Pascale Berthillon

Impact of patatin‐like phospholipase‐3 (rs738409 C>G) polymorphism on fibrosis progression and steatosis in chronic hepatitis C

Only 20% of patients with chronic hepatitis C (CHC) will develop cirrhosis, and fibrosis progression remains highly unpredictable. A recent genome‐wide association study identified a genetic variant in the patatin‐like phospholipase‐3 (PNPLA3) gene (rs738409 C>G) associated with steatosis that was further demonstrated to influence severity of fibrosis in nonalcoholic fatty liver disease. The aim of this study was to assess the impact of this polymorphism on histological liver damage and response to antiviral therapy in CHC. We recruited 537 Caucasian CHC patients from three European centers (Brussels, Belgium [n = 229]; Hannover, Germany [n = 171]; Lyon, France [n = 137]); these patients were centrally genotyped for the PNPLA3 (rs738409 C>G) polymorphism. We studied the influence of rs738409 and other variants in the PNPLA3 region on steatosis and fibrosis assessed both in a cross‐sectional and longitudinal manner. Seven other variants previously associated with fibrosis progression were included. Finally, we explored the impact of rs738409 on response to standard antiviral therapy using the interferon lambda 3 (IL28B) [rs12979860 C>T] variant both as a comparator and as a positive control. After adjustment for age, sex, body mass index, alcohol consumption, and diabetes, rs738409 mutant G allele homozygote carriers remained at higher risk for steatosis (odds ratio [OR] 2.55, 95% confidence interval [CI] 1.08‐6.03, P = 0.034), fibrosis (OR 3.13, 95% CI 1.50‐6.51, P = 0.002), and fibrosis progression (OR 2.64, 95% CI 1.22‐5.67, P = 0.013). Conversely, rs738409 was not independently associated with treatment failure (OR 1.07, 95% CI 0.46‐2.49, P = 0.875) and did not influence clinical or biological variables. Conclusion: The PNPLA3 (rs738409 C>G) polymorphism favors steatosis and fibrosis progression in CHC. This polymorphism may represent a valuable genetic predictor and a potential therapeutic target in CHC liver damage. (HEPATOLOGY 2011;)

Infection with Hepatitis B and C Viruses and Risk of Lymphoid Malignancies in the European Prospective Investigation into Cancer and Nutrition (EPIC)

Background: Case–control studies suggested a moderate, but consistent, association of hepatitis C virus (HCV) infection with lymphoid tissue malignancies, especially non–Hodgkin lymphoma (NHL). More limited data suggested that hepatitis B virus (HBV) infection might also be associated with NHL. However, prospective studies on the topic are few. Methods: A nested case–control study was conducted in eight countries participating in the EPIC prospective study. Seven hundred thirty-nine incident cases of NHL, 238 multiple myeloma (MM), and 46 Hodgkin lymphoma (HL) were matched with 2,028 controls. Seropositivity to anti-HCV, anti-HBc, and HBsAg was evaluated and conditional logistic regression was used to estimate odds ratios (OR) and corresponding 95% confidence intervals (CI) for NHL, MM, or HL, and their combination. Results: Anti-HCV seropositivity among controls in different countries ranged from 0% to 5.3%; HBsAg from 0% to 2.7%; and anti-HBc from 1.9% to 45.9%. Similar nonsignificant associations were found with seropositivity to HBsAg for NHL (OR = 1.78; 95% CI: 0.78–4.04), MM (OR = 4.00; 95% CI: 1.00–16.0), and HL (OR = 2.00; 95% CI: 0.13–32.0). The association between HBsAg and the combination of NHL, MM, and HL (OR = 2.21; 95% CI: 1.12–4.33) was similar for cancer diagnosed less than 3 and 3 or more years after blood collection. No significant association was found between anti-HCV and NHL, MM, or HL risk, but the corresponding CIs were very broad. Conclusions: Chronic HBV infection may increase the risk of lymphoid malignancies among healthy European volunteers. Impact: Treatment directed at control of HBV infection should be evaluated in HBsAg-seropositive patients with lymphoid tissue malignancies. Cancer Epidemiol Biomarkers Prev; 20(1); 208–14. ©2011 AACR.

Role of a cirrhosis risk score for the early prediction of fibrosis progression in hepatitis C patients with minimal liver disease.

BACKGROUND & AIMS Fibrosis progression in patients with chronic hepatitis C (CHC) is highly variable. A Cirrhosis Risk Score (CRS) based on seven genetic variants has been recently developed for identifying patients at risk for cirrhosis. The objective of this study was to assess the role of the CRS for the early prediction of fibrosis progression in CHC patients with mild liver fibrosis. In addition, we evaluated the potential benefit, for prediction accuracy, of a recently described non-invasive fibrosis staging assay, the Enhanced Liver Fibrosis (ELF) test. METHODS Two separate cohorts of HCV patients (Brussels, Belgium/Hannover, Germany) were retrospectively analyzed. Only patients with a fibrosis Ishak or METAVIR score of F0-F1 at baseline were included. Patients were classified as progressors if they showed an increase ≥2 fibrosis stages at the second histological evaluation after a follow-up ≥5years. The CRS was calculated locally. Genotyping was performed by PCR and oligonucleotide ligation with the resulting signal detected with a Luminex® 200TM and computer analysis. RESULTS In Brussels, 12/25 patients progressed (48%); similarly in Hannover, 16/31 (52%) patients progressed. In both sample sets, the CRS was significantly associated with fibrosis progression (p=0.050 in Brussels; p=0.018 in Hannover). The ELF test was only a significant predictor in Hannover (p=0.015). In multivariate analysis the CRS remained the only variable associated with fibrosis progression (odds-ratio=2.23, 95%CI 1.21-4.11 p=0.01). CONCLUSIONS Although conducted on a limited number of patients, this study in two independent centres confirms that the CRS predicts fibrosis progression in initially mild CHC.

Clinical relevance of total HCV Core antigen testing for hepatitis C monitoring and for predicting patients’ response to therapy

Summary. To study the correlation between total Hepatitis C virus (HCV) Core antigen (Ag) and HCV‐RNA, and to assess the proficiency of HCV Core Ag testing in monitoring and predicting virologic response during and after pegylated interferon (PEG‐IFN) and ribavirin combination therapy.

Intrahepatic innate immune response pathways are downregulated in untreated chronic hepatitis B

BACKGROUND & AIMS Hepatitis B virus (HBV) persistence and the pathobiology of chronic HBV (CHB) infections result from the interplay between viral replication and host immune responses. We aimed to comprehensively analyse the expression of intrahepatic host genes as well as serum and liver HBV markers in a large cohort of untreated CHB patients. METHODS One-hundred and five CHB patients untreated at the time of liver biopsy (34 HBeAg[+] and 71 HBeAg[-]) were analysed for the intrahepatic expression profile of 67 genes belonging to multiple innate immunity pathways. Results were correlated to serological (quantification of HBsAg [qHBsAg] and HBV DNA) and intrahepatic viral markers (total HBV DNA, pre-genomic RNA and covalently closed circular HBV DNA). RESULTS Intrahepatic gene expression profiling revealed a strong downregulation of antiviral effectors, interferon stimulated genes, Toll-like and pathogen recognition receptor pathways in CHB patients as compared to non-infected controls, which was not directly correlated to HBV replication. A subset of genes [CXCL10, GBP1, IFITM1, IFNB1, IL10, IL6, ISG15, TLR3, SOCS1, SOCS3] was more repressed in HBeAg(-) respect to HBeAg(+) patients (median of serum HBV DNA 7.9×103vs. 7.9×107IU/ml, respectively). Notably, HBeAg(-) patients with lower qHBsAg (<5×103IU/ml) showed a relief of repression of genes belonging to multiple pathways. CONCLUSIONS Our results show a strong impairment of innate immune responses in the liver of CHB patients. The association of low levels of qHBsAg with gene repression, if confirmed, might prove useful for the identification of patients who would most benefit from immune-modulators and/or HBsAg targeting agents as strategies to restore immune responsiveness. LAY SUMMARY Chronic hepatitis B virus (HBV) infections represent a major public health problem worldwide. Over 200 million people are chronically infected and at risk of developing chronic hepatitis, liver cirrhosis and cancer. Our work aimed to understand the molecular consequences of chronic hepatitis B in the infected liver. It was conducted in a large cohort of untreated chronically infected HBV patients and analysed the expression of immunity and liver disease-related genes in the liver, with respect to markers of viral replication and persistence. Our results indicate that chronic HBV infection has a suppressive effect on immune responses, which was more pronounced with high levels of hepatitis B virus surface antigen (HBsAg). These data provide novel insight into the mechanisms of HBV persistence in the liver and suggest that approaches aimed at reducing HBsAg levels, may restore immune responsiveness against the virus.

Long-term propagation of serum hepatitis C virus (HCV) with production of enveloped HCV particles in human HepaRG hepatocytes†‡

HepaRG human liver progenitor cells exhibit morphology and functionality of adult hepatocytes. We investigated the susceptibility of HepaRG hepatocytes to in vitro infection with serum‐derived hepatitis C virus (HCV) particles (HCVsp) and the potential neutralizing activity of the E1E2‐specific monoclonal antibody (mAb) D32.10. The infection was performed using HCVsp when the cells actively divided at day 3 postplating. HCV RNA, E1E2, and core antigens were quantified in HCV particles recovered from culture supernatants of differentiated cells for up to 66 days. The density distributions of particles were analyzed on iodixanol or sucrose gradients. Electron microscopy (EM) and immune‐EM studies were performed for ultrastructural analysis of cells and localization of HCV E1E2 proteins in thin sections. HCV infection of HepaRG cells was documented by increasing production of E1E2‐core‐RNA(+) HCV particles from day 21 to day 63. Infectious particles sedimented between 1.06 and 1.12 g/mL in iodixanol gradients. E1E2 and core antigens were expressed in 50% of HCV‐infected cells at day 31. The D32.10 mAb strongly inhibited HCV RNA production in HepaRG culture supernatants. Infected HepaRG cells frozen at day 56 were reseeded at low density. After only 1‐3 subcultures and induction of a cell differentiation process the HepaRG cells produced high titer HCV RNA and thus showed to be sustainably infected. Apolipoprotein B‐associated empty E1E2 and complete HCV particles were secreted. Characteristic virus‐induced intracellular membrane changes and E1E2 protein‐association to vesicles were observed. Conclusion: HepaRG progenitor cells permit HCVsp infection. Differentiated HepaRG cells support long‐term production of infectious lipoprotein‐associated enveloped HCV particles. The E1E2‐specific D32.10 mAb neutralizes the infection and this cellular model could be used as a surrogate infection system for the screening of entry inhibitors. (HEPATOLOGY 2011;)

Removal of cell-bound lipoproteins: a crucial step for the efficient infection of liver cells with hepatitis C virus in vitro

Hepatitis C virus (HCV) is of major social, medical and economic importance. The prevalence of HCV is approximatively 1% in most developed countries, and much higher in developing countries. HCV infection is the second major cause, after hepatitis B virus infection, for the generation of chronic liver disease and hepatocellular carcinoma. To date, the only reliable model for the study of HCV infection is the chimpanzee. Indeed, there is no robust in vitro infection system, yet. There is thus an urgent need for such an in vitro infection system in order to evaluate therapeutic agents. Here, a process is provided for infecting hepatocyte cell lines with hepatitis C virus in vitro. It is strongly suggested that cell-bound lipoproteins are playing a crucial role during the infection process. In order to obtain a robust infection, the cell-bound lipoproteins have first to be removed from their cellular receptor prior to the addition of viral inocula originating from human sera, the latter being made originally of a virus-lipoprotein complex.

Interferon therapy for hepatitis C.

Initial trials indicated that around 50% of patients respond to recombinant alpha interferon by normalizing alanine aminotransferase (ALT) at the end of therapy and that half of these relapsed within 6 months following cessation of treatment. Both dose and duration of treatment are critical in the response to therapy. Higher doses and longer duration have been suggested to be more effective than the current recommendations of 3 MUI thrice weekly for 6 months based on results of these initial studies which used ALT and histological scores to evaluate the efficacy of interferon therapy. Following studies using virological markers have shown that improvements in clinical features of disease are associated with decrease or loss of hepatitis C virus (HCV) from serum and liver. The heterogeneity of the response rates between clinical centers using identical protocol emphasizes that the selection of the patients treated was as important for the outcome that the therapy regimen itself with better responses in cases without cirrhosis and with low levels of HCV RNA. Furthermore, the genotype of HCV seems to be also critical for the response rate. Virological evaluations appears therefore crucial to assess not only HCV infection but also for the indication and monitoring of therapy.

Association of anti‐E1E2 antibodies with spontaneous recovery or sustained viral response to therapy in patients infected with hepatitis C virus

The monoclonal antibody (mAb) D32.10 recognizes a discontinuous epitope encompassing three regions E1 (amino acids 297‐306), E2A (amino acids 480‐494), and E2B (amino acids 613‐621) juxtaposed on the surface of serum‐derived hepatitis C virus (HCV) particles (HCVsp). The mAb D32.10 inhibits efficiently and specifically the binding of HCVsp to human hepatocytes. Therefore, we investigated the clinical relevance of anti‐E1E2A,B response in the serum of patients infected with HCV. To this end, an enzyme‐linked immunosorbent assay (ELISA) using synthetic E1‐, E2A‐, and E2B‐derived peptides was used. The ELISA was validated in terms of sensitivity, specificity, and test efficiency. The detection of the anti‐E1E2 D32.10 epitope‐binding antibodies during natural HCV infection in more than 300 HCV‐positive sera demonstrated significantly (P < 0.001) higher prevalence of these antibodies: (1) in patients who spontaneously cured HCV infection (46 of 52, 88.5%) showing high titers (70% ≥ 1/1000) compared to never‐treated patients with chronic hepatitis C (7 of 50, 14%) who actively replicated the virus, and (2) in complete responders (20 of 52, 38.5%) who cleared virus following treatment and achieved a sustained viral response compared to nonresponders (4 of 40, 10%). Serum anti‐E1E2 antibodies were monitored before, during, and after the current standard‐of‐care therapy (pegylated interferon plus ribavirin) in responder and nonresponder patients. Optimal cutoff values were assessed by receiver operating characteristic curve analysis. One month prior to therapy initiation, the threshold of 1131 (optical density × 1000) gave 100% and 86% positive and negative predictive values, respectively, for achieving or not achieving a sustained viral response. Conclusion: The anti‐E1E2 D32.10 epitope‐binding antibodies are associated with control of HCV infection and may represent a new relevant prognostic marker in serum. This unique D32.10 mAb may also have immunotherapeutic potential. (HEPATOLOGY 2010)

Interactions between hepatitis B virus and aflatoxin B(1): effects on p53 induction in HepaRG cells.

Infection by hepatitis B virus (HBV) and dietary exposure to aflatoxin B(1) (AFB(1)) are the main risk factors for the development of chronic liver disease and hepatocellular carcinoma (HCC). How these factors cooperate is still largely unknown. AFB(1) activation leads to DNA adduction and mutagenesis, with a specific mutation at codon 249 in TP53 (p.R249S). So far, only limited studies have addressed the effects of AFB(1) on HBV replication. We have analysed the effects of both risk factors on p53 induction during HBV infection in HepaRG, a cell line with hepatocyte-like morphology that metabolizes AFB(1) and supports HBV infection. Exposure to AFB(1) up to 5 µM induced a downregulation of HBV replication after 48 h, as measured by a decrease in viral antigens in the culture medium (HBsAg, HBeAg and large envelope protein) and in intracellular levels of HBV transcripts, DNA and HBsAg. Conversely, HBV infection did not significantly modify AFB(1)-DNA adduct formation or repair as assessed by immunodot-blot assay, and the induction of p53 in response to AFB(1) was similar in infected and non-infected HepaRG cells. Overall, our results suggest that AFB(1) exposure decreases HBV replication, whereas DNA damage by AFB(1) and subsequent p53 induction is not affected by the presence of the virus. Thus, in HepaRG cell line, AFB(1) and HBV do not cooperate to increase DNA damage by AFB(1). Further studies on the effects of both factors in a context of chronicity are needed to better understand synergistic effects.