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399 The hepatic transcriptome in hepatitis C virus cirrhosis and hepatitis C virus cirrhosis with hepatocellular carcinoma
Category 5b: Viral Hepatitis: Hepatitis C – Basic SVM-classification-algorithms may predict MC by mutational frequencies of specific residues within HVR1 and the CD81-binding-sites. Increased CD81 expression on B-cells is associated with MC.
399 THE HEPATIC TRANSCRIPTOME IN HEPATITIS C VIRUS CIRRHOSIS AND HEPATITIS C VIRUS CIRRHOSIS WITH HEPATOCELLULAR CARCINOMA
X.X. Huang 1,2,3 , M.D. Gorrell 1,2,3 , R.B.H. Williams 1,3 , D. Seth 1 , G.W. McCaughan 1,2,3 . 1 A. W. Morrow Gastroenterology and Liver Centre At Royal Prince Alfred Hospital, Newtown, Australia; 2 Centenary Institute of Cancer Medicine and Cell Biology, Australia; 3 The University of Sydney, Sydney, Australia Background: The pathogenesis of hepatitis C virus (HCV) associated cirrhosis and HCV cirrhosis with hepatocellular carcinoma (HCC) is poorly understood. Aim: The aim of this work was to analyse the liver transcriptome in HCV cirrhosis and HCV cirrhosis with HCC. Methods: Utilizing oligonucleotide plastic arrays of 8,300 genes, HCV cirrhosis (n=14) was compared with HCV cirrhosis with HCC (n=5) and non-diseased (ND) liver (n=14). Results: More than 35% of immune, apoptosis and fibrosis-related genes were differentially expressed (DE) in HCV cirrhosis. The greatest DE is genes related stress in HCV cirrhosis with HCC versus HCV cirrhosis, with predominant DE in genes related immune response and fibrosis. RT-PCR analyses confirmed the array data that the oncogenes, BCL9, ETV1 and PIM2 were increased in HCV cirrhosis with HCC versus HCV cirrhosis alone. In contrast placental growth factor (PLGF) and its receptor Flt1 although showing upregulation in HCV cirrhosis versus ND liver showed decreased expression in HCV cirrhosis with HCC compared to HCV alone. PLGF and Flt1 expression were on small proliferating vessels and bile ductules in portal tract in cirrhotic liver. Less PLGF and Flt1 immunopositivity occurred in ND liver. Conclusion: The greater DE of genes involved in immune activation, fibrosis, cellular proliferation and cell signalling indicated that these processes are more active in the cirrhotic liver that has developed HCC than the cirrhotic liver without HCC development. The PLGF/Flt1 signalling has an important role in neo-vascular development within organs and may be important in development of HCV cirrhosis and HCC.
400 SMALL INTERFERING RNAS TARGETING HIGHLY CONSERVED REGIONS INHIBIT HEPATITIS C VIRUS TRANSLATION AND REPLICATION
M. Korf, D. Jarczak, M.P. Manns, M. Kruger. Department of Gastroenterology, Hepatology and Endocrinology, Medizinische Hochschule Hannover, Hannover, Germany Hepatitis C Virus (HCV) is an important cause of chronic liver disease. Therapeutic options are still limited in a significant proportion of patients. Recently discovered small hairpin RNAs are an efficient tool to inhibit gene expression by RNA interference mechanism. Since HCV RNA replicates in the cytoplasm of liver cells without integration into the genome, RNAdirected antiviral strategies are likely to successfully block replication cycle. We designed a panel of siRNAs directed against the HCV Internal Ribosomal Entry Site (HCV IRES) and the 3 untranslated region (3 UTR) of the HCV genome. These regions are highly conserved and therefore represent ideal target sites for inhibition of all HCV genotypes. SiRNAs were driven from a U6 promoter expressed from a retroviral vector transcript. To test siRNA efficacy, Huh7 cells were transfected with bicistronic reporter construct (Renilla-HCV IRES-Firefly-poly(U/C) tract, 3 UTR) and siRNAexpression plasmids. Several siRNAs directed against 5 UTR specifically reduced HCV IRES activity up to 30%, whereas reduction in luciferase activities of up to 45% was observed by siRNAs targeting the 3 UTR. SiRNAs were expressed in HuH7 cells expressing monocistronic subgenomic
119
HCV replicon. Northern blot analysis, revealed inhibition of HCV replicon RNA levels of up to 65% by acitve siRNAs compared with control siRNA. Significant reduction (of up to 65%) in NS5B protein was observed by western blot analysis. These results demonstrate significant as well as specific inhibition of HCV IRES-mediated translation and HCV subgenomic RNA replication by selected siRNA and support the development of siRNA as a novel strategy against HCV infection.
401 INCREASED RANTES STEADY STATE MRNA LEVELS IN LIVER BIOPSIES OF HIV/HCV COINFECTED PATIENTS
T. Kuntzen 1 , C. Tural 2 , G. Feldmann 1 , L. Leifeld 1 , M. Wolff 3 , F.L. Dumoulin 1 , H.D. Nischalke 1 , B. Clotet 2 , T. Sauerbruch 1 , U. Spengler 1 , J.K. Rockstroh 1 . 1 Department of Internal Medicine I, Rheinische Friedrich-Wilhelms-Universitat Bonn, Bonn, Germany; 2 Clinical HIV-Unit, Hospital Universitari Germans Trias I Pujol, Universitat Autonoma De Barcelona, Barcelona, Spain; 3 Department of Surgery, Rheinische Friedrich-Wilhelms-Universitat Bonn, Bonn, Germany Background: HCV-infection takes a much more rapid and severe course in HIV-infected patients than in patients with HCV infection alone. Cell culture experiments, measurements of cytokine levels in peripheral blood and immunohistochemistry of liver specimens indicate that RANTES production may be up-regulated in HCV infection in close correlation to inflammatory activity. RANTES is an antiviral chemokine, which also plays an important role in HIV infection. However, thus far, intrahepatic cytokine levels have not been analysed in HIV/HCV co-infection. Objectives: Semi-quantitative determination of RANTES steady state mRNA levels in liver-tissue of HIV/HCV-coinfected patients as compared to HCV-monoinfected individuals. Methods: RANTES steady state mRNA was determined in biopsies of 8 HIV/HCV-co-infected (age 28-58, 7 male, 1 female, CD4+ 254-1289/µl) and 11 HCV mono-infected patients (age 20-62, 7 male, 4 female) using a highly sensitive and reproducible real-time RT-PCR. All but one patients in the HIV/HCV co-infected group were free of antiretroviral therapy. In either group, none of the patients had received interferon. Mean RANTES mRNA was quantified relative to mRNA levels of the housekeeping gene β-actin. All samples were measured together in the same run. Runs were repeated three times. Results: HIV/HCV-co-infected patients had significantly higher intrahepatic RANTES mRNA (mean RANTES/β-actin ratio 0,086 ± 0,035) than HCV-mono-infected subjects (ratio 0,055 ± 0,041; p = 0,039). Conclusion: We here report increased intrahepatic RANTES levels in HIV/HCV co-infected patients as compared to patients with HCV infection alone, which may importantly contribute to enhanced hepatic inflammation and accelerated progression towards cirrhosis in double infected patients.
402 HUMAN HEPATIC SINUSOIDAL ENDOTHELIAL CELLS (HSEC) INTERACT WITH HEPATITIS C VIRUS (HCV) E2 GLYCOPROTEIN VIA DCSIGN AND LSIGN
W.K. Lai 1 , P.J. Sun 1 , J. Zhang 2 , J. Youster 1 , J. McKeating 2 , D.H. Adams 1 . 1 Liver Resarch Laboratories, Univesity of Birmingham, Birmingham, UK; 2 Center For Study of Hepatitis C, The Rockefeller University, New York NY, USA The viral attachment factors conferring liver tropism to HCV are unknown. Recent data suggest LSIGN, a type 2 C type lectin expressed on HSEC can bind with high affinity to HCV E2 glycoprotein. A closely related homologue DCSIGN, expressed only on dendritic cells and a subset of macrophages has similar binding affinity to HCV E2 glycoprotein. These receptors function as adhesion and antigen presentation molecules and may play an important role in facilitating the viral uptake and the nature of the immune response to the virus. We report for the first time, DCSIGN expression on liver tissue and on isolated HSEC. Tissue staining
399 THE HEPATIC TRANSCRIPTOME IN HEPATITIS C VIRUS CIRRHOSIS AND HEPATITIS C VIRUS CIRRHOSIS WITH HEPATOCELLULAR CARCINOMA
X.X. Huang 1,2,3 , M.D. Gorrell 1,2,3 , R.B.H. Williams 1,3 , D. Seth 1 , G.W. McCaughan 1,2,3 . 1 A. W. Morrow Gastroenterology and Liver Centre At Royal Prince Alfred Hospital, Newtown, Australia; 2 Centenary Institute of Cancer Medicine and Cell Biology, Australia; 3 The University of Sydney, Sydney, Australia Background: The pathogenesis of hepatitis C virus (HCV) associated cirrhosis and HCV cirrhosis with hepatocellular carcinoma (HCC) is poorly understood. Aim: The aim of this work was to analyse the liver transcriptome in HCV cirrhosis and HCV cirrhosis with HCC. Methods: Utilizing oligonucleotide plastic arrays of 8,300 genes, HCV cirrhosis (n=14) was compared with HCV cirrhosis with HCC (n=5) and non-diseased (ND) liver (n=14). Results: More than 35% of immune, apoptosis and fibrosis-related genes were differentially expressed (DE) in HCV cirrhosis. The greatest DE is genes related stress in HCV cirrhosis with HCC versus HCV cirrhosis, with predominant DE in genes related immune response and fibrosis. RT-PCR analyses confirmed the array data that the oncogenes, BCL9, ETV1 and PIM2 were increased in HCV cirrhosis with HCC versus HCV cirrhosis alone. In contrast placental growth factor (PLGF) and its receptor Flt1 although showing upregulation in HCV cirrhosis versus ND liver showed decreased expression in HCV cirrhosis with HCC compared to HCV alone. PLGF and Flt1 expression were on small proliferating vessels and bile ductules in portal tract in cirrhotic liver. Less PLGF and Flt1 immunopositivity occurred in ND liver. Conclusion: The greater DE of genes involved in immune activation, fibrosis, cellular proliferation and cell signalling indicated that these processes are more active in the cirrhotic liver that has developed HCC than the cirrhotic liver without HCC development. The PLGF/Flt1 signalling has an important role in neo-vascular development within organs and may be important in development of HCV cirrhosis and HCC.
400 SMALL INTERFERING RNAS TARGETING HIGHLY CONSERVED REGIONS INHIBIT HEPATITIS C VIRUS TRANSLATION AND REPLICATION
M. Korf, D. Jarczak, M.P. Manns, M. Kruger. Department of Gastroenterology, Hepatology and Endocrinology, Medizinische Hochschule Hannover, Hannover, Germany Hepatitis C Virus (HCV) is an important cause of chronic liver disease. Therapeutic options are still limited in a significant proportion of patients. Recently discovered small hairpin RNAs are an efficient tool to inhibit gene expression by RNA interference mechanism. Since HCV RNA replicates in the cytoplasm of liver cells without integration into the genome, RNAdirected antiviral strategies are likely to successfully block replication cycle. We designed a panel of siRNAs directed against the HCV Internal Ribosomal Entry Site (HCV IRES) and the 3 untranslated region (3 UTR) of the HCV genome. These regions are highly conserved and therefore represent ideal target sites for inhibition of all HCV genotypes. SiRNAs were driven from a U6 promoter expressed from a retroviral vector transcript. To test siRNA efficacy, Huh7 cells were transfected with bicistronic reporter construct (Renilla-HCV IRES-Firefly-poly(U/C) tract, 3 UTR) and siRNAexpression plasmids. Several siRNAs directed against 5 UTR specifically reduced HCV IRES activity up to 30%, whereas reduction in luciferase activities of up to 45% was observed by siRNAs targeting the 3 UTR. SiRNAs were expressed in HuH7 cells expressing monocistronic subgenomic
119
HCV replicon. Northern blot analysis, revealed inhibition of HCV replicon RNA levels of up to 65% by acitve siRNAs compared with control siRNA. Significant reduction (of up to 65%) in NS5B protein was observed by western blot analysis. These results demonstrate significant as well as specific inhibition of HCV IRES-mediated translation and HCV subgenomic RNA replication by selected siRNA and support the development of siRNA as a novel strategy against HCV infection.
401 INCREASED RANTES STEADY STATE MRNA LEVELS IN LIVER BIOPSIES OF HIV/HCV COINFECTED PATIENTS
T. Kuntzen 1 , C. Tural 2 , G. Feldmann 1 , L. Leifeld 1 , M. Wolff 3 , F.L. Dumoulin 1 , H.D. Nischalke 1 , B. Clotet 2 , T. Sauerbruch 1 , U. Spengler 1 , J.K. Rockstroh 1 . 1 Department of Internal Medicine I, Rheinische Friedrich-Wilhelms-Universitat Bonn, Bonn, Germany; 2 Clinical HIV-Unit, Hospital Universitari Germans Trias I Pujol, Universitat Autonoma De Barcelona, Barcelona, Spain; 3 Department of Surgery, Rheinische Friedrich-Wilhelms-Universitat Bonn, Bonn, Germany Background: HCV-infection takes a much more rapid and severe course in HIV-infected patients than in patients with HCV infection alone. Cell culture experiments, measurements of cytokine levels in peripheral blood and immunohistochemistry of liver specimens indicate that RANTES production may be up-regulated in HCV infection in close correlation to inflammatory activity. RANTES is an antiviral chemokine, which also plays an important role in HIV infection. However, thus far, intrahepatic cytokine levels have not been analysed in HIV/HCV co-infection. Objectives: Semi-quantitative determination of RANTES steady state mRNA levels in liver-tissue of HIV/HCV-coinfected patients as compared to HCV-monoinfected individuals. Methods: RANTES steady state mRNA was determined in biopsies of 8 HIV/HCV-co-infected (age 28-58, 7 male, 1 female, CD4+ 254-1289/µl) and 11 HCV mono-infected patients (age 20-62, 7 male, 4 female) using a highly sensitive and reproducible real-time RT-PCR. All but one patients in the HIV/HCV co-infected group were free of antiretroviral therapy. In either group, none of the patients had received interferon. Mean RANTES mRNA was quantified relative to mRNA levels of the housekeeping gene β-actin. All samples were measured together in the same run. Runs were repeated three times. Results: HIV/HCV-co-infected patients had significantly higher intrahepatic RANTES mRNA (mean RANTES/β-actin ratio 0,086 ± 0,035) than HCV-mono-infected subjects (ratio 0,055 ± 0,041; p = 0,039). Conclusion: We here report increased intrahepatic RANTES levels in HIV/HCV co-infected patients as compared to patients with HCV infection alone, which may importantly contribute to enhanced hepatic inflammation and accelerated progression towards cirrhosis in double infected patients.
402 HUMAN HEPATIC SINUSOIDAL ENDOTHELIAL CELLS (HSEC) INTERACT WITH HEPATITIS C VIRUS (HCV) E2 GLYCOPROTEIN VIA DCSIGN AND LSIGN
W.K. Lai 1 , P.J. Sun 1 , J. Zhang 2 , J. Youster 1 , J. McKeating 2 , D.H. Adams 1 . 1 Liver Resarch Laboratories, Univesity of Birmingham, Birmingham, UK; 2 Center For Study of Hepatitis C, The Rockefeller University, New York NY, USA The viral attachment factors conferring liver tropism to HCV are unknown. Recent data suggest LSIGN, a type 2 C type lectin expressed on HSEC can bind with high affinity to HCV E2 glycoprotein. A closely related homologue DCSIGN, expressed only on dendritic cells and a subset of macrophages has similar binding affinity to HCV E2 glycoprotein. These receptors function as adhesion and antigen presentation molecules and may play an important role in facilitating the viral uptake and the nature of the immune response to the virus. We report for the first time, DCSIGN expression on liver tissue and on isolated HSEC. Tissue staining