Nibhriti Das

Complement Receptor 1: disease associations and therapeutic implications.

Abstract Exaggerated complement activation is a key event in the pathogenesis of a range of autoimmune and inflammatory diseases. Complement Receptor 1 (CR1) has emerged as a molecule of immense interest in gaining insight to the susceptibility, pathophysiology, diagnosis, prognosis and therapy of such diseases. This review brings forth a composite view of the current understanding on the structure, functions, genetics, disease associations and therapeutic implications of CR1.

Expression of complement regulatory proteins in diffuse proliferative glomerulonephritis.

This study assessed the expression of complement receptor 1 (CR1), decay accelerating factor (DAF) and membrane inhibitor of reactive lysis (CD59) on the erythrocytes and glomerulus of diffuse proliferative glomerulonephritis (DPGN) of systemic lupus erythematosus (SLE) patients using flow cytometry and immunofluorescence techniques to elucidate their role in the pathogenesis of DPGN. Expression of CR1 on the erythrocytes and glomerulus of DPGN patients was reduced compared with expression in normal subjects. However, expression of DAF and CD59 was increased on both erythrocytes and glomerulus of DPGN patients, suggesting the generation of a protective response against complement-mediated injury.

Immunomodulatory effect of DDT (bis[4-chlorophenyl]-1,1,1-trichloroethane) on complement system and macrophages.

DDT (bis[4-chlorophenyl]-1,1,1-trichloroethane) is responsible for many immuno-dysregulatory functions in exposed animals, but data particularly on complement system and macrophages are limited. In this study we have shown that DDT activates the complement system through the alternative pathway in the absence of any pathogen. A significant (p<0.05) increase in C3b, C3d and C3a generation, and decline in complement hemolytic activity was observed in insecticide exposed sera. The uncontrolled complement consumption reduces the lytic activity of the complement, which enhances the susceptibility to pyogenic infection if the exposure to DDT remains unabated. Further, DDT induced the significant (p<0.05) production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in macrophages and thus contributes inflammatory reactions, cytokine imbalance and immune-dysregulation. These molecular changes in macrophages lead to structural aberrations like heterochromatin condensation, loss of pseudopodia, cytoplasmic vacuolization, DNA fragmentation and hypodiploid nuclei as seen in our study, suggesting apoptosis. However, in presence lipopolysaccharide, DDT induced significant (p<0.05) suppression of TNF-alpha and NO generation, suggestive of impairment of macrophage microbiocidal effects. This study concludes that the functional and structural derangements of macrophages in association with uncontrolled and excessive complement consumption by DDT are perhaps one of the major mechanisms contributing to the immunosuppressive effects of insecticide.

Apolipoprotein E polymorphism in Northern Indian patients with coronary heart disease: phenotype distribution and relation to serum lipids and lipoproteins.

Apolipoprotein E (apo E), a genetic determinant of plasma lipid levels and coronary heart disease (CHD) needs to be investigated in Asian Indians since they have a propensity to develop dyslipidemia and accelerated atherosclerosis. We studied apo E phenotypes and plasma lipid levels in 52 Northern Indian male patients (aged 38–71 years) with angiographically proven CHD, and compared them to 50 healthy blood donors taken as the control group. High levels of Lp(a), (p < 0.05), and a definite trend towards lower levels of HDL-C (p < 0.05), was observed in the CHD patients as compared to the control subjects. The frequency of apo E allele ε3 was 0.86 and 0.862, and ε4 allele was 0.12 and 0.08 in the patients and controls, respectively. However, a lower frequency of the E2 allele was observed in the patient group (ε2 = 0.02) as compared to the controls (ε2 = 0.06) (p = ns). In individuals with apo E3/E3 phenotype, significantly lower HDL-C levels was observed in the CHD patients as compared to the control subjects (p < 0.05). A positive correlation was observed between apo E phenotypes and Lp(a) levels in the CHD subjects as compared to the controls (p < 0.05), the level being significantly high in CHD subjects with at least one E4 allele. To conclude, in this sample of Northern Indian subjects with CHD, there is a significant correlation between apo E3/E3 phenotype and low levels of HDL-C as compared to the control subjects. Further, apo E phenotype is positively correlated with high Lp(a) levels in the CHD subjects having at least one E4 allele. However, these relationships need to be explored in a larger sample of subjects.

Cytokine imbalance in Systemic Lupus Erythematosus: A study on northern Indian subjects

Objective: The phenotype of systemic lupus erythematosus (SLE) in Asian Indians is more severe as compared with that in Caucasians. The reason is not clear. In this context, we studied serum levels of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin-4 (IL-4) and interlekin-10 (IL-10). Their interrelations and correlation with SLEDAI scores were evaluated. Materials and methods: Forty patients with active SLE and 40 controls were studied. The mean SLEDAI score and anti-ds DNA level of the patients were 21.45 ± 8.61 and 176.68 ± 81.31 (IU/ml), respectively. Cytokines were estimated by enzyme-linked immunosorbent assay. Results: In controls, the levels of IFN-γ were highest, followed by TNF-α, IL-10 & IL-4. In patients, however, the levels of TNF-α were highest, followed by IFN-γ, IL-10 & IL-4. IL-10 and IL-4 correlated negatively, and IFN-γ and TNF-α correlated positively with the SLEDAI scores. As compared with controls, in patients, the mean values of TNF-α, IL-10 and TNF-α/IL-10 ratio were higher by 6.9, 2.9 and 2.3 times, respectively (p < 0.001). Significant positive correlation was found between these two cytokines in patients (r = 0.327, p < 0.05) but not in controls. The levels and ratio of IL-4 and IFN-γ were comparable between patients and controls. These two cytokines correlated negatively both in controls (r = −0.358, p < 0.05) and patients (r = −0.990, p < 0.001). The ratio of TNF-α/IL-4 was 4.2 times higher, and those of IFN-γ/IL-4 and IFN-γ/IL-10 were 1.89 and 3.40 times lower in patients as compared with controls. A positive correlation between IL-10 and IL-4 (r = 0.345, p < 0.05) and a negative correlation between IL-10 and IFN-γ (r = −0.382, p < 0.05) were observed only in patients. Conclusion: This study showed a distinct profile of cytokine imbalance in patients with SLE from the northern plains of India. The levels, ratios and correlations of cytokines in patients suggested significant deviation from normal. Correlations of cytokines with SLEDAI scores indicated that TNF-α contributes significantly to the pathological manifestations of SLE in patients from the region. A detailed study is warranted.

Prevalence of eNOS Glu298Asp Polymorphism in Healthy Volunteers from a Region of Northern India

Objective: Endothelial nitric oxide synthase (eNOS) Glu298Asp polymorphisms are under extensive study worldwide due to their suggested role in cardiovascular disorders. This polymorphism had gained more attention since several reports suggest its association with hypertension and coronary artery disease (CAD). Asian Indians are highly susceptible to ischemic heart dis eases. We determined the prevalence of eNOS Glu298 Asp polymorphism in 139 healthy volunteers from Delhi and the surrounding areas. The subjects were recruited from those who willingly participated in this study in response to a publicized call and a standard questionnaire. Male to female ratio was 2.7:1 due to the larger number of male participants in this investigation. This, however, does not represent normal male to female distribution in the area. Despite the male bias, this investigation was justified. The prevalence of CAD in males is about 3 times higher in this region and no data had so far been available on the distribution of this polymorphism from India. Method: The eNOS Glu298Asp polymorphism was studied by PCR-RFLP. Results: Distribution of genotype GG, GT and TT in the study subjects was found to be 71.22, 28.06 and 0.72%, respectively, and allele frequency was G,0.853;T,0.147. Conclusion: T allele had been described as susceptibility allele for CAD in several population studies. The frequency of the T allele was found to be two times higher in our subjects than that reported for Japanese and Korean populations. This study does not provide any direct evidence for eNOS gene disease associations but is the first report on the prevalence of eNOS Glu298Asp gene polymorphism in Indian subjects. Whether the observed pattern of eNOS Glu298Asp polymorphism contributes to the greater susceptibility of Asian Indians to CAD as compared to the other population groups, needs to be investigated.

Genetic and structural polymorphism of complement receptor 1 in normal Indian subjects.

The human complement receptor 1 (C3b/C4b receptor, CD35, CR1), a polymorphic membrane bound glycoprotein, is differentially expressed on erythrocytes, eosinophils, monocytes, B and T-lymphocytes, dendritic cells and kidney podocytes. It also occurs in the plasma as soluble CR1 (sCR1) and in urine as urinary CR1 (uCR1). Different population studies have suggested the functional and physiological significance of the structural (CR1-A/190, CR1-B/220, CR1-C/160 and CR1-D/250 kDa) and genomic (HH, high erythrocyte CR1 expression, HL, intermediate and LL, low expression) polymorphisms in health and disease. However, simultaneous study on the structural and genomic polymorphism in the same group of study subjects is lacking. This is the first study on both quantitative and structural polymorphism in 101 healthy volunteers from different parts of India by random sampling. In our study, AA phenotype was found to be expressed in 84.2% of individuals and 14.8% carried AB phenotype. One individual (0.9%) was found to possess BB phenotype. Homozygous BB pattern was identified for the first time in Indian subjects. The relative gene frequencies for A and B allele were found to be 0.916 and 0.084, respectively. Pertaining to quantitative polymorphism, percentage distribution for HH, HL and LL phenotypes was found to be 23.7, 54.45 and 21.79%, respectively and the gene frequencies were 0.51 and 0.49 for H and L allele, respectively. The observations for quantitative as well as structural polymorphism showed a good probability of fitness with Hardy-Weinberg equilibrium, thus proving that both the types of CR1 polymorphic forms are encoded by autosomal co-dominant alleles. We found a higher frequency of HL and AA phenotypes in the study subjects. Our findings are unique as we found that gene frequencies for structural and quantitative polymorphism in our study subjects were a combination of those found in Caucasian and Oriental populations.

Apolipoprotein C3 SstI polymorphism in the risk assessment of CAD

Various population studies have reported the association of rare S2 allele of apolipoprotein C3 (APOC3) SstI polymorphism with hypertriglyceridemia (HTG) and coronary artery disease (CAD). We were the first to report an association of S2 allele with high triglyceride (TG) levels in healthy volunteers from Northern India. Since HTG is suggested to be a predominant risk factor for CAD among Indians, we have elucidated the relationship of APOC3 SstI polymorphism with the lipid profile and CAD. A total of 158 patients with ≥ 70% stenosis in one or more coronary artery (angiographically proven CAD patients), 35 subjects with < 70% stenosis (NCAD) and 151 normal controls (free of heart disease) from Northern plains of India were recruited in the study. DNA samples were analyzed by polymerase chain reaction (PCR) followed by SstI digestion. Lipid profile was estimated by enzymatic kit. We found a strong association of S2 allele with high TG levels, which was more significant in patients. Prevalence of S2 allele in normal controls and CAD patients were comparable, despite the fact that mean TG level was significantly higher in patients. A greater insight into this observation revealed that the prevalence of high TG, if not coupled with other risk factors (like high total cholesterol, low HDL), was comparable in patients and controls. Thus, our study reveals that rare S2 allele may be employed as a susceptibility marker for high TG. However, high TG or S2 allele alone may not contribute to the etiology of CAD.

Effect of ascorbic acid on prevention of hypercholesterolemia induced atherosclerosis.

The notion that oxidation of lipids and propagation of free radicals may contribute to the pathogenesis of atherosclerosis is supported by a large body of evidence. To circumvent the damage caused by oxygen free radicals, antioxidants are needed which provide the much needed neutralization of free radical by allowing the pairing of electrons. In this study we have investigated the effect of ascorbic acid, a water soluble antioxidant on the development of hypercholesterolemia induced atherosclerosis in rabbits. Rabbits were made hypercholesterolemic and atherosclerotic by feeding 100 mg cholesterol/day. Different doses of ascorbic acid were administered to these rabbits. Low dose of ascorbic acid (0.5 mg/100 g body weight/day) did not have any significant effect on the percent of total area covered by atherosclerotic plaque. However, ascorbic acid when fed at a higher dose (15 mg/100 g body weight/day) was highly effective in reducing the atherogenecity. With this dose the percent of total surface area covered by atherosclerotic plaque was significantly less (p < 0.001). This suggests that use of ascorbic acid may have great promise in the prevention of hypercholesterolemia induced atherosclerosis.

Two- and three-locus haplotypes of the paraoxonase (PON1) gene are associated with coronary artery disease in Asian Indians.

INTRODUCTION In view of the reported association of SNPs in the paraoxonase (PON1) gene with coronary artery disease (CAD), and the absence of conclusive data from India, we investigated the relationship of three SNPs at different loci (-108C/T, L55M and Q192R) of the PON1 gene and their haplotypes with CAD among people residing in the northern plains of India. MATERIALS AND METHODS One hundred and seventy-eight healthy controls and two hundred and four angiographically-proven CAD patients were genotyped using PCR-RFLP. RESULTS Of the three SNPs, only the R allele of Q192R polymorphism was associated with CAD (p<0.05). Two locus haplotypes QT (OR 0.55, p=0.0004, 95% CI 0.39-0.77, significant) and LQ (odds ratio 0.73, p=0.03, 95% CI 0.55-0.97, trend) showed protective effects, while haplotypes MR (OR=5.36, p=0.0001, 95% CI 2.045-14.049) and MC (OR=2.71, p=0.011, 95% CI 1.221-6.046) were associated with increased risk of CAD. MRT, a minor three-locus haplotype also displayed significant association (OR 4.93, 95% CI 1.7-13.5) with the disease. Significance was assessed after applying Bonferronis correction. CONCLUSIONS Our study revealed that only one SNP at a single locus but several haplotype combinations of PON1 coding and promoter-region polymorphisms were associated with the risk of or protection against CAD. Thus, haplotype analysis brought better insights into the association of PON1 gene polymorphisms with CAD in Asian Indians.