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Mo1533 Analysis of Differentially Expressed Genes and MicroRNAs in Alcoholic Liver Disease
CONCLUSIONS: In chronic hepatitis B patients with incomplete virologic response to telbivudine, HBV DNA level of <2000 IU/ml predicts a favorable outcome in achieving maintained virologic response after switching to entecavir.
cirrhosis (median 0.20, range 0.05-1.11) (P< 0.0001). There was no statistically significant difference in RTL based on known risk factors for cirrhosis including history of alcohol use, age, or family history of cirrhosis. History of tobacco use also had no statistically significant impact on RTL. Telomere lengths were then divided by the median telomere length, into short and long. Based on conditional logistic regression analysis, patients with a long RTL were significantly more likely to be cirrhotic than those with short RTL (OR=2.76, CI: 1.55.1, P=0.001). Quartile analysis further indicated a dose-response effect for this association. Compared to patients with the lowest quartile of RTL, the OR for cirrhosis in the second, third, and highest quartile of RTL was 2.68 (0.91-7.87, P=0.073), 3.37 (1.32-10.54, P= 0.013), and 6.64 (2.41-18.32, P<0.0001), respectively (Ptrend<0.0001). Conclusion: Longer RTL measured from circulating cell-free serum DNA is associated with cirrhosis in Korean American patients with chronic hepatitis B. Telomere length may be useful as a non-invasive biomarker for risk prediction or early detection of cirrhosis and in treatment decision making. Mo1535
Relations of HBV DNA level and proportion of maintained virologic response after switching to entecavir over time in patients with incomplete virologic response to telbivudine
Background & Aims: Analysis of the molecular pathways linking inflammation and hepatocyte survival is essential to identify novel therapies for inflammatory liver disease. Release of extracellular ATP and subsequent activation of purinergic P2 receptors (P2R) is increasingly recognized as important mediator of pro-inflammatory responses, raising the question of how ATP and P2R signaling contribute to the pathogenesis of acute hepatitis. Methods: To address this issue, acute hepatitis was induced in P2R knockout mice and controls by injection of Concanavalin A, an established model of fulminant and TNFα-dependent hepatitis. Moreover, hepatocyte-specific functions of P2R signaling in response to TNFα-mediated cell death were analyzed in primary mouse hepatocytes. Results: Induction of acute hepatitis in control animals resulted in a strong ATP release from the liver and induction of P2Y2R expression. Concanavalin A-mediated liver damage and necrosis were dramatically reduced in wild type mice co-injected with the P2YR inhibitor Suramin as well as in P2Y2R-/- mice. Analysis of chimeric mice revealed that P2Y2R expression in the bone marrow was required for efficient neutrophil infiltration to the liver. On the other hand, P2Y2R expression strongly promoted hepatocyte death In Vitro. On the molecular level, improved hepatocyte survival in the absence of P2Y2R correlated with increased activation of NF-κB and subsequent transcription of hepatoprotective genes. Conclusions: These findings indicate that extracellular ATP and P2Y2R have cell type-specific but synergistic functions during acute hepatitis by regulating cellular immune responses as well as hepatocyte survival. Targeting of P2Y2R may therefore be a promising approach to treat inflammatory liver disease.
Mo1533 Analysis of Differentially Expressed Genes and MicroRNAs in Alcoholic Liver Disease Ying Liu, You M. Li Abstract Background and Aim: To screen differentially expressed genes and MicroRNAs hoping bring us a new target spot to accurate diagnosis and effective therapy in gene and MicroRNA level of alcoholic liver disease(ALD).Methods: The total RNA of peripheral blood was extracted from four groups of three subjects.And all patients signed the informed consents. Microarrays were utilized to detect differentially expressed genes and MicroRNAs. According to gene values, significance level (p values) and false discovery rate with a random variance model, combining the GO and KEGG database, node genes and key MicroRNAs in network were obtained and analyzed.Results: A total of 878 differentially expressed genes and 26 MicroRNAs were discovered. Not all of these genes have biological effects on the development of ALD. By combining the gene ontology (GO) database and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, 16 significantly different pathways were found, where 31 corresponding genes participated in.Calculating betweenness centrality of each gene in network, which can describe the degree of importance of the nodes in the whole net. In co-expression network of genes, the node genes modulating the network were ACSF3, FZD5, LOC727987 and C1orf222. And in MicroRNA-Gene network the key MicroRNAs were hsa-miR-570, hsa-miR-122, hsa-miR-34b, hsa-miR-29c, hsa-miR-922 and hsa-miR-185 they negatively regulated about 79 genes locate their downstream.Conclusions: In the course of the ALD, we found 4 differentially expressed node genes in network and analyzed Acyl-coenzyme A synthetase-3 (ACSF3) , the maximal betweenness centrality in the entire gene-modulating network was 0.102935, indicating its controlling capability and significance level. Continuous up-regulation of ACSF3 maintains transcriptional activation and subsequently strengthens the binding of ACSF3 to thioester and CoA to activate fatty acids, which results in the irreversible advancement of ALD from hepatitis to cirrhosis. The gene values of Frizzled-5(FZD5) in alcoholic hepatitis and cirrhosis were clearly downregulated. The second highest value of betweenness centrality in the entire gene-modulating network was 0.087. A possible mechanism may be that continuous down-regulation of FZD5 during advancement of alcoholic hepatitis to cirrhosis decreased gene activities and syntheses of multi-transmembrane transport proteins. This reduced the number of β-catenin molecules in hepatocytes and therefore promoted hepatocyte apoptosis. The other two genes' functions were unknown. And the 6 key MicroRNAs perhaps controlled numerous biology functions such as immune response, activity of cancer gene, inflammatory mediated response, cell cycle, glutathione metabolism. Maybe the discovery will provide valuable directions to diagnosis and treatment of ALD.
Mo1536 Human Hepatocytes Produce Inflammatory Cytokines and Type I, II and III Interferons in Response to Toll-Like Receptor Activation, Resulting in IFN-λMediated Suppression of HCV In Vitro Ruth Bröring, Melanie Lutterbeck, Kathrin Kleinehr, Andreas Paul, Guido Gerken, Joerg F. Schlaak Background: The function of the hepatic innate immune system and its role in the defence against Hepatitis C Virus infection are not well understood. Recent publications suggested that type-III interferons (IFN; Interleukin-28A, -28B and -29) play a crucial role in the host response against HCV. Aim of this study was to investigate the Toll-like receptor (TLR) signaling in primary human hepatocytes, the capacity of these cells to produce type-III IFNs and their ability to control HCV replication. Methods: Human liver samples were obtained after tumor resection (n=16) or liver transplantation (n=14). Human hepatocytes (n=30) were isolated after perfusion and digestion of the liver. Cells were stimulated with TLR1-9 ligands for 6h to 24h, expression of Tumor necrosis factor α (TNF-α), Interleukin 6 (IL6), IL-10, IFN-α, -β, -γ and -λ (IL-28A, IL-28B and IL-29) were determined by qRT-PCR and ELISA. Gene expression of interferon sensitive genes (ISGs) was determined by qRTPCR. Supernatants from PHH treated with TLR ligands were pre-incubated with neutralizing antibodies and co-cultured with a subgenomic HCV replicon system (con1). Results: In human hepatocytes stimulation with TLR1-9 ligands, except TLR9, led to induction of proinflammatory (TNF-α, IL-6) and anti-inflammatory cytokines (IL-10). In contrast, expression of type-I, -II and -III interferons (IFN-α, -β, -γ, IL-28A, IL-28B, IL-29) could only be induced after TLR3 stimulation, herein IL-29 and IL-28A are significantly higher expressed than IFN-α and IFN-β, resulting in enhanced expression of ISGs (ISG15, IFI-T1, RSAD2, MXA). Therefore, only supernatants from TLR3-activated hepatocytes suppressed HCV replication in co-cultured con1 cells. This antiviral effect could not be neutralized by antibodies against IFN-α, -β or -γ. The different underlying diseases, the type of surgery, fibrosis stage as well as liver function tests (ALT, AST, GGT, AP) did not correlate with TLR signaling and antiviral activity in these cells. Conclusions Primary isolated human hepatocytes respond to TLR ligands by production of inflammatory cytokines as well as type-I, -II and -III IFNs, whereas the poly I:C-induced antiviral effect is exclusively mediated by type-III interferons. These findings shed new light on the importance of IFN-λ in the pathogenesis of HCV.
Mo1534 Association Between Telomere Length and Risk of Cirrhosis in Patients With Chronic Hepatitis B Jennifer S. Au, Hie-Won L. Hann, Shaogui Wan, Ronald E. Myers, Xiaoying Fu, Victor J. Navarro, Su H. Kim, Hushan Yang Background: The decision to treat chronic hepatitis B is based on clinical guidelines which utilize HBeAg status, transaminases, HBV DNA, and occasionally liver biopsy. The presence of known risk factors for the progression to cirrhosis, including older age, HDV or HIV coinfection, and alcohol consumption, influence the decision to treat. Telomeres consist of small tandem nucleotide repeats that form the physical ends of eukaryotic chromosomes. Shortened hepatocyte telomeres have been implicated in the development and progression to cirrhosis. Therefore, telomere length may serve as a potential constitutive biomarker for risk prediction or early detection of cirrhosis. Purpose: To examine telomere length in cellfree serum DNA as a non-invasive predictor of cirrhosis risk in patients with chronic hepatitis B. Methods: Serum samples were obtained from patients who visited The Liver Disease Prevention Center at Thomas Jefferson University Hospital between 1988 and 2010. One hundred patients with HBV cirrhosis were selected as cases and 100 chronic HBV noncirrhotic patients were selected as controls. All patients were of Korean ethnicity and controls were matched to cases by age, and sex. All patients were sero-negative for HCV and HDV. Demographic information including history of alcohol and tobacco use, as well as family history of cirrhosis were collected. Circulating DNA was extracted from 200μL of serum and then qRT-PCR was conducted in duplicate to measure the number of telomere repeats as compared to a gene with a single copy. Results: Cirrhotic HBV patients had a significantly longer (median 0.36; range 0.08-1.87) relative telomere length (RTL) than those without
Mo1537 TLR3-Dependent Immunological Properties of Liver Cells are Controlled by Anti-Inflammatory Cytokines Through Modulation of miR-155 Expression Min Jiang, Martin Trippler, Ruth Bröring, Jun Wu, Guido Gerken, Mengji Lu, Joerg F. Schlaak Background & Aims: The hepatitis C virus (HCV) can establish a persistent infection despite a strong activation of the innate immune system through TLR3 and other sensors, the so-called “IFN-paradox”. We analyzed regulatory mechanisms of TLR3-mediated immune responses in human liver and in murine non-parenchymal liver cells (NPC) Methods: Hepatic expression of Interleukin 10 (IL-10) and transforming growth factor beta (TGF-β) in biopsies
S-989
AASLD Abstracts
AASLD Abstracts
P2Y2 Receptor Function Aggravates Acute Hepatitis by Regulating Neutrophil Infiltration and Hepatocyte Survival Korcan Ayata, Marco Idzko, Peter Hasselblatt
cirrhosis (median 0.20, range 0.05-1.11) (P< 0.0001). There was no statistically significant difference in RTL based on known risk factors for cirrhosis including history of alcohol use, age, or family history of cirrhosis. History of tobacco use also had no statistically significant impact on RTL. Telomere lengths were then divided by the median telomere length, into short and long. Based on conditional logistic regression analysis, patients with a long RTL were significantly more likely to be cirrhotic than those with short RTL (OR=2.76, CI: 1.55.1, P=0.001). Quartile analysis further indicated a dose-response effect for this association. Compared to patients with the lowest quartile of RTL, the OR for cirrhosis in the second, third, and highest quartile of RTL was 2.68 (0.91-7.87, P=0.073), 3.37 (1.32-10.54, P= 0.013), and 6.64 (2.41-18.32, P<0.0001), respectively (Ptrend<0.0001). Conclusion: Longer RTL measured from circulating cell-free serum DNA is associated with cirrhosis in Korean American patients with chronic hepatitis B. Telomere length may be useful as a non-invasive biomarker for risk prediction or early detection of cirrhosis and in treatment decision making. Mo1535
Relations of HBV DNA level and proportion of maintained virologic response after switching to entecavir over time in patients with incomplete virologic response to telbivudine
Background & Aims: Analysis of the molecular pathways linking inflammation and hepatocyte survival is essential to identify novel therapies for inflammatory liver disease. Release of extracellular ATP and subsequent activation of purinergic P2 receptors (P2R) is increasingly recognized as important mediator of pro-inflammatory responses, raising the question of how ATP and P2R signaling contribute to the pathogenesis of acute hepatitis. Methods: To address this issue, acute hepatitis was induced in P2R knockout mice and controls by injection of Concanavalin A, an established model of fulminant and TNFα-dependent hepatitis. Moreover, hepatocyte-specific functions of P2R signaling in response to TNFα-mediated cell death were analyzed in primary mouse hepatocytes. Results: Induction of acute hepatitis in control animals resulted in a strong ATP release from the liver and induction of P2Y2R expression. Concanavalin A-mediated liver damage and necrosis were dramatically reduced in wild type mice co-injected with the P2YR inhibitor Suramin as well as in P2Y2R-/- mice. Analysis of chimeric mice revealed that P2Y2R expression in the bone marrow was required for efficient neutrophil infiltration to the liver. On the other hand, P2Y2R expression strongly promoted hepatocyte death In Vitro. On the molecular level, improved hepatocyte survival in the absence of P2Y2R correlated with increased activation of NF-κB and subsequent transcription of hepatoprotective genes. Conclusions: These findings indicate that extracellular ATP and P2Y2R have cell type-specific but synergistic functions during acute hepatitis by regulating cellular immune responses as well as hepatocyte survival. Targeting of P2Y2R may therefore be a promising approach to treat inflammatory liver disease.
Mo1533 Analysis of Differentially Expressed Genes and MicroRNAs in Alcoholic Liver Disease Ying Liu, You M. Li Abstract Background and Aim: To screen differentially expressed genes and MicroRNAs hoping bring us a new target spot to accurate diagnosis and effective therapy in gene and MicroRNA level of alcoholic liver disease(ALD).Methods: The total RNA of peripheral blood was extracted from four groups of three subjects.And all patients signed the informed consents. Microarrays were utilized to detect differentially expressed genes and MicroRNAs. According to gene values, significance level (p values) and false discovery rate with a random variance model, combining the GO and KEGG database, node genes and key MicroRNAs in network were obtained and analyzed.Results: A total of 878 differentially expressed genes and 26 MicroRNAs were discovered. Not all of these genes have biological effects on the development of ALD. By combining the gene ontology (GO) database and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, 16 significantly different pathways were found, where 31 corresponding genes participated in.Calculating betweenness centrality of each gene in network, which can describe the degree of importance of the nodes in the whole net. In co-expression network of genes, the node genes modulating the network were ACSF3, FZD5, LOC727987 and C1orf222. And in MicroRNA-Gene network the key MicroRNAs were hsa-miR-570, hsa-miR-122, hsa-miR-34b, hsa-miR-29c, hsa-miR-922 and hsa-miR-185 they negatively regulated about 79 genes locate their downstream.Conclusions: In the course of the ALD, we found 4 differentially expressed node genes in network and analyzed Acyl-coenzyme A synthetase-3 (ACSF3) , the maximal betweenness centrality in the entire gene-modulating network was 0.102935, indicating its controlling capability and significance level. Continuous up-regulation of ACSF3 maintains transcriptional activation and subsequently strengthens the binding of ACSF3 to thioester and CoA to activate fatty acids, which results in the irreversible advancement of ALD from hepatitis to cirrhosis. The gene values of Frizzled-5(FZD5) in alcoholic hepatitis and cirrhosis were clearly downregulated. The second highest value of betweenness centrality in the entire gene-modulating network was 0.087. A possible mechanism may be that continuous down-regulation of FZD5 during advancement of alcoholic hepatitis to cirrhosis decreased gene activities and syntheses of multi-transmembrane transport proteins. This reduced the number of β-catenin molecules in hepatocytes and therefore promoted hepatocyte apoptosis. The other two genes' functions were unknown. And the 6 key MicroRNAs perhaps controlled numerous biology functions such as immune response, activity of cancer gene, inflammatory mediated response, cell cycle, glutathione metabolism. Maybe the discovery will provide valuable directions to diagnosis and treatment of ALD.
Mo1536 Human Hepatocytes Produce Inflammatory Cytokines and Type I, II and III Interferons in Response to Toll-Like Receptor Activation, Resulting in IFN-λMediated Suppression of HCV In Vitro Ruth Bröring, Melanie Lutterbeck, Kathrin Kleinehr, Andreas Paul, Guido Gerken, Joerg F. Schlaak Background: The function of the hepatic innate immune system and its role in the defence against Hepatitis C Virus infection are not well understood. Recent publications suggested that type-III interferons (IFN; Interleukin-28A, -28B and -29) play a crucial role in the host response against HCV. Aim of this study was to investigate the Toll-like receptor (TLR) signaling in primary human hepatocytes, the capacity of these cells to produce type-III IFNs and their ability to control HCV replication. Methods: Human liver samples were obtained after tumor resection (n=16) or liver transplantation (n=14). Human hepatocytes (n=30) were isolated after perfusion and digestion of the liver. Cells were stimulated with TLR1-9 ligands for 6h to 24h, expression of Tumor necrosis factor α (TNF-α), Interleukin 6 (IL6), IL-10, IFN-α, -β, -γ and -λ (IL-28A, IL-28B and IL-29) were determined by qRT-PCR and ELISA. Gene expression of interferon sensitive genes (ISGs) was determined by qRTPCR. Supernatants from PHH treated with TLR ligands were pre-incubated with neutralizing antibodies and co-cultured with a subgenomic HCV replicon system (con1). Results: In human hepatocytes stimulation with TLR1-9 ligands, except TLR9, led to induction of proinflammatory (TNF-α, IL-6) and anti-inflammatory cytokines (IL-10). In contrast, expression of type-I, -II and -III interferons (IFN-α, -β, -γ, IL-28A, IL-28B, IL-29) could only be induced after TLR3 stimulation, herein IL-29 and IL-28A are significantly higher expressed than IFN-α and IFN-β, resulting in enhanced expression of ISGs (ISG15, IFI-T1, RSAD2, MXA). Therefore, only supernatants from TLR3-activated hepatocytes suppressed HCV replication in co-cultured con1 cells. This antiviral effect could not be neutralized by antibodies against IFN-α, -β or -γ. The different underlying diseases, the type of surgery, fibrosis stage as well as liver function tests (ALT, AST, GGT, AP) did not correlate with TLR signaling and antiviral activity in these cells. Conclusions Primary isolated human hepatocytes respond to TLR ligands by production of inflammatory cytokines as well as type-I, -II and -III IFNs, whereas the poly I:C-induced antiviral effect is exclusively mediated by type-III interferons. These findings shed new light on the importance of IFN-λ in the pathogenesis of HCV.
Mo1534 Association Between Telomere Length and Risk of Cirrhosis in Patients With Chronic Hepatitis B Jennifer S. Au, Hie-Won L. Hann, Shaogui Wan, Ronald E. Myers, Xiaoying Fu, Victor J. Navarro, Su H. Kim, Hushan Yang Background: The decision to treat chronic hepatitis B is based on clinical guidelines which utilize HBeAg status, transaminases, HBV DNA, and occasionally liver biopsy. The presence of known risk factors for the progression to cirrhosis, including older age, HDV or HIV coinfection, and alcohol consumption, influence the decision to treat. Telomeres consist of small tandem nucleotide repeats that form the physical ends of eukaryotic chromosomes. Shortened hepatocyte telomeres have been implicated in the development and progression to cirrhosis. Therefore, telomere length may serve as a potential constitutive biomarker for risk prediction or early detection of cirrhosis. Purpose: To examine telomere length in cellfree serum DNA as a non-invasive predictor of cirrhosis risk in patients with chronic hepatitis B. Methods: Serum samples were obtained from patients who visited The Liver Disease Prevention Center at Thomas Jefferson University Hospital between 1988 and 2010. One hundred patients with HBV cirrhosis were selected as cases and 100 chronic HBV noncirrhotic patients were selected as controls. All patients were of Korean ethnicity and controls were matched to cases by age, and sex. All patients were sero-negative for HCV and HDV. Demographic information including history of alcohol and tobacco use, as well as family history of cirrhosis were collected. Circulating DNA was extracted from 200μL of serum and then qRT-PCR was conducted in duplicate to measure the number of telomere repeats as compared to a gene with a single copy. Results: Cirrhotic HBV patients had a significantly longer (median 0.36; range 0.08-1.87) relative telomere length (RTL) than those without
Mo1537 TLR3-Dependent Immunological Properties of Liver Cells are Controlled by Anti-Inflammatory Cytokines Through Modulation of miR-155 Expression Min Jiang, Martin Trippler, Ruth Bröring, Jun Wu, Guido Gerken, Mengji Lu, Joerg F. Schlaak Background & Aims: The hepatitis C virus (HCV) can establish a persistent infection despite a strong activation of the innate immune system through TLR3 and other sensors, the so-called “IFN-paradox”. We analyzed regulatory mechanisms of TLR3-mediated immune responses in human liver and in murine non-parenchymal liver cells (NPC) Methods: Hepatic expression of Interleukin 10 (IL-10) and transforming growth factor beta (TGF-β) in biopsies
S-989
AASLD Abstracts
AASLD Abstracts
P2Y2 Receptor Function Aggravates Acute Hepatitis by Regulating Neutrophil Infiltration and Hepatocyte Survival Korcan Ayata, Marco Idzko, Peter Hasselblatt