P. Kar

Role of aflatoxin B1 as a risk for primary liver cancer in north Indian population.

OBJECTIVES The present study was designed to determine whether aflatoxin B1 (AFB1) exposure has any role to play in hepatocellular carcinoma (HCC) patients from northern India. DESIGN AND METHODS A total of 266 HCC patients and 251 patients of chronic liver disease without-HCC were enrolled into the study. All samples were screened for serological markers for hepatitis B and C infections and levels of AFB1 in food and urine samples. RESULTS A threefold (OR=3.43) and five-fold (OR=5.47) increased risk of HCC was observed amongst HBV infection and AFB1-levels in food and urine samples, respectively. However, a non-significant risk was observed with respect to HCV infection (OR=1.27) and alcohol consumption (OR=1.18). A threefold (OR=3.15) increased risk of HCC was observed amongst cases of non-viral etiology with respect to urinary AFB1. CONCLUSION The data provides an exposure and disease risk information for establishing intervention studies to diminish the impact of aflatoxin exposure in Indian population.

Genetic polymorphism of glutathione S transferases M1 and T1 in Indian patients with hepatocellular carcinoma.

Objective: Our aim was to evaluate whether the association of GSTM1/T1 gene polymorphisms modifies the risk of Hepatocellular carcinoma (HCC) and what is its correlation with other predisposing risk factors like alcohol intake, cigarette smoking and hepatitis B and C infections. Study design/setting: It was a case-control study, included 254 HCC cases compared with 525 hospital-based age and sex matched cases of chronic liver disease without HCC as controls from Indian population. The GSTM1 and GSTT1 genotypes were detected using conventional multiplex PCR method. Results: In this case-control study, we observed a positive correlation between age, HBV and HCV infection, smoking habit of > 20 packs/year, alcohol consumption of > 100 g/day and risk of liver cancer. We found significantly increased risk associated with GSTM1 null genotype (OR = 3.49; 95% CI = 2.52–4.84) as well as GSTT1 null genotype (OR = 3.12; 95% CI = 2.19–4.45), respectively. However, an increased risk of HCC was observed among heavy drinkers with GSTM1 (OR = 2.01; 95% CI = 1.11–3.66). Further, cigarette smoking showed a non-significant association with GSTT1 (OR = 1.49; CI = 0.69–3.25). Conclusion: Our results suggest that the variants in low penetrance gene such as GSTM1 and GSTT1 are associated with an increased liver cancer risk. Further, an influence of GSTM1/T1 null genotypes may contribute in the etiology of HCC in patients with higher cigarette and alcohol consumption.

Genetic variants of heat shock protein A1L2437 and A1B1267 as possible risk factors for hepatocellular carcinoma in India.

To study the role of heat shock protein A1L (HSPA1L) and A1B (HSPA1B) polymorphisms and subsequent risk of hepatocellular carcinoma (HCC) in India. Subjects enrolled included 185 cases of HCC, 182 cases of chronic hepatitis (CH) and 200 healthy controls. Genomic DNA was typed for HSPA1L2437 and HSPA1B1267 SNP using polymerase chain reaction with restriction fragment length polymorphism. Other risk factors were also analysed. Hepatitis B virus (HBV) infection, older age >35 years and high aflatoxin level in urine increased the risk of HCC. The frequencies of HSPA1L BB genotype and B allele in HCC were more than in CH [odds ratio (OR): 9.83; P = 0.000], but also in HBV‐related HCC than Chronic Hepatitis B (CHB) [OR: 3.44; P = 0.004] and HCV‐related HCC compared to CHC [OR: 6.32; P = 0.010]. The frequency of HSPA1B genotype in the homozygous state was more in CH [OR: 6.01; P = 0.001] and is a good marker to predict the risk of HCV‐related CH (CHC) compared to controls. HCV‐related HCC has a higher frequency of the B allele of HSPA1B than healthy controls [OR: 3.95; P = 0.000] and CHC [OR: 2.35; P = 0.000], respectively. The frequencies increased further significantly in CHC compared to healthy controls [OR: 9.26; P = 0.000]. The risk for the development of CH and HCC compared to healthy controls irrespective of the aetiology was significant in terms of the HSPA1B marker than HSPA1L in the Indian population.

Role of nitric oxide synthase genes in hepatitis E virus infection.

Hepatitis E virus (HEV) is the most common cause of endemic and epidemic acute hepatitis. A correlation between iNOS, eNOS polymorphisms, levels and severity of disease has been reported, and here, we examined the role of iNOS and eNOS gene polymorphisms and their levels in HEV‐related acute viral hepatitis and acute liver failure. Hepatitis E virus‐related cases of acute hepatitis (294 patients) and liver failure (82 patients) and age‐ and sex‐matched healthy controls (331 subjects) were included in the study. PCR‐RFLP was performed to identify the polymorphisms in the iNOS and eNOS genes. iNOS and eNOS levels were studied using ELISA assays and HEV viral load, genotype and combined effects of iNOS genotype, levels and parameters for disease severity were examined. The frequency of iNOS (CT + TT) and eNOS (GT + TT) genotypes was higher in subjects with liver failure compared with controls. iNOS and eNOS levels in patients with acute liver failure (55.51 ± 6.33 IU/mL, 60.2 ± 3.69) cases were significantly increased as compared to patients with acute viral hepatitis (17.8 ± 6.08 IU/mL, 23.7 ± 6.57) and controls (P < 0.05). A significant positive correlation was observed between the iNOS and eNOS levels in our study population when compared with the severity of disease parameters. Hence, the iNOS C150T polymorphism and the eNOS G894T polymorphism and high levels of iNOS and eNOS are associated with an increased risk of HEV‐related acute hepatitis and liver failure. This study supports the possible role of nitric oxide synthase genes (iNOS and eNOS) in determining the severity of HEV infection.

Predictive value of serum actin‐free Gc‐globulin for complications and outcome in acute liver failure

This prospective study was designed to evaluate whether early changes in actin‐free Gc‐globulin levels were associated with complications and outcomes and to identify factors associated with persistent low actin‐free Gc‐globulin levels in acute liver failure (ALF). Thirty‐two consecutive ALF patients admitted from October 2011 to December 2012 were followed up until death or complete recovery. All had serum actin‐free Gc‐globulin estimation at admission and at day three or expiry. Logistic regression analysis was performed to identify independent predictors of mortality. A receiver operating characteristic curve analysis was also performed. Nonsurvivors had significantly lower median actin‐free Gc‐globulin levels than survivors (87.32 vs 180 mg/L; P < 0.001). A receiver operating characteristic curve analysis revealed an area under curve (AUC) of 0.771 and showed that serum actin‐free Gc‐globulin level of ≤124 mg/L would predict mortality with 92% sensitivity and 71.4% specificity. Patients with lower serum actin‐free Gc‐globulin levels and decreasing trend in serum actin‐free Gc‐globulin levels were found to have more mortality and developed more complications. Logistic regression analysis showed that serum actin‐free Gc‐globulin, total leucocyte count and serum creatinine at admission were independent predictors of mortality. Incorporating these variables, a score predicting mortality risk at admission was derived. The scoring system was compared to MELD score and Kings College Criteria as individual predictor of mortality. Serum actin‐free Gc‐globulin level at presentation is predictive of outcome and can be used for risk stratification. Its persistent low‐level predicts mortality and is correlated with various complications.

Analysis of carriers of hepatitis B virus from a tertiary referral hospital: Does the viral load change during the natural course of infection?

This study was designed to determine the prevalence, forms of transmission, mutational profile and viral load at baseline of hepatitis B virus (HBV) carriers in Delhi. HBV surface antigen (HBsAg)‐positive patients were enrolled and evaluated clinically for liver function, serological markers for hepatitis B and HBV DNA quantitation. Tests were carried out again 1 year later and the results were compared. Liver biopsy was carried out on all carriers with active viral replication. HBV DNA‐positive samples were subjected to polymerase chain reaction single‐stranded conformation polymorphism (PCR‐SSCP) to screen mutations in the Precore, core, and the X‐gene prior to sequencing analysis. Among the 100 patients examined, HBeAg was detected in 23% and 40% were HBV DNA‐positive. Of the 40 HBV DNA‐positive cases, 8 had precore/core mutations, [G1896A (10%), T2066A (12.5%), T2050C (10%), and G1888A (7.5%)]. No X gene mutants were detected. Reduction in viral load was higher in HBeAg‐positive patients, as compared to HBeAg‐negative patients, over 1 year. At follow‐up, 2/8 HBV mutants corresponded with altered liver function and morphological changes suggestive of chronic hepatitis. One patient was re‐designated as DNA‐negative on follow‐up and had wild‐type virus infection with a relatively low viral load. The predominant route for HBV transmission was determined to be parenteral. Twenty percent of the HBV carriers were infected with precore and core mutant HBV. Although the clinical and biochemical profiles of these HBV carriers remained largely stable on follow‐up, there was evidence of spontaneous reduction in the mean viral load over the 1‐year study period. J. Med. Virol. 83:1151–1158, 2011.

rs2230201 polymorphism may dictate complement C3 levels and response to treatment in chronic hepatitis C patients

The basis of response of chronic hepatitis C (CHC) patients to treatment is still unclear, and there may be many other factors which influence treatment outcome other than the existing ones. The serum concentration of C3 closely reflects the total complement activity, and individuals affected by C3 deficiency suffer from recurrent pyogenic infections. This study aims to find out relationship between levels of C3 in serum and its functional SNPs with response to treatment. The study included 132 CHC patients of which 48 received Pegylated IFN+Ribavirin and 81 controls. C3 levels and its three known functional SNPs genotyped by ELISA and SSP PCR, respectively. C3 Level of the healthy group was significantly higher (88.5 ± 19 mg/dL) when compared to CHC group (56 ± 18 mg/dL; P < 0.001). Thirty‐three of 36 responders were rs2230201 CC genotype carriers, whereas 9 of 12 nonresponders were non‐CC genotype. The ‘C’ allele of rs2230201 was found to be associated with increased serum C3 levels when compared to other genotypes in healthy group, whereas CT genotype was associated with lowered serum C3 in CHC group. A serum C3 value of <53 mg/dL was predictive of SVR with sensitivity 63.89% and specificity 66.67%. The study supports the observation that rs2230201 ‘C’ allele is associated with increase of serum C3 levels when compared to ‘T’ allele which may confer advantage in attaining SVR when present in homozygous condition. The study suggests that patients with serum C3 value <53 mg/dL and non‐CC genotypes may not respond to treatment.

Role of complement component C4 in treatment response and disease progression in chronic hepatitis C patients.

The basis of hepatitis C virus (HCV) evasion of immune system and its response to treatment is still elusive. There have been studies where the level of C4 has been found to be associated with HCV persistence and disease progression. This study aims to find out relationship between levels of C4 in serum, and its functional SNPs with response to treatment. The study included 84 patients with CHC who received treatment and 75 healthy controls. C4 expression, both at mRNA and protein level, was estimated by Real time and ELISA respectively. Its functional SNPs genotyped by AS‐PCR. The mean ± SD baseline C4 levels between the disease and healthy cases was significantly different (1075.74 ± 65.25 vs 1593 ± 24.55 ng/mL, P < 0.001). The mean ± SD baseline C4 levels of CC, GC and GG genotype of rs2857009 in the healthy group were 1540.97 ± 7.87, 1599.53 ± 11.75 and 1604.86 ± 10.79 ng/mL, respectively (P < 0.001), whereas the levels in the CHC group were 1022.81 ± 32.95, 1058.19 ± 55.02 and 1150.26 ± 14.64 ng/mL, respectively (P < 0.001). CC genotype resulted in decreased C4 mRNA levels compared to GG genotype in healthy group (3.81‐fold) and CHC group (1.4‐fold). The CC genotype of rs2857009 is associated with reduced expression of C4, both at mRNA and protein level. The C4 serum level at baseline and total protein were found to be independent predictors for treatment response. New predictive score using the above factors, a value of ≥0.542 was found to predict positive response to treatment. Increased age, rs2857009 SNP and HCV genotype were associated with disease progression.

Hepatitis G virus (HGV) infection and its pathogenic significance in patients of cirrhosis

In the present study the hepatitis G virus (HGV) infection and its pathogenic significance in patients of cirrhosis were assessed using reverse transcription plus nested polymerase chain reaction (RT-PCR). Serum samples were collected from a total of 50 patients of histologically proven non-alcoholic cirrhosis and from a control group consisting of 50 healthy voluntary blood donors. HGV RNA was detected by RT-PCR using primer sequences located in the conserved NS3 helicase region of HGV genome. Serological evaluation for markers of chronic infection with HBV (HBsAg, IgG anti-HBc, HBeAg) and HCV (anti-HCV) was carried out using commercially available kits. HBV DNA and HCV RNA were also tested by PCR in those samples that were found to be non-B, non-C by serological assays. Serological evidence of exposure to HBV was found in 31 (62%) and to HCV in 15 (30%) patients. HGV RNA was detected in 6 (12%) cirrhosis patients and in 2 (4%) healthy blood donors but the difference between the two groups was not statistically significant. Of the 6 HGV positive patients, 2 were coinfected with HBV, 1 with HCV, while the remaining 3 belonged to non-B, non-C category. No significant difference was observed in the clinical and biochemical profiles of HGV-positive and HGV-negative patients except that a history of blood transfusion was significantly (P < 0.005) more common in the former. The findings indicate that the HGV infection is commonly observed in both cirrhosis patients as well as healthy blood donors. A significant association of the virus with blood transfusion is indicative of a parenteral route of transmission. The observations of this study also suggest that the pathogenic role of HGV in the causation of liver disease may be insignificant.

1001 EXPRESSION ANALYSIS OF NF-κB IN PATIENTS WITH ACUTE LIVER FAILURE DURING PREGNANCY

Background and Aims: There is paucity of information on the pathophysiological mechanisms of the hepatic damage during acute liver failure caused by hepatitis viruses. Nuclear Factor Kappa B transcription factor, a key regulator of genes involved in response to infection, inflammation and stress leads to production of inflammatory cytokines and apoptotic stimuli. The study was aimed to determine any role of NF-uB in the death of acute liver failure women during pregnancy. Methods: A total of 60 patients were included in the study which constituted of 25 acute liver failure patients and controls consisting of 20 healthy blood donors, 10 healthy pregnant females and 5 autopsy cases. All the samples were subjected to serological analysis, PCR and nuclear protein extraction was carried out. Western blotting analysis was carried out to detect the expression of NF-uB using primary antibodies specific for p65 and p50 subunits. Results: Mortality rate in the pregnant female acute liver failure patient was very high 75% (9/12) but no significant difference was observed between the pregnant and nonpregnant patients. The expression of p50 protein in blood was very high78.94% (15/19) in the HEV infected acute liver failure cases while in majority of the cases 52.89% (11/19), p65 showed nil expression in the HEV infected cases. In the pregnant acute liver failure patients, expression of essential p65 was nil in 58.3% (7/12) cases and low in majority 60% (6/10) of the healthy pregnant patients showing nil expression in the HEV infected cases. The expression of p50 protein was very high in 6 cases (6/10, 60%) of the post mortem liver tissue whereas 3 (3/5, 60%) controls showed normal expression whereas in the surviving cases moderate expression of p50 was observed in the blood of all the six cases compared to p65 which showed low to nil expression in all the cases. Conclusion: p65 is an essential component for normal functioning of the NF-uB complex and its absence causes increased apoptosis and liver degeneration leading to liver damage and death in acute liver failure patients confirming the anti-apoptotic role of NF-uB.