a multifunctional cytokine endowed with immunosuppressive and fibrogenic properties. Our results demonstrate that cyclosporine (i) stimulates TGF-beta 1 promoter-dependent transcription of chloramphenicol acetyl transferase gene in transiently transfected human A-549 cells, (ii) stimulates the synthesis of TGF-beta 1 RNA transcripts in human T cells, and (iii) permits the expression/emergence of DNA regulatory proteins (retinoblastoma control factor-1 (RCF-1) and RCF-2) that bind and regulate TGF-beta 1 promoter activity. Our studies demonstrate for the first time that cyclosporine stimulates TGF-beta 1 gene transcription and suggest a novel mechanism of action of cyclosporine.

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The basic immunosuppressive protocol involves the use of multiple drugs, each directed at a discrete site in the T-cell activation cascade. The prevailing paradigm regarding the mechanisms of action of immunosuppressants is that they function to prevent allograft rejection by preventing cell activation, proliferation and/or cytokine production. A new hypothesis is that some of the immunosuppressants might function by stimulating the expression of immunosuppressive molecules and/or cells.

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During normal aging and in chronic diabetes the excessive accumulation of reactive glucose-protein or glucose-lipid adducts known as advanced glycosylation endproducts (AGEs) has been shown to induce tissue dysfunction, in part through interaction with AGE-specific receptors on monocyte/macrophages and other cells. Recognizing that circulating lymphocytes trafficking through tissues interact with tissue AGEs, we searched for the expression of AGE-binding sites on peripheral blood T lymphocytes. Resting rat and human T cells bound 125I-AGE-albumin with an affinity of 7.8 x 10(7) M-1, whereas, after stimulation with phytohemagglutinin (PHA) for 48 h, binding affinity increased to 5.8 x 10(8) M-1. Flow cytometric analysis of resting rat T cells using polyclonal antibodies raised against rat liver AGE-binding proteins (p60 and p90) revealed the constitutive expression of both immunoreactivities. The number of resting CD4+ and CD8+ T cells positive for anti-p60 antibody binding (34.2 and 58.5%, respectively) increased to 92 and 90% of cells after 48-h stimulation with PHA. Exposure of PHA-activated T lymphocytes to AGE-albumin enhanced expression of interferon gamma (IFN-gamma) mRNA 10-fold and induced greater elaboration of the mature protein than did exposure to unmodified protein or PHA treatment alone. These data indicate that T cells contain an inducible system of surface receptors for AGE-modified proteins, and that receptor occupancy is linked to lymphokine production. This T cell AGE-receptor system might serve to target lymphocytes to AGE-rich tissues and involve them in the regulation of tissue homeostasis either by assisting in macrophage-dependent clearance of AGE-proteins, or by exerting direct antiproliferative action on mesenchymal cells. Under conditions of excessive AGE-protein and AGE lipid accumulation (e.g., aging and diabetes), enhanced production of AGE-induced IFN-gamma may accelerate immune responses that contribute to tissue injury.

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We have previously reported various inductive effects of nitric oxide on human PBMC. We describe a novel and potentially important mechanism of nitric oxide signaling-through direct activation of guanine nucleotide-binding proteins (G proteins). We have found that nitric oxide treatment of membranes isolated from fresh human PBMC enhances the ability of these membranes to hydrolyze [gamma-32P]GTP and bind [gamma-35S]GTP. In addition, treatment of whole cells with nitric oxide yielded membranes with enhanced GTPase activity. Furthermore, the GTPase activity of pure, recombinant Gs alpha, Gi alpha 1, and p21ras was greatly enhanced by nitric oxide. In support of the existence of this pathway in whole cells, we found that the G protein inhibitor, GDP-beta-S, blocked NF-kappa B translocation induced by nitric oxide or LPS in permeabilized cells. In addition, nitric oxide greatly reduced the pertussis toxin-mediated ADP-ribosylation of 45- and 41-kDa proteins in membranes of these cells. Because G proteins play a central role in many diverse signaling systems, activation by an endogenous and inducible oxidant may represent a novel signaling pathway.

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We have demonstrated earlier that the crosslinkage of the CD3/TCR complex with the CD2 antigen results in the proliferation of normal human T cells. The effect of this synergism was perceptible at the level of induction of the IL-2 gene, a process critical for T cell growth. To further understand the molecular and nuclear basis for this synergism, we have explored the induction of DNA-binding proteins in highly purified normal human T cells signaled via the CD3 and/or CD2 proteins. The effect of transmembrane signaling of T cells with ionomycin, and/or sn-1,2 dioctanoyl glycerol, was also determined. The emergence of nuclear binding proteins was investigated using interleukin-2 sequence specific oligonucleotide probes in the electrophoretic mobility shift assay. Our studies demonstrate for the first time that CD3 antigen-derived signals and CD2 antigen-derived signals are synergistic in inducing the emergence of transcription factors that bind to the NF-AT1, AP-1, and NF-kB sites located in the promoter/enhancer region of the IL-2 gene. Moreover, cyclosporine, at concentrations readily accomplished in clinical practice, was found to inhibit the emergence of these DNA-binding proteins in normal human T cells signaled via cell surface proteins implicated in antigen-dependent T cell activation and in T cells stimulated by mobilization of cellular calcium and activation of protein kinase C.

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