Association of interleukin-18 genotypes (-607C > A) and (-137 G > C) with the hepatitis B virus disease progression to hepatocellular carcinoma.

Chronic infection with HBV has been reported to be associated with the development of HCC. The inflammation mounted by cytokine-mediated immune system plays an important role in the pathogenesis of HBV-associated HCC. IL-18 is a pro-inflammatory cytokine whose role in the development of HBV-associated chronic to malignant disease state has not been much studied. The present study was conceived to determine the role of genetic polymorphisms in IL-18, serum levels of IL-18, and expression level of its signal transducers in the HBV disease progression. A total of 403 subjects were enrolled for this study including 102 healthy subjects and 301 patients with HBV infection in different diseased categories. Polymorphism was determined using PCR-RFLP. Genotypic distributions between the groups were compared using odd's ratio and 95% CI were calculated to express the relative risk. Circulating IL-18 levels were determined by ELISA. Expression levels of pSTAT-1 and pNFƙB was determined by western blotting. In case of IL-18(- 607C > A), the heterozygous genotype (CA) was found to be a protective factor while in case of IL-18(- 137G > C) the heterozygous genotype (GC) acted as a risk factor for disease progression from HBV to HCC. Moreover, serum IL-18 levels were significantly increased during HBV disease progression to HCC as compared to controls. Also the levels of activated signal transducers (pSTAT-1 and pNF-κB) of IL-18 in stimulated PBMCs were significantly increased during HBV to HCC disease progression. These findings suggest that IL-18 has the potential to act as a biomarker of HBV-related disease progression to HCC.

Association of rno-miR-183-96-182 cluster with diethyinitrosamine induced liver fibrosis in Wistar rats.

Chronic liver injury due to various etiological factors including environmental carcinogens results in development of liver fibrosis. Numerous studies showed role of miRNAs in liver fibrosis. In the present study, we determined the rno-miR-183-96-182 cluster expression during hepatic fibrosis induced by diethylnitrosamine (DEN) treated Wistar rats and its association with plasma levels of circulating rno-miR-96, rno-miR-182, rno-miR-183, liver function test and lipid profile, aiming to identify their potential for histological stratification and early diagnosis of liver fibrosis. We found significant upregulation in the hepatic expression of rno-miR-183-96-182 cluster upon development of fibrosis in a DEN treated rats. Interestingly, the hepatic expression of this miRNA cluster correlates positively with the progression of fibrosis. Univariate analysis showed that hepatic expression of rno-miR-182-5p and rno-miR-183-5p and plasma activity of ALT are significant predictors of fibrosis. Multivariate logistic regression analysis revealed a panel of rno-miR-182-5p and ALT that can discriminate F2-F3 from F0-F1 (AUC = 0.87; P-value < 0.001), F4-F5-F6 from F0 to F1 (AUC = 0.981; P-value < 0.001), and F4-F5-F6 from F2 to F3 (AUC = 0.824; P-value < 0.001). A significant positive correlation of rno-miR-183-96-182 cluster members was also observed with plasma activities of ALT, AST, ALP, and levels of total cholesterol, HDLc and LDLc during fibrosis progression in DEN treated Wistar rats. Thus, it can be concluded that rno-miR-183-96-182 cluster being significantly up regulated and associated with chronic liver disease might play a role in fibrosis maintenance and progression. A panel of rno-miR-182-5p and ALT being significant predictors of fibrosis might improve histological stratification of fibrosis staging.

IFN-γ (+874) and not TNF-α (-308) is associated with HBV-HCC risk in India.

Tumor necrosis factor (TNF)-α and interferon (IFN)-γ, the pro-inflammatory Th1 cytokines are the indispensable coordinators of the inflammatory responses involved in hepatitis B virus (HBV) pathogenesis. This study attempted to evaluate any possible association among TNF-α (-308G>A) and IFN-γ (+874T/A) genotypes, the spontaneous blood and mRNA levels and expression of their major signal transducers, namely STAT1 and NF-кB with hepatitis B virus-induced hepatocellular carcinoma (HCC) susceptibility in India. For this, 398 subjects (146 controls, 68 inactive-HBV-carriers, 64 chronic-active HBV patients, 61 HBV-cirrhotics, and 59 HBV-HCC subjects) were enrolled. Polymerase chain reaction-restriction fragment length polymorphism, allele-specific PCR, enzyme-linked immunosorbent assay, reverse transcriptase-PCR, and Western blot analysis were done for assessing polymorphism, blood levels, mRNA expression, and protein expression of signal transducers, respectively, of TNF-α and IFN-γ. The study revealed no significant association of TNF-α (-308) GA genotype, while a significant negative association of IFN-γ (+874) TA and AA genotypes, in HBV-HCC risk. Moreover, blood levels of TNF-α were significantly elevated as disease progresses to HCC, while IFN-γ levels were raised in HCC patients only. Besides, IFN-γ mRNA levels were significantly elevated in cirrhotics, with no change observed in TNF-α transcript levels. Moreover, NF-кB expression also consistently increased during HCC progression. These observations suggest a vital negative association of IFN-γ (+874) with HBV-HCC risk, with no significant association evident in TNF-α (-308). However, the TNF-α and IFN-γ levels markedly increased in HCC development.

IL-6(-572/-597) polymorphism and expression in HBV disease chronicity in an Indian population.

OBJECTIVES: This study evaluated the association among IL-6(-572) and IL-6(-597) genotypes, haplotypes, mRNA, and protein levels with hepatitis B virus (HBV)-Hepatocellular carcinoma (HCC) risk in India. METHODS: For this, 403 participants (153 controls, 61 inactive HBV-carriers, 65 chronic-active HBV patients, 63 HBV-cirrhotics, and 61 HBV-HCC participants) were enrolled in the study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), ELISA, and RT-PCR methods were used for assessing polymorphism, protein, and the mRNA levels, respectively, of IL-6. RESULTS: The study revealed that the IL-6(-572) GC genotype shared a positive association with hepatitis among controls, and a negative association with cirrhosis and consequent HCC development among carriers. However, the CC genotype shared a significant negative association with cirrhosis among controls and carriers. The IL-6(-597G>A), GA genotype acted as a potential protective factor for hepatitis, cirrhosis, and subsequent HCC development among carriers. The GA and CG haplotypes acted as a vital risk factor for HCC among controls and carriers. On the contrary, the CA haplotype was found to be a potential protective factor for HCC among carriers. Besides, the IL-6 levels significantly increased with cirrhosis development, as compared to carriers and hepatitis subjects. CONCLUSIONS: These preliminary findings indicate a potential role of IL-6(-572/-597) genotypes in HBV disease pathogenesis in an Indian population.

Association of interleukin-10 with hepatitis B virus (HBV) mediated disease progression in Indian population.

BACKGROUND & OBJECTIVES: Interleukin (IL)-10, an anti-inflammatory Th2 cytokine, is one of the key coordinators of the inflammatory responses involved. The present study was designed to evaluate the impact of IL-10 (-819/-592) genotypes, haplotypes, mRNA and the protein levels with risk for hepatitis B virus (HBV) related hepatocellular carcinoma (HCC) development in India. METHODS: A total of 390 subjects (145 controls, 62 inactive HBV-carriers, 64 chronic-active HBV patients, 60 HBV related cirrhotics and 59 HBV- HCC subjects) were enrolled in the study. Allele specific (AS)-PCR, ELISA and RT-PCR methods were used for assessing polymorphism, spontaneous blood levels and the mRNA expression, respectively of IL-10. RESULTS: The study revealed that the CC/TA genotype acted as a risk factor for cirrhosis (OR a =2.02; P<0.05) and the subsequent HCC development (OR a =2.20; P<0.05), with controls as reference. However, no significant association was found between the two haplotypes (CC and TA) observed and HCC risk. Moreover, the IL-10 protein and mRNA levels in peripheral blood mono nuclear cells (PBMCs) showed a significant elevation as the disease progressed to cirrhosis. But, no variation was observed in the IL-10 levels in subjects with different IL-10 genotypes. INTERPRETATION & CONCLUSIONS: These preliminary results suggest a strong association of IL10 (-819/-592) with the HBV infection mediated disease progression, from inactive carrier state to malignancy, in Indian population.

Indocyanine green clearance test (using spectrophotometry) and its correlation with model for end stage liver disease (MELD) score in Indian patients with cirrhosis of liver.

BACKGROUND: Child Turcotte Pugh (CTP) score and Model for End Stage Liver Disease (MELD) are used commonly to assess the prognosis of liver disease but the disadvantage of these static tests is their inability to identify the functional reserve of the liver. Among all quantitative liver function tests indocyanine green (ICG) clearance test is most widely used and has been used to determine operative risk before hepatectomy and to assess prognosis of patients with cirrhosis. AIM: To correlate indocyanine green (ICG) clearance test with MELD score in patients with cirrhosis of liver. METHODS: Forty patients with cirrhosis of liver were included and divided into two groups according to their CTP scores. Group A had 20 patients with CTP class A and group B had 20 patients with CTP class B. After ICG injection, ICG retention at 15 minutes (ICGR 15) and ICG clearance rate were calculated. RESULTS: In group A, the mean ICGR15 was 32.86% +/- 6.4% while in group B it was 51.08% +/-12.8% (p <0.001). ICG clearance rates were 4.3% +/- 2.8% and 3.5% +/- 3.8% per minute in group A and B respectively. MELD score had a strong positive correlation with ICGR15 but a negative correlation with ICG clearance rate. On ROC curve analysis, AUC for MELD was 0.805 vs. 0.88 for ICGR15 in assessing prognosis of patients with cirrhosis. The sensitivity and specificity of MELD score was 60% and 80% respectively while that of ICGR15 was 85% and 90% respectively. CONCLUSION: ICGR15 has a higher sensitivity and specificity than MELD score in assessing the prognosis of patients with cirrhosis of liver.

Distribution of XRCC1 genotypes in north Indian population.

BACKGROUND & OBJECTIVES: XRCC1, a major DNA repair gene, acts as a scaffold of different activities involved in repair by interacting with components of base excision repair (BER) at the site of damage. Polymorphisms in this gene are associated with variations in the repair efficiency which might predispose an individual to cancer risk. To associate a gene polymorphism with disease risk, it is imperative to have the data for its genotype distribution in normal population. The present study was therefore carried out to find distribution of XRCC1 polymorphisms (codons 194, 280 and 399) in normal north Indian population. METHODS: Healthy volunteers hailing from north India (150) were enrolled in the study. DNA was isolated from blood samples and genotyping of codons 194, 280 and 399 of XRCC1 gene was done by PCR- restriction fragment length polymorphism (RFLP), using specific primers. RESULTS: The frequencies obtained for heterozygous genotype of codons 194 and 399 were 45 and 49 per cent respectively and were higher than wild and variant genotypes. For codon 280, the highest frequency (59%) was obtained for the wild genotype. Frequencies of the variant genotypes of codons 194 and 399 were higher in males and females respectively. The allele frequencies also followed the similar trends. INTERPRETATION & CONCLUSIONS: A significant distribution of variant and heterozygous XRCC1 genotypes was noticed that warrants further studies on the association between these genotypes and disease risk in our study population.

Polymorphism of DNA repair gene XRCC1 and hepatitis-related hepatocellular carcinoma risk in Indian population.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the life-threatening malignancies worldwide with hepatitis B and C virus infection as the major risk factor. The risk of HCC might also increase because of the hereditary genetic defects in DNA repair genes. In this regard, X-ray cross-complementing group 1 gene (XRCC1) is a major DNA repair gene involved in base excision repair (BER). AIM: The present study was designed with an aim to find out any possible association between XRCC1 (codons 194, 280, and 399) polymorphisms and the risk of developing hepatitis virus-related HCC in Indian population. METHODS: A total of 407 subjects comprising (170 controls, 174 chronic viral hepatitis, and 63 HCC subjects) were included in the study. PCR-RFLP was used for the genotyping of the three codons of XRCC1. RESULTS: The study revealed that two genotypes Arg194Trp and Arg280His increased the risk of HCC by 2.27- (95% CI = 1.01-5.08; P < 0.001) and 4.95-folds (95% CI = 2.48-9.89; P < 0.001), respectively. Interestingly, the risk for HCC was further enhanced by 35.96 (95% CI = 11.64-110.91; P < 0.001) and 5.28 times (95% CI = 2.81-9.09; P < 0.001) when the genotype Arg280His was found in association with Arg194Trp and Arg399Gln, respectively. CONCLUSION: These preliminary results suggest a positive association of XRCC1 genotypes and risk of hepatitis virus-related HCC in India.

Th1/Th2 cytokines and their genotypes as predictors of hepatitis B virus related hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), the predominant type of primary liver cancer, is one of the most serious life-threatening malignancies, worldwide. In majority of the cases, HCC develops after prolonged and persistent chronic liver disease. hepatitis B virus (HBV) or HCV infection is prominent etiological factors, attributing to this condition. It has been well documented that HBV, being the inducer of chronic inflammation, is the main causative agent in causing HCC, particularly in Asian countries. The HBV infection leads to a wide range of clinical symptoms from carrier state to malignancy. Cytokines being immune-modulatory molecules, are the key mediators in the defense mechanism against viral infection. In this regard, this review will detail the substantial role of key Th1: interleukin 1 (IL-1), IL-2, IL-12, tumor necrosis factor-α, interferon-γ; Th2: IL-4, IL-10 and non Th1/Th2: IL-6, transforming growth factor-ß1 cytokines genotypes in analyzing the variability in the clinical manifestations in an HBV-afflicted individual, which might finally, culminates into HCC. Since cytokine production is regulated genetically, the cytokine promoter region single-nucleotide polymorphisms induced changes, greatly affects the cytokine production, thus resulting into differential outcome of immune balance.

Effect of IL-12B, IL-2, TGF-ß1, and IL-4 polymorphism and expression on hepatitis B progression.

The hepatitis B virus (HBV) infection-induced chronic inflammation is considered to be the major etiological factor for HBV-related disease chronicity. Cytokines act as the key coordinators of the inflammatory responses involved in HBV disease pathogenesis. The present study assessed association among IL-12B(+1188), IL-2(-330), TGF-ß1(-509), and IL-4(-590) genotypes; mRNA; and protein levels with HBV-hepatocellular carcinoma (HCC) risk in India. For this, 403 subjects (153 controls, 67 inactive HBV-carriers, 62 chronic-active HBV patients, 62 HBV-cirrhotics, and 59 HBV-HCC ssubjects) were enrolled in the study. The genotyping was carried by polymerase chain reaction (PCR)-restriction fragment length polymorphism (IL-12+1188A/C, IL-2-330T/G, and TGF-ß1-509C/T), and allele specific (AS)-polymerase chain reaction (IL-4-590C/T). Enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction methods were used for assessing protein and the mRNA expression, respectively, of the mentioned cytokines. The study revealed that the IL-12B(+1188) CC genotype shared a significant positive association with hepatitis, among controls. While, in the case of IL-2(-330), both the TG and GG genotypes were not significantly associated with HCC risk. The TGF-ß1(-509) TT genotype acted as a potential protective factor for cirrhosis and the HCC risk, among carriers. On the contrary, the IL-4(-590) CT genotype was found to be a vital protective factor for the development of hepatitis, among carriers. Besides, IL-12B, TGF-ß1, and IL-2 seem to be majorly involved in the development of HCC, while, IL-4 might be responsible for the progression of the HBV disease till cirrhosis development. These initial findings are indicative of the vital role of genotypes and/or levels of IL-12B, IL-2, IL-4, and TGF-ß1 in HBV disease chronicity in Indian population.

Interleukin-1 polymorphism and expression in hepatitis B virus-mediated disease outcome in India.

The present study evaluated any possible association among Interleukin-1-beta (IL-1B)-511 and IL-1 receptor antagonist (IL-1RN) variable number tandem-repeat (VNTR) genotypes, haplotypes, and IL-1B expression with risk for hepatitis B virus-related hepatocellular carcinoma (HBV-HCC) development in India. For this, 406 subjects (153 controls, 67 inactive HBV-carriers, 65 patients with chronic-active HBV, 62 HBV-cirrhotics, and 59 subjects with HBV-HCC) were enrolled in the study. Polymerase chain reaction (PCR)-restriction fragment length polymorphism, reverse transcriptase-PCR, and enzyme-linked immunosorbent assay methods were used for assessing polymorphism, mRNA, and protein levels, respectively, of IL-1. The study revealed no significant association of IL-1B(-511) CT and TT genotypes, while a significant positive association of the IL-1RN (VNTR) 1/2 genotype with HCC development, among controls and carriers. Besides, 2/2 genotypes acted as a potential risk factor for hepatitis and subsequent cirrhosis development, among the same groups. Furthermore, the IL-1 haplotypes 2 and 3 were found to be significant protective factors for hepatitis and subsequent HCC development, among controls. However, haplotype 4 shared a significant negative association with hepatitis only. Moreover, proinflammatory IL-1B levels significantly and steadily elevated with the disease progression to HCC, as compared to controls. These preliminary findings indicate a key role of IL-1 in the HBV-mediated disease chronicity, in the Indian population.

Microsomal Epoxide Hydrolase Polymorphisms and Haplotypes as Determinants of Hepatitis B Virusand Hepatitis C Virus-related Liver Disease in Indian Population.

BACKGROUND: Hepatocellular carcinoma (HCC) is the fifth most common cancer and third leading cause of death worldwide. Main causes of HCC are hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. mEPHX, a xenobiotic metabolizing enzyme, exhibits a dual role of procarcinogen detoxification and activation, hence considered as a cancer risk factor as well as a protective factor. Two known polymorphic forms of mEPHX, exon in exon 3 and 4, are associated with the development of HCC. OBJECTIVE: To determine the association of genotypes and haplotypes of mEPHX with risk of HCC developments separately in HBV- and HCV-infected carriers and patients with hepatitis. METHODS: Polymerase chain reactions (PCR) were carried out using primers to amplify exon 3 (113 Tyr→His variant) and exon 4 (139 His→Arg) polymorphic sites. To distinguish the wild and variant forms, PCR amplification products were digested with restriction endonucleases EcoRV and Rsa1 for exons 3 and 4, respectively. RESULT: Exon 3 genotypes, Y113H and H113H, shared a protective association with HBV-chronic hepatitis infection (P < 0.001 and P< 0.01, respectively) as well as HBV-HCC development (P < 0.001) among HBV-carrier group, while Y113H acts as a risk factor for HCV-chronic hepatitis development (P < 0.001) as well as for HCC development (P < 0.01) with HCV-carrier group as reference. Both H139R and R139R, exon 4 genotypes, acted as a risk factor for HBV/HCV-chronic hepatitis infection and for HBV/HCV-HCC development (P ranges from < 0.05 to < 0.001) with HBV/HCV carriers as reference. 113His-139His and 113His-139Arg haplotypes shared a significant negative and positive association, respectively, with HBV hepatitis and HBV-HCC risk. 113Tyr-139Arg haplotype acted as a risk for HCV-HCC development. CONCLUSION: Polymorphic and haplotypic variant forms of mEPHX exon 3 and 4 variably determine the susceptibility to develop HCC in HBV- and HCV-carrier subjects.