Search

Aim: In patients with liver cirrhosis, high levels of serum myostatin are associated with poor prognosis. We aimed to clarify the influence of myostatin on the prognosis of patients with non-alcoholic fatty liver disease-hepatocellular carcinoma (NAFLD-HCC) without cirrhosis and on the progression of liver fibrosis., Methods: Serum myostatin levels were evaluated in 234 patients who underwent primary surgical resection for single HCC. To clarify the impact of myostatin on liver fibrosis, we established human primary liver fibroblasts from resected livers, and cultured them in the presence of myostatin., Results: The median age was 67.4 years, the median L3 skeletal muscle mass index was 44.4 cm 2 /m 2 , and the median body mass index was 23.4 kg/m 2 . Eighty-two (35.0%) patients had sarcopenia (L3 skeletal muscle mass index: men <42, women <38 cm 2 /m 2 ). The etiologies of liver disease were hepatitis B virus (n = 61), hepatitis C virus (n = 86), and non-B non-C hepatitis (n = 87) including NAFLD (n = 74). High preoperative serum myostatin and vascular invasion were independent predictors of poor overall survival (OS). High serum myostatin was associated with poor OS in patients with no sarcopenia (n = 152). In patients without advanced liver fibrosis (Fibrosis stage, 0-2; n = 58), high levels of serum myostatin were also associated with poor OS, regardless of sarcopenia. Serum myostatin levels were increased with the progression of liver fibrosis. Liver fibroblasts were activated and produced collagen following stimulation with myostatin., Conclusions: In patients with NAFLD-HCC without advanced liver fibrosis, high levels of serum myostatin were associated with poor OS. Myostatin activated primary fibroblasts and stimulated collagen production., (© 2021 The Japan Society of Hepatology.)

Aims: Exercise is effective for the prevention of liver cancer. Exercise exerts biological effects through the regulation of microRNAs (miRNAs) and cytokines/myokines. We aimed to investigate the effects of low-intensity resistance exercise on serum miRNA and cytokine/myokine expressions in subjects with no exercise habits., Methods: We enrolled seven male subjects with no exercise habits in this prospective before-after study. All subjects performed a low-intensity resistance exercise program (three metabolic equivalents, approximately 20 min/session). Serum miRNA expressions were evaluated using microarrays. We performed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed miRNAs before and after exercise. Serum cytokine/myokine expressions were evaluated using a multiplex panel., Results: All subjects completed the exercise program with no adverse events. In the microarray analysis, seven miRNAs showed a significant change between before and after exercise. Of these, microRNA (miR)-630 and miR-5703 showed a >1.5-fold increase (miR-630: 40.7 vs. 69.3 signal intensity, p = 0.0133; miR-5703: 30.7 vs. 55.9 signal intensity, p = 0.0051). KEGG pathway enrichment analysis showed that miR-630- and miR-5703-related genes were enriched in 37 and 5 pathways, including transforming growth factor-beta and Wnt signaling pathways, respectively. In the multiplex analysis, 12 cytokines/myokines showed significant alteration after exercise compared to before exercise. Of these, fractalkine/CX3CL1 showed the most significant up-regulation by exercise (94.5 vs. 109.1 pg/ml, p = 0.0017)., Conclusions: A low-intensity resistance exercise program was associated with upregulation of serum miR-630, miR-5703, and fractalkine/CX3CL1 expressions in subjects with no exercise habits. Thus, even low-intensity exercise may alter miRNA and cytokine/myokine expressions in humans., (© 2021 The Japan Society of Hepatology.)

Background: Hepatitis B surface antigen (HBsAg) loss is an ideal goal for chronic hepatitis B patients. Antiretroviral therapy (ART) in hepatitis B virus/human immunodeficiency virus-1 (HBV/HIV-1)-coinfected patients can lead to hepatic flare (HF) caused by immune reconstitution-induced inflammatory syndrome (IRIS). Here, we investigated the impact of IRIS-HF on HBsAg loss., Methods: This was a retrospective study of 58 HBV/HIV-1-coinfected subjects HBsAg-positive for ≥6 months before ART initiation and followed for ≥1 year (median 9.9 years) after ART initiation. We examined humoral factors in sera from healthy volunteers, HIV-monoinfected patients, and HBV/HIV-1-coinfected patients with IRIS-HF or acute hepatitis B infection., Results: During ART, HBsAg loss was observed in 20 of 58 HBV/HIV-1-coinfected patients (34.5%). Of the 58 patients, 15 (25.9%) developed IRIS-HF within 12 months of ART initiation. HBsAg loss was more frequent among patients who developed IRIS-HF (11/15, 73.3%) than those who did not (9/43, 20.9%). Multivariate analysis showed IRIS-HF was an independent predictor of subsequent HBsAg loss. Younger age and higher baseline HBV DNA titer were associated with IRIS-HF. Elevation of sCD163, not CXCL9, CXC10, CXCXL11, or CXCL13, was observed at IRIS-HF., Conclusions: IRIS-HF was associated with HBsAg loss in HBV/HIV-1-coinfected patients., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Overall response rates of systemic therapies against advanced hepatocellular carcinoma (HCC) remain unsatisfactory. Thus, searching for new immunotherapy targets is indispensable. NK cells are crucial effectors and regulators in the tumor microenvironment and a determinant of responsiveness to checkpoint inhibitors. We revealed the landscape of NK cell phenotypes in HCC patients to find potential immunotherapy targets. Using single cell mass cytometry, we analyzed 32 surface markers on CD56 dim and CD56 bright NK cells, which included Sialic acid-binding immunoglobulin-type lectins (Siglecs). We compared peripheral NK cells between HCC patients and healthy volunteers. We also compared NK cells, in terms of their localizations, on an individual patient bases between peripheral and intrahepatic NK cells from cancerous and noncancerous liver tissues. In the HCC patient periphery, CD160 + CD56 dim NK cells that expressed Siglec-7, NKp46, and NKp30 were reduced, while CD49a + CD56 dim NK cells that expressed Siglec-10 were increased. CD160 and CD49a on CD56 dim NK cells were significantly correlated to other NK-related markers in HCC patients, which suggested that CD160 and CD49a were signature molecules. CD49a + CX3CR1 + Siglec-10 + NK cells had accumulated in HCC tissues. Considering further functional analyses, CD160, CD49a, CX3CR1, and Siglec-10 on CD56 dim NK cells may be targets for immunotherapies of HCC patients.

Immunoevasins are viral proteins that prevent antigen presentation on major histocompatibility complex (MHC) class I, thus evading host immune recognition. Hepatitis C virus (HCV) evades immune surveillance to induce chronic infection; however, how HCV-infected hepatocytes affect immune cells and evade immune recognition remains unclear. Herein, we demonstrate that HCV core protein functions as an immunoevasin. Its expression interfered with the maturation of MHC class I molecules catalyzed by the signal peptide peptidase (SPP) and induced their degradation via HMG-CoA reductase degradation 1 homolog, thereby impairing antigen presentation to CD8 + T cells. The expression of MHC class I in the livers of HCV core transgenic mice and chronic hepatitis C patients was impaired but was restored in patients achieving sustained virological response. Finally, we show that the human cytomegalovirus US2 protein, possessing a transmembrane region structurally similar to the HCV core protein, targets SPP to impair MHC class I molecule expression. Thus, SPP represents a potential target for the impairment of MHC class I molecules by DNA and RNA viruses., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Background and Aims: The usefulness of APRI or FIB-4 is well established as a non-invasive liver fibrosis marker at a point of diagnosis in patients with chronic liver disease. However, their applicability for the monitoring of progression of liver fibrosis over time is yet to be determined. We aimed to clarify the feasibility of APRI and FIB-4 for the longitudinal evaluation of liver fibrosis in patients with chronic hepatitis B and C., Methods: This is a multi-center retrospective and prospective cohort study, enrolling 1029 patients with HCV and 384 patients with HBV who were histologically diagnosed by liver biopsy. The observation period of retrospective and prospective study was 14 and 12 years, respectively. The APRI and FIB-4 were traced back in cases of histologically diagnosed cirrhosis, and those were prospectively analyzed after biopsy in cases diagnosed as F3 of METAVIR score, respectively., Results: The averaged APRI and FIB-4 exhibited time-dependent increase in the retrospective study of hepatitis C patients (increase by 0.09/year in APRI and 0.29/year in FIB-4). In the prospective study of untreated hepatitis C patients, such increases were 0.14/year in APRI and 0.40/year in FIB-4, respectively. Neither the average of APRI nor FIB-4 showed a specific tendency with hepatitis B patients and treatment-experienced hepatitis C patients., Conclusion: The APRI and FIB-4 may serve as a transition indicator of liver fibrosis in anti-viral treatment-naïve patients with chronic hepatitis C.

Congestive hepatopathy (CH) with chronic passive congestion is characterized by the progression of liver fibrosis without prominent inflammation and hepatocellular damage. Currently, the lack of reliable biomarkers for liver fibrosis in CH often precludes the clinical management of patients with CH. To explore fibrosis biomarkers, we performed proteome analysis on serum exosomes isolated from patients with CH after the Fontan procedure. Exosomal cluster of differentiation (CD)44 levels were increased in patients with CH compared to healthy volunteers and was accompanied by increases in serum levels of soluble CD44 and CD44 expression in the liver. To address the roles of CD44 in CH, we established a mouse model of chronic liver congestion by partial inferior vena cava ligation (pIVCL) that mimics CH by fibrosis progression with less inflammation and cellular damage. In the pIVCL mice, enhanced CD44 expression in hepatic stellate cells (HSCs) and deposition of its ligand hyaluronan were observed in the liver. Blood levels of soluble CD44 were correlated with liver fibrosis. The blockade of CD44 with specific antibody inhibited liver fibrosis in pIVCL mice and was accompanied by a reduction in S100 calcium-binding protein A4 expression following activation of HSCs. Conclusion: Chronic liver congestion promotes fibrosis through CD44. This identifies CD44 as a novel biomarker and therapeutic target of liver fibrosis in patients with CH., (© 2021 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Non-alcoholic fatty liver disease (NAFLD) is a progressive disorder that can develop into liver fibrosis and hepatocellular carcinoma. Natural killer (NK) cells have been shown to protect against liver fibrosis and tumorigenesis, suggesting that they may also play a role in the pathogenesis of NAFLD. Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of inhibitory and activating receptors expressed by many cell types, including NK cells. Here, we investigated the phenotypic profiles of peripheral blood and intrahepatic NK cells, including expression of Siglecs and immune checkpoint molecules, and their association with NK cell function in patients with NAFLD. Immune cells in the peripheral blood of 42 patients with biopsy-proven NAFLD and 13 healthy volunteers (HVs) were identified by mass cytometry. The function of various NK cell subpopulations was assessed by flow cytometric detection of intracellular IFN-γ and CD107a/LAMP-1, a degranulation marker, after in vitro stimulation. We found that peripheral blood from NAFLD patients, regardless of fibrosis stage, contained significantly fewer total CD56 + NK cell and CD56 dim NK cell populations compared with HVs, and the CD56 dim cells from NAFLD patients were functionally impaired. Among the Siglecs examined, NK cells predominantly expressed Siglec-7 and Siglec-9, and both the expression levels of Siglec-7 and Siglec-9 on NK cells and the frequencies of Siglec-7 + CD56 dim NK cells were reduced in NAFLD patients. Notably, Siglec-7 levels on CD56 dim NK cells were inversely correlated with PD-1, CD57, and ILT2 levels and positively correlated with NKp30 and NKp46 levels. Further subtyping of NK cells identified a highly dysfunctional Siglec-7 - CD57 + PD-1 + CD56 dim NK cell subset that was increased in patients with NAFLD, even those with mild liver fibrosis. Intrahepatic NK cells from NAFLD patients expressed elevated levels of NKG2D and CD69, suggesting a more activated phenotype than normal liver NK cells. These data identify a close association between NK cell function and expression of Siglec-7, CD57, and PD-1 that could potentially be therapeutically targeted in NAFLD., Competing Interests: TK received lecture fees from Gilead Sciences and Merck Sharp & Dohme. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sakamoto, Yoshio, Doi, Mori, Matsuda, Kawai, Shimagaki, Yoshikawa, Aoki, Osawa, Yoshida, Arai, Itokawa, Atsukawa, Ito, Honda, Mise, Ono, Takahashi, Saiura, Taketomi and Kanto.)

Background: Pathological angiogenesis is involved in the development of hepatocellular carcinoma. In patients with chronic hepatitis C (CHC), the level of angiogenic factor angiopoietin (ANGP)-2 is reported to be increased in the blood, correlating with fibrosis. In this study, we aimed to clarify whether blood ANGP-2 is useful as a biomarker for liver angiogenesis and fibrosis in CHC patients and to further reveal the relationship between such pathology in a carbon tetrachloride (CCl 4 )-treated liver fibrosis mouse model., Methods: Plasma levels of ANGP-2, expression of a liver sinusoidal endothelial cell (LSEC) marker (CD31), collagen deposition (Sirius Red staining) in the liver, clinical fibrosis markers (Mac-2 binding protein glycosylation isomer, virtual touch quantification, and liver stiffness measurement), and liver function (albumin bilirubin score) were examined in CHC patients. To determine the effects of an anti-angiogenic agent on liver fibrosis in vivo, sorafenib was administered to the CCl 4 -treated mice (BALB/c male)., Results: The plasma levels of ANGP-2 were increased in CHC patients compared to healthy volunteers and decreased by the eradication of hepatitis C with direct-acting antivirals. In addition, plasma ANGP-2 levels were correlated with CD31 expression, collagen deposition, clinical fibrosis markers, and liver function. Sorafenib inhibited liver angiogenesis and fibrosis in the CCl 4 -treated mice and was accompanied by decreased ANGP-2 expression in LSECs., Conclusions: ANGP-2 may serve as a useful biomarker for liver angiogenesis and fibrosis in CHC patients. In addition, angiogenesis and fibrosis may be closely related.

Background: We recently reported the real-world changes in the etiologies of liver cirrhosis (LC) based on nationwide survey data and assessed the etiologies of LC with hepatocellular carcinoma (HCC)., Methods: Fifty-five participants from 68 institutions provided data on 23,637 patients with HCC-complicated LC. The changing trends in etiologies were assessed. We further analyzed the data from 29 hospitals that provided the annual number of newly identified HCC-complicated LC patients from 2008 to 2016 (N = 9362) without any missing years and assessed the transition in the real number of newly identified HCC-complicated LC cases., Results: In the overall cohort, hepatitis C virus (HCV) infection (60.3%) and hepatitis B virus (HBV) infection (12.9%) were the leading and third-most common causes of HCC-complicated LC in Japan, respectively. HCV infection was found to be the leading cause throughout Japan. The rate of viral hepatitis-related HCC decreased from 85.3 to 64.4%. Among non-viral etiologies, notable increases were observed in nonalcoholic steatohepatitis (NASH)-related HCC (from 1.5 to 7.2%) and alcoholic liver disease (ALD)-related HCC (from 8.5 to 18.6%). Regarding the real number of newly diagnosed patients, the number of patients with viral hepatitis-related HCC decreased, while the number of patients with non-viral HCC, particularly NASH-related HCC, increased., Conclusions: Viral hepatitis has remained the main cause of HCC in Japan. However, the decrease in viral hepatitis-related HCC, particularly HCV-related HCC highly contributed to the etiological changes. In addition, the increased incidence of non-viral HCC, particularly NASH-related HCC, was involved in the changing etiologies of HCC-complicated LC in Japan.

COVID-19, a novel coronavirus-related illness, has spread worldwide. Patients with apparently mild/moderate symptoms can suddenly develop severe pneumonia. Therefore, almost all COVID-19 patients require hospitalization, which can reduce limited medical resources in addition to overwhelming medical facilities. To identify predictive markers for the development of severe pneumonia, a comprehensive analysis of serum chemokines and cytokines was conducted using serial serum samples from COVID-19 patients. The expression profiles were analyzed along the time axis. Serum samples of common diseases were enrolled from a BioBank to confirm the usefulness of predictive markers. Five factors, IFN-λ3, IL-6, IP-10, CXCL9, and CCL17, were identified as predicting the onset of severe/critical symptoms. The factors were classified into two categories. Category A included IFN-λ3, IL-6, IP-10, and CXCL9, and their values surged and decreased rapidly before the onset of severe pneumonia. Category B included CCL17, which provided complete separation between the mild/moderate and the severe/critical groups at an early phase of SARS-CoV-2 infection. The five markers provided a high predictive value (area under the receiver operating characteristic curve (AUROC): 0.9-1.0, p < 0.001). Low expression of CCL17 was specifically observed in pre-severe COVID-19 patients compared with other common diseases, and the predictive ability of CCL17 was confirmed in validation samples of COVID-19. The factors identified could be promising prognostic markers to distinguish between mild/moderate and severe/critical patients, enabling triage at an early phase of infection, thus avoiding overwhelming medical facilities., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Real-world data on the efficacy and safety of sofosbuvir plus velpatasvir (SOF/VEL) treatment for patients with hepatitis C virus (HCV)-related decompensated cirrhosis are limited in Japan., Methods: A total of 190 patients with compensated (108) or decompensated (82) cirrhosis who initiated direct-acting antiviral (DAA) treatment between February 2019 and August 2019 were enrolled. Sustained virologic response (SVR) was defined as undetectable serum HCV-RNA at 12 weeks after the end of treatment (EOT)., Results: The SVR12 rates were 92.6% in patients with compensated cirrhosis and 90.2% in patients with decompensated cirrhosis (p = 0.564), and the treatment completion rates were 98.1% and 96.3%, respectively (p = 0.372). In patients with decompensated cirrhosis, 3 patients discontinued treatment and 2 patients died because of liver-related events. In patients with decompensated cirrhosis with SVR12, 50% of patients with Child-Pugh class B at baseline showed improvement to class A at SVR12, and 27% and 9% of patients with Child-Pugh class C at baseline showed improvement to class B and class A at SVR12, respectively. Patients who achieved SVR12 showed elevated serum albumin levels at the EOT, which were further elevated at SVR12, but no elevated serum albumin levels after the EOT were observed in patients with baseline serum albumin levels less than 2.8 g/dl., Conclusions: Real-world efficacy of SOF/VEL treatment for patients with decompensated cirrhosis was similar to Japanese phase 3 study, although treatment discontinuation and death related to liver disease occurred. In patients with poor hepatic reserve, whether it improves continuously after viral clearance requires further evaluation.

Aim: Direct-acting antivirals for hepatitis C virus have reduced the decompensation risk. Immunosuppressants for transplantation raise the risk of occurrence of de novo malignancies. We assessed the probabilities of and risk factors for de novo hepatocellular carcinoma (HCC) development post-living donor liver transplantation (LDLT)., Methods: We retrospectively evaluated the data of developed HCC in a graft including metastatic HCC post-LDLT from 2779 adult cases collected from nine major liver transplantation centers in Japan., Results: Of 2779 LDLT adult recipients, 34 (1.2%) developed HCCs in their grafts. Of 34, five HCCs appeared to be de novo because of a longer period to tumor detection (9.7 [6.4-15.4] years) and no HCC within the native liver of the two recipients. The donor origin of three of five de novo HCCs was confirmed using microsatellite analysis in resected tissue. Primary disease of all five was hepatitis C virus-related cirrhosis, of which two were treated with direct-acting antivirals. Four of five developed HCC de novo in the hepatitis B core antibody-positive grafts. De novo HCCs had favorable prognosis; four of five were cured with complete remission. However, recurrent HCC (n = 29) in the graft had a poorer outcome, especially in patients with neutrophil to lymphocyte ratio scores above 4 (median survival time, 262 [19-463] days)., Conclusion: Analysis of the database from major liver transplantation institutes in Japan revealed that de novo HCCs determined by microsatellite analysis were rarely detected, but the majority were successfully treated. LDLT recipients with higher risks of de novo HCC, including those with hepatitis B core antibody-positive grafts, should be carefully followed by surveillance of the liver graft., (© 2020 The Japan Society of Hepatology.)

Hepatocellular carcinoma (HCC) occurs in the chronic liver inflammation such as viral hepatitis, alcoholic and non-alcoholic steatohepatitis. While anti-viral treatment has been significantly improved, the prevalence of HCC remains high and treatment is still challenging. The continuation of hepatocyte death, inflammation, and fibrosis leads to the accumulation of gene alterations, which may trigger carcinogenesis. Hepatocytes are a unique cell type having more than one complete set of 23 chromosomes, termed polyploidy. Due to gene redundancy, hepatocytes may tolerate lethal mutations. Next generation sequencing technology has revealed gene alterations in HCC related to telomere maintenance, Wnt/β-catenin pathway, p53 cell-cycle pathway, epigenetic modifiers, oxidative stress pathway, PI3K/AKT/mTOR, and RAS/RAF/MAPK pathway with or without a chromosomal instability. Some type of driver gene mutations accumulates in hepatocytes and breaks the orchestration of excessive copies of chromosomes, which may lead to unfavorable gene expressions and fuel tumorigenesis. Recently, molecular targeted drugs, developed with the aim of interfering with these signaling pathways, are being used for HCC patients in the clinics. Therefore, a deeper understanding of hepatocyte ploidy and genetic or epigenetic alterations is indispensable for the establishment of novel therapeutic strategies against HCC., Competing Interests: T.K. receives lecture fees from Gilead Sciences, MSD and Abbvie. The other authors have no conflict of interest to disclose., (2020, National Center for Global Health and Medicine.)

Chronic hepatitis B virus (HBV) genotype C infection is unlikely to show a good response to interferon (IFN). However, it is unknown whether a high dose of pegylated IFN (PEG-IFN) treatment would be effective for hepatitis B e antigen (HBeAg)-positive children with chronic HBV genotype C infection., Methods: HBeAg-positive children and adolescents with chronic HBV genotype C infection were eligible for this study. To increase the dose of PEG-IFN, all patients received PEG-IFN-α-2a (180 μg) without dose adjustment on the basis of body surface area for 48 weeks and were followed up for 24 weeks after the completion of treatment., Results: Thirteen patients (median age, 9 years) were enrolled prospectively for this study. One patient dropped out, and the remaining 12 patients were evaluated. Of the 12 patients, 11 received PEG-IFN of 180 μg/1.73 m 2 or more (median, 287 μg/1.73 m 2 ). Eight (67%) experienced HBeAg seroconversion, and 1 (8%) achieved hepatitis B surface antigen (HBsAg) loss at the end of follow-up. There was a significant difference in the decrease of hepatitis B surface antigen levels from the baseline to week 24 of treatment between the responders and the nonresponders. Serum cytokines and chemokines were measured in 10 patients. The levels of C-X-C motif chemokine ligand 9, 10, 11, and 13 in the responders tended to be higher than those in the nonresponders during the first 24 weeks of treatment., Conclusions: A high dose of PEG-IFN treatment was effective and safe. A decrease in the hepatitis B surface antigen level from baseline to week 24 of treatment might be a predictor of HBeAg seroconversion., Competing Interests: T. Kanto received lecture fees from Gilead Sciences and Merck, Sharp, & Dohme. The remaining authors report no conflicts of interests. The corresponding author is responsible for submitting a competing statement on behalf of all authors of this article., (Copyright © 2020 The Author(s). Published by Wolters Kluwer on behalf of European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition.)

Hepatitis B virus (HBV) reactivation reportedly occurs frequently after hematopoietic stem cell transplantation (HSCT) in resolved HBV-infected patients. Here, 50 patients with resolved HBV infections and scheduled to undergo HSCT were enrolled; all subjects were vaccinated with three doses of hepatitis B vaccine 12 months after HSCT and the incidence of HBV reactivation was monitored. The patients' characteristics were: median age, 61 (34-72) years; male/female, 27/19; allogeneic/autologous, 40/6; bone marrow/peripheral blood stem cells/cord blood, 26/16/4. Of the 46 patients who underwent HSCT, 19 were excluded and did not make it to vaccination due to relapse of underlying disease, HBV reactivation within 12 months of HSCT, or transfer of patients. The remaining 27 were vaccinated 12 months after HSCT and monitored for 2 years. Six showed HBV reactivation, with a 2-year cumulative reactivation incidence of 22.2%; the same incidence was 27.3% only in allogeneic HSCT patients. Factors associated with HBV reactivation included the discontinuation of immunosuppressants (P = 0.0379) and baseline titers of antibody against hepatitis B surface antigen (P = 0.004). HBV reactivation with vaccination following HSCT could occur despite maintenance of serum anti-HBs at more than protective levels.

Background & Aims: We aimed to clarify the features of resistance-associated substitutions (RASs) after failure of multiple interferon (IFN)-free regimens in HCV genotype 1b infections., Methods: A total of 1,193 patients with HCV for whom direct-acting antiviral (DAA) treatment had failed were enrolled from 67 institutions in Japan. The RASs in non-structural protein (NS)3, NS5A, and NS5B were determined by population sequencing., Results: Failure of 1, 2, and 3 regimens was observed in 1,101; 80; and 12 patients, respectively. Among patients with failure of 1 regimen, Y56H and D168V in NS3 were more frequently detected after failure of paritaprevir, whereas D168E was more frequently detected after failure of regimens including asunaprevir. R30H and L31-RAS in NS5A were frequently detected after failure of regimens including daclatasvir. The prevalence of Y93-RAS was high irrespective of the regimen. S282T RAS in NS5B was detected in 3.9% of ledipasvir/sofosbuvir failures. The prevalence of D168-RAS increased significantly according to the number of failed regimens ( p <0.01), which was similar to that seen with L31-RAS and Y93-RAS. The prevalence of patients with RASs in either NS3 or NS5A, or in both, increased significantly with increasing numbers of failed regimens. The P32del, which is unique to patients for whom DAA had failed, was linked to the absence of Y93H, the presence of L31F, and previous exposure to IFN plus protease inhibitor regimens., Conclusions: Failure of multiple DAA regimens can lead to the generation of multiple RASs in the NS3 and NS5A regions of the HCV 1b genome. These mutations contribute to viral resistance to multiple treatment regimens and, therefore, should be considered during decision making for treatment of chronic HCV., Lay Summary: Resistance-associated substitutions (RAS) in the genome of the hepatitis C virus are 1 of the major causes for failed treatment. We investigated RASs after failure of various treatments for chronic hepatitis C, and found that more complicated RASs accumulated in the viral genome with successive failed treatments. The highly resistant P32del RAS at NS5A region was uniquely found in patients for whom DAA treatments had failed, and was linked to the presence and absence of specific RASs., Competing Interests: The authors declare no conflicts of interest. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2020 The Author(s).)

Aim: Non-alcoholic fatty liver disease (NAFLD) is a leading cause of liver disease worldwide. Because liver fibrosis is associated with the long-term prognosis of patients with NAFLD, there is an urgent need for non-invasive markers of liver fibrosis. Sialic acid-binding immunoglobulin-like lectin-7 (Siglec-7) is an immunomodulatory molecule expressed on various immune cells, including macrophages, which plays a key role in liver inflammation and fibrosis in NAFLD. We aimed to determine whether serum levels of soluble Siglec-7 (sSiglec-7) could have utility at a marker of fibrosis in this patient population., Methods: We examined serum samples from 93 NAFLD patients and 19 healthy donors for macrophage-associated protein, including sSiglec-7, soluble CD163, and YKL-40, and examined their correlation with liver fibrosis scores, tissue elastography, and histological findings. Independent factors associated with advanced fibrosis were analyzed using a logistic regression model and a decision tree. To clarify the source of sSiglec-7, we examined its expression in liver tissue-derived macrophages and cultured monocyte-derived macrophages., Results: Serum sSiglec-7 levels were significantly higher in NAFLD patients compared with healthy donors, and correlated positively with sCD163 and YKL-40 levels. Serum sSiglec-7 was an independent diagnostic marker with high specificity (96.3%) for advanced fibrosis (F3 and F4) in NAFLD patients. Siglec-7 was mainly expressed on CCR2 + macrophages in the liver, and sSiglec-7 production by monocyte-derived macrophages in vitro was increased after stimulation by pro-inflammatory factors., Conclusions: Elevated serum sSiglec-7 could serve as an independent marker with high specificity for advanced liver fibrosis in patients with NAFLD., (© 2019 The Japan Society of Hepatology.)

Fibrosis is an incurable disorder of unknown etiology. Segregated-nucleus-containing atypical monocytes (SatMs) are critical for the development of fibrosis. Here we examined the mechanisms that recruit SatMs to pre-fibrotic areas. A screen based on cytokine expression in the fibrotic lung revealed that the chemokine Cxcl12, which is produced by apoptotic nonhematopoietic cells, was essential for SatM recruitment. Analyses of lung tissues at fibrosis onset showed increased expression of Rbm7, a component of the nuclear exosome targeting complex. Rbm7 deletion suppressed bleomycin-induced fibrosis and at a cellular level, suppressed apoptosis of nonhematopoietic cells. Mechanistically, Rbm7 bound to noncoding (nc)RNAs that form subnuclear bodies, including Neat1 speckles. Dysregulated expression of Rbm7 resulted in the nuclear degradation of Neat1 speckles, the dispersion of the DNA repair protein BRCA1, and the triggering of apoptosis. Thus, Rbm7 in epithelial cells plays a critical role in the development of fibrosis by regulating ncRNA decay and thereby the production of chemokines that recruit SatMs., Competing Interests: Declaration of Interests The authors declare competing financial interests: details are available in the online version of the paper. Readers are welcome to comment on the online version of the paper. Correspondence and requests for materials should be addressed to S.A. (sakira@biken.osaka-u.ac.jp) and T.S. (sohsatoh@biken.osaka-u.ac.jp)., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: To assess the recent real-world changes in the etiologies of liver cirrhosis (LC) in Japan, we conducted a nationwide survey in the annual meeting of the Japan Society of Hepatology (JSH)., Methods: We investigated the etiologies of LC patients accumulated from 68 participants in 79 institutions (N = 48,621). We next assessed changing trends in the etiologies of LC by analyzing cases in which the year of diagnosis was available (N = 45,834). We further evaluated the transition in the real number of newly identified LC patients by assessing data from 36 hospitals with complete datasets for 2008-2016 (N = 18,358)., Results: In the overall data, HCV infection (48.2%) was the leading cause of LC in Japan, and HBV infection (11.5%) was the third-most common cause. Regarding the transition in the etiologies of LC, the contribution of viral hepatitis-related LC dropped from 73.4 to 49.7%. Among the non-viral etiologies, alcoholic-related disease (ALD) and nonalcoholic steatohepatitis (NASH)-related LC showed a notable increase (from 13.7 to 24.9% and from 2.0 to 9.1%, respectively). Regarding the real numbers of newly diagnosed patients from 2008 to 2016, the numbers of patients with viral hepatitis-related LC decreased, while the numbers of patients with non-viral LC increased., Conclusions: HCV has remained the main cause of LC in Japan; however, the contribution of viral hepatitis as an etiology of LC is suggested to have been decreasing. In addition, non-viral LC, such as ALD-related LC and NASH-related LC, is suggested to have increased as etiologies of LC in Japan.

Aim: Regional core centers for the management of liver disease, which are located in every prefecture in Japan, not only take the lead in hepatitis care in their respective regions, but also serve a wide range of other functions, such as education, promotion of hepatitis testing, treatment, and research., Method: Since fiscal year 2010, the Hepatitis Information Center has conducted surveys of regional core centers throughout Japan regarding information about their facilities, programs for patient support, training, and education of medical personnel., Results: By compiling and analyzing the results of these surveys, we have elucidated the status of regional core centers and the issues they currently have. We found that regional core centers have come to play widely varied roles in hepatitis treatment and have expanded their programs. These surveys also suggest that uniform accessibility of hepatitis treatment has been implemented throughout Japan., Conclusion: To continue serving their diverse roles, regional core centers require further development of hepatitis care networks that include specialized institutions, primary care physicians, and local and central governments; as well as collaboration with other professions and groups., (© 2019 The Authors. Hepatology Research published by John Wiley & Sons Australia, Ltd on behalf of Japan Society of Hepatology.)

Background: Cancer-associated fibroblasts (CAFs) are essential constituents of cancer-supportive microenvironments. The high incidence of hepatocellular carcinoma (HCC) in advanced fibrosis patients implies that fibroblasts have a promoting effect on HCC development. We aimed to explore the regulators of phenotypes and function of CAFs in the liver., Methods: We established primary cancer-associated fibroblasts (CAFs) and non-cancerous liver fibroblasts (NFs) from 15 patients who underwent HCC resection. We compared phenotypes, capacity of cytokine/chemokine production and gene expression profiles between pairs of CAFs and NFs from the same donors. We examined resected tissue from additional 50 patients with HCC for immunohistochemical analyses., Results: The CAFs expressed more ACTA2 and COL1A1 than the NFs, suggesting that CAFs are more activated phenotype. The CAFs produced larger amounts of IL-6, IL-8 and CCL2 than the NFs, which led to invasiveness of HuH7 in vitro. We found that Bone Morphogenetic Protein-4 (BMP4) is up-regulated in CAFs compared to NFs. The CAF phenotype and function were gained by BMP4 over-expression or recombinant BMP4 given to fibroblasts, all of which decreased with BMP4 knockdown. In tissues obtained from the patients, BMP4-positive cells are mainly observed in encapsulated fibrous lesions and HCC. Positive expression of BMP4 in HCC in resected tissues, not in fibroblasts, was associated with poorer postoperative overall survival in patients with HCC., Conclusion: Endogenous and exogenous BMP4 activate liver fibroblasts to gain capacity of secreting cytokines and enhancing invasiveness of cancer cells in the liver. BMP4 is one of the regulatory factors of CAFs functioning in the microenvironment of HCC.

One of the important missions of the Hepatitis Information Center is to disseminate information regarding liver disease. The Hepatitis Information Center, National Center for Global Health and Medicine (NCGM) has been endeavoring to ensure that reliable and up-to-date information on liver disease is accessible to all people, regardless of age, disability, and background. Described here are several initiatives with regard to the dissemination of information about liver disease including: i ) Education tool for youth, ii ) Conversion of materials on liver diseases into audio format for the visually impaired, and iii ) Hepatic Disease Medical Navigation System (Hepatic Navi). Hepatic Navi is a web-based search tool that informs users of the location and other information concerning medical centers where people can be tested for the hepatitis virus for free or at reduced cost. Hepatic Navi consolidates data from 47 prefectures into one database. The system depicts data via an interface that can be accessed anywhere with a PC, tablet, smart phone, or mobile phone. As a result, it has become possible for anyone from anywhere to access information on hepatitis virus testing. By using Hepatic Navi, it is anticipated that general people in need feel free to access to the testing and further treatment for virus hepatitis., (2019, National Center for Global Health and Medicine.)

Current serum hepatocellular carcinoma (HCC) biomarkers are insufficient for early diagnosis. We aimed to clarify whether serum MFG-E8 can serve as a diagnostic or prognostic biomarker of HCC. Serum MFG-E8 levels of 282 HCC patients, who underwent primary hepatectomy, were examined by ELISA. We also quantified serum MFG-E8 levels in patients with chronic hepatitis (CH), liver cirrhosis (LC), as well as in healthy volunteers (HVs). Serum MFG-E8 levels were significantly lower in HCC patients than in HVs regardless of the etiology of liver disease (3.6 ± 0.1 vs 5.8 ± 0.2 ng/mL, p < 0.0001), and recovered after treatment of HCC. Serum MFG-E8 levels in CH and LC patients were comparable to those in HVs. Serum MFG-E8 could detect HCCs, even α-fetoprotein (AFP)-negative or des-γ-carboxy prothrombin (DCP)-negative HCCs, in CH and LC patients. Our new HCC prediction model using MFG-E8 and DCP (Logit(p) = 2.619 - 0.809 × serum MFG-E8 + 0.0226 × serum DCP) distinguished HCC patients from CH and LC patients with an area under the curve of 0.923, a sensitivity of 81.1%, and a specificity of 89.8%. Futhermore, low preoperative serum MFG-E8 was an independent predictor of poor overall survival. Thus, serum MFG-E8 could serve as a feasible diagnostic and prognostic biomarker for HCC.

During the last two decades, there has been a dramatic increase in so-called non-B non-C hepatocellular carcinoma (NBNC HCC) in Japan. Majority of NBNC HCC are considered as so-called metabolic HCC and some could be related to occult HBV infection. Although there have been some reports on histological features predominant in metabolic HCC, very few specific driver genes for NBNC HCC have been reported. Most of the NBNC HCC are found incidentally and are relatively large in size. Since liver function is generally normal or subnormal, such patients have a higher chance for undergoing curative surgery. Although there has been slightly conflicting long-term outcomes reported for NBNC HCC, slightly better outcomes may be expected compared to other etiologies after curative surgery. However, risk of recurrence depends on the background liver. NBNC HCC in cirrhotic patients have a persistently higher risk of tumor recurrence requiring a long-term postoperative surveillance. It would be safe to conclude at this moment that NBNC HCCs should be treated using the same surgical strategy as HCCs with viral origin, same operative indications and same follow-up protocol., (2019, National Center for Global Health and Medicine.)

Aims: We aimed to investigate the impact of interleukin (IL)-34 and YKL-40, regulators of hepatic fibrosis and tumor growth, on the prognosis of patients with non-viral hepatocellular carcinoma (HCC)., Methods: We enrolled 159 non-viral HCC patients (age, 70.8 ± 8.5 years; female/male, 43/116). Of these, 86 patients were alive and 73 patients had died at the censor time point. Serum IL-34 and YKL-40 levels were quantified by enzyme-linked immunosorbent assay. Patients were stratified by the median level of serum IL-34 to examine its effect on survival. Multivariate analysis and random forest analysis were used to evaluate the impact of IL-34 and YKL-40 on the prognosis of non-viral HCC patients., Results: Interleukin-34 (hazard ratio [HR] 1.30; 95% confidence interval [CI], 1.13-1.49; P ≤ 0.01), tumor size (HR 1.63; 95% CI, 1.37-1.94; P ≤ 0.01), and tumor number (HR 1.53; 95% CI, 1.25-1.87; P ≤ 0.01) were independent predictive factors for survival. Furthermore, the survival rates were significantly lower in the high IL-34 group than in the low IL-34 group (5-year survival rates, 34.7% vs. 59.8%, respectively; P < 0.05). In the random forest analysis for survival, IL-34 was the third-highest ranking factor, following tumor size and number. In a stratification analysis, serum α-fetoprotein level and Fibrosis-4 index were independent positive risk factors for high serum IL-34 level. YKL-40 was not associated with prognosis in either the multivariate or random forest analysis., Conclusion: Interleukin-34 was an independent factor for survival of non-viral HCC patients. Interleukin-34 might be associated with prognosis through tumor and hepatic fibrosis factors., (© 2019 The Japan Society of Hepatology.)

Introduction : Hepatic fibrosis and hepatocellular carcinoma (HCC) can develop in children with congenital heart disease. Although hepatic fibrosis and HCC are prone to develop after the Fontan operation, they can also develop in patients suffering from congenital heart disease who have not undergone Fontan operation. Area covered : The history of cardiac hepatopathy including Fontan-associated liver disease is described. Patient characteristics, liver histology, imaging examinations and blood tests are reviewed to elucidate the mechanism of cardiac hepatopathy. In addition, a flowchart for the follow-up management of cardiac hepatopathy in children with congenital heart disease is proposed. Expert opinion : Congestion and low cardiac output are the main causes of cardiac hepatopathy. Advanced hepatic fibrosis is presumed to be associated with HCC. HCC can develop in both adolescents and young adults. Regardless of whether the Fontan operation is performed, children with a functional single ventricle and chronic heart failure should be regularly examined for cardiac hepatopathy. There is no single reliable laboratory parameter to accurately detect cardiac hepatopathy; hepatic fibrosis indices and elastography have shown inconsistent results for detection of this disease. Further studies using liver specimen-confirmed patients and standardization of evaluation protocols are required to clarify the pathogenesis of cardiac hepatopathy.

Immune checkpoint blockade with specific antibodies can accelerate anti-tumor immunity, resulting in clinical responses in patients with various types of cancer. However, these antibodies achieve only partial tumor regression. Thus, a wide variety of treatment combinations based on programmed death-ligand 1 (PD-L1) pathway inhibition are under development to enhance such therapeutic effects. In this study, the effects of combination treatment using PRI-724, a selective inhibitor of CBP/β-catenin, and an anti-PD-L1 antibody were examined in a mouse model of colon cancer liver metastasis. Mice were inoculated with SL4 colon cancer cells to produce metastatic liver tumors. The combination treatment resulted in regression of tumor growth, whereas monotherapy with each treatment individually failed to exhibit any anti-tumor activity. In addition, co-administration of the inhibitor and antibody induced CD8 + CD44 low CD62L low cells and interferon (IFN)-γ production in CD8 + T-cells in the liver compared with that in control mice. Administration of an anti-CD8 antibody mitigated the anti-tumor effects of the combined treatment of PRI-724 and anti-PD-L1 antibody. In conclusion, targeting CBP/β-catenin, combined with PD-1/PD-L1 immune checkpoint blockade, shows potential as a new therapeutic strategy for treating liver metastasis during colon cancer., Competing Interests: CONFLICTS OF INTEREST There is no conflicts of interest.

Global implementation of a birth-dose hepatitis B (HB) vaccine has significantly reduced the prevalence of hepatitis B virus (HBV) carriers. Durable and sufficient titers of antibodies to hepatitis B surface antigen (anti-HBs) are desirable for vaccinees to gain resistance to HBV exposure. However, the existence of primary nonresponders and vaccinees who lost anti-HBs over time remains a challenge for the strategy of HBV elimination. We thus aim to clarify the mechanisms of acquisition and maintenance of vaccine-induced anti-HBs in healthy adults. We retrospectively analyzed the vaccination records of 3,755 first-time HB-vaccinated students and also traced the acquired antibody transition of 392 first-time vaccinees for 10 consecutive years. To understand the cellular and humoral immune response, we prospectively examined peripheral blood from 47 healthy first-time HB-vaccinated students, 62 booster-vaccinated health care workers, and 20 individuals who maintained their anti-HBs. In responders, a significant increase of follicular helper T (Tfh) cells, activated plasmablasts, and plasma cells was observed in first-time-vaccinated but not booster-vaccinated persons. We also discovered memory B cells and antibody-secreting cells were more abundant in individuals who maintained anti-HBs. According to vaccination records, higher anti-HBs antibody titer acquisition was related to the longer term maintenance of anti-HBs, the level of which was positively correlated with prevaccination levels of serum interferon-γ and related chemokines. The second series of vaccination as a booster provided significantly higher anti-HBs antibody titers compared to the initial series. Conclusion : Coordinated activation of Tfh and B-cell lineages after HB vaccination is involved in the acquisition and maintenance of anti-HBs. Our findings support the rationale of preconditioning the immune status of recipients to ensure durable vaccine responses.

Hepatitis C virus (HCV) utilizes cellular factors for efficient propagation. Ubiquitin is covalently conjugated to the substrate to alter its stability or to modulate signal transduction. In this study, we examined the importance of ubiquitination for HCV propagation. We found that inhibition of deubiquitinating enzymes (DUBs) or overexpression of nonspecific DUBs impaired HCV replication, suggesting that ubiquitination regulates HCV replication. To identify specific DUBs involved in HCV propagation, we set up RNA interference (RNAi) screening against DUBs and successfully identified ubiquitin-specific protease 15 (USP15) as a novel host factor for HCV propagation. Our studies showed that USP15 is involved in translation of HCV RNA and production of infectious HCV particles. In addition, deficiency of USP15 in human hepatic cell lines (Huh7 and Hep3B/miR-122 cells) but not in a nonhepatic cell line (293T cells) impaired HCV propagation, suggesting that USP15 participates in HCV propagation through the regulation of hepatocyte-specific functions. Moreover, we showed that loss of USP15 had no effect on innate immune responses in vitro and in vivo We also found that USP15-deficient Huh7 cells showed reductions in the amounts of lipid droplets (LDs), and the addition of palmitic acids restored the production of infectious HCV particles. Taken together, these data suggest that USP15 participates in HCV propagation by regulating the translation of HCV RNA and the formation of LDs. IMPORTANCE Although ubiquitination has been shown to play important roles in the HCV life cycle, the roles of deubiquitinating enzymes (DUBs), which cleave ubiquitin chains from their substrates, in HCV propagation have not been investigated. Here, we identified USP15 as a DUB regulating HCV propagation. USP15 showed no interaction with viral proteins and no participation in innate immune responses. Deficiency of USP15 in Huh7 cells resulted in suppression of the translation of HCV RNA and reduction in the amounts of lipid droplets, and the addition of fatty acids partially restored the production of infectious HCV particles. These data suggest that USP15 participates in HCV propagation in hepatic cells through the regulation of viral RNA translation and lipid metabolism., (Copyright © 2019 American Society for Microbiology.)

Background: The clearance of hepatitis B surface antigen (HBsAg) loss, defined as functional cure, is a clinical target in patients with chronic hepatitis B (CH). To understand the immune responses underlying functional cure, we evaluated cytokine and chemokine expression profiles from patients with resolving and nonresolving acute hepatitis B (AH)., Methods: We cross-sectionally evaluated 41 chemokines and cytokines at the peak of hepatitis in the sera from 41 self-limited AH patients who achieved HBsAg seroconversion, 8 AH patients who failed to clear HBsAg within 1 year after the diagnosis, 8 CH patients with hepatic flare, and 14 healthy volunteers. We longitudinally examined 41 chemokines and cytokines in the sera from 4 self-limited AH patients, 3 chimpanzees inoculated with hepatitis B virus (HBV), and 2 CH patients treated with nucleotide analogs and PEG-IFN-α, one resulting in functional cure., Results: In AH patients and HBV-inoculated chimpanzees with HBsAg loss, CXCL9, CXCL10, CXCL11, CXCL13, and IL-21 were elevated at hepatitis with subsequent decline of HBsAg. Interestingly, IL-21 elevation was observed only in resolving AH patients but not in nonresolvers. CXCL13 and IL-21 elevation was not observed in CH patients who failed to attain HBsAg loss, even at hepatic flare. A concomitant increase of CXCL13 and IL-21 was significant in CH patients who attained HBsAg seroconversion with a sequential therapy., Conclusion: Elevation of serum CXCL9, CXCL10, CXCL11, CXCL13, and IL-21 might be a hallmark of functional cure of AH or CH patients.

BCL2 family proteins including pro-survival proteins, BH3-only proteins and BAX/BAK proteins control mitochondria-mediated apoptosis to maintain cell homeostasis via the removal of damaged cells and pathogen-infected cells. In this study, we examined the roles of BCL2 proteins in the induction of apoptosis in cells upon infection with flaviviruses, such as Japanese encephalitis virus, Dengue virus and Zika virus. We showed that survival of the infected cells depends on BCLXL, a pro-survival BCL2 protein due to suppression of the expression of another pro-survival protein, MCL1. Treatment with BCLXL inhibitors, as well as deficient BCLXL gene expression, induced BAX/BAK-dependent apoptosis upon infection with flaviviruses. Flavivirus infection attenuates cellular protein synthesis, which confers reduction of short-half-life proteins like MCL1. Inhibition of BCLXL increased phagocytosis of virus-infected cells by macrophages, thereby suppressing viral dissemination and chemokine production. Furthermore, we examined the roles of BCLXL in the death of JEV-infected cells during in vivo infection. Haploinsufficiency of the BCLXL gene, as well as administration of BH3 mimetic compounds, increased survival rate after challenge of JEV infection and suppressed inflammation. These results suggest that BCLXL plays a crucial role in the survival of cells infected with flaviviruses, and that BCLXL may provide a novel antiviral target to suppress propagation of the family of Flaviviridae viruses., Competing Interests: The authors have declared that no competing interests.

Background: Direct-acting anti-viral agents have improved the treatment of chronic hepatitis C virus (HCV) infection, but this treatment is challenging for patients using co-medications because of potential drug-drug interactions. This study aimed to examine the comorbidities and co-medications of Japanese chronic HCV patients by age group, compared with a non-HCV patient population., Methods: This was a retrospective observational study using a hospital-based medical claims database. We extracted data of patients with chronic HCV aged ≥18 years, and age-, sex-, and hospital-matched patients without HCV, for the period from January 2015 to November 2016, and then examined chronic comorbidities, long-term co-medications, and medications prescribed at least once during the study period., Results: We analysed data from 128,967 chronic HCV patients and 515,868 non-HCV patients. The median age was 70 years, and 51.0% of patients were male. More chronic HCV patients than non-HCV patients (70.5% vs. 47.1%) had at least one comorbidity, and older patients had more comorbidities than younger patients. The most common comorbidities in chronic HCV patients were diseases of oesophagus, stomach and duodenum (41.7%), followed by hypertensive diseases (31.4%). Chronic HCV patients used co-medications more commonly than non-HCV patients, and older patients used more co-medications. The most common long-term co-medications in chronic HCV patients were proton pump inhibitors (14.0%), which were prescribed to 31.9% of chronic HCV patients at least once during the study period., Conclusions: Patients with chronic HCV in Japan had more comorbidities than patients without chronic HCV regardless of age. Particularly older patients, who constitute the majority of the HCV patient population in Japan, commonly had multiple comorbidities and used co-medications. To optimise HCV treatment, physicians need to know the exact medication profiles of patients and take appropriate action to manage drug-drug interactions.

Aim: Early hepatocellular carcinoma (HCC) recurrence after curative resection is a known poor prognostic factor. We aimed to identify microRNAs associated with recurrence after curative HCC resection., Methods: To identify risk factors for early recurrence and metastasis, 694 patients who underwent primary curative HCC resection were analyzed. We evaluated microRNA expression in cancerous and non-cancerous tissues by microarray and quantitative PCR analyses using 16 HCC samples. We defined patients who had a recurrence within 1 year of resection as the early recurrence (ER) group, patients who had a recurrence within 1-5 years as the late recurrence (LR) group, and patients who did not recur during the 5-year observation period as the no recurrence (NR) group. We examined the relationship between microRNA expression and clinical features., Results: Multivariate analysis revealed that α-fetoprotein >31 ng/mL, tumor size >4 cm, and intrahepatic metastasis (IM) were significant factors. Afterwards, microarray analyses revealed that microRNA (miR)-125b-5p and miR-148a-3p were significantly downregulated in recurrent cases. The ratio of miR-125b-5p expression in cancerous versus non-cancerous tissue (miR-125b ratio), but not miR-148a-3p, was significantly lower in the ER group. Early recurrence was associated with reduced overall survival compared with the LR and NR group. The miR-125b ratio was significantly lower in the ER group than in the LR and NR groups. Multivariate analysis showed that a low miR-125b ratio and IM were independently associated with ER and disease-free survival., Conclusions: Assessing tissue miR-125b-5p expression and IM is useful for stratifying patients at risk of early HCC recurrence after curative resection., (© 2017 The Japan Society of Hepatology.)

Hepatocyte apoptosis has been implicated in the progression of nonalcoholic steatohepatitis. However, it is unclear whether the induction of tumor necrosis factor (TNF)-α-mediated hepatocyte apoptosis in the simple fatty liver triggers liver fibrosis. To address this question, high-fat diet-fed mice were repeatedly administered D-galactosamine, which increases the sensitivity of hepatocytes to TNF-α-mediated apoptosis. In mice treated with a high-fat diet plus D-galactosamine, hepatocyte apoptosis and liver fibrosis were induced, whereas both apoptosis and fibrosis were inhibited in these mice following gut sterilization with antimicrobials or knockout of TNF-α. Furthermore, liver fibrosis was diminished when hepatocyte apoptosis was inhibited by expressing a constitutively active inhibitor of nuclear factor κB kinase subunit β. Thus, hepatocyte apoptosis induced by intestinal dysbiosis or TNF-α up-regulation in the steatotic liver caused fibrosis. Organ fibrosis, including liver fibrosis, involves the interaction of cyclic adenosine monophosphate-response element-binding protein-binding protein (CBP) and β-catenin. Here, hepatocyte-specific CBP-knockout mice showed reduced liver fibrosis accompanied by hepatocyte apoptosis diminution; notably, liver fibrosis was also decreased in mice in which CBP was specifically knocked out in collagen-producing cells because the activation of these cells was now suppressed. Conclusion : TNF-α-mediated hepatocyte apoptosis induced fibrosis in the steatotic liver, and inhibition of CBP/β-catenin signaling attenuated the liver fibrosis due to the reduction of hepatocyte apoptosis and suppression of the activation of collagen-producing cells. Thus, targeting CBP/β-catenin may represent a new therapeutic strategy for treating fibrosis in nonalcoholic steatohepatitis. ( Hepatology Communications 2018;2:407-420).

Background and Aim: The mechanism underlying hyperglobulinemia in cirrhosis, a long appreciated phenomenon, has never been clearly understood. The aim of this study is to investigate the basis for changes in humoral immunity observed in cirrhosis., Methods: We retrospectively reviewed our medical record to analyze serum immunoglobulin (Ig) levels in patients with liver disease. We also prospectively analyzed peripheral blood mononuclear cells and sera from liver disease patients. Peripheral blood mononuclear cell surface marker expressions were measured by flow cytometry and serum B-cell-activating factor was measured by enzyme-linked immunosorbent assay. Expression of specific gene expression in magnetically separated B cells was also analyzed by real-time polymerase chain reaction., Results: In retrospective analysis, we found that advancing cirrhosis, irrespective of underlying etiology or hepatocellular carcinoma, resulted in progressively increasing levels of serum IgG and IgA. In prospective analysis using clinical samples, we demonstrated that advancing cirrhosis stage was associated with increased toll-like-receptor (TLR)9 expression in CD27 + B cell and serum B-cell-activating factor levels but decreased CD27 + memory B-cell frequency. The remaining CD27 + B cells in peripheral blood exhibited increased activation-induced cytidine deaminase mRNA expression. Finally, we also demonstrated isolated B cells from advanced cirrhosis were more reactive to TLR9 stimulation that drove antibody secreting cells differentiation leading to hyperimmunoglobulinemia in vitro., Conclusions: Enhanced TLR9-induced differentiation into antibody secreting cell may explain peripheral reductions of circulating CD27 + memory B cells as well as increased serum Ig levels in cirrhosis., (© 2018 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Lactic acid bacteria (LAB) are one of the major commensal species in the small intestine and known for contributing to maintenance of protective immunity and immune homeostasis. However, currently there has been no evidence regarding the cellular mechanisms involved in the probiotic effects of LAB on human immune cells. Here, we demonstrated that LAB double-stranded RNA (dsRNA) triggered interferon-β (IFN-β) production by human dendritic cells (DCs), which activated IFN-γ-producing T cells. Interleukin-12 (IL-12) secretion from human DCs in response to LAB was abrogated by depletion of bacterial dsRNA, and was attenuated by neutralizing IFN-β, indicating LAB dsRNA primarily activated the IFN-β/IL-12 pathway. Moreover, the induction of IL-12 secretion from DCs by LAB was abolished by the inhibition of endosomal acidification, confirming the critical role of the endosomal digestion of LAB. In a coculture of human naïve CD4 + T cells and BDCA1 + DCs, DCs stimulated with LAB containing dsRNA induced IFN-γ-producing T cells. These results indicate that human DCs activated by LAB enhance Th1 immunity depending on IFN-β secretion in response to bacterial dsRNA.

Sorafenib, a multi-kinase inhibitor, inhibits tumor angiogenesis and is the first-line systemic therapy for patients with advanced hepatocellular carcinoma (HCC). However, due to its limited effects and frequent occurrence of side effects, biomarkers are needed to predict the effects of sorafenib. We considered the possibility of using TIE-2-expressing monocytes (TEMs) to predict the response in sorafenib-treated patients with advanced HCC. TEMs serve as a diagnostic marker of HCC and are related to angiogenesis. We analyzed 25 advanced HCC patients and prospectively evaluated TEMs before (Pre TEMs) and at 1 month after initial therapy (T1m TEMs). The radiologic response was evaluated by modified Response Evaluation Criteria in Solid Tumors (mRECIST). Median survival time (MST) was significantly longer in the partial response/stable disease (PR/SD) group (21.8 months) than in the PD group (8.7 months). ΔTEMs (changes of T1m TEMs compared to Pre TEMs) were significantly lower in the PR/SD group than in the PD group. MST of the ΔTEMs low group (14.2 months) was significantly longer than that of the high group (8.7 months). Univariate and multivariate Cox regression analyses showed that ΔTEMs [hazard ratio (HR) = 8.53, 95% confidence interval (CI) = 1.51-48.16, p = 0.015] and Child-Pugh class (HR = 5.59, 95% CI = 1.06-29.63, p = 0.043) were independently associated with overall survival. Our results suggest that ΔTEMs could serve as a biomarker for predicting radiologic response and overall survival in sorafenib-treated patients with advanced HCC., (© 2017 UICC.)

Background: There is currently no anti-fibrotic drug therapy available to treat hepatitis C virus (HCV) cirrhosis. The aim of this study was to assess the safety, tolerability, and anti-fibrotic effect of PRI-724, a small-molecule modulator of Wnt signaling, in patients with HCV cirrhosis., Methods: In this single-center, open-label, phase 1 trial, we sequentially enrolled patients with HCV cirrhosis who were classified as Child-Pugh (CP) class A or B. PRI-724 was administered as a continuous intravenous infusion of 10, 40, or 160mg/m 2 /day for six cycles of 1week on and 1week off. The primary endpoints were frequency and severity of adverse events. The secondary endpoint was efficacy of PRI-724 in treating cirrhosis based on CP score and liver biopsy. This study is registered with ClinicalTrials.gov (no. NCT02195440)., Findings: Between Sept 3, 2014 and May 2, 2016, 14 patients were enrolled: CP class A:B, 6:8; median age, 62 (range: 43 to 74) years; male:female, 10:4. Twelve of the 14 patients completed six cycles of treatment; one was withdrawn from the study due to possible study drug-related liver injury (grade 3) in the 160mg/m 2 /day dose cohort and one withdrew for personal reasons. Serious adverse events occurred in three patients [21% (3/14)], one of which was possibly related to PRI-724. The most common adverse events were nausea [29% (4/14)] and fatigue [21% (3/14)]. After PRI-724 administration, the CP scores worsened by 1 point in two patients in the 10mg/m 2 /day cohort, improved in three patients at 1, 1, and 2 points in the 40mg/m 2 /day cohort, and improved in one patient by 3 points in the 160mg/m 2 /day cohort. The histology activity index scores of the liver tissue improved in two patients and exacerbated in two patients in the 10mg/m 2 /day cohort, and improved in one patient in the 40mg/m 2 /day cohort., Interpretation: This study showed that administration of 10 or 40mg/m 2 /day intravenous PRI-724 over 12weeks was well-tolerated by patients with HCV cirrhosis; however, liver injury as a possible related serious adverse event was observed in the 160mg/m 2 /day cohort., Funding Source: AMED., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

In Japan, hepatitis B or C virus infection has been a major health issue. For the prevention of liver-related deaths, multifaceted strategies have been taken against hepatitis virus. In fiscal year (FY) 2002, nationwide screening for hepatitis was started as a part of health examinations provided by municipal governments. From FY2007, the hepatitis treatment network has been strengthened by the nationwide establishment of regional government-based hepatitis treatment systems, comprising linked regional core centers, specialized institutions for hepatitis treatment, primary care physicians, and regional governments. Special subsidy program for patients with viral hepatitis was started at FY2008. The range of coverage has been expanding from patients treated with interferon to those on nucleotide analogs or interferon-free therapies, including drug prices and examination expenses. The Basic Act on Hepatitis Measures was established in 2009. The Basic Guidelines for Promotion of Control Measures for hepatitis was issued in 2011, comprising nine principles in order to promote measures for hepatitis B and C. The Hepatitis Information Center was established in 2008. Its mission is to provide up-to-date hepatitis-related information, supporting collaboration between regional core centers, and training medical personnel. The revision of the above-mentioned Basic Guidelines in 2016 set the target as the reduction of patients progressing to cirrhosis and/or liver cancer. Achieving this goal definitely requires active collaboration among the national and local governments, regional core centers, and the Hepatitis Information Center, and participation by medical personnel, patients, and people with awareness., (© 2017 The Authors Hepatology Research published by John Wiley & Sons Australia, Ltd on behalf of Japan Society of Hepatology.)

In Japan, the estimated number of chronic hepatitis B virus infections was 1.1 to 1.4 million, and that of chronic hepatitis C virus was 1.9 to 2.3 million in 2000. The mortality of hepatocellular carcinoma had been increasing and hit the peak at around 2002, which subsequently started to decrease. Japan has a national action plan for addressing viral hepatitis called Basic Act on Hepatitis Measures, established in 2009. In 2011, basic guidelines for promotion of control measures for hepatitis were issued, comprising nine principles in order to promote measures to prevent hepatitis B and C. According to these guidelines, national and local governments share screening costs for testing hepatitis B and C in residents who are over 40 years old. Thus, out-of-pocket expenses from examinees are nil or reduced to the minimum. In addition, for patients with chronic hepatitis B or C and on treatment, drug prices of nucleotide analogs, interferon (IFN) treatment, or IFN-free direct antiviral agents along with examination expenses should be covered by special programs for viral hepatitis. The national and local governments cover the amount in excess of 100 to 200 USD of the cost of treatment. The proportion of liver cancer with nonviral etiology has been increasing in Japan. For the screening and follow-up of patients with nonalcoholic fatty liver disease, we demonstrated that interleukin 34 is a feasible fibrosis marker. Several advantages have prevailed in the Japanese health care systems for patients with viral liver disease compared with those in countries in the Western Pacific region. Therefore, Japan should take a lead in helping the implementation of practical hepatitis action plans in every country when in need. How to cite this article: Kanto T, Yoshio S. Hepatitis Action Plan and Changing Trend of Liver Disease in Japan: Viral Hepatitis and Nonalcoholic Fatty Liver Disease. Euroasian J Hepato-Gastroenterol 2017;7(1):60-64., Competing Interests: Source of support: Nil Conflict of interest: None

Aim: Telaprevir (TVR) remarkably improves the efficacy of interferon treatment for chronic hepatitis C. Interleukin-28B (IL28B) genotype and interferon-gamma-inducible protein-10 (IP-10) level predict virologic response to peg-interferon (Peg-IFN)/ribavirin (RBV) therapy. We aimed to investigate the usefulness of pretreatment serum IP-10 levels and IL28B genotyping in predicting sustained virologic response (SVR) to TVR-based triple therapy., Methods: In this multi-center study, patients infected with hepatitis C virus genotype 1 with high viral load (⩾5.0logIU/mL) were treated with TVR for 12weeks and Peg-IFN/RBV for 24weeks in Japan. IL28B genotype, serum IP-10 levels, other clinical parameters, and drug dosages were assessed before treatment., Results: We included 121 patients who were treated with TVR for at least 8weeks and Peg-IFN/RBV for 24weeks. The median IP-10 levels were significantly lower in rapid virologic response (RVR) or SVR in the IL28B non-TT genotype group, with no significant difference in the TT genotype group. RVR rates were significantly lower in the group with higher serum IP-10 levels (>450pg/mL). In the non-TT IL28B genotype group, RVR and SVR rates were significantly lower in the group with higher IP-10 levels. SVR rates in the group with lower IP-10 levels (<450pg/mL) increased to 82% for those showing RVR, but reduced to 27% in the group with higher IP-10 levels for those not showing RVR., Conclusions: Determination of serum IP-10 levels before treatment could be useful for predicting favorable virologic response to TVR-based triple therapy, especially in patients with IL28B non-TT genotype., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Non-alcoholic fatty liver disease (NAFLD) is a common cause of chronic non-viral liver disease. YKL-40, chitinase-like protein expressed in multiple tissues including liver, is involved in cell proliferation, inflammation and remodeling of the extracellular matrix. The aim of this study was to assess whether serum YKL-40 levels are associated with liver fibrosis in NAFLD patients. Serum YKL-40 levels were quantified in 111 NAFLD patients and 23 HCC patients with NAFLD. To identify the source of YKL-40, immunofluorescence staining of liver specimens from NAFLD patients was performed. Serum YKL-40 levels in NAFLD patients increased in accordance with the progression of liver fibrosis. Multivariate analysis revealed that YKL-40 was one of the independent factors significantly associated with severe fibrosis (F3-4). We established a new predictive model for fibrosis of NAFLD, using logistic regression analysis: YKL-40 based fibrosis score = -0.0545 + type IV collagen 7s * 0.3456 + YKL-40 * 0.0024. Serum YKL-40 levels of HCC patients with non-cirrhotic NAFLD were significantly higher than those without HCC. Immunofluorescence staining showed that YKL-40 was expressed by macrophages in liver tissue of NAFLD patients. In conclusion, macrophage-derived YKL-40 is a feasible biomarker of liver fibrosis in NAFLD patients.

Despite the breadth of knowledge that exists regarding the function of long noncoding RNAs (lncRNAs) in biological phenomena, the role of lncRNAs in host antiviral responses is poorly understood. Here, we report that lncRNA#32 is associated with type I IFN signaling. The silencing of lncRNA#32 dramatically reduced the level of IFN-stimulated gene (ISG) expression, resulting in sensitivity to encephalomyocarditis virus (EMCV) infection. In contrast, the ectopic expression of lncRNA#32 significantly suppressed EMCV replication, suggesting that lncRNA#32 positively regulates the host antiviral response. We further demonstrated the suppressive function of lncRNA#32 in hepatitis B virus and hepatitis C virus infection. lncRNA#32 bound to activating transcription factor 2 (ATF2) and regulated ISG expression. Our results reveal a role for lncRNA#32 in host antiviral responses., Competing Interests: The authors declare no conflict of interest.

Development of spider mite management technology other than chemical control is desired because of the serious development of acaricide resistance worldwide. Recent studies have evidenced the lethal effects of ultraviolet-B (UVB) radiation on spider mites. To develop the technology on how to use UVB irradiation for spider mite control, we tested whether UVB lamp-light reflection sheet (LRS) combinations suppressed the population size of Tetranychus urticae Koch on strawberry in a greenhouse from December to May (2012-2013, 2013-2014) in Japan. We designed four combinations of UVB lamps and LRSs: 1) neither UVB lamps nor LRSs (UV-LRS-); 2) a UVB lamp without an LRS (UV+LRS-; 2012-2013 only); 3) a UVB lamp and a mulch-type LRS (UV+LRSm); and 4) a UVB lamp and a wing-type LRS (UV+LRSw). The number of adult females peaked at 438.0 and 222.0 per plant in UV-LRS- of 2012-2013 and 2013-2014, respectively, and peaked at 191.6 females in UV+LRS- of 2012-2013. In contrast, the peak abundance was 20.9-98.0 females in UV+LRSm, and fewer than 15 females were noted in UV+LRSw over either experimental period. UVB irradiance on lower leaf surfaces was higher in UV+LRSm and UV+LRSw than UV-LRS- and UV+LRS-, and the mite densities were significantly correlated with UVB irradiance on lower leaf surfaces. Consequently, we conclude that the combinations of UVB lamp-LRS have an excellent capacity to control T. urticae on greenhouse strawberry, and that the LRS was an essential component in this technological approach., (© The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Non-alcoholic fatty liver disease (NAFLD) is a common cause of chronic non-viral liver disease. Activation of macrophages and hepatic stellate cells is a critical step that promotes liver fibrosis. We aimed to explore the feasibility of interleukin-34 (IL-34), a key regulator of macrophages, as a fibrosis marker in patients with NAFLD. We enrolled 197 liver biopsy-proven NAFLD patients. We evaluated the serum levels of IL-34, macrophage-colony stimulating factor (M-CSF), soluble CD163 (sCD163), 40 cytokines/chemokines, hyaluronic acid, type IV collagen 7s, and clinically-approved fibrosis scores. IL-34 increased with the progression of fibrosis and was an independent marker for liver fibrosis. Immunostaining experiments, using resected liver specimens from NAFLD patients, revealed that IL-34 was mainly expressed on liver fibroblasts. IL-34 based fibrosis score (0.0387*IL-34 (pg/ml) + 0.3623*type IV collagen 7s (ng/ml) + 0.0184*age (year)-1.1850) was a practical predictive model of liver fibrosis. Using receiver-operating characteristic analyses, the area under the curve, sensitivity, and specificity of IL-34 based fibrosis score were superior or comparable to the other fibrosis biomarkers and scores. In conclusion, the IL-34 based fibrosis score, including serum IL-34, type IV collagen 7s and age, is a feasible diagnostic marker of liver fibrosis in NAFLD patients.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) are among the most endemic pathogens worldwide, with more than 500 million people globally currently infected with these viruses. These pathogens can cause acute and chronic hepatitis that progress to liver cirrhosis or hepatocellular carcinoma. Both viruses utilize multifaceted strategies to evade the host surveillance system and fall below the immunological radar. HBV has developed specific strategies to evade recognition by the innate immune system and is acknowledged to be a stealth virus. However, extensive research has revealed that HBV is recognized by dendritic cells (DCs) and natural killer (NK) cells. Indoleamine-2, 3-dioxygenase is an enforcer of sequential immune reactions in acute hepatitis B, and this molecule has been shown to be induced by the interaction of HBV-infected hepatocytes, DCs, and NK cells. The interleukin-28B genotype has been reported to influence HCV eradication either therapeutically or spontaneously, but the biological function of its gene product, a type-III interferon (IFN-λ3), remains to be elucidated. Human BDCA3(+)DCs have also been shown to be a potent producer of IFN-λ3 in HCV infection, suggesting the possibility that BDCA3(+)DCs could play a key role in developing therapeutic HCV vaccine. Here we review the current state of research on immune responses against HBV and HCV infection, with a specific focus on innate immunity. A comprehensive study based on clinical samples is urgently needed to improve our understanding of the immune mechanisms associated with viral control and thus to develop novel immune modulatory therapies to cure chronic HBV and HCV infection.

Background: Appropriate utilization of different diagnostic modalities is essential for the accurate liver stiffness measurements (LSM) in patients with chronic liver diseases. The aim of this study was to evaluate the efficacy of Virtual Touch Quantification (VTQ) and the FibroScan M and XL probes in term of accurate LSM and to identify factors associated with inadequate measurements in obese and non-obese Japanese patients., Methods: A total of 664 consecutive patients with chronic liver disease were prospectively enrolled. LSM were evaluated concurrently with VTQ and the FibroScan M and XL probes. LSM quality was categorized as inadequate (success rate <60% and/or interquartile range/median value of ≥30%) or adequate., Results: No significant differences in the rate of inadequate LSM were observed among the three diagnostic modalities. In multivariate analysis, skin capsule distance (SCD) was strongly associated with inadequate rates obtained with VTQ and the M probe [odds ratio (OR) 1.28, P < 0.0001 and OR 1.20, P < 0.0001, respectively]. Inadequate LSM rates with both VTQ and the M probe increased with longer SCD, with a significant difference between subgroups at an SCD of ≥22.5 mm (VTQ 54.0%; M probe 51.1%; XL probe 25.2%; P < 0.0001). The rates of inadequate LSM rates with VTQ were significantly lower than those with the XL probe at an SCD of <17.5 mm. A total of 15 liver biopsy specimens obtained from nonalcoholic fatty liver disease patients confirmed the diagnostic accuracy and high applicability of the XL probe., Conclusions: Long SCD reduced the diagnostic performance of the FibroScan® M probe and VTQ. LSM modalities should be selected according to SCD.

Associations of variants located in the HLA class II region with chronic hepatitis B (CHB) infection have been identified in Asian populations. Here, HLA imputation method was applied to determine HLA alleles using genome-wide SNP typing data of 1,975 Japanese individuals (1,033 HBV patients and 942 healthy controls). Together with data of an additional 1,481 Japanese healthy controls, association tests of six HLA loci including HLA-A, C, B, DRB1, DQB1, and DPB1, were performed. Although the strongest association was detected at a SNP located in the HLA-DP locus in a SNP-based GWAS using data from the 1,975 Japanese individuals, HLA genotyping-based analysis identified DQB1*06:01 as having the strongest association, showing a greater association with CHB susceptibility (OR = 1.76, P = 6.57 × 10(-18)) than any one of five HLA-DPB1 alleles that were previously reported as CHB susceptibility alleles. Moreover, HLA haplotype analysis showed that, among the five previously reported HLA-DPB1 susceptibility and protective alleles, the association of two DPB1 alleles (DPB1*09:01, and *04:01) had come from linkage disequilibrium with HLA-DR-DQ haplotypes, DRB1*15:02-DQB1*06:01 and DRB1*13:02-DQB1*06:04, respectively. The present study showed an example that SNP-based GWAS does not necessarily detect the primary susceptibility locus in the HLA region.