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Background & Aims: Targeting exhausted immune systems would be a promising therapeutic strategy to achieve a functional cure for HBV infection in patients with chronic hepatitis B (CHB). However, animal models recapitulating the immunokinetics of CHB are very limited. We aimed to develop an immunocompetent mouse model of CHB for intrahepatic immune profiling. Methods: CHB mice were created by intrahepatic delivery of the Sleeping Beauty transposon vector tandemly expressing the hepatitis B virus (HBV) genome and fumarylacetoacetate hydrolase (FAH) cDNA into C57BL/6J congenic FAH knockout mice via hydrodynamic tail vein injection. We profiled the viral and intrahepatic immune kinetics in CHB mice with or without treatment with recombinant IFNα or the hepatotropic Toll-like receptor 7 agonist SA-5 using single-cell RNA-seq. Results: CHB mice exhibited sustained HBV viremia and persistent hepatitis. They showed intrahepatic expansion of exhausted CD8+ T (Tex) cells, the frequency of which was positively associated with viral load. Recruited macrophages increased in number but impaired inflammatory responses in the liver. The cytotoxicity of mature natural killer (NK) cells also increased in CHB mice. IFNα and SA-5 treatment both resulted in viral suppression with mild hepatic flares in CHB mice. Although both treatments activated NK cells, SA-5 had the capacity to revitalize the impaired function of Tex cells and liver-recruited macrophages. Conclusion: Our novel CHB mouse model recapitulated the intrahepatic exhausted antiviral immunity in patients with CHB, which might be able to be reinvigorated by a hepatotropic TLR7 agonist.

Abstract The number of cancer cases diagnosed during the coronavirus disease 2019 (COVID-19) pandemic has decreased. This study investigated the impact of the pandemic on the clinical practice of hepatocellular carcinoma (HCC) using a novel nationwide REgistry for Advanced Liver diseases (REAL) in Japan. We retrieved data of patients initially diagnosed with HCC between January 2018 and December 2021. We adopted tumor size as the primary outcome measure and compared it between the pre-COVID-19 (2018 and 2019) and COVID-19 eras (2020 and 2021). We analyzed 13,777 patients initially diagnosed with HCC (8074 in the pre-COVID-19 era and 5703 in the COVID-19 era). The size of the maximal intrahepatic tumor did not change between the two periods (mean [SD] = 4.3 [3.6] cm and 4.4 [3.6] cm), whereas the proportion of patients with a single tumor increased slightly from 72.0 to 74.3%. HCC was diagnosed at a similar Barcelona Clinic Liver Cancer stage. However, the proportion of patients treated with systemic therapy has increased from 5.4 to 8.9%. The proportion of patients with a non-viral etiology significantly increased from 55.3 to 60.4%. Although the tumor size was significantly different among the etiologies, the subgroup analysis showed that the tumor size did not change after stratification by etiology. In conclusion, the characteristics of initially diagnosed HCC remained unchanged during the COVID-19 pandemic in Japan, regardless of differences in etiology. A robust surveillance system should be established particularly for non-B, non-C etiology to detect HCC in earlier stages.

Abstract Background and Aim Magnetic resonance elastography (MRE) is used for the evaluation of liver fibrosis; however, it remains unclear whether MRE‐based liver stiffness is associated with hepatocellular carcinoma (HCC) development, particularly in patients with chronic hepatitis B. Methods A total of 504 patients with chronic hepatitis B receiving MRE were enrolled. The primary endpoint was the association between MRE‐based liver stiffness and HCC. Results In a cross‐sectional analysis at the time of MRE measurement, the median (interquartile range) liver stiffness values in patients with presence or history of HCC and those without HCC were 3.68 (2.89–4.96) and 2.60 (2.22–3.45) kPa, respectively, and liver stiffness was significantly higher in patients with presence or history of HCC than in those without HCC (P

IntroductionNatural killer (NK) cells play a pivotal role in immune surveillance in the liver. We aimed to identify potential targets for NK cell-mediated immune intervention by revealing the functional molecules on NK cells in HCC patients.MethodsTo evaluate the impact of aging on NK cell phenotypes, we examined NK cells from healthy volunteers (HVs) of various ages. Because ILT2 expression on CD56dim NK cells increased with increasing age, we enrolled age-matched HCC patients and HVs. We determined the NK cell phenotypes in blood mononuclear cells (PBMCs) and intrahepatic lymphocytes (IHLs) from cancerous and non-cancerous tissues. We evaluated cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) of NK cells in vitro.ResultsILT2-positive CD56dim NK cells in PBMCs were increased in HCC patients compared with HVs. In HCC patients, ILT2-positive CD56dim NK cells were increased in cancerous IHLs compared with non-cancerous IHLs and PBMCs. We examined the impact of macrophage migration inhibitory factor (MIF) on ILT2 expression in co-cultures of HCC cells and NK cells. The enhanced expression of ILT2 on CD56dim NK cells from HCC patients was inhibited by masking antibodies against MIF and CXCR4. ILT2-positive CD56dim NK cells exhibited lower capacities for cytotoxicity and ADCC than ILT2-negative cells, which were partially restored by ILT2 blockade.ConclusionsIn HCC patients, ILT2 is a signature molecule for cancerous CD56dim NK cells with impaired cytolytic capacity. The MIF-CXCR4 interaction is associated with ILT2 induction on CD56dim NK cells and ILT2 serves as a target for functional NK cell restoration.

AimMetabolic dysfunction-associated steatohepatitis (MASH) is one of the most prevalent liver diseases and is characterized by steatosis and the accumulation of bioactive lipids. This study aims to understand the specific lipid species responsible for the progression of liver fibrosis in MASH.MethodsChanges in bioactive lipid levels were examined in the livers of MASH mice fed a choline-deficient diet (CDD). Additionally, sphingosine kinase (SphK)1 mRNA, which generates sphingosine 1 phosphate (S1P), was examined in the livers of patients with MASH.ResultsCDD induced MASH and liver fibrosis were accompanied by elevated levels of S1P and increased expression of SphK1 in capillarized liver sinusoidal endothelial cells (LSECs) in mice. SphK1 mRNA also increased in the livers of patients with MASH. Treatment of primary cultured mouse hepatic stellate cells (HSCs) with S1P stimulated their activation, which was mitigated by the S1P receptor (S1PR)2 inhibitor, JTE013. The inhibition of S1PR2 or its knockout in mice suppressed liver fibrosis without reducing steatosis or hepatocellular damage.ConclusionS1P level is increased in MASH livers and contributes to liver fibrosis via S1PR2.

The Japanese archipelago is a terminal location for human migration, and the contemporary Japanese people represent a unique population whose genomic diversity has been shaped by multiple migrations from Eurasia. We analyzed the genomic characteristics that define the genetic makeup of the modern Japanese population from a population genetics perspective from the genomic data of 9,287 samples obtained by high-coverage whole-genome sequencing (WGS) by the National Center Biobank Network. The dataset comprised populations from the Ryukyu Islands and other parts of the Japanese archipelago (Hondo). The Hondo population underwent two episodes of population decline during the Jomon period, corresponding to the Late Neolithic, and the Edo period, corresponding to the Early Modern era, while the Ryukyu population experienced a population decline during the shell midden period of the Late Neolithic in this region. Haplotype analysis suggested increased allele frequencies for genes related to alcohol and fatty acid metabolism, which were reported as loci that had experienced positive natural selection. Two genes related to alcohol metabolism were found to be 12,500 years out of phase with the time when they began to increase in the allele frequency; this finding indicates that the genomic diversity of Japanese people has been shaped by events closely related to agriculture and food production.

Abstract We developed a low‐intensity 10‐min resistance exercise program for nonalcoholic fatty liver disease (NAFLD). We report a case of NAFLD with elevated hepatic fibrosis indices, which were improved by a 60‐week daily exercise program. A 71‐year‐old female patient with NAFLD whose hepatic fibrosis stage corresponded to F2 was referred to our hospital. She performed the exercise once a day with no changes in other lifestyle habits and medications. The homeostasis model assessment‐insulin resistance value and NAFLD‐liver fat score, the Hepamet fibrosis score, and the enhanced liver fibrosis score decreased. The FIB‐4 index and serum levels of Mac‐2 binding protein glycosylation isomer decreased to the reference values. We investigated the changes in chemokines/cytokines. The serum granulocyte‐colony stimulating factor level was increased, and serum interferon‐gamma‐induced protein‐10 and platelet‐derived growth factor‐BB levels were decreased. Our program may be beneficial for improving hepatic fibrosis in patients with NAFLD.

Once-weekly semaglutide is a widely used glucagon-like peptide-1 receptor agonist (GLP-1RA) used for the treatment of type 2 diabetes (T2D). In clinical trials, semaglutide improved glycemic control and obesity, and reduced major cardiovascular events. However, the reports are limited on its real-world efficacy relating to various metabolic factors such as dyslipidemia or metabolic dysfunction-associated steatotic liver disease (MASLD) in Asian patients with T2D. In our retrospective longitudinal study, we selected patients with T2D who were given once-weekly semaglutide and compared metabolic parameters before and after the start of semaglutide. Seventy-five patients were eligible. HbA1c decreased significantly, by 0.7–0.9%, and body weight by 1.4–1.7 kg during the semaglutide treatment. Non-HDL cholesterol decreased significantly at 3, 6 and 12 months after the initiation of semaglutide; LDL cholesterol decreased at 3 and 6 months; and HDL cholesterol increased at 12 months. The effects on body weight, HbA1c and lipid profile were pronounced in patients who were given semaglutide as a first GLP-1RA (GLP-1R naïve), whereas improvements in HbA1c were also observed in patients who were given semaglutide after being switched from other GLP-1RAs. During a 12-month semaglutide treatment, the hepatic steatosis index (HSI) tended to decrease. Moreover, a significant decrease in the AST-to-platelet ratio index (APRI) was observed in GLP-1RA naïve patients. Our real-world study confirmed the beneficial effects of once-weekly semaglutide, namely, improved body weight, glycemic control and atherogenic lipid profile. The beneficial effects on MASLD were also suggested.

Despite treatment, hepatitis B surface antigen (HBsAg) persists in patients with chronic hepatitis B (CHB), suggesting the likely presence of the virus in the body. CD8+ T cell responses are essential for managing viral replication, but their effect on HBsAg levels remains unclear. We studied the traits of activated CD8+ T cells and HBV-specific CD8+ T cells in the blood of CHB patients undergoing nucleos(t)ide analog (NUC) therapy. For the transcriptome profiling of activated CD8+ T cells in peripheral blood mononuclear cells (PBMCs), CD69+ CD8+ T cells were sorted from six donors, and single-cell RNA sequencing (scRNA-seq) analysis was performed. To detect HBV-specific CD8+ T cells, we stimulated PBMCs from 26 donors with overlapping peptides covering the HBs, HBcore, and HBpol regions of genotype A/B/C viruses, cultured for 10 days, and analyzed via multicolor flow cytometry. scRNA-seq data revealed that CD8+ T cell clusters harboring the transcripts involved in the cytolytic functions were frequently observed in donors with high HBsAg levels. Polyfunctional analysis of HBV-specific CD8+ T cells utilized by IFN-γ/TNFα/CD107A/CD137 revealed that HBcore-specific cells exhibited greater polyfunctionality, suggesting that the quality of HBV-specific CD8+ T cells varies among antigens. Moreover, a subset of HBcore-specific CD8+ T cells with lower cytolytic potential was inversely correlated with HBsAg level. Our results revealed a stimulant-dependent qualitative difference in HBV-specific CD8+ T cells in patients with CHB undergoing NUC therapy. Hence, the induction of HBcore-specific CD8+ T cells with lower cytolytic potential could be a new target for reducing HBsAg levels.

Abstract Chronic cholestatic liver diseases are characterized by injury of the bile ducts and hepatocytes caused by accumulated bile acids (BAs) and inflammation. Wnt/β‐catenin signaling is implicated in organ fibrosis; however, its role in cholestatic liver fibrosis remains unclear. Therefore, we explored the effect of a selective cAMP response element‐binding protein‐binding protein (CBP)/β‐catenin inhibitor, PRI‐724, on murine cholestatic liver fibrosis. PRI‐724 suppressed liver fibrosis induced by multidrug resistance protein 2 knockout (KO), bile duct ligation, or a 3.5‐diethoxycarbonyl‐1.4‐dihydrocollidine (DDC) diet; it also suppressed BA synthesis and macrophage infiltration. The expression of early growth response‐1 (Egr‐1), which plays a key role in BA synthesis, was increased in the hepatocytes of patients with cholestatic liver disease. PRI‐724 inhibited Egr‐1 expression induced by cholestasis, and adenoviral shEgr‐1‐mediated Egr‐1 knockdown suppressed BA synthesis and fibrosis in DDC diet–fed mice, suggesting that PRI‐724 exerts its effects, at least in part, by suppressing Egr‐1 expression in hepatocytes. Hepatocyte‐specific CBP KO in mice suppressed BA synthesis, liver injury, and fibrosis, whereas hepatocyte‐specific KO of P300, a CBP homolog, exacerbated DDC‐induced fibrosis. Intrahepatic Egr‐1 expression was also decreased in hepatocyte‐specific CBP‐KO mice and increased in P300‐KO mice, indicating that Egr‐1 is located downstream of CBP/β‐catenin signaling. Conclusion: PRI‐724 inhibits cholestatic liver injury and fibrosis by inhibiting BA synthesis in hepatocytes. These results highlight the therapeutic effect of CBP/β‐catenin inhibition in cholestatic liver diseases.

IntroductionWe examined the neutralizing antibody production efficiency of the second and third severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine doses (2nd- and 3rd-dose) and neutralizing activity on mutant strains, including, the Ancestral, Beta and Omicron strains using green fluorescent protein-carrying recombinant SARS-CoV-2, in living-donor liver transplantation (LDLT) recipients.MethodsThe patients who were administered vaccines other than Pfizer- BioNTechBNT162b2 and who had coronavirus disease 2019 in this study period were excluded. We enrolled 154 LDLT recipients and 50 healthy controls.ResultThe median time were 21 days (between 1st and 2nd vaccination) and 244 days (between 2nd and 3rd vaccination). The median neutralizing antibody titer after 2nd-dose was lower in LDLT recipients than in controls (0.46 vs 1.00, P

ABSTRACT This study aimed to calibrate hepatitis E virus (HEV) serological assays. We optimized the previously developed in-house HEV antibody enzyme-linked immunosorbent assay (ELISA) by setting the cutoff with an in-house serological performance panel consisting of broad HEV antibody titers and subtracting nonspecific background values for anti-HEV IgM, IgA, and IgG. We also compared the assay’s performance with that of commercial serological assay kits (four kits for IgM, one for IgA, and two for IgG). Although all serological assays readily detected HEV antibodies at high titers in the symptomatic hepatitis E population, considerable variations between assays were observed in the asymptomatic population. The in-house ELISA showed a higher sensitivity for HEV IgM, IgA, and IgG than the commercial kits and detected the seroconversion of HEV IgM and IgG earlier when testing a commercially available HEV seroconversion panel. The low sensitivity of the commercial kits was due to the high setting of the original cutoff, which was demonstrated by receiver operating characteristic analysis. However, the corrected cutoff value reduced assay specificity. Background subtraction is essential to achieve high specificity because the in-house ELISA without background subtraction reduced its specificity. These results indicate that asymptomatic specimens and background subtraction contribute to the optimization of HEV serological assays. IMPORTANCE Accurate diagnosis of hepatitis E virus (HEV) infection is essential for public health surveillance and for preventing HEV-contaminated blood transfusion. Anti-HEV IgM or IgA is used as a reliable marker of recent HEV infection. However, considerable variability in the sensitivity and specificity of HEV antibody detection is observed among several commercially available assay kits. In addition, none of the HEV antibody detection methods have been approved by the U.S. Food and Drug Administration (FDA). Here, we show that the in-house enzyme-linked immunosorbent assay (ELISA) could detect HEV IgM and IgA more sensitively than commercial kits in the asymptomatic population. We also suggest that the assay performance of commercial kits might be improved by optimizing the cutoff and reducing nonspecific background noise. A sensitive serological (IgM or IgA) assay in addition to HEV RNA testing will contribute to accurate diagnosis of acute HEV infection because HEV RNA-positive duration is relatively short.

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.

Non-alcoholic fatty liver disease/steatohepatitis (NAFLD/NASH) are becoming major liver diseases worldwide. Liver fibrosis and cirrhosis are among the most significant risk factors of hepatocellular carcinoma (HCC) and associated with the long-term prognosis of NAFLD patients. To stratify the risk of HCC in NAFLD patients clinically, the discovery of non-invasive fibrosis markers is needed urgently. Liver macrophages play critical roles in the regulation of inflammation and fibrosis by interacting with hepatic stellate cells (HSCs) and other immune cells. Thus, it is rational to explore feasible biomarkers for liver fibrosis by focusing on macrophage-related factors. We examined serum factors comprehensively in multiple cohorts of NAFLD/NASH patients to determine whether they were correlated with the biopsy-proven fibrosis stage. We found that the serum levels of interleukin (IL)-34, YKL-40 and soluble Siglec-7 (sSiglec7) were closely associated with liver fibrosis and served as diagnostic biomarkers in patients with NAFLD/NASH. In the NAFLD liver, IL-34 was produced by activated fibroblasts, and YKL-40 and sSiglec-7 were secreted from macrophages. The sensitivity and specificity of these markers to detect advanced liver fibrosis varied, supporting the notion that the combination of these markers with other modalities is an option for clinical application.

Summary: Background: We conducted an exploratory study to assess the safety tolerability, and anti-fibrotic effects of PRI-724, a CBP/β-catenin inhibitor, in patients with hepatitis C virus (HCV)- and hepatitis B virus (HBV)-induced cirrhosis. Methods: This multicentre, open-label, non-randomised, non-placebo-controlled phase 1/2a trial was conducted at three hospitals in Japan. Between July 27, 2018, and July 13, 2021, we enrolled patients with HCV- and HBV-induced cirrhosis classified as Child–Pugh (CP) class A or B. In phase 1, 15 patients received intravenous infusions of PRI-724 at escalating doses of 140, 280, and 380 mg/m2/4 h twice weekly for 12 weeks. In phase 2a, 12 patients received the recommended PRI-724 dose. The primary endpoints of phases 1 and 2a were the frequency and severity of adverse events and efficacy in treating cirrhosis based on liver biopsy. This study was registered at ClinicalTrials.gov (no. NCT 03620474). Findings: Three patients from phase 1 who received the recommended PRI-724 dose were evaluated to obtain efficacy and safety data in phase 2a. Serious adverse events occurred in three patients, one of which was possibly related to PRI-724. The most common adverse events were diarrhoea and nausea. PRI-724 did not decrease hepatic fibrosis with any statistical significance, either by ordinal scoring or measurement of collagen proportionate area at 12 weeks; however, we observed statistically significant improvements in liver stiffness, Model for End-stage Liver Disease score, and serum albumin level. Interpretation: Intravenous administration of 280 mg/m2/4 h PRI-724 over 12 weeks was preliminarily assessed to be well tolerated; however, further evaluation of anti-fibrotic effects in patients with cirrhosis is warranted. Funding: AMED, Ohara Pharmaceutical

Abstract 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 (CCl4)-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 CCl4-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 CCl4-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.

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.

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 CD56dim NK cell populations compared with HVs, and the CD56dim 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+CD56dim NK cells were reduced in NAFLD patients. Notably, Siglec-7 levels on CD56dim 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+CD56dim 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.

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

Abstract 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.

Hepatitis A virus (HAV) causes transient acute infection, and little is known of viral shedding via the duodenum and into the intestinal environment, including the gut microbiome, from the period of infection until after the recovery of symptoms. Therefore, in this study, we aimed to comprehensively observe the amount of virus excreted into the intestinal tract, the changes in the intestinal microbiome, and the level of inflammation during the healing process. We used blood and stool specimens from patients with human immunodeficiency virus who were infected with HAV during the HAV outbreak in Japan in 2018. Moreover, we observed changes in fecal HAV RNA and quantified the plasma cytokine level and gut microbiome by 16S rRNA analysis from clinical onset to at least 6 months after healing. HAV was detected from clinical onset up to a period of more than 150 days. Immediately after infection, many pro-inflammatory cytokines were elicited, and some cytokines showed different behaviors. The intestinal microbiome changed significantly after infection (dysbiosis), and the dysbiosis continued for a long time after healing. These observations suggest that the immunocompromised state is associated with prolonged viral shedding into the intestinal tract and delayed recovery of the intestinal environment.

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 CD56dim and CD56bright 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+CD56dim NK cells that expressed Siglec-7, NKp46, and NKp30 were reduced, while CD49a+CD56dim NK cells that expressed Siglec-10 were increased. CD160 and CD49a on CD56dim 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 CD56dim NK cells may be targets for immunotherapies of HCC patients.

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 160 mg/m2/day for six cycles of 1 week on and 1 week 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 160 mg/m2/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 10 mg/m2/day cohort, improved in three patients at 1, 1, and 2 points in the 40 mg/m2/day cohort, and improved in one patient by 3 points in the 160 mg/m2/day cohort. The histology activity index scores of the liver tissue improved in two patients and exacerbated in two patients in the 10 mg/m2/day cohort, and improved in one patient in the 40 mg/m2/day cohort. Interpretation: This study showed that administration of 10 or 40 mg/m2/day intravenous PRI-724 over 12 weeks was well-tolerated by patients with HCV cirrhosis; however, liver injury as a possible related serious adverse event was observed in the 160 mg/m2/day cohort. Funding Source: AMED.

Despite treatment, hepatitis B surface antigen (HBsAg) persists in patients with chronic hepatitis B (CHB), suggesting the likely presence of the virus in the body. CD8+ T cell responses are essential for managing viral replication, but their effect on HBsAg levels remains unclear. We studied the traits of activated CD8+ T cells and HBV-specific CD8+ T cells in the blood of CHB patients undergoing nucleos (t)ide analog (NUC) therapy. For the transcriptome profiling of activated CD8+ T cells in peripheral blood mononuclear cells (PBMCs), CD69+ CD8+ T cells were sorted from six donors, and single-cell RNA sequencing (scRNA-seq) analysis was performed. To detect HBV-specific CD8+ T cells, we stimulated PBMCs from 26 donors with overlapping peptides covering the HBs, HBcore, and HBpol regions of genotype A/B/C viruses, cultured for 10 days, and analyzed via multicolor flow cytometry. scRNA-seq data revealed that CD8+ T cell clusters harboring the transcripts involved in the cytolytic functions were frequently observed in donors with high HBsAg levels. Polyfunctional analysis of HBVspecific CD8+ T cells utilized by IFN-γ/TNFα/CD107A/CD137 revealed that HBcore-specific cells exhibited greater polyfunctionality, suggesting that the quality of HBV-specific CD8+ T cells varies among antigens. Moreover, a subset of HBcore-specific CD8+ T cells with lower cytolytic potential was inversely correlated with HBsAg level. Our results revealed a stimulant-dependent qualitative difference in HBV-specific CD8+ T cells in patients with CHB undergoing NUC therapy. Hence, the induction of HBcore-specific CD8+ T cells with lower cytolytic potential could be a new target for reducing HBsAg levels. [ABSTRACT FROM AUTHOR]

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.

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.