P. Muñoz de Rueda

Genetic Variants of Interferon-Stimulated Genes and IL-28B as Host Prognostic Factors of Response to Combination Treatment for Chronic Hepatitis C

Chronic hepatitis C (CHC) is a worldwide health problem that is highly related to liver fibrosis, cirrhosis, and hepatocellular carcinoma. The achievement of response to the current standard of care—pegylated interferon plus ribavirin—has recently been described to be associated with single‐nucleotide polymorphisms (SNPs) near the IL‐28B gene. Additionally, baseline expression levels of genes involved in interferon (IFN)‐stimulated genes (ISGs) have been found to be related to treatment outcome. In the present study, 285 patients were genotyped for 63 SNPs within genes of the IFN signaling pathway (IPGs) and ISGs. Two ISG polymorphisms—OASL rs12819210 (odds ratio (OR) = 2.1, P = 0.03) and IFIT1 rs304478 (OR = 2.5, P = 0.01)—were found to be independent predictive factors of sustained virological response (SVR) after adjusting for other clinical covariates. Furthermore, the predictive value of IL‐28B SNP was notably improved by simultaneous genotyping of rs12819210 and rs304478, particularly in patients with the worst prognosis (viral genotype 1, area under the curve (AUC) = 0.74). In conclusion, ISG SNPs could constitute a valuable tool for individualizing CHC therapy.

The antigenic variability of HCV in viral HLA-Ag binding is related to the activation of the host immune response

Our previous data show that hepatitis C virus (HCV) genotype 1 patients expressing the HLA-DQB1 * 0301 allele have a combined response probability of 69%, while the remaining 31% do not respond, probably because the HCV immunodominant epitope (IE) against the DQB1 * 0301 allele is mutated. HCV IE (region sequenced in NS3 is a region encoding aa 1253–1272) from 37 patients (21 Sustained Virological Response, SVR; 16 non-SVR) HLA-DQB1 * 0301+, were analysed by pyrosequencing. In vitro cultures were also determined by CD4+ proliferation, using non-mutated IE (wild-type synthetic peptide) and synthetic mutated peptide. The pyrosequencing study revealed 34 different haplotypes. The SVR patients had fewer haplotypes (P = 0.07), mutations/haplotypes (P = 0.01) and polymorphic sites (P = 0.02) than non-SVR. Three polymorphic sites were associated with the non-SVR patients: haplotype 7 (L5P); haplotype 11 (L7P); and haplotype 15, (L15S) (P = 0.02). The in vitro study (n = 7) showed that in 4/7 patients (Group 1) the CD4+ proliferation obtained with wild-type synthetic peptide was higher than that obtained with the negative control and with the synthetic mutated peptide (P = 0.039). However, in the remaining 3/7 patients (Group 2) this pattern was not observed (P = 0.7). Our findings suggest that HLA-DQB1 * 0301+ patients with high antigenic variability in HCV IE (NS31253-1272) have a lower SVR rate, due to reduced CD4+ proliferation as a result of incorrect viral HLA-Ag binding.

P1200 EFFECTIVENESS OF TRIPLE THERAPY WITH BOCEPREVIR OR TELAPREVIR IN A MULTICENTRE CLINICAL PRACTICE COHORT OF HCV TREATMENT-EXPERIENCED PATIENTS WITH ADVANCED HEPATIC FIBROSIS. SVR-12W AFTER TREATMENT

P1200 EFFECTIVENESS OF TRIPLE THERAPY WITH BOCEPREVIR OR TELAPREVIR IN A MULTICENTRE CLINICAL PRACTICE COHORT OF HCV TREATMENT-EXPERIENCED PATIENTS WITH ADVANCED HEPATIC FIBROSIS. SVR-12W AFTER TREATMENT C. Fernandez, P. Munoz de Rueda, S. Alonso, M. Prieto, A. Martin-Alvarez, J. Pons, J. Pascasio, M. Serra, M. Romero, P. Conde, I. Carmona, M. Diago, M. Testillano, J. NavarroJarabo, J. Sanchez Ruano, J. Sousa, F. Nogueras, R. Granados, G. Sanchez Antolin, R. Andrade, H. Hallal, M. Marti Arribas, M. del Moral, J. Salmeron. Servicio de Aparato Digestivo, HU Fundacion de Alcorcon, Madrid, Unidad de Apoyo a la Investigacion, HU San Cecilio, Ciber de Enfermedades Hepaticas y Digestivas (CIBERehd), Granada, Unidad de Gestion Cĺinica de Enfermedades Digestivas, H La Fe, Valencia, Unidad de Hepatoloǵia, H Virgen de Arrixaca, Murcia, Unidad de Hepatoloǵia, H Virgen del Rocio, Sevilla, H Cĺinico de Valencia, Valencia, H Nuestra Senora de Valme, CIBERehd, Sevilla, H Virgen de la Concha, Zamora, Servicio de Aparato Digestivo, HU Virgen de la Macarena, Sevilla, H General de Valencia, Valencia, HU de CRUCES, Bilbao, Vizcaya, Unidad de Aparato Digestivo, Agencia Sanitaria Costa del Sol, Marbella, Malaga, Servicio de Aparato Digestivo, Complejo Hospitalario de Toledo, Toledo, Servicio de Digestivo, HU Virgen de las Nieves, Granada, HU Gran Canaria Dr. Negŕin, Las Palmas de Gran Canaria, Unidad de Hepatologia, HU Ŕio Hortega, Valladolid, HU Virgen de la Victoria, Malaga, H Morales Meseguer, Murcia, HU Salamanca, Salamanca, Unidad de Gestion Cĺinica de Aparato Digestivo, HU San Cecilio, Ciber de Enfermedades Hepaticas y Digestivas (Ciberehd), Granada, Spain E-mail: fsalmero@ugr.es

P214 SPECIFIC MUTATIONS (L5P; L7P; L15S) IN THE IMMUNODOMINANT EPITOPE HCV-NS3 (AA 1253–1272) AGAINST HLA-DQB1*0301, PROVOKE NON-RESPONSE TO ANTIVIRAL TREATMENT WITH PEGIFN/RBV IN PATIENTS WITH CHC-1

Background and Aims: Recent studies indicate that metformin has growth inhibitory and antiangiogenic effects, reducing the risk of some tumors. In this work, we have investigated the molecular mechanisms underlying HCC development promoted by HCV infection. Methods: Huh7.5 and HepG2 cells were grown in supplemented DMEM culture medium. Huh7.5 cells were infected with JFH1 particles (one particle/cell). Human hepatocytes were prepared from the liver biopsies obtained from 3 patients and hepatocyte isolation was based on the two-step collagenase procedure and treated with metformin (2 mM) for 48 hours. Gene expression was analyzed by qPCR.Proteins were analyzed by western-blot using primary antibodies against AKT, MTOR, PTEN and PTP1B. Results: HepG2 and Huh7.5 cell-growth was affected by metformin treatment at the indicated concentrations. A significant reduction (20%-Huh7.5 and 30%-HepG2) in cell confluence was seen after 48 and 72 hours of metformin treatment, respectively. This effect was observed to be dose-dependent. In Huh7.5 cultures, MAP3K (1.5±0.3 fold), AKT (2.3±0.5), PI3K (1.7±0.3), PTEN1 (1.8±0.2), TPT (3.3±0.6) and PTPN2 (2.3±0.5) gene expression were upregulated. Metformin treatment downregulated PTP1B and PTEN protein expression in JFH1-infected cells compared to Huh7.5 noninfected cells (control). MTOR protein expression was increased. In primary hepatocytes treated with metformin, PTP1B and PTEN proteins were also found down-regulated, together with AKT and mTOR.Metformin activated apoptosis through caspase-3. Conclusions: PTP1B and PTEN, involved in tumor progression were found down-regulated in HCV infection and primary hepatocytes treated with metformin, suggesting a role for these proteins in HCC development. Akt/mTOR pathway is activated in HCV infected cells and was found down-regulated after metformin treatment. These findings could explain the tumor suppressor effect exerted by metformin.

1162 ASSOCIATION OF SINGLE NUCLEOTIDE POLYMORPHISMS IN INTERFERON STIMULATED GENES WITH CHRONIC HEPATITIS C TREATMENT OUTCOME

insulin resistance and hepatic steatosis. Steatosis may increase susceptibility to apoptosis, inflammation and fibrosis by triggering hepatocytes to up-regulate CD95/Fas. We investigated this hypothesis and potential role of adipocytokines in modulating the progression of liver disease in patients with HCV-4. Methods: In 147 HCV patients and 89 controls we measured serum adiponectin, HMW adiponectin, leptin, TNF-a, IL-6 and CK-18. Liver biopsies were evaluated for steatosis/inflammation/ fibrosis, adiponectin mRNA/protein, AdipoR1/-R2 mRNA and phosphoenolpyruvate carboxykinase (PEPCK) gene expression; and adiponectin and CD95 immunoreactivity. The potential associations with hepatic steatosis and fibrosis were analyzed. Results: CD95 immunoreactivity and adiponectin immunoreactivity were readily detected in all biopsies examined and scored as grade 3 in 24 (16.3%) of patients, and as bright in 20 (13.6%) of patients, respectively. Adiponectin immunostaining within the liver correlated positively with the intensity of hepatic CD95/Fas immunostaining within the liver (r = 424; p =0.001). A significant association between high serum adiponectin and HMW adiponectin levels with CD95/Fas immunoreactivity (r = −0.16, p = 0.04, r = 0.21, p = 0.001; respectively), but not with adiponectin immunoreactivity in the liver was also identified. Adiponectin and HMW adiponectin were negatively correlated with the expression of AdipoR1, but positively correlated with the expression of AdipoR2. Hepatocyte CD95/Fas up-regulation correlated with steatosis, inflammation and fibrosis (p = 0.001). Significant correlation of serum adiponectin, HMW adiponectin and AdipoR1 and 2 mRNA expression, as well as liver adiponectin immunostaining within the liver were identified with steatosis. mRNA transcription for PEPCK was also significantly correlated with the amount of steatosis. A positive association between adiponectin and HMW adiponectin and hepatic inflammation was identified. This correlation remained significant even after following adjusting for age, gender and BMI (r = 0.17; p = 0.03; r = 0.45; p = 0.0001) respectively. Factors independently associated with the stage of fibrosis were HOMA-IR, inflammation score and age. Conclusions: Our findings in HCV-4 infection shows that adiponectin correlates with the different stages of liver injury. Steatosis up-regulates hepatocyte CD95/Fas and thus increases apoptosis, which facilitates inflammation and fibrosis. These findings may offer potential clues for future therapeutic intervention.