Suman Kalyan Paine

Association of tumor necrosis factor α, interleukin 6, and interleukin 10 promoter polymorphism with proliferative diabetic retinopathy in type 2 diabetic subjects.

Purpose: New blood vessel formation in the retina because of prolonged hypoxia is believed to be directly associated with increased expression of several growth factors and angiogenic cytokines. In the present study, we made an attempt to investigate the possible association of the promoter polymorphisms of interleukin 6, tumor necrosis factor &agr;, and interleukin 10 for the pathogenesis of proliferative diabetic retinopathy (PDR). Methods: This case–control study comprised 493 volunteers (253 PDR cases and 240 diabetic controls). Cases and controls were ascertained such that age, sex, nutrition, and glycemic status were matched. Genotypes were determined by polymerase chain reaction–based methods. Results: Interleukin 10–1082GG (P = 0.0037; odds ratio [OR] = 2.232), tumor necrosis factor &agr;–238AA (P = 0.0001; OR = 5.791), and GA (P = 0.0015; OR = 1.909) genotypes were significantly associated with PDR occurrence. The interleukin 10–1082G allele (P = 0.0048, OR = 1.4442) and the tumor necrosis factor &agr;–238A allele (P = 0.0001; OR = 2.2897) were significantly increased among PDR cases. Conclusion: From our study, it may be concluded that the genetic variation, that is, tumor necrosis factor &agr;–238A and interleukin 10–1082G alleles are the potent risk factors for the pathogenesis of PDR.

Multiplex PCR technique could be an alternative approach for early detection of leprosy among close contacts--a pilot study from India.

BackgroundImplementation of Multi drug Therapy (MDT) regimen has resulted in the decline of the total number of leprosy cases in the world. Though the prevalence rate has been declining, the incidence rate remains more or less constant and high in South East Asian countries particularly in India, Nepal, Bangladesh, Pakistan and Srilanka. Leprosy, particularly that of multibacillary type spreads silently before it is clinically detected. An early detection and treatment would help to prevent transmission in the community. Multiplex PCR (M-PCR) technique appears to be promising towards early detection among contacts of leprosy cases.MethodsA total of 234 paucibacillary (PB) and 205 multibacillary (MB) leprosy cases were studied in a community of an endemic area of Bankura district of West Bengal (Eastern India). They were assessed by smear examination for acid-fast bacilli (AFB) and M-PCR technique. These patients were treated with Multidrug Therapy (MDT) as prescribed by WHO following detection. A total of 110 MB and 72 PB contacts were studied by performing M-PCR in their nasal swab samples.Results83.4% of MB patients were observed to be positive by smear examination for AFB and 89.2% by M-PCR. While 22.2% of PB patients were found to be positive by smear examination for AFB, 80.3% of these patients were positive by M-PCR. Among leprosy contacts (using M-PCR), 10.9% were found to be positive among MB contacts and 1.3% among PB contacts. Interestingly, two contacts of M-PCR positive MB cases developed leprosy during the period of two years follow up.ConclusionThe M-PCR technique appears to be an efficient tool for early detection of leprosy cases in community based contact tracing amongst close associates of PB and MB cases. Early contact tracing using a molecular biology tool can be of great help in curbing the incidence of leprosy further.

Role of hyperglycemia-mediated erythrocyte redox state alteration in the development of diabetic retinopathy.

Purpose: To evaluate erythrocyte redox state and its surrogates in patients with different stages of diabetic retinopathy and their association with cellular metabolic derangement developed in retinal microvascular cells. Methods: Sixty type 2 diabetic patients with nonproliferative diabetic retinopathy (NPDR), 85 patients with proliferative diabetic retinopathy (PDR), and 70 patients with diabetes but without retinopathy were considered as diabetic control (DC) for the study. In addition, 65 normal individuals without diabetes were enrolled as healthy control in this study. Erythrocyte oxidized nicotinamide adenine dinucleotide phosphate / reduced nicotinamide adenine dinucleotide phosphate (NADP+ / NADPH), oxidized nicotinamide adenine dinucleotide / reduced nicotinamide adenine dinucleotide (NAD+ / NADH) glutathione, plasma and vitreous lactate, and pyruvate levels were determined by enzymatic reaction–based spectrophotometric assay for the patients and individuals. Result: Erythrocyte NADP+ to NADPH ratio to NADPH ratio was found to be significantly higher among NPDR and PDR patients compared with DC subjects (P < 0.0001). Erythrocyte-reduced glutathione was significantly decreased in patients of NPDR (P = 0.0004) and patients of PDR (P = 0.0157) compared to DC. Erythrocyte NAD+ to NADH ratio was also significantly decreased in patients of NPDR (P < 0.0001) and PDR (P < 0.0001) compared to DC subjects. Lactate to pyruvate ratio of plasma was elevated significantly in patients with NPDR compared with DC (P < 0.0001) and those having PDR (P = 0.0046). In the vitreous fluid, the lactate to pyruvate ratios were found to be significantly lower in normal individuals without diabetes compared with patients having PDR (P < 0.0001). Conclusion: Hyperglycemia-mediated erythrocyte redox state alterations might be a potential risk factor for the development of NPDR in poorly controlled diabetic subjects.

Rapid Identification of Mycobacterium Species with the Aid of Multiplex Polymerase Chain Reaction (PCR) From Clinical Isolates

Mycobacteria are aerobic, nonspore forming, non-motile,single-cell bacteria.Of more than 40 currently recognized species of mycobacteria, Mycobacterium tuberculosis, the causative agent of human TB is the commonest pathogen for pulmonary and extra pulmonary tuberculosis cases. The other members of the Mycobacterium tuberculosis complex (MTC) or the nontubercular mycobacterium (NTM) produces similar diseases which cannot be differentiated from tuberculosis by clinical symptoms and signs. But this differentiation is important as the chemotherapy varies widely according to the strain of mycobacterium. The burden of morbidity and mortality of tuberculosis is rapidly growing worldwide, particularly with the HIV/AIDS epidemic. The strain identification of Mycobacterium remains a cumbersome, labor intensive and expensive procedure, which requires 3 to 12 weeks of time. The conventional methods of strain identification lack proper standardization and precise diagnosis. The prime objective of this study is to overcome these problems. A multiplex PCR using 3 amplicons of 165,365, and 541 base pair target sequences was done with a total number of 165 clinical isolates of suspected Koch’s patients. Strain identification was compared both by conventional methods and multiplex PCR. The results of the study show that this multiplex PCR is supposed to be less complicated, less time consuming, cost-effective and superior to the conventional methods. It is also applicable for culture negative samples where strain identification is not possible by conventional approach.

Assessment of gelatinase and tumor necrosis factor-α level in the vitreous and serum of patients with Eales disease: role of inflammation-mediated angiogenesis in the pathogenesis of Eales disease.

Background: Eales disease (ED) is an idiopathic, inflammatory, venoocclusive disorder of peripheral retina resulting in retinal angiogenesis and vitreous hemorrhage. The objective of the present study is to investigate the expression and activation of gelatinase associated with the retinal neovascularization in ED and the relation between the levels of gelatinase and the cytokine tumor necrosis factor-α, known to upregulate matrix metalloproteinase (MMP) expression on various cells. Methods: Vitreous and serum samples from 19 patients with ED who underwent retinal surgery were estimated for levels of MMP-2, MMP-9, tissue inhibitor of metalloproteinase-1, tissue inhibitor of metalloproteinase-2, and tumor necrosis factor-α by enzyme-linked immunosorbent assay method. Matrix metalloproteinase-2 and MMP-9 activities in serum and vitreous samples were evaluated by gelatin zymography method. Vitreous samples from 16 patients with macular hole undergoing vitrectomy were used as controls. Results: Among the 2 gelatinase examined in vitreous and serum samples, only level and activity of MMP-9 were significantly higher in serum (P = 0.0001) and vitreous (P = 0.0002) samples of patients with ED than those of control subjects. Simultaneously, a positive correlation was found between intraocular tumor necrosis factor-α and MMP-9 concentration (Spearman correlation coefficient, r = 0.7040, P = 0.0023) in patients with ED. Conclusion: Increase in MMP-9 activity and its concentration in serum and vitreous of patients with ED compared with that of control subjects and correlation between intraocular levels of MMP-9 and tumor necrosis factor-α in patients with ED seem to provide a plausible explanation for inflammation-mediated angiogenesis during the development of this condition.

Reply: To PMID 22653543.

To the Editor: I would like to thank Dr. Rupak Roy for his interest in our recent article entitled “Role of hyperglycemiamediated erythrocyte redox state alteration in the development of diabetic retinopathy.” In our previous study, we had collected control vitreous samples from 36 normal individuals without diabetes and retinopathy for the estimation of vitreous lactate and pyruvate level. Among these 36 controls, 15 subjects had traumatic dislocation of crystalline lens and 21 subjects had peroperative complication of phacoemulsification. We had excluded trauma cases with vitreous hemorrhage because hemorrhage into the vitreous body results in rapid clot formation and clears at a rate of 1% per day. Erythrocytes exit through the trabecular meshwork, undergo hemolysis and phagocytosis within the vitreous, and may impair in the chemical nature of the vitreous. Emergency vitrectomy was performed for all the subjects, and vitreous were collected through pars plana vitrectomy during removal of dropped nucleus, which occurred accidentally after blunt trauma and peroperative complication of phacoemulsification. Part of researchers believe that vitrectomy in accidental cases were not true normal controls as trauma itself induces various biochemical changes in the vitreous fluid along with high chances of contamination of free radicals in the vitreous. However, it is well known that normal vitreous body of human eye contains hyaluronic acid (HA), which is a potent biopolymer that appears to have an impressive range of biologic functions. Hyaluronic acid has unique hygroscopic, rheological, and viscoelastic properties. It is well accepted that hyaluronan draws water into the fibril network of vitreous to achieve the Donnan equilibrium, adds chemical stability to the vitreous collagen, and separates the fibrils. Wu et al reported that HA is an effective protective agent that serves as an antioxidant in the vitreous body of eye. Previous studies demonstrated that HA could protect cells against cell death, inflammation, and oxidative stress in ocular surface epithelial cells. In cases of accidental blunt trauma, operations were performed on the same day within 4 hours of trauma, and for remaining cases, the vitrectomy was performed immediately after the occurrence of complication of phacoemulsification. Although in our previous work, we have not measured the HA content of control vitreous, but based on earlier study reports and findings, we believe that vitreous HA content of normal controls might protect them against trauma-related inflammation or oxidant reactions. Moreover, the vitrectomies were performed within very short period from the occurrence of an accident. So, these cases did not get enough time to develop inflammation or any oxidation reactions, which were further excluded by anterior and posterior segment evaluation by slit-lamp biomicroscopy and indirect ophthalmoscopy. Hence, we believe that the control vitreous samples, which were used in our previous study, were chemically stable and had less chance of contamination with free radicals.