ABSTRACT
AIM: Gamma-glutamyltransferase (GGT) is known as an oxidative stress marker, induced by alcohol consumption and metabolic disorders, and is reported as a predictor of hepatocellular carcinoma (HCC) development after hepatitis C virus (HCV) elimination. However, it is not clear whether GGT serves simply as a surrogate marker for overlapping metabolic diseases or reflects HCV-specific carcinogenicity. We investigated the association between GGT and hepatocarcinogenesis after achieving a sustained viral response (SVR), accounting for drinking habits or diabetes, and examined predisposing factors associated with GGT levels after SVR. METHODS: This is a prospective, multicenter, and observational study using the database of 1001 patients after HCV eradication with direct-acting antiviral agents. The association of GGT at SVR with cumulative HCC development was examined in a multivariate analysis using Cox proportional hazard models after adjustment for covariates including alcohol and diabetes. The association between oxidative stress markers or genetic factors and GGT levels was analyzed. RESULTS: High GGT levels at SVR were associated with HCC development (HR] 2.38, 95% CI 1.10-5.17). This association was also significant when restricted to patients without alcohol consumption or diabetes (HR 8.38, 95% CI 2.87-24.47). GGT levels were correlated with serum growth differentiation factor 15 levels, a marker of mitochondrial dysfunction. Single-nucleotide polymorphisms of ZNF827 and GDF15 were associated with high GGT levels. CONCLUSIONS: High GGT levels at SVR were associated with HCC development after accounting for alcohol consumption and diabetes. GGT levels are influenced by genetic predisposition and may reflect mitochondrial dysfunction after HCV eradication.
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Hepatic stellate cells (HSCs) are responsible for liver fibrosis accompanied by its activation into myofibroblasts and the abundant production of extracellular matrix. However, the HSC contribution to progression of liver inflammation has been less known. We aimed to elucidate the mechanism in HSCs underlying the inflammatory response and the function of tumor necrosis factor α-related protein A20 (TNFAIP3). We established A20 conditional knockout (KO) mice crossing Twist2-Cre and A20 floxed mice. Using these mice, the effect of A20 was analyzed in mouse liver and HSCs. The human HSC line LX-2 was also used to examine the role and underlying molecular mechanism of A20. In this KO model, A20 was deficient in >80% of HSCs. Spontaneous inflammation with mild fibrosis was found in the liver of the mouse model without any exogenous agents, suggesting that A20 in HSCs suppresses chronic hepatitis. Comprehensive RNA sequence analysis revealed that A20-deficient HSCs exhibited an inflammatory phenotype and abnormally expressed chemokines. A20 suppressed JNK pathway activation in HSCs. Loss of A20 function in LX-2 cells also induced excessive chemokine expression, mimicking A20-deficient HSCs. A20 overexpression suppressed chemokine expression in LX-2. In addition, we identified DCLK1 in the genes regulated by A20. DCLK1 activated the JNK pathway and upregulates chemokine expression. DCLK1 inhibition significantly decreased chemokine induction by A20-silencing, suggesting that A20 controlled chemokine expression in HSCs via the DCLK1-JNK pathway. In conclusion, A20 suppresses chemokine induction dependent on the DCLK1-JNK signaling pathway. These findings demonstrate the therapeutic potential of A20 and the DCLK1-JNK pathway for the regulation of inflammation in chronic hepatitis.
Subject(s)
Chemokines , Hepatic Stellate Cells , MAP Kinase Signaling System , Mice, Knockout , Protein Serine-Threonine Kinases , Tumor Necrosis Factor alpha-Induced Protein 3 , Animals , Hepatic Stellate Cells/metabolism , Tumor Necrosis Factor alpha-Induced Protein 3/metabolism , Tumor Necrosis Factor alpha-Induced Protein 3/genetics , Mice , Humans , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Chemokines/metabolism , Chemokines/genetics , Hepatitis, Chronic/metabolism , Hepatitis, Chronic/pathology , Hepatitis, Chronic/genetics , Doublecortin-Like Kinases , Mice, Inbred C57BL , Cell Line , MaleABSTRACT
BACKGROUND/AIM: Antiviral hepatitis and systemic therapies for hepatocellular carcinoma (HCC) remarkably progressed in the recent 10 years. This study aimed to reveal the actual transition and changes in the prognosis and background liver disease in non-advanced HCC in the past 20 years. METHODS: This retrospectively recruited 566 patients who were diagnosed with non-advanced HCC from February 2002 to February 2022. The prognosis was analyzed by subdividing according to the diagnosis date (period I: February 2002-April 2009 and period â ¡: May 2009-February 2022). RESULTS: Patients in period II (n = 351) were significantly older, with lower albumin-bilirubin (ALBI) scores and alpha-fetoprotein (AFP) and more anti-viral therapy, systemic therapy, and hepatic arterial infusion chemotherapy as compared with those in period I (n = 215). The etiology ratio of the background liver disease revealed decreased hepatitis C virus from 70.6% to 49.0% and increased non-B, non-C from 17.7% to 39.9% from periods I to â ¡. The multivariate analysis revealed older age and higher ALBI score in Barcelona Clinic Liver Cancer (BCLC) 0/A stage, AFP of >20 ng/mL, and higher ALBI score in BCLC B stage as independent prognosis factors. Fine-Gray competing risk model analysis revealed that liver-related deaths significantly decreased in period II as compared to period I, especially for BCLC stage 0/A (HR: 0.656; 95%CI: 0.442-0.972, P = 0.036). CONCLUSION: The characteristics of patients with non-advanced HCC have changed over time. Appropriate background liver management led to better liver-related prognoses in BCLC 0/A.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Humans , Carcinoma, Hepatocellular/pathology , Liver Neoplasms/pathology , alpha-Fetoproteins , Retrospective Studies , PrognosisABSTRACT
BACKGROUND: It is crucial to identify risk factors for life prognosis after hepatitis C virus (HCV) eradication among patients with or without a high risk of liver cancer or complications. METHODS: This is a prospective, multicenter and observational study using the database of 1031 patients after HCV eradication by direct-acting antiviral agents (DAAs) to evaluate the development of hepatocellular carcinoma (HCC) and patients' survival after a sustained virological response (SVR). The Cox proportional hazards regression model was used to estimate hazard ratios associated with HCC development and survival. RESULTS: AFP at SVR was significantly associated with HCC recurrence in the adjusted model. Liver fibrosis, Mac-2 binding protein glycosylation isomer (M2BPGi) at SVR and smoking status before treatment were positively associated with the development of HCC and M2BPGi was positively associated with HCC recurrence, although not reaching statistical significance. Among patients without a history of HCC, M2BPGi and estimated glomerular filtration rate (eGFR) at SVR were significantly associated with death after viral eradication [M2BPGi (HR 4.07, 95% CI 1.22, 13.57), eGFR (HR 0.97, 95% CI 0.94, 0.99)]. Strikingly, of 16 patients who died, among participants without a history of HCC, only two died of liver cancer associated with HCV, whereas 11 died of non-HCV- related cancer or cardiovascular diseases. CONCLUSION: M2BPGi at SVR is a potential predictor for patients' survival and a candidate biomarker for detecting individuals who are at greater risk of death due to cancer-related and unrelated to HCV, as well as cardiovascular diseases, after viral eradication.
Subject(s)
Antigens, Neoplasm/metabolism , Antiviral Agents/administration & dosage , Biomarkers, Tumor/metabolism , Carcinoma, Hepatocellular/epidemiology , Hepatitis C, Chronic/drug therapy , Liver Neoplasms/epidemiology , Aged , Aged, 80 and over , Carcinoma, Hepatocellular/virology , Female , Hepatitis C, Chronic/complications , Humans , Liver Neoplasms/virology , Male , Middle Aged , Prognosis , Prospective Studies , Risk Factors , Survival Rate , Sustained Virologic ResponseABSTRACT
Formation of intrahepatic bile ducts (IHBDs) proceeds in accordance with their microenvironment. Particularly, mesenchymal cells around portal veins regulate the differentiation and ductular morphogenesis of cholangiocytes in the developing liver; however, further studies are needed to fully understand the arrangement of IHBDs into a continuous hierarchical network. This study aims to clarify the interaction between biliary and liver mesenchymal cells during IHBD formation. To identify candidate factors contributing to this cell-cell interaction, mesenchymal cells were isolated from embryonic day 16.5 matrix metalloproteinase 14 (MMP14)-deficient (knockout [KO]) mice livers, in which IHBD formation is retarded, and compared with those of the wild type (WT). WT mesenchymal cells significantly facilitated the formation of luminal structures comprised of hepatoblast-derived cholangiocytes (cholangiocytic cysts), whereas MMP14-KO mesenchymal cells failed to promote cyst formation. Comprehensive analysis revealed that expression of vasoactive intestinal peptide (VIP) was significantly suppressed in MMP14-KO mesenchymal cells. VIP and VIP receptor 1 (VIPR1) were mainly expressed in periportal mesenchymal cells and cholangiocytic progenitors during IHBD development, respectively, in vivo. VIP/VIPR1 signaling significantly encouraged cholangiocytic cyst formation and up-regulated tight junction protein 1, cystic fibrosis transmembrane conductance regulator, and aquaporin 1, in vitro. VIP antagonist significantly suppressed the tight junction assembly and the up-regulation of ion/water transporters during IHBD development in vivo. In a cholestatic injury model of adult mice, exogenous VIP administration promoted the restoration of damaged tight junctions in bile ducts and improved hyperbilirubinemia. Conclusion: VIP is produced by periportal mesenchymal cells during the perinatal stage. It supports bile duct development by establishing tight junctions and up-regulating ion/water transporters in cholangiocytes. VIP contributes to prompt recovery from cholestatic damage through the establishment of tight junctions in the bile ducts.
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AIM: Hepatitis E virus (HEV) can cause chronic infection in immunocompromised hosts. However, the dynamics of HEV during persistent infection is not well understood. To elucidate time course alterations in virus sequences and anti-HEV antibodies during persistent infection, we analyzed the HEV sequences and titers of anti-HEV antibodies from a chronic hepatitis E patient. METHODS: Serum samples were obtained from a chronic hepatitis E patient under corticosteroid therapy for neurological disease. The titers of anti-HEV antibodies (immunoglobulin A, immunoglobulin M, and immunoglobulin G) in serum samples were detected by enzyme immunoassay. The full or near-full nucleotide sequences of HEV isolated from consecutive serum samples were identified and compared. Phylogenetic analysis was also performed. RESULTS: Alterations of anti-HEV antibodies from a chronic hepatitis E patient were different from those previously reported in acute hepatitis E patients. The virus sequence was unchanged in the period without treatment, but nucleotide mutations were observed after ribavirin treatment was started. In addition, the sequence of this strain had extremely high identity to that isolated from swine liver in Japan. CONCLUSIONS: Virus mutations in HEV emerged after ribavirin treatment was started. Sequence analysis may useful for deciding the treatment strategy for chronic hepatitis E patients who did not eliminate the virus with 3 months of RBV treatment and inferring the origin of the infection. This report provides insights into the chronicity of hepatitis E, and the impact of persistent infection and ribavirin treatment on the emergence of virus mutations.
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AIM: The genetic profile of cholangiolocellular carcinoma (CLC) and its origin in relation to hepatocellular carcinoma (HCC) remain unclear. To elucidate the genetic profile of CLC, a comprehensive analysis of genetic mutations was carried out in a case of CLC with an HCC-like focal area and metachronous HCC. METHOD: Liver tissue was obtained from CLC, a co-existent HCC-like area, and metachronously developed HCC by laser capture microdissection of formalin-fixed paraffin-embedded specimens obtained by hepatectomy. Gene mutational profiles were analyzed comprehensively by next-generation sequencing and digital PCR. Relationships among gene profiles, immunohistochemistry, and clinicopathological findings were investigated. RESULTS: Mutations in EGFR, PTEN, RB1, TP53, and ERBB2 were found in CLC, whereas mutations in KIT, BRAF, PTEN, TP53, and SMAD4 were found in the coexistent HCC-like area. Only the mutation in PTEN has a common Catalogue of Somatic Mutations in Cancer ID in the CLC and coexistent HCC-like area, and is related to the kinase-RAS module. In contrast, no cancer-related mutations were found in the metachronous HCC. No TERT mutations were found in any of the regions by digital PCR. Immunohistochemical staining for p53 was negative in CLC, although ≤10% positive in the coexistent HCC-like area. Immunostaining of C-kit, HER2, PTEN, and SMAD4 were negative. CONCLUSION: The genomic features of CLC and the focal area of an HCC-like region differ, but are related to the kinase-RAS module. The development of carcinogenesis in the CLC and HCC-like areas in this case might differ, following a common PTEN mutation, although alteration of the kinase-RAS module is the most common molecular event in CLC.
ABSTRACT
Resistance-associated substitutions (RASs) of hepatitis C virus (HCV) in the NS5A region impair the efficacy of NS5A inhibitors. In this study, we evaluated the characteristics of the novel RASs observed in treatment-failure patients, A92K and a deletion at P32 (P32del), and the susceptibility of viruses with these RASs to various anti-HCV reagents by using JFH-1 based recombinant HCV with NS5A from a genotype 1b Con1 strain (JFH1/5ACon1). We introduced A92K or P32del solely or in combination with Q24K, L28M, R30Q or L31F into the NS5A of JFH1/5ACon1. Viruses harboring R30Q/A92K showed high extracellular core antigens and infectivity titers, whereas the other viruses with RASs showed low replication levels and infectivity titers. All the viruses with A92K or P32del were markedly resistant to ledipasvir, velpatasvir and elbasvir. Interestingly, viruses with R30Q/A92K were more susceptible to grazoprevir than viruses without RAS. All the viruses had a similar susceptibility to ribavirin and sofosbuvir. In conclusion, combination RASs R30Q/A92K enhanced virus production whereas other RASs impaired virus replication. Both A92K and P32del conferred severe resistance even to second generation NS5A inhibitors. However, these viruses were susceptible to grazoprevir, ribavirin and sofosbuvir. Thus, combination regimens with these reagents may eradicate viruses harboring A92K or P32del.
Subject(s)
Antiviral Agents/therapeutic use , Drug Resistance, Viral/genetics , Hepacivirus/genetics , Hepatitis C/drug therapy , Viral Nonstructural Proteins/genetics , Antiviral Agents/pharmacology , Cell Line , Genotype , Hepacivirus/drug effects , Humans , Sustained Virologic Response , Treatment FailureABSTRACT
BACKGROUND & AIMS: Congenital hepatic fibrosis (CHF) is a genetic liver disease resulting in abnormal proliferation of cholangiocytes and progressive hepatic fibrosis. CHF is caused by mutations in the PKHD1 gene and the subsequent dysfunction of the protein it encodes, fibrocystin. However, the underlying molecular mechanism of CHF, which is quite different from liver cirrhosis, remains unclear. This study investigated the molecular mechanism of CHF pathophysiology using a genetically engineered human induced pluripotent stem (iPS) cell model to aid the discovery of novel therapeutic agents for CHF. METHODS: PKHD1-knockout (PKHD1-KO) and heterozygously mutated PKHD1 iPS clones were established by RNA-guided genome editing using the CRISPR/Cas9 system. The iPS clones were differentiated into cholangiocyte-like cells in cysts (cholangiocytic cysts [CCs]) in a 3D-culture system. RESULTS: The CCs were composed of a monolayer of cholangiocyte-like cells. The proliferation of PKHD1-KO CCs was significantly increased by interleukin-8 (IL-8) secreted in an autocrine manner. IL-8 production was significantly elevated in PKHD1-KO CCs due to mitogen-activated protein kinase pathway activation caused by fibrocystin deficiency. The production of connective tissue growth factor (CTGF) was also increased in PKHD1-KO CCs in an IL-8-dependent manner. Furthermore, validation analysis demonstrated that both the serum IL-8 level and the expression of IL-8 and CTGF in the liver samples were significantly increased in patients with CHF, consistent with our in vitro human iPS-disease model of CHF. CONCLUSIONS: Loss of fibrocystin function promotes IL-8-dependent proliferation of, and CTGF production by, human cholangiocytes, suggesting that IL-8 and CTGF are essential for the pathogenesis of CHF. IL-8 and CTGF are candidate molecular targets for the treatment of CHF. LAY SUMMARY: Congenital hepatic fibrosis (CHF) is a genetic liver disease caused by mutations of the PKHD1 gene. Dysfunction of the protein it encodes, fibrocystin, is closely associated with CHF pathogenesis. Using an in vitro human induced pluripotent stem cell model and patient samples, we showed that the loss of fibrocystin function promotes proliferation of cholangiocytes and the production of connective tissue growth factor (CTGF) in an interleukin 8 (IL-8)-dependent manner. These results suggest that IL-8 and CTGF are essential for the pathogenesis of CHF.
Subject(s)
Connective Tissue Growth Factor/metabolism , Epithelial Cells/metabolism , Genetic Diseases, Inborn/metabolism , Liver Cirrhosis/metabolism , Bile Ducts/pathology , Cell Proliferation , Gene Editing/methods , Humans , Induced Pluripotent Stem Cells , Interleukin-8/metabolism , Mutagenesis, Site-Directed/methods , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolismABSTRACT
Human induced pluripotent stem (iPS) cells can differentiate into hepatocyte lineages, although the phenotype of the differentiated cells is immature compared to adult hepatocytes. Improvement of cell-cell interactions between epithelium and mesenchyme is a potential approach to address this phenotype issue. In this study, we developed a model system for improving interactions between human iPS-derived hepatic progenitor cells (iPS-HPCs) and human iPS-derived hepatic stellate cell-like cells (iPS-HSCs). The phenotype of iPS-HSCs, including gene and protein expression profiles and vitamin A storage, resembled that of hepatic stellate cells. Direct co-culture of iPS-HSCs with iPS-HPCs significantly improved hepatocytic maturation in iPS-HPCs, such as their capacity for albumin production. Next, we generated iPS cell lines overexpressing LIM homeobox 2 (LHX2), which suppresses myofibroblastic changes in HSCs in mice. Hepatocytic maturation in iPS-HPCs was significantly increased in direct co-culture with iPS-HSCs overexpressing LHX2, but not in co-culture with a human hepatic stellate cell line (LX-2) overexpressing LHX2. LHX2 regulated the expression of extracellular matrices, such as laminin and collagen, in iPS-HSCs. In conclusion, this study provides an evidence that LHX2 upregulation in iPS-HSCs promotes hepatocytic maturation of iPS-HPCs, and indicates that genetically modified iPS-HSCs will be of value for research into cell-cell interactions.
Subject(s)
Hepatic Stellate Cells/metabolism , Induced Pluripotent Stem Cells/metabolism , LIM-Homeodomain Proteins/metabolism , Liver/metabolism , Transcription Factors/metabolism , Animals , Cell Communication/physiology , Cell Culture Techniques/methods , Cell Differentiation/physiology , Cell Line , Coculture Techniques/methods , Hepatocytes/metabolism , Humans , Mice , Up-Regulation/physiologyABSTRACT
BACKGROUND: The risk factors associated with the development of hepatocellular carcinoma (HCC) in nonalcoholic fatty liver disease (NAFLD) are still unclear. The aim of the present study was to identify such risk factors in NAFLD patients who developed HCC. METHODS: Between April 2000 and -December 2016, a total of 182 patients with NAFLD were enrolled in this study; of these, only 22 patients had HCC. To identify risk factors, univariate and multivariate analyses were performed. To identify risk factors other than the degree of fibrosis, propensity matched analysis adjusted by the NAFLD fibrosis score (NFS) was carried out on 44 patients. Multivariate and survival analyses were also performed in HCC patients. RESULTS: In 182 patients, multivariate analysis highlighted the NFS (OR 2.275; p < 0.001) and hypertension (OR 5.868; p = 0.037) as independent factors that were significantly associated with the development of HCC. After adjustment for the NFS, multivariate analysis identified diabetic retinopathy (OR 8.654; p = 0.017) as an independent factor that was significantly associated with the development of HCC. For predicting the development of HCC, the area under the receiver operating characteristic curve of diabetic retinopathy was significantly higher than that of diabetes (0.731 vs. 0.615; p < 0.001). In patients with HCC, multivariate analysis indicated that the NFS were significantly associated with diabetic retinopathy. CONCLUSIONS: Diabetic retinopathy as well as liver fibrosis is a risk factor that associates with the development of HCC in NAFLD patients. Therefore, NAFLD patients with diabetic retinopathy should undergo careful screening for HCC.
Subject(s)
Carcinoma, Hepatocellular/complications , Diabetic Retinopathy/etiology , Liver Neoplasms/complications , Non-alcoholic Fatty Liver Disease/complications , Adult , Aged , Aged, 80 and over , Carcinoma, Hepatocellular/pathology , Female , Humans , Liver Cirrhosis/pathology , Liver Neoplasms/pathology , Male , Middle Aged , Multivariate Analysis , Non-alcoholic Fatty Liver Disease/diagnosis , Propensity Score , ROC Curve , Risk FactorsABSTRACT
AIM: Although the efficacy of hepatitis C virus (HCV) treatment is improved dramatically by direct-acting antiviral agents (DAAs), the assessment of hepatocellular carcinoma (HCC) remains important. Interferon lambda 3 (IFN-λ3) is associated with liver fibrosis and inflammation in chronic hepatitis C (CHC) patients, but its impact on carcinogenesis remains controversial and little is known about its effects after viral clearance. To determine the contribution of IFN-λ3 to hepatocarcinogenesis after HCV clearance, we analyzed IFNL3 genotypes and serial serum IFN-λ3 levels in CHC patients who achieved sustained virologic responses (SVR). METHODS: This study comprised 201 CHC patients treated with DAAs. Serum samples were collected sequentially and IFN-λ3 levels were quantified by chemiluminescence enzyme immunoassay. The IFNL3 polymorphism (rs8099917) was genotyped in 195 patients. RESULTS: One hundred and twenty-five patients were rs8099917 T/T and 70 were non-T/T. Serum IFN-λ3 levels did not differ significantly with IFNL3 genotype, dropped markedly by 1 week and remained low up to 24 weeks after the end of treatment. Interferon-λ3 levels were significantly higher after viral clearance in patients who developed HCC and were associated with a higher potential for hepatocarcinogenesis, such as a higher frequency of non-hypervascular hypointensive nodules (P = 0.046), higher stages of liver fibrosis (P < 0.001), and higher post-treatment levels of Wisteria floribunda agglutinin positive Mac-2 binding protein (P < 0.001) and alanine aminotransferase (P < 0.001). CONCLUSIONS: Serum IFN-λ3 levels after HCV clearance are associated with the potential for HCC development. Interferon-λ3 could be helpful for elucidating the relationships among immunologic status, liver fibrosis, liver inflammation, and hepatocarcinogenesis, after achieving SVR.
ABSTRACT
BACKGROUND AND AIMS: Although treatment for hepatitis C virus has been dramatically improved by the development of direct-acting antiviral agents (DAAs), whether interferon (IFN)-free therapy reduces hepatocarcinogenesis in an equivalent manner to IFN-based therapy remains controversial. The aims of this study were to evaluate the occurrence and recurrence of hepatocellular carcinoma (HCC) in chronic hepatitis C (CHC) patients treated with DAAs and to identify biomarkers of HCC development after antiviral treatment. METHODS: A restrospective review of a prospective database of 1,897 CHC patients who were treated with IFN-based (1,145) or IFN-free therapies (752) was carried out. Cumulative HCC occurrence and recurrence rates were compared using propensity score-matched analysis. Predictors of HCC development after viral eradication were identified by multivariate analysis. RESULTS: Propensity score-matched analysis showed no significant difference in HCC occurrence (p=0.49) and recurrence rates (p=0.54) between groups treated with IFN-based or IFN-free therapies. In multivariate analysis, higher levels of post-treatment α-fetoprotein (AFP) or Wisteria floribunda agglutinin positive Mac-2 binding protein (WFA+M2BP) were independently associated with HCC occurrence and recurrence after viral eradication. Only post-treatment WFA+M2BP level was significantly associated with HCC occurrence and recurrence among patients without severe fibrosis. The area under the receiver operating characteristic (ROC) curve for WFA+M2BP levels was greater than that for AFP levels in ROC analysis. CONCLUSION: The risks of early HCC occurrence and recurrence after viral eradication were similar between IFN-based and IFN-free therapies. Post-treatment levels of WFA+M2BP may be helpful screening biomarkers for assessing the risk of HCC after IFN-free therapy. Patients with high WFA+M2BP levels after antiviral treatment, even without severe fibrosis, must be followed up carefully for HCC development. Lay summary: The risks of early HCC occurrence and recurrence after viral eradication were similar between IFN-based and IFN-free therapies. Post-treatment levels of WFA+M2BP may be helpful screening biomarkers for assessing the risk of HCC after IFN-free therapy.
Subject(s)
Antiviral Agents/administration & dosage , Carcinoma, Hepatocellular/prevention & control , Hepatitis C, Chronic , Interferons/administration & dosage , Liver Neoplasms/prevention & control , Neoplasm Recurrence, Local , Adult , Aged , Antigens, Neoplasm/analysis , Biomarkers, Tumor/analysis , Carcinoma, Hepatocellular/epidemiology , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/physiopathology , Female , Hepatitis C, Chronic/complications , Hepatitis C, Chronic/drug therapy , Humans , Japan/epidemiology , Liver Neoplasms/etiology , Liver Neoplasms/pathology , Liver Neoplasms/physiopathology , Membrane Glycoproteins/analysis , Middle Aged , Neoplasm Recurrence, Local/diagnosis , Neoplasm Recurrence, Local/etiology , Neoplasm Recurrence, Local/virology , Plant Lectins/analysis , Receptors, N-Acetylglucosamine/analysis , Risk Assessment/methods , Risk Factors , alpha-Fetoproteins/analysisABSTRACT
AIM: Sofosbuvir (SOF) and ribavirin (RBV) combination therapy produces a sustained response in many patients with genotype 2 chronic hepatitis C. However, RBV-induced anemia is a troublesome side-effect that may limit this treatment. Genetic variation leading to inosine triphosphatase (ITPA) deficiency is known to protect against RBV-induced hemolytic anemia. This study aimed to evaluate the relationships between the efficacy and safety of SOF/RBV treatment and ITPA gene variants. METHODS: Ninety patients with genotype 2 chronic hepatitis C treated with SOF/RBV were studied. The relationships among genetic polymorphisms of ITPA and the decline in hemoglobin levels from baseline, RBV dose reduction, and sustained virological response (SVR) rates were analyzed. RESULTS: Overall SVR at 12 weeks was 94.4% (85/90). Patients with the ITPA CA/AA genotypes had a lower degree of anemia throughout the therapy than those with the ITPA CC genotype. The percentage of patients requiring RBV dose reduction was significantly lower for those with the ITPA CA/AA variation, a difference even more apparent when the pretreatment hemoglobin level was <12 g/dL. The dose reduction of RBV and serum albumin level were significantly associated with SVR. CONCLUSIONS: Patients with the ITPA CA/AA genotype were less likely to develop anemia than those with the ITPA CC genotype and were more likely to complete SOF/RBV therapy. These results may provide a valuable pharmacogenetic diagnostic tool to predict drug-induced adverse events, particularly in patients with pre-existing anemia.
ABSTRACT
Fetal hepatic stem/progenitor cells, called hepatoblasts, play central roles in liver organogenesis; however, molecular mechanisms regulating proliferation and terminal differentiation of such cells have not been completely elucidated. Bone morphogenetic protein-4 (BMP-4) is essential for the development of stem cells in various tissues, but its function in regulating the phenotype of hepatoblasts after the mid-gestational fetal stage remains unclear. The aim of this study is to clarify a functional role for BMP-4 in proliferation and terminal differentiation of murine hepatoblasts in mid-gestational fetal livers. METHODS: A functional role for BMP-4 in proliferation and terminal differentiation of murine hepatoblasts was validated by assay of colony formation, biliary luminal formation, and hepatic maturation using primary hepatoblasts in vitro. Molecular mechanisms regulating such effects of BMP-4 on primary hepatoblasts were also analyzed. RESULTS: Stimulation of BMP-4 upregulated phosphorylation of Smad1/5 in hepatoblasts. Bone morphogenetic protein-4 significantly suppressed colony formation of primary hepatoblasts in a dose-dependent manner, significantly suppressed cholangiocytic luminal formation of hepatoblasts, and promoted hepatic maturation of primary hepatoblasts. Stimulation of BMP-4 regulated the activation of several mitogen-activated protein kinases, such as extracellular signal-regulated kinase, Akt, p38 mitogen-activated protein kinase, and calcium/calmodulin-dependent protein kinase IIα in primary hepatoblasts. Moreover, Wnt5a, a molecule regulating cholangiocytic luminal formation, and BMP-4 coordinately suppressed proliferation and cholangiocytic luminal formation of hepatoblasts. CONCLUSION: This study shows that BMP-4-mediated signaling controls proliferation and terminal differentiation of fetal hepatic stem/progenitor cells.
ABSTRACT
Single nucleotide polymorphisms (SNPs) within or near interferon lambda 4 (IFNL4) gene located upstream of IFNL3 are associated with response to anti-HCV therapy both in interferon (IFN)-based and IFN-free regimens. IFNL4 encodes IFNλ4, a newly discovered type III IFN, and its expression is controlled by rs368234815-TT/ΔG, which is in strong linkage disequilibrium (LD) with other tag SNPs within or near IFNL4 such as rs12979860 and rs8099917. Intrahepatic expression levels of IFN-stimulated genes (ISGs) affect the responsiveness to IFNα and are also associated with IFNL4 genotype. However, IFNL4 expressions and its role in intrinsic antiviral innate immunity remain unclear. This study evaluated the effect of IFNL4 on intrahepatic ISG expression and investigated its relationship with treatment outcomes in liver samples obtained from 49 chronic hepatitis C patients treated with pegylated (PEG)-IFN/ribavirin therapy. IFNL4 mRNA was detected in 11 of 22 patients with IFNL4-unfavorable SNPs but not in patients with favorable genotypes. IFNL4 expression was associated with non-response to PEG-IFN/ribavirin therapy. Intrahepatic expression of antiviral ISGs (ISG15 and MX1) was significantly higher in IFNL4-unfavorable patients with detectable IFNL4 mRNA than in patients with undetectable IFNL4 mRNA, whereas the expression of suppressive ISGs (RNF125, SOCS1, SOCS3, and RNF11) was lower in patients with detectable IFNL4 mRNA. In summary, intrahepatic expression of IFNL4 was associated with increased antiviral ISG expression and decreased suppressive ISG expression at baseline, resulting in poor responsiveness to IFNα-based therapy in HCV infection.
Subject(s)
Hepatitis C, Chronic/drug therapy , Hepatitis C, Chronic/immunology , Interferons/therapeutic use , Interleukins/genetics , Liver/drug effects , Adult , Aged , Cytokines/genetics , Drug Resistance, Viral , Female , Gene Expression Regulation , Genotype , Hepacivirus/genetics , Hepatitis C, Chronic/genetics , Humans , Interferon-alpha/therapeutic use , Interleukins/metabolism , Liver/immunology , Liver/virology , Male , Middle Aged , Myxovirus Resistance Proteins/genetics , Polymorphism, Single Nucleotide , RNA, Messenger , Ribavirin/adverse effects , Ribavirin/therapeutic use , Suppressor of Cytokine Signaling 1 Protein/genetics , Ubiquitins/geneticsABSTRACT
Hepatitis B virus (HBV) is not eradicated by current antiviral therapies due to persistence of HBV covalently closed circular DNA (cccDNA) in host cells, and thus development of novel culture models for productive HBV infection is urgently needed, which will allow the study of HBV cccDNA eradication. To meet this need, we developed culture models of HBV infection using human induced pluripotent stem cell-derived hepatocyte lineages, including immature proliferating hepatic progenitor-like cell lines (iPS-HPCs) and differentiated hepatocyte-like cells (iPS-Heps). These cells were susceptible to HBV infection, produced HBV particles, and maintained innate immune responses. The infection efficiency of HBV in iPS-HPCs predominantly depended on the expression levels of sodium taurocholate cotransporting polypeptide (NTCP), and was low relative to iPS-Heps: however, long-term culture of iPS-Heps was difficult. To provide a model for HBV persistence, iPS-HPCs overexpressing NTCP were established. The long-term persistence of HBV cccDNA was detected in iPS-HPCs overexpressing NTCP, and depended on the inhibition of the Janus-kinase signaling pathway. In conclusion, this study provides evidence that iPS-derived hepatic cell lines can be utilized for novel HBV culture models with genetic variation to investigate the interactions between HBV and host cells and the development of anti-HBV strategies.
Subject(s)
DNA, Circular/genetics , Hepatitis B virus/physiology , Hepatitis B/virology , Hepatocytes/cytology , Induced Pluripotent Stem Cells/cytology , Models, Biological , Organic Anion Transporters, Sodium-Dependent/metabolism , Symporters/metabolism , Cell Differentiation , Cell Line , Cell Proliferation , DNA, Circular/immunology , DNA, Viral/genetics , DNA, Viral/immunology , Hep G2 Cells , Hepatitis B/genetics , Hepatitis B/immunology , Hepatitis B/metabolism , Hepatitis B virus/genetics , Hepatitis B virus/immunology , Hepatocytes/immunology , Hepatocytes/metabolism , Hepatocytes/virology , Humans , Immunity, Innate , Induced Pluripotent Stem Cells/immunology , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/virology , MAP Kinase Signaling System , Organic Anion Transporters, Sodium-Dependent/genetics , Symporters/genetics , Transfection , Virus ReplicationABSTRACT
AIM: Wisteria floribunda agglutinin positive (WFA+) Mac-2-binding protein (M2BPGi) is a noninvasive glyco-marker for liver fibrosis. This study evaluated the utility of serial measurement of serum M2BPGi and total M2BP as a predictor of fibrosis and the development of hepatocellular carcinoma (HCC). METHODS: This study included 119 patients with chronic hepatitis C (CHC). Of these patients, 97 were treated with IFN-based therapy and 22 were treated with daclatasvir and asunaprevir. Serum M2BPGi values were measured prior to, at the end of, and at 24 weeks after the completion of treatment. As subanalysis, serum total M2BP levels were measured in patients treated with pegylated-interferon and ribavirin. RESULTS: In patients treated with IFN-based therapy, M2BPGi levels were elevated at the end of treatment but decreased afterwards. In contrast, M2BPGi levels in patients treated with IFN-free therapy decreased immediately after starting the treatment without transient elevation. Though pre-treatment M2BPGi levels significantly correlated with fibrosis in both patients with a sustained virological response (SVR) and non-SVR, post-treatment M2BPGi levels decreased regardless of the degree of fibrosis in patients with SVR. In multivariate analysis, non-SVR and HCC development were independent factors associated with M2BPGi level ≥2.2. In patients treated with pegylated-interferon and ribavirin, total M2BP levels were positively correlated with fibrosis and HCC development. CONCLUSION: Real-time monitoring of the serum M2BPGi level after antiviral therapy for CHC patients could be a helpful screening tool for assessing the risk of HCC. M2BP and its glycan structure could be associated together with hepatocarcinogenesis.
Subject(s)
Antigens, Neoplasm/blood , Antiviral Agents/therapeutic use , Carcinoma, Hepatocellular/diagnosis , Hepatitis C, Chronic/drug therapy , Liver Cirrhosis/diagnosis , Liver Neoplasms/diagnosis , Membrane Glycoproteins/blood , Plant Lectins/blood , Biomarkers, Tumor/blood , Carbamates , Carcinoma, Hepatocellular/metabolism , Carrier Proteins , Early Diagnosis , Female , Glycoproteins , Hepatitis C, Chronic/complications , Hepatitis C, Chronic/metabolism , Humans , Imidazoles/therapeutic use , Interferon-alpha/therapeutic use , Isoquinolines/therapeutic use , Liver Cirrhosis/metabolism , Liver Neoplasms/metabolism , Male , Pyrrolidines , Sulfonamides/therapeutic use , Valine/analogs & derivativesABSTRACT
BACKGROUND AND AIMS: The hepatitis C virus (HCV) genotype 1b is known to exhibit treatment resistance with respect to interferon (IFN) therapy. Substitution of amino acids 70 and 91 in the core region of the 1b genotype is a significant predictor of liver carcinogenesis and poor response to pegylated-IFN-α and ribavirin therapy. However, the molecular mechanism has not yet been clearly elucidated because of limitations of the HCV genotype 1b infectious model. Recently, the TPF1-M170T HCV genotype 1b cell culture system was established, in which the clone successfully replicates and infects Huh-7-derived Huh7-ALS32.50 cells. Therefore, the purpose of this study was to compare IFN resistance in various HCV clones using this system. METHODS: HCV core amino acid substitutions R70Q and L91M were introduced to the TPF1-M170T clone and then transfected into Huh7-ALS32.50 cells. To evaluate the production of each virus, intracellular HCV core antigens were measured. RESULTS were confirmed with Western blot analysis using anti-NS5A antibodies, and IFN sensitivity was subsequently measured. RESULTS: Each clone was transfected successfully compared with JFH-1, with a significant difference in intracellular HCV core antigen (p < 0.05), an indicator of continuous HCV replication. Among all clones, L91M showed the highest increase in the HCV core antigen and HCV protein. There was no significant resistance against IFN treatment in core substitutions; however, IFN sensitivity was significantly different between the wildtype core and JFH-1 (p < 0.05). CONCLUSIONS: A novel genotype 1b HCV cell culture was constructed with core amino acid substitutions, which demonstrated IFN resistance of genotype 1b. This system will be useful for future analyses into the mechanisms of HCV genotype 1b treatment.
ABSTRACT
Fetal hepatic stem/progenitor cells, called hepatoblasts, play central roles in liver development; however, the molecular mechanisms regulating the phenotype of these cells have not been completely elucidated. Matrix metalloproteinase (MMP)-14 is a type I transmembrane proteinase regulating pericellular proteolysis of the extracellular matrix and is essential for the activation of several MMPs and cytokines. However, the physiological functions of MMP-14 in liver development are unknown. Here we describe a functional role for MMP-14 in hepatic and biliary differentiation of mouse hepatoblasts. MMP-14 was upregulated in cells around the portal vein in perinatal stage liver. Formation of bile duct-like structures in MMP-14-deficient livers was significantly delayed compared with wild-type livers in vivo. In vitro biliary differentiation assays showed that formation of cholangiocytic cysts derived from MMP-14-deficient hepatoblasts was completely impaired, and that overexpression of MMP-14 in hepatoblasts promoted the formation of bile duct-like cysts. In contrast, the expression of molecules associated with metabolic functions in hepatocytes, including hepatic nuclear factor 4α and tryptophan 2,3-dioxygenase, were significantly increased in MMP-14-deficient livers. Expression of the epidermal growth factor receptor and phosphorylation of mitogen-activated protein kinases were significantly upregulated in MMP-14-deficient livers. We demonstrate that MMP-14-mediated signaling in fetal hepatic progenitor cells promotes biliary luminal formation around the portal vein and negatively controls the maturation of hepatocytes.