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Persistent Increase in Lipid Levels in Patients With Hepatitis C Virus After Achieving Sustained Virologic Response
medications. All patients who have received anti-HBV treatment (Entecavir, Tenofovir or Tenofovir-Emtricitabine) have undetectable viral load at the present time. Conclusion: 1) Cirrhosis, advanced HCC and co-infection with HIV are not uncommon in patients with chronic HBV who have emigrated from Africa. 2) The majority of these patients are eligible for treatment of HBV infection. 3) Early identification, treatment, and screening for liver cancer in patients who have emigrated from Africa is warranted based on our findings
Mo1644
Background and aim: Recent studies have shown that excess hepatic iron accumulation contributes to liver injury in chronic hepatitis C (CHC) patients. Free iron in the liver is believed to facilitate the formation of reactive oxygen species (ROS), including hydroxyl radicals , which cause oxidative damage of numerous cellular components such as lipids, proteins, and nucleic acids, and also upregulate collagen synthesis. Further, the hydroxyl are known to generate promutagenic bases such as 8-hydroxy-2-deoxyguanosine (8-OHdG), which has been implicated in DNA mutagenesis and carcinogenesis. In general, 8-OHdG lesions may be promptly repaired by human 8-oxo-guanine DNA glycosylase (hOGG1) and human MutY homolog (MutYH). Therefore, it is plausible that the development of hepatocellular carcinoma (HCC) from CHC is determined by the balance between DNA damage and repair. In this study, we conducted comparative analyses of single nucleotide polymorphisms (SNPs) in hOGG1 and MutYH genes on patient of CHC with or without development of HCC. Methods: Genomic DNA and total RNA were extracted from peripheralblood mononuclear cells obtained from 65 patients of CHC without HCC (non-HCC group) and 31 patients with HCC (HCC group). We analyzed the several SNPs and mRNA of hOGG1 and MutYH by direct sequence and Taqman RT-PCR. The HCC incidence rate (hepatocarcinogenesis rate) was calculated based on the period between the diagnosis of CHC and the appearance of HCC, using the Kaplan-Meier technique. All factors found to be at least marginally associated with liver carcinogenesis (P< 0.15) were tested by the multivariate Cox proportional hazard model. P< 0.05 was considered to be significant. Results: The frequency of SNPs in hOGG1 did not differ between HCC group and nonHCC group. In one of MutYH SNPs (under patent application), the frequency of minor homo or hetero allele carrier in the HCC group significantly higher than that in HCC group. In addition, the expression of MutYH mRNA in minor homo allele tended to be lower than that in major homo allele. Multivariate Cox regression analysis indicated that Age (≥70yr) and the minor allele carrier significantly associated with hepatocarcinogenesis independently. Conclusions: These results indicated that the minor hetero allele of this SNP in MutYH could be a significant risk factor of liver carcinogenesis from CHC and could be a predictive marker of HCC development in CHC patients.
Mo1642 Characterization of Step-Wise Accumulation of DNA Methylation Alterations During Human Hepatocarcinogenesis Naoshi Nishida, Takeshi Nagasaka, C. Richard Boland, Tsutomu Chiba, Ajay Goel Background: Epigenetic aberrations are essential for the pathogenesis of hepatocellular carcinoma (HCC), and DNA methylation alterations are among the best markers that adequately represent this phenotype. However, there is an essential gap in knowledge since the timing of individual DNA methylation changes in HCC are unclear, and an understanding of such events could provide important insights into HCC pathogenesis. Aim: We characterized the role of step-wise accumulation of hypermethylation in a panel of HCC-related putative tumor suppressor genes (TSGs) as well as hypomethylation at repetitive DNA elements in a comprehensive spectrum of healthy, benign and neoplastic liver tissues. Methods: We analyzed a total of 180 HCCs and 178 matched non-tumor tissues. HCCs were classified as early, intermediate or advanced stage according to their size, differentiation status and tumor vascularity as determined by imaging studies. Methylation levels of 30 CpG loci consisting of 24 TSG promoters, 3 MINT loci and 3 repetitive DNA elements (LINE1, Alu and Sat2) were measured by quantitative COBRA and MethyLight assays. Results: Among 30 CpG loci, 22 revealed significant differences in methylation levels between HCC and non-tumor liver. We discovered that 8 TSGs showed a prominent increase in methylation levels between non-cancerous liver and early-stage tumors (p < 0.0001), while 4 CpGs represented significant differences in methylation between non-cancerous liver and intermediate-stage tumors, and 7 methylation loci robustly discriminated between advanced tumors and all other stages. We also observed a significant increase in hypomethylation of all repetitive DNA elements with disease progression. Of interest, hypomethylation of Sat2, which predominantly associates with heterochromatin disorganization and increased chromosomal instability, was especially prominent in early-stage tumors (p < 0.0001). The progressive hypermethylation of TSGs was predominant in HCV-positive cases, while the hypomethylation on repetitive DNA elements was more common in HCV-negative cases. Conclusions: Our comprehensive and systematic analyses in a large series of HCC allowed us to determine important sets of methylated genes at each step of hepatocarcinogenesis. Significantly increased methylation of 8 genes in early-stage tumors suggests that these are potential drivers for the clonal expansion of neoplastic cells that promote early HCC. Furthermore, our discovery of novel methylated genes in the intermediate and advancedstage tumors suggests that these genes might facilitate tumor growth in later stages of HCC. Hypomethylation of repetitive DNA elements, particularly Sat2, provides additional insights into the dynamic overlap of the simultaneous increase in chromosomal instability with epigenetic alterations in the pathogenesis of HCC.
Mo1645 Persistent Increase in Lipid Levels in Patients With Hepatitis C Virus After Achieving Sustained Virologic Response Marion-Anna Protano, Katherine Small, Douglas T. Dieterich, Marie-Louise C. Vachon Background and Aims: Hepatitis C virus (HCV) and low-density lipoprotein (LDL) are closely linked. HCV can enter hepatocytes via LDL receptors; LDL receptor expression correlates with HCV RNA levels. LDL levels increase at the time of achieving sustained virologic response (SVR) compared to baseline levels both in HCV mono-infected and HIV/HCV coinfected patients. Whether the increase in lipid levels is sustained beyond the time of SVR is unknown. Methods: A database of patients with chronic HCV infection who achieved SVR was retrospectively reviewed in a liver practice at a large university medical center between 06/2008 and 09/2010. Patients co-infected with hepatitis B virus were excluded. Paired Student t-test and Wilcoxon signed ranks test were used as appropriate to compare lipid levels at treatment initiation and after SVR among all patients and within subgroups of patients. Results: Of the 117 patients who achieved SVR after treatment with peg-interferon and ribavirin, 28 had lipid levels measured both at the time of treatment initiation and after achieving SVR. Their mean age was 54 years (SD=8), 18/28 (67%) were male and 18/28 (67%) had HCV genotype 1. Of the 28, 8 (29%) were co-infected with HIV. The mean time at which lipid levels were measured after SVR was 19 months (SD=14). Mean lipid levels at the time of treatment initiation and after achieving SVR were determined (Table 1). Total cholesterol (TC) and LDL increased significantly from the time of treatment initiation to a mean of 19 months after achieving SVR (respectively p=0.05 and p=0.01). No statistical significance was seen in high-density lipoprotein (HDL) or triglyceride levels. The increase in TC and LDL after achieving SVR was the most significant in those with HIV/HCV coinfection [mean increase in TC was 40.9 mg/dl (SD=36.7), p=0.02; and in LDL was 34.5 mg/dl (SD=28.4), p=0.01] and in those with higher baseline HCV RNA levels (>800,000 IU/ml) [mean increase in TC was 23.6 mg/dl (SD=41.5), p=0.05; and in LDL was 20.8 mg/ dl (SD=35.7), p=0.05]. Traditional risk factors for hyperlipidemia (smoking, hypertension, obesity, and diabetes) had no consistent effect on the change in lipids from baseline to a mean of 19 months after achieving SVR. Conclusions: TC and LDL levels are significantly and persistently elevated compared to baseline at a mean of 19 months after achieving SVR. Patients with higher baseline HCV RNA levels are more likely to have significant elevation of TC and LDL levels after achieving SVR. This may explain the more pronounced lipid increase observed in HIV-infected patients achieving SVR. These results support continued study of the relationship between LDL, HCV and SVR. Table 1. Mean lipid levels before treatment and at a mean of 19 months after achieving SVR
Mo1643 The Toll-Like Receptor Mediated Induction of Interferons is Not Impaired in Primary Liver Cells From HCV-Positive Patients Ruth Bröring, Kathrin Kleinehr, Andreas Paul, Guido Gerken, Joerg F. Schlaak Aims and Background: The function of the innate immune system of the human liver and its role in the local defense against HCV are not well understood. Recent data from our lab suggested that murine hepatocytes and non-parenchymal liver cells (NPC) can elicit potent antiviral responses and immune modulation. On the contrary it has been reported that TLR induced IFN production in HCV-bearing hepatoma cells is blunted by a NS3/4 dependent mechanism. Therefore, the aim of this study was to investigate the TLR signaling in primary isolated human hepatocytes as well as non parenchymal liver cells from HCV-positive and HCV-negative individuals. Materials and methods: Human liver samples were obtained after tumor resection (n=10) or liver transplantation (n=19; HCV n=8), respectively. Human hepatocytes (n=22) were isolated after liver perfusion and digestion of the liver. In addition, endothelial cells (LSEC, n=5), stellate cells (HSC, n=4) and a mix of remaining NPC (n= 13) were purified by density centrifugation and MACS separation. Cells were stimulated with TLR1-9 ligands for 6h, RNA was extracted and expression of TNF-α, IL-6, IL-10, a subset of interferons and interferon sensitive genes (ISGs) was determined by qRT-PCR. Results: In human hepatocytes as well as in different NPCs stimulation with TLR1-9 ligands, except TLR9, led to cell type specific induction of type-I interferons, pro-inflammatory (TNF-α, IL-6) and anti-inflammatory cytokines (IL-10). In contrast, expression of type-I and -II interferons (IFN-α, -β, -γ, IL28A, IL28B and IL29) could only be induced after TLR3 stimulation, resulting in enhanced expression of ISGs (ISG15, IFI-T1, RSAD2 and MXA). The different underlying diseases, the type of surgery, fibrosis stage as well as liver function tests (ALT, AST, GGT and AP) were not correlated with TLR signaling in these cells. Primary hepatocytes, but not NPC, from HCV infected patients (n=6) showed lower IL-10 expression levels after TLR stimulation. Conclusions: In general, liver cells isolated from HCV infected individuals respond to TLR ligands in a similar fashion as compared to HCV-negative controls. In contrast to data generated in hepatoma cell lines, primary hepatocytes from HCV-infected patients are able to produce a variety of IFNs after TLR stimulation.
S-971
AASLD Abstracts
AASLD Abstracts
The Comparative Analyses of Single Nucleotide Polymorphism of Oxidative DNA Repair Genes in Patients With Chronic Hepatitis C Koji Miyanishi, Masayoshi Kobune, Shingo Tanaka, Hiroyuki Nagashima, Tsutomu Sato, Yasushi Sato, Rishu Takimoto, Junji Kato
Mo1644
Background and aim: Recent studies have shown that excess hepatic iron accumulation contributes to liver injury in chronic hepatitis C (CHC) patients. Free iron in the liver is believed to facilitate the formation of reactive oxygen species (ROS), including hydroxyl radicals , which cause oxidative damage of numerous cellular components such as lipids, proteins, and nucleic acids, and also upregulate collagen synthesis. Further, the hydroxyl are known to generate promutagenic bases such as 8-hydroxy-2-deoxyguanosine (8-OHdG), which has been implicated in DNA mutagenesis and carcinogenesis. In general, 8-OHdG lesions may be promptly repaired by human 8-oxo-guanine DNA glycosylase (hOGG1) and human MutY homolog (MutYH). Therefore, it is plausible that the development of hepatocellular carcinoma (HCC) from CHC is determined by the balance between DNA damage and repair. In this study, we conducted comparative analyses of single nucleotide polymorphisms (SNPs) in hOGG1 and MutYH genes on patient of CHC with or without development of HCC. Methods: Genomic DNA and total RNA were extracted from peripheralblood mononuclear cells obtained from 65 patients of CHC without HCC (non-HCC group) and 31 patients with HCC (HCC group). We analyzed the several SNPs and mRNA of hOGG1 and MutYH by direct sequence and Taqman RT-PCR. The HCC incidence rate (hepatocarcinogenesis rate) was calculated based on the period between the diagnosis of CHC and the appearance of HCC, using the Kaplan-Meier technique. All factors found to be at least marginally associated with liver carcinogenesis (P< 0.15) were tested by the multivariate Cox proportional hazard model. P< 0.05 was considered to be significant. Results: The frequency of SNPs in hOGG1 did not differ between HCC group and nonHCC group. In one of MutYH SNPs (under patent application), the frequency of minor homo or hetero allele carrier in the HCC group significantly higher than that in HCC group. In addition, the expression of MutYH mRNA in minor homo allele tended to be lower than that in major homo allele. Multivariate Cox regression analysis indicated that Age (≥70yr) and the minor allele carrier significantly associated with hepatocarcinogenesis independently. Conclusions: These results indicated that the minor hetero allele of this SNP in MutYH could be a significant risk factor of liver carcinogenesis from CHC and could be a predictive marker of HCC development in CHC patients.
Mo1642 Characterization of Step-Wise Accumulation of DNA Methylation Alterations During Human Hepatocarcinogenesis Naoshi Nishida, Takeshi Nagasaka, C. Richard Boland, Tsutomu Chiba, Ajay Goel Background: Epigenetic aberrations are essential for the pathogenesis of hepatocellular carcinoma (HCC), and DNA methylation alterations are among the best markers that adequately represent this phenotype. However, there is an essential gap in knowledge since the timing of individual DNA methylation changes in HCC are unclear, and an understanding of such events could provide important insights into HCC pathogenesis. Aim: We characterized the role of step-wise accumulation of hypermethylation in a panel of HCC-related putative tumor suppressor genes (TSGs) as well as hypomethylation at repetitive DNA elements in a comprehensive spectrum of healthy, benign and neoplastic liver tissues. Methods: We analyzed a total of 180 HCCs and 178 matched non-tumor tissues. HCCs were classified as early, intermediate or advanced stage according to their size, differentiation status and tumor vascularity as determined by imaging studies. Methylation levels of 30 CpG loci consisting of 24 TSG promoters, 3 MINT loci and 3 repetitive DNA elements (LINE1, Alu and Sat2) were measured by quantitative COBRA and MethyLight assays. Results: Among 30 CpG loci, 22 revealed significant differences in methylation levels between HCC and non-tumor liver. We discovered that 8 TSGs showed a prominent increase in methylation levels between non-cancerous liver and early-stage tumors (p < 0.0001), while 4 CpGs represented significant differences in methylation between non-cancerous liver and intermediate-stage tumors, and 7 methylation loci robustly discriminated between advanced tumors and all other stages. We also observed a significant increase in hypomethylation of all repetitive DNA elements with disease progression. Of interest, hypomethylation of Sat2, which predominantly associates with heterochromatin disorganization and increased chromosomal instability, was especially prominent in early-stage tumors (p < 0.0001). The progressive hypermethylation of TSGs was predominant in HCV-positive cases, while the hypomethylation on repetitive DNA elements was more common in HCV-negative cases. Conclusions: Our comprehensive and systematic analyses in a large series of HCC allowed us to determine important sets of methylated genes at each step of hepatocarcinogenesis. Significantly increased methylation of 8 genes in early-stage tumors suggests that these are potential drivers for the clonal expansion of neoplastic cells that promote early HCC. Furthermore, our discovery of novel methylated genes in the intermediate and advancedstage tumors suggests that these genes might facilitate tumor growth in later stages of HCC. Hypomethylation of repetitive DNA elements, particularly Sat2, provides additional insights into the dynamic overlap of the simultaneous increase in chromosomal instability with epigenetic alterations in the pathogenesis of HCC.
Mo1645 Persistent Increase in Lipid Levels in Patients With Hepatitis C Virus After Achieving Sustained Virologic Response Marion-Anna Protano, Katherine Small, Douglas T. Dieterich, Marie-Louise C. Vachon Background and Aims: Hepatitis C virus (HCV) and low-density lipoprotein (LDL) are closely linked. HCV can enter hepatocytes via LDL receptors; LDL receptor expression correlates with HCV RNA levels. LDL levels increase at the time of achieving sustained virologic response (SVR) compared to baseline levels both in HCV mono-infected and HIV/HCV coinfected patients. Whether the increase in lipid levels is sustained beyond the time of SVR is unknown. Methods: A database of patients with chronic HCV infection who achieved SVR was retrospectively reviewed in a liver practice at a large university medical center between 06/2008 and 09/2010. Patients co-infected with hepatitis B virus were excluded. Paired Student t-test and Wilcoxon signed ranks test were used as appropriate to compare lipid levels at treatment initiation and after SVR among all patients and within subgroups of patients. Results: Of the 117 patients who achieved SVR after treatment with peg-interferon and ribavirin, 28 had lipid levels measured both at the time of treatment initiation and after achieving SVR. Their mean age was 54 years (SD=8), 18/28 (67%) were male and 18/28 (67%) had HCV genotype 1. Of the 28, 8 (29%) were co-infected with HIV. The mean time at which lipid levels were measured after SVR was 19 months (SD=14). Mean lipid levels at the time of treatment initiation and after achieving SVR were determined (Table 1). Total cholesterol (TC) and LDL increased significantly from the time of treatment initiation to a mean of 19 months after achieving SVR (respectively p=0.05 and p=0.01). No statistical significance was seen in high-density lipoprotein (HDL) or triglyceride levels. The increase in TC and LDL after achieving SVR was the most significant in those with HIV/HCV coinfection [mean increase in TC was 40.9 mg/dl (SD=36.7), p=0.02; and in LDL was 34.5 mg/dl (SD=28.4), p=0.01] and in those with higher baseline HCV RNA levels (>800,000 IU/ml) [mean increase in TC was 23.6 mg/dl (SD=41.5), p=0.05; and in LDL was 20.8 mg/ dl (SD=35.7), p=0.05]. Traditional risk factors for hyperlipidemia (smoking, hypertension, obesity, and diabetes) had no consistent effect on the change in lipids from baseline to a mean of 19 months after achieving SVR. Conclusions: TC and LDL levels are significantly and persistently elevated compared to baseline at a mean of 19 months after achieving SVR. Patients with higher baseline HCV RNA levels are more likely to have significant elevation of TC and LDL levels after achieving SVR. This may explain the more pronounced lipid increase observed in HIV-infected patients achieving SVR. These results support continued study of the relationship between LDL, HCV and SVR. Table 1. Mean lipid levels before treatment and at a mean of 19 months after achieving SVR
Mo1643 The Toll-Like Receptor Mediated Induction of Interferons is Not Impaired in Primary Liver Cells From HCV-Positive Patients Ruth Bröring, Kathrin Kleinehr, Andreas Paul, Guido Gerken, Joerg F. Schlaak Aims and Background: The function of the innate immune system of the human liver and its role in the local defense against HCV are not well understood. Recent data from our lab suggested that murine hepatocytes and non-parenchymal liver cells (NPC) can elicit potent antiviral responses and immune modulation. On the contrary it has been reported that TLR induced IFN production in HCV-bearing hepatoma cells is blunted by a NS3/4 dependent mechanism. Therefore, the aim of this study was to investigate the TLR signaling in primary isolated human hepatocytes as well as non parenchymal liver cells from HCV-positive and HCV-negative individuals. Materials and methods: Human liver samples were obtained after tumor resection (n=10) or liver transplantation (n=19; HCV n=8), respectively. Human hepatocytes (n=22) were isolated after liver perfusion and digestion of the liver. In addition, endothelial cells (LSEC, n=5), stellate cells (HSC, n=4) and a mix of remaining NPC (n= 13) were purified by density centrifugation and MACS separation. Cells were stimulated with TLR1-9 ligands for 6h, RNA was extracted and expression of TNF-α, IL-6, IL-10, a subset of interferons and interferon sensitive genes (ISGs) was determined by qRT-PCR. Results: In human hepatocytes as well as in different NPCs stimulation with TLR1-9 ligands, except TLR9, led to cell type specific induction of type-I interferons, pro-inflammatory (TNF-α, IL-6) and anti-inflammatory cytokines (IL-10). In contrast, expression of type-I and -II interferons (IFN-α, -β, -γ, IL28A, IL28B and IL29) could only be induced after TLR3 stimulation, resulting in enhanced expression of ISGs (ISG15, IFI-T1, RSAD2 and MXA). The different underlying diseases, the type of surgery, fibrosis stage as well as liver function tests (ALT, AST, GGT and AP) were not correlated with TLR signaling in these cells. Primary hepatocytes, but not NPC, from HCV infected patients (n=6) showed lower IL-10 expression levels after TLR stimulation. Conclusions: In general, liver cells isolated from HCV infected individuals respond to TLR ligands in a similar fashion as compared to HCV-negative controls. In contrast to data generated in hepatoma cell lines, primary hepatocytes from HCV-infected patients are able to produce a variety of IFNs after TLR stimulation.
S-971
AASLD Abstracts
AASLD Abstracts
The Comparative Analyses of Single Nucleotide Polymorphism of Oxidative DNA Repair Genes in Patients With Chronic Hepatitis C Koji Miyanishi, Masayoshi Kobune, Shingo Tanaka, Hiroyuki Nagashima, Tsutomu Sato, Yasushi Sato, Rishu Takimoto, Junji Kato