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Congestive hepatopathy often leads to liver fibrosis and hepatocellular carcinoma. Imaging modalities provided clinical evidence that elevation of liver stiffness and tumor occurrence are mainly induced in liver periphery of patients with congestive hepatopathy. However, clinical relevance of liver stiffness and liver fibrosis is unclear because liver congestion itself increases liver stiffness in congestive hepatopathy. It also unclear which factors configure such regional disparity of tumor development in patients with congestive hepatopathy. To answer these questions, we evaluated the macroscopic spatial distribution of liver fibrosis and tumors in murine model of congestive hepatopathy.Chronic liver congestion was induced by partial ligation of the suprahepatic inferior vena cava (pIVCL). Distribution of liver congestion, fibrosis, and tumors in pIVCL mice was assessed by histological findings, laser microdissection (LMD)-based qPCR and enhanced computed tomography. LMD-based RNA-seq was performed to identify causal factors which promotes tumor development in congestive hepatopathy.Liver fibrosis was mainly induced in liver periphery and co-localized with distribution of liver congestion. Liver tumors were also induced in liver periphery where liver congestion and fibrosis occurred. LMD-based RNA-seq revealed the upregulation of extracellular matrix/collagen fibril related-, wound healing related-, angiogenesis related-, morphogenesis related-, and cell motility-related signaling pathways in liver periphery compared to liver center.Our findings showed experimental relevance of liver congestion, fibrosis and tumor development in congestive hepatopathy, and may provide important locational information. Macroscopic regional disparity observed in this murine model should be considered to manage patients with congestive hepatopathy. This article is protected by copyright. All rights reserved.

No study has analyzed more than100 cases of eosinophilic gastroenteritis (EGE) in children in a single center. We aimed to describe the clinical features of pediatric EGE.This retrospective study was conducted at a single center. Between April 2007 and December 2017, 860 children between the ages of 1 year and 15 years underwent endoscopy for gastrointestinal symptoms of unknown cause. Among them, 109 (12.7%) were diagnosed with EGE according to the diagnostic criteria for EGE developed by the research group of the Ministry of Health, Labour and Welfare of Japan for eosinophilic gastrointestinal disorder in 2015. We investigated their symptoms, comorbidities, endoscopic findings, pathological findings, treatments, and outcomes.Seventy-one boys (65.1%) and 38 girls (34.9%) were diagnosed with EGE. The median age at diagnosis was 11 years (range, 1-15 years). The chief complaints were abdominal pain in 83 (76.1%) and diarrhea in 26 (23.9%). Upper and lower gastrointestinal endoscopies showed normal findings in 32 patients (29.4%). The most common treatment was a combination of elimination of foods suspected of causing EGE and anti-allergic agents in 50 cases (45.9%). The outcomes were symptom disappearance in 43 patients (39.4%) and symptom improvement in 53 patients (48.6%).For gastrointestinal symptoms of unknown cause in children, EGE should be considered as a differential diagnosis. Although the symptoms and endoscopic findings are nonspecific, cracked mucosa may be a specific endoscopic finding for pediatric EGE. An elimination diet and/or anti-allergic drugs were effective in most patients with pediatric EGE.

Background & aims Chronic liver congestion reflecting right-sided heart failure (RHF), Budd-Chiari syndrome, or Fontan-associated liver disease (FALD) is involved in liver fibrosis and hepatocellular carcinoma (HCC). However, molecular mechanisms of fibrosis and HCC in chronic liver congestion remain poorly understood. Approach & results Here, we first demonstrated that chronic liver congestion promoted HCC and metastatic liver tumor growth using murine model of chronic liver congestion by partial inferior vena cava ligation (pIVCL). As the initial step triggering HCC promotion and fibrosis, gut-derived lipopolysaccharide (LPS) appeared to induce liver sinusoidal endothelial cells (LSECs) capillarization in mice and in vitro. LSECs capillarization was also confirmed in FALD patients. Mitogenic factor, sphingosine-1-phosphate (S1P) was increased in congestive liver and expression of sphingosine kinase 1 (SphK1), a major synthetase of S1P, was increased in capillarized LSECs after pIVCL. Inhibition of S1P receptor (S1PR)1 (Ex26) and S1PR2 (JTE013) mitigated HCC development and liver fibrosis, respectively. Antimicrobials treatment lowered portal blood LPS concentration, LSECs capillarization, and liver S1P concentration accompanied by reduction of HCC development and fibrosis in the congestive liver. Conclusions In conclusion, chronic liver congestion promotes HCC development and liver fibrosis by S1P production from LPS-induced capillarized LSECs. Careful treatment of both RHF and liver cancer might be necessary for RHF patients with primary or metastatic liver cancer.

Hepatitis B virus (HBV) DNA is detectable in the nails and hair of patients with chronic HBV infection. However, it remains unclear whether HBV DNA can be detectable in the nails and hair of patients with acute HBV infection. We encountered two cases of children with acute HBV infection. HBV DNA in the nails and hair from the two children was evaluated by real-time PCR. To clarify the characteristics of HBV DNA, full-length HBV genome sequencing and phylogenetic tree analysis were performed. The levels of serum HBV DNA in children of cases 1 and 2 at day 0 were 7.6 Log IU/mL and 7.4 Log IU/mL, respectively. Nail HBV DNA was detected in both children (case 1: 4.6 Log IU/mL at day 0, case 2: 5.5 Log IU/mL at day 14). Moreover, hair HBV DNA was detectable in case 2 (4.0 Log IU/mL at day 14). Serum HBV DNA became undetectable within approximately 3-4 months after the first hospital visit. After the resolution of HBV viremia, nail and hair HBV DNA became undetectable. The sequence analysis of serum, nail and hair HBV DNA showed the same HBV genotype in each case (case1: genotype C, case 2: genotype A). In case 1, 3 nucleotides were different in the full-genome HBV sequence between the serum and nails. In case 2, the full-genome HBV sequences were identical among the serum, nails and hair. In conclusion, HBV DNA was detectable in nails and hair of children with acute HBV infection.

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., 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 also contributes to prompt recovery from cholestatic liver damage through the establishment of tight junctions in the bile ducts.

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.

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.

Background: Hepatitis B virus (HBV) is detected in extrahepatic tissues of individuals with HBV infection. Whether nails and hair contain HBV has been unknown. Methods: We examined two patient groups: those with chronic HBV infection alone (n=71), and those with both chronic HBV and hepatitis delta virus (HDV) infections (n=15). HBV DNA in the patients' fingernails and hair were measured by real-time PCR. Hepatitis B surface antigen (HBsAg) of fingernails was evaluated by an enzyme immunoassay. HDV RNA in fingernails was measured by real-time PCR. Immunochemical staining was performed on nails. We used chimeric mice with humanized livers to evaluate the infectivity of nails.Results: Of the 71 pairs of HBV-alone nail and hair samples, 70 (99%) nail and 60 (85%) hair samples were positive for β-actin DNA. Of those 70 nail samples, 65 (93%) were HBV DNA-positive. Of the 60 hair samples, 49 (82%) were HBV DNA-positive. The serum HBV DNA level of the nail HBV DNA-positive patients was significantly higher than that of the nail HBV DNA-negative patients. The hair HBV DNA-positive patients' serum HBV DNA level was significantly higher compared to the hair HBV DNA-negative patients. The nail HBV DNA level was significantly higher than the hair HBV DNA level. The nails and hair HBV DNA levels were correlated (r=0.325, pConclusions: Nails and hair were the reservoir of HBV DNA. Moreover, nails can contain HBsAg, HDV RNA, and HD antigen.

Background Castleman's disease (CD) is a lymphoproliferative disorder. TAFRO syndrome is classified as a variant of CD based on its key clinical manifestations of thrombocytopenia, anasarca (generalized edema and pleural effusion), fever (pyrexia), reticulin fibrosis in the bone marrow and the proliferation of megakaryocytes, and organomegaly (such as hepatosplenomegaly and multiple lymphadenopathies); TAFRO syndrome is mainly reported in Japanese patients. To our knowledge, this is the first pediatric case report detailing a CD-associated disorder progressing to cirrhosis. Case summary A 10-year old male patient presented with fever and anemia. Six months before hospitalization, he had remarkable abdominal distention. Subsequently, he visited a clinic for a fever that lasted 5 d. The physical findings were marked hepatosplenomegaly and cervical lymphadenopathy. A blood test revealed leukocytosis, microcytic anemia, aspartate aminotransferase-dominant transaminase elevation, high levels of C-reactive protein, polyclonal hypergammaglobulinemia, and high levels of interleukin-6 and vascular endothelial growth factor. Abdominal contrast computed tomography and magnetic resonance imaging suggested cirrhosis, which was confirmed by liver histology. Histological findings in the enlarged hepatic lymph nodes revealed both hyperplasia and atrophy of lymphoid follicles with some vascular hyperplasia and moderate plasmacytosis between the lymphoid follicles, which is compatible with lymph node histology in TAFRO syndrome. Prednisolone was not effective in reducing the patient's symptoms; therefore, the patient was prescribed tocilizumab. To date, the patient remains free of fever and continues to receive tocilizumab. Conclusion We described the clinicopathological features of TAFRO syndrome to highlight the clinical presentation of this rare disease in a pediatric case.

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.

Background The aim of the present study was to clarify the roles of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV-6) in immunocompetent children with acute liver dysfunction not resulting from hepatitis virus. Methods Sixty-eight children (median age, 3 years) hospitalized as a result of acute liver dysfunction were enrolled in this study. Hepatitis A, B, and C were excluded. The prevalence of CMV, EBV, and HHV-6 and viral DNA load in whole blood was prospectively evaluated on multiplex real-time polymerase chain reaction (PCR). Results Of the 68 children with acute liver dysfunction, multiplex real-time PCR was positive in 30 (44%). CMV, EBV, and HHV-6 DNA were detected in 13 (19%), 14 (21%), and seven (10%), respectively. Serum CMV immunoglobulin (Ig)G/IgM and EBV viral capsid antigen IgG/IgM were measured in 40 (CMV DNA positive, n = 10; negative, n = 30) and 45 (EBV DNA positive, n = 14; negative, n = 31) of the 68 children, respectively. Eighteen percent (CMV, 7/40) and 9% (EBV, 4/45) were positive for both PCR and viral-specific IgM. There was no significant difference in CMV and EBV viral load between IgM-positive and -negative children with viremia. Conclusions CMV, EBV, and HHV-6 DNA were frequently detected in immunocompetent children with acute liver dysfunction, but primary CMV and EBV infection were confirmed in 10-20% of the children with acute liver dysfunction. The combination of PCR assay and serology is necessary to make a diagnosis of acute liver dysfunction due to primary CMV, EBV and/or HHV-6 infection in immunocompetent children.

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 (Hb) 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 Hb level was

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.

Aim Primary sclerosing cholangitis (PSC) is very rare in Japan. Although a large-scale cohort study of 781 pediatric-onset PSC patients in Europe and North America showed that the 5-year survival with native liver was 88%, the long-term outcomes of pediatric-onset PSC in Japan are unknown. Here, we evaluated the clinical outcomes of pediatric-onset PSC in Japan. Methods We carried out a retrospective cohort study with a medical records review of pediatric PSC patients diagnosed between 1986 and 2017 at a single center. The PSC diagnoses were based on cholangiography, liver histology, and biochemical findings. The patients' survival was analyzed using the Kaplan-Meier method. Prognostic factors were determined by univariate and multivariate analyses using the Cox proportional hazards regression model. Results We identified 39 pediatric-onset PSC patients (22 boys, 17 girls). The median age at diagnosis was 9 years (interquartile range 6.0-13.5 years). The median follow-up period was 5.5 years (interquartile range 3.4-8.7 years). The phenotypes of PSC-autoimmune hepatitis, PSC-inflammatory bowel disease, and small-duct PSC were diagnosed in 13 (33.3%), 36 out of 38 (94.8%), and three (7.7%) patients, respectively. The 5-year liver transplantation-free survival of the whole cohort was 93.5%. Nine patients underwent liver transplantation, and four of these nine cases resulted in death. Both the univariate and multivariate analyses showed that the phenotype of "PSC-autoimmune hepatitis overlap" was an independent poor prognostic factor. Conclusions The overall survival of pediatric-onset PSC in Japan was comparable to those in Western countries. The phenotype of PSC-autoimmune hepatitis was identified as a prognostic factor associated with a poorer long-term outcome.

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.

Aim 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 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 BMP-4 stimulation upregulated phosphorylation of Smad1/5 in hepatoblasts. BMP-4 significantly suppressed colony formation of primary hepatoblasts in a dose-dependent manner. BMP-4 significantly suppressed cholangiocytic luminal formation of hepatoblasts, and promoted hepatic maturation of primary hepatoblasts. BMP-4 stimulation regulated the activation of several mitogen-activated protein kinases (MAPK), such as extracellular signal-regulated kinase, Akt, p38 MAPK, 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 demonstrates that BMP-4–mediated signaling controls proliferation and terminal differentiation of fetal hepatic stem/progenitor cells. This article is protected by copyright. All rights reserved.

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.

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

Background Vaccination against hepatitis B virus (HBV) infection in infants born to hepatitis B surface antigen (HBsAg)-positive mothers using HB immunoglobulin (HBIG) and hepatitis B (HB) vaccine was launched in Japan in 1985. Infants testing positive for HBsAg at 1 month of age are considered to have prenatally acquired the infection and are usually excluded from the prevention program. Infants born to HB e antigen (HBeAg)-positive mothers are at a high risk of perinatally acquiring the infection. In this study, long-term outcome was evaluated in children with prenatal HBV infection who received the HBIG and HB vaccine in Japan. Methods Newborns of both HBsAg- and HBeAg-positive carrier mothers received HBIG within 48 h of birth and at 2 months of age. Subsequently, three doses of recombinant HB vaccine were given at 2, 3, and 5 months of age. Outcome was compared between the following two groups: infants who completed the vaccination program, even if they were HBsAg positive at 1 month of age (n = 15), and infants who did not (n = 51). Results Seroconversion from HBeAg to anti-HBe antibody (HBeAb) before 3 years of age was observed in five children (33%) who completed the vaccination program and in two (4%) who did not (P = 0.005). In 2/5 children who completed the vaccination program and achieved HBeAb seroconversion, seroconversion from HBsAg to anti-HBs antibody was also noted. Conclusion This specific vaccination program for children with prenatal HBV infection has the potential to alter immune tolerance to HBV.

BACKGROUND Hepatitis B e antigen (HBeAg) seroconversion is an important event in patients with chronic hepatitis B virus (HBV) infection. This study aimed to clarify the outcome of long-term follow-up of chronic HBV infection and the factors affecting HBeAg seroconversion in children in Japan. METHODS Patients who were first examined at our institution between 1980 and 2012, who were

Dear Sir, Primary sclerosing cholangitis (PSC) is a liver disease often associated with IBD.1 Dysbiosis of the gut microbiota is implicated in PSC aetiology in adults,2–4 but less is known about paediatric-onset PSC. We analysed the faecal microbiota of 27 Japanese patients with paediatric-onset PSC as well as 16 age-matched patients with UC and 23 healthy controls (HCs) (see online supplementary table S1) with pyrosequencing data of 16S rRNA gene V1-V2 region (accession #DRA004773). We assessed the influence of medications on the gut microbiota and found that salazosulfapyridine (SASP) treatment affected the microbiota structure with significant changes in the abundance of six major genera between the treated and untreated patients with PSC, perhaps due to its bactericidal property (see online supplementary figure S1). We thus report the analysis of 13 patients with PSC and 15 patients with UC and 23 HCs, all SASP untreated (see online supplementary table S2). ### Supplementary tables [gutjnl-2016-312533supp_tables.pdf] ### Supplementary figure [gutjnl-2016-312533supp_figure.pdf] Clustering analysis of the 16S reads revealed that the microbiota in the PSC and HC groups had significantly high species richness compared with the UC group, and the species richness of PSC samples was lower than that of HCs. The PSC group also exhibited an intermediate trend in the Shannon's index between the UC and HC groups (figure 1A). The unweighted UniFrac metric revealed a significant difference in the overall microbiota structure among the three groups, in which the PSC samples tended to aggregate between the HC and UC samples (figure 1B, C). Collectively, the data suggested that the patients with …

Although neonatal hemochromatosis (NH) is a well-known cause of liver failure during the neonatal period and iron deposition in extrahepatic tissues is considered essential in the diagnosis of NH, there is no consensus regarding the pathology or diagnostic criteria of NH. Recent studies of immunohistochemical assays have shown that the C5b-9 complex (the terminal membrane attack complement complex) is strongly expressed in the liver of NH cases, suggesting that a gestational alloimmune mechanism is the cause of liver injury. The patient was a low birthweight primiparous male born at 37 weeks of gestation by vaginal delivery. Blood tests 3 h after birth showed signs of liver failure, including high transferrin saturation, resembling the clinical characteristics of NH. However, magnetic resonance imaging and a lip biopsy showed no obvious iron deposition outside the liver. The patient was refractory to exchange transfusion and immunoglobulin therapy but was successfully treated by liver transplantation. Histologically, the explanted liver showed established cirrhosis, with large amounts of human C5b-9 in the residual hepatocytes, suggesting the alloimmune mechanism of liver injury was the cause of his liver failure. Liver failure caused by a gestational alloimmune mechanism should be considered in patients with antenatal liver failure, even without obvious extrahepatic siderosis.

Aim The relationship between pegylated interferon (PEG IFN)-α-2a and growth of children with chronic hepatitis C (CHC) remains unclear. This study was to evaluate the effects of PEG IFN-α-2a on growth. Methods From 2003–2012, we prospectively analyzed the data of children with CHC through mother-to-infant transmission. They were all treatment naive and were treated with PEG IFN-α-2a monotherapy. Results Among 31 children (19 boys, 12 girls; median age, 6 years) treated with monotherapy during the study period, 21 children (13 boys, eight girls; median age, 7 years) were statistically analyzed. The median treatment period of the 21 children was 48 weeks (range, 48–72). Z-scores of height and weight before treatment, at the end of treatment and 1 year after treatment were −0.05, −0.24 and −0.12 (height), and +0.11, −0.23 and −0.05 (weight). Both Z-scores were significantly decreased at the end of the treatment. One year after treatment, Z-scores of height and bodyweight were significantly improved compared with that of end of treatment but were still lower than those before treatment, with statistical significance. Z-scores of height growth velocity was significantly increased after the treatment (+0.71), compared with that during treatment (−2.25). Conclusion PEG IFN-α-2a has an inhibitory effect on children's growth, and Z-scores of height and bodyweight were decreased at the end of treatment. Although Z-scores improved after the treatment, they had not returned to the baseline level 1 year after the treatment.

Plasma mannose is suggested to be largely generated from liver glycogen-oriented glucose-6-phosphate. This study examined plasma mannose in glycogen storage disease type Ia (GSD Ia) lacking conversion of glucose-6-phosphate to glucose in the liver. We initially examined fasting-and postprandial 2 h-plasma mannose and other blood carbohydrates and lipids for seven GSD Ia children receiving dietary interventions using cornstarch and six healthy age-matched children. Next, one-day successive intra-individual parameter changes were examined for six affected and two control children. Although there were no significant differences in fasting-and postprandial 2 h-glucose and insulin levels, the mannose level of the affected group was invariably much higher than that of the control group (p

To clarify the physiological and pathophysiological roles of intestinal urate excretion via ABCG2 in humans, we genotyped ABCG2 dysfunctional common variants, Q126X (rs72552713) and Q141K (rs2231142), in end-stage renal disease (hemodialysis) and acute gastroenteritis patients, respectively. ABCG2 dysfunction markedly increased serum uric acid (SUA) levels in 106 hemodialysis patients (P = 1.1 × 10−4), which demonstrated the physiological role of ABCG2 for intestinal urate excretion because their urate excretion almost depends on intestinal excretion via ABCG2. Also, ABCG2 dysfunction significantly elevated SUA in 67 acute gastroenteritis patients (P = 6.3 × 10−3) regardless of the degree of dehydration, which demonstrated the pathophysiological role of ABCG2 in acute gastroenteritis. These findings for the first time show ABCG2-mediated intestinal urate excretion in humans, and indicates the physiological and pathophysiological importance of intestinal epithelium as an excretion pathway besides an absorption pathway. Furthermore, increased SUA could be a useful marker not only for dehydration but also epithelial impairment of intestine.

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.

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.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.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.This study shows that BMP-4-mediated signaling controls proliferation and terminal differentiation of fetal hepatic stem/progenitor cells.

Objectives To assess the feasibility and safety of same day regimen of low-volume polyethylene glycol solution with ascorbic acid for bowel cleansing before colonoscopy in children. Methods The data on children who received polyethylene glycol solution with ascorbic acid for bowel cleansing in our department were retrospectively analyzed. On the day before the procedure, patients ate a low-residue diet and received sodium picosulfate in the evening. The following day patients took polyethylene glycol solution with ascorbic acid in the morning; the procedure was performed in the afternoon. Dosages of sodium picosulfate and polyethylene glycol solution with ascorbic acid were adjusted based on body weight. Bowel cleansing efficacy was rated on a scale of 1–5 by the colonoscopist. Results Between July 2013 and November 2014, polyethylene glycol solution with ascorbic acid was used in 112 cases (96 patients; male:female 73:39; median age 10.9 years old, range 4 – 19 years). 91 cases (81%) were able to orally ingest the prescribed amount of polyethylene glycol solution with ascorbic acid. Satisfactory bowel cleansing (cleansing grade ≥3) was attained in 87% on intention to treat analysis and 85% on per protocol analysis. Cleansing grade was significantly better in children ingested polyethylene glycol solution with ascorbic acid within 60 minutes (P < 0.05). There were no serious adverse events. Conclusion Same day regimen of polyethylene glycol solution with ascorbic acid is effective and safe for bowel cleansing in children. This article is protected by copyright. All rights reserved.

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). 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. 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. 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.

Aim The relation between interleukin-28B (IL-28B) genotypes and treatment-induced hepatitis C virus (HCV) clearance in children is unknown. This was a retrospective study to evaluate the association between an IL-28B genotype (rs8099917) and pegylated (PEG) interferon (IFN) response. Methods Sixty-three children (median age, 7 years; range, 3–17 years; 22 with HCV genotype 1 and 41 with genotype non-1) with chronic HCV infection who were treated with response-guided PEG IFN on the basis of viral load were evaluated. Results The duration of treatment with PEG IFN was 24 weeks in one child (2%), 36 weeks in eight children (13%), 48 weeks in 36 children (57%), 60 weeks in 11 children (17%) and 72 weeks in seven children (11%). Of the total 63 children, 54 (86%) were initially treated with PEG IFN-α-2a monotherapy. The remaining nine (14%) received PEG IFN plus ribavirin as the initial therapy. Of the 54 children initially treated with monotherapy, 35 (65%) continued receiving monotherapy until the end of treatment. In the remaining 19 (35%), monotherapy was changed to PEG IFN plus ribavirin at 12 or 24 weeks of treatment. Of the total 63 children, 54 (86%) achieved a sustained virological response (SVR). In univariate analysis, rs8099917 genotype TT (P = 0.075) showed a weak association with SVR. However, the multivariate analysis revealed no predictive factors which had a significant association with SVR. Conclusion The IL-28B genotype was not a strong pretreatment predictor for SVR in a mixed genotype cohort of children treated with response-guided PEG IFN therapy.

Aim: There is little information available on the efficacy of pegylated interferon (PEG IFN) therapy for children with chronic hepatitis C. The aim of this study was to evaluate the efficacy and tolerability of PEG IFN-α2a monotherapy for children infected by chronic hepatitis C virus (HCV). Methods: From 2004–2006, we conducted a prospective, open-label, multicenter study of 22 patients aged 4–18 years, including eight with genotype 1 and 14 with genotype 2. None had previously received IFN. The patients were treated with s.c. PEG IFN-α2a at a dose of 3 µg/kg once a week for 48 weeks. Rapid virological response (RVR) was defined as: undetectable serum HCV RNA at week 4; early viral response (EVR) as a 2 or more log reduction or undetectable serum HCV RNA at week 12; and sustained viral response (SVR) as undetectable serum HCV RNA at 24 weeks after the cessation of treatment. Results: SVR was achieved in 10 (45%) of the 22 patients (three with genotype 1, seven with genotype 2). Retrospectively, the patients with SVR included five with RVR (one with genotype 1, four with genotype 2) and five with EVR (two with genotype 1, three with genotype 2). PEG IFN-α2a monotherapy was well tolerated, except in one patient in whom alanine aminotransferase activity flared (>500 IU/L) during treatment. Conclusion: The efficacy of PEG IFN-α2a monotherapy in children is similar to that for adults, while tolerability seems to be better in children than in adults.

We investigated whether we could applied the new "criteria of guideline for liver transplantation in patients with fulminant hepatic failure (FHF)" to children. Eight children with FHF were evaluated by new criteria at the onset of hepatic encephalopathy (day 0) and 5 days after encephalopathy (day 5), retrospectively. At day 0, only a patient who was received liver transplantation was predicted as dead accurately. At day 5, 4 patients alive without liver transplantation were judged as alive, and 3 patients received liver transplantation were judged as dead, accurately. New criteria permit accurate prognosis for children with FHF as well as adults. However, scoring according to new criteria should be evaluated sequentially, not only at day 0 in children.

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.

A 7-year-old girl was referred to our hospital for severe abdominal pain and elevated serum levels of amylase and aminotransferase. Radiological examinations revealed choledocholithiasis. EPBD was performed under intravenous anesthesia using midazolam and ketamine chloride. After fluoroscopic confirmation of a bile duct stone, a dilation balloon (30 mm-long, 8 mm-diameter) was passed over the guidewire and placed across the papilla. The balloon was gradually inflated until the notch created by the papillary sphincter disappeared. At 4 atm, the notch disappeared and the pressure was maintained for 15 s. Stone extraction was performed by using a retrieval balloon catheter. Abdominal pain disappeared immediately after EPBD, and she resumed oral intake 2 days after EPBD. In conclusion, EPBD may be a safe and effective technique for the treatment of choledocholithiasis in pediatric patients. EPDB should be selected as a treatment for pediatric choledocholithiasis in view of its advantages of preserving papillary function.