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Background: The branched-chain amino acids (BCAAs) to tyrosine (Tyr) ratio (BTR) test is used to evaluate the progression of chronic liver disease (CLD). However, the differences across sex, age, body mass index (BMI) and etiologies are still unclear. Methods: We retrospectively reviewed data from 2,529 CLD cases with free amino acids (FAAs) in peripheral blood from four hospitals and 16,421 general adults with FAAs data from a biobank database. In total, 1,326 patients with CLD (covering seven etiologies) and 8,086 healthy controls (HCs) were analyzed after exclusion criteria. We investigated the change of BTR in HCs by sex, age and BMI and then compared these to patients divided by modified ALBI (mALBI) grade after propensity score matching. Results: BTR is significantly higher in males than females regardless of age or BMI and decreases with aging in HCs. In 20 types of FAAs, 7 FAAs including BCAAs were significantly decreased, and 11 FAAs including Tyr were significantly increased by mALBI grade in total CLD. The decreasing timings of BTR were at mALBI grade 2b in all CLD etiologies compared to HCs, however in chronic hepatitis C (CHC), chronic hepatitis B (CHB) and alcoholic liver disease (ALD), BTR started to decrease at 2a. There was a positive correlation between BCAAs and albumin among parameters in BTR and mALBI. The correlation coefficients in PBC, ALD and MASLD were higher than those of other etiologies. Conclusions: BTR varies by sex and age even among healthy adults, and decreasing process and timing of BTR during disease progression is different among CLD etiologies. [ABSTRACT FROM AUTHOR]

Aim: Reports of patients with hepatitis B have highlighted associations between polymorphisms in the human leukocyte antigen (HLA)‐DPB1, CXCL13, and CXCR5 genes and disease pathology. Owing to its potential to contribute to the development of new diagnostic and therapeutic methods, we aimed to establish a reliable host genome analysis technique that can be used in countries with inadequate infrastructure. Method: We compared multiple commercially available kits for dried blood spot (DBS)‐based sample collection to develop a basic DBS‐based host genome analysis technique. We then collected blood samples from Cambodian patients with hepatitis B and performed single‐nucleotide polymorphism genotyping and HLA allele typing by the DBS system. Result: We were able to perform single‐nucleotide polymorphism genotyping and HLA allele typing with host DNA samples obtained using a combination of a HemaSpot™ filter paper‐based device and a SMITEST® EX‐R&D DNA extraction kit. The accuracy of genotyping using samples obtained by this method was not inferior to one using samples obtained by venipuncture. In the Cambodian population, significant associations of HLA‐DPB1*04:01 with protection against chronic hepatitis B virus (HBV) infection, and HLA‐DPB1*05:01 and HLA‐DPB1*13:01 with susceptibility to chronic HBV infection were identified. Conclusion: Based on the DBS system, we clarified the associations of HLA‐DPB1 alleles with chronic HBV infection in the Cambodian population for the first time. Because the DBS is a low‐cost, durable, transportable, and easy‐to‐handle modality, genetic analysis based on the DBS system is a feasible strategy for obtaining a deeper understanding of HBV epidemiology, especially in middle‐ or low‐income countries. [ABSTRACT FROM AUTHOR]

Background: The antidiabetic drugs sodium glucose cotransporter 2 inhibitors (SGLT2is) and thiazolidinediones have beneficial effects on the liver dysfunction of patients with nonalcoholic fatty liver disease and type 2 diabetes mellitus (T2DM). We aimed to determine the efficacy of these drugs for the treatment of liver disease in patients with metabolic dysfunction‐associated fatty liver disease (MAFLD) and T2DM. Methods: We undertook a retrospective study of 568 patients with MAFLD and T2DM. Of these, 210 were treating their T2DM with SGLT2is (n = 95), 86 with pioglitazone (PIO), and 29 with both. The primary outcome was the change in Fibrosis‐4 (FIB‐4) index between baseline and 96 weeks. Results: At 96 weeks, the mean FIB‐4 index had significantly decreased (from 1.79 ± 1.10–1.56 ± 0.75) in the SGLT2i group, but not in the PIO group. The aspartate aminotransferase to platelet ratio index, serum aspartate and alanine aminotransferase (ALT), hemoglobin A1c, and fasting blood sugar significantly decreased in both groups (ALT: SGLT2i group, −17 ± 3 IU/L; PIO group, −14 ± 3 IU/L). The bodyweight of the SGLT2i group decreased, but that of the PIO group increased (−3.2 kg and +1.7 kg, respectively). When the participants were allocated to two groups according to their baseline ALT (>30 IU/L), FIB‐4 index significantly decreased in both groups. In patients taking pioglitazone, the addition of SGLT2i improved liver enzymes but not FIB‐4 index for 96 weeks. Conclusions: Treatment with SGLT2i causes a larger improvement in FIB‐4 index than PIO in patients with MAFLD over 96 weeks. [ABSTRACT FROM AUTHOR]

Background: Combination therapy with anti-programmed death-ligand 1 and anti-vascular endothelial growth factor (VEGF) antibodies has become the standard treatment for un-resectable hepatocellular carcinoma (uHCC). We aimed to identify predictive circulating biomarkers for the outcome/response of the combination therapy in uHCC patients. Methods: This prospective multicenter study enrolled 70 patients with uHCC who received atezolizumab and bevacizumab (Atez/Bev). We evaluated 47 circulating proteins in sera before and after 1 and 6 weeks of Atez/Bev therapy by multiplex bead-based immunoassay and ELISA. As controls, we analyzed the sera from 62 uHCC patients before treatment of lenvatinib (LEN) and healthy volunteers (HVs). Results: The disease control rate was 77.1%. Median progression-free survival (PFS) was 5.7 months (95% confidence interval [CI] = 3.8–9.5). The pretreatment levels of osteopontin (OPN), angiopoietin-2, VEGF, S100–calcium-binding protein A8/S100–calcium-binding protein A9, soluble programmed cell death-1, soluble CD163, and 14 cytokines/chemokines were higher in patients with uHCC than in HVs. Among these, pretreatment OPN levels were higher in PD group than in non-PD group for Atez/Bev. The PD rate was higher in high OPN group than in low OPN group. Multivariate analysis identified high pretreatment OPN and high α‐fetoprotein levels as independent predictors of PD. In the sub-analysis of Child–Pugh class A patients, PFS was also shorter in the high OPN group than in the low OPN group. Pretreatment OPN level was not associated with treatment response for LEN. Conclusion: High serum OPN levels were associated with poor response to Atez/Bev in patients with uHCC. [ABSTRACT FROM AUTHOR]

Backgrounds and Aims: Toll‐like receptor (TLR) agonists have been developed as adjuvants to efficiently induce antiviral immune responses. Specificity and potency of these compounds are essential requirements for clinical trial applications. In patients with hepatitis B virus (HBV) infections, sustained loss of hepatitis B surface antigen (HBsAg) is a therapeutic goal, which may be achievable by the sequential activation of follicular helper T cells (Tfh) and antibody‐secreting B cells. We aimed to elucidate whether novel TLR7 agonist, GS‐986, could activate immune responses involved in HBV elimination. Methods: To clarify the impact of GS‐986 on pDCs, we quantified the expression levels of surface markers and evaluated for Tfh induction in a culture model consisting of human pDCs with allogeneic naïve CD4+ T cells. In addition, we examined whether GS‐986 could enhance HBs antibody production capacity using PBMC from CHB patients. Results: pDCs from CHB patients had lower OX40L expression and as well as impaired capacity for Tfh induction compared with those from healthy donors. However, GS‐986–stimulated pDCs from CHB patients expressed OX40L and produced IL‐6 and IL‐12, resulting in the induction of IL‐21–producing Tfh cells (CXCR5+PD‐1+CD4+) from naïve CD4+ T cells. The Tfh‐inducing capacity of GS‐986 was reduced in the presence of an anti‐OX40L blocking antibody. Furthermore, GS‐986 promoted HBsAg‐specific antibody production in PBMCs from CHB patients. Conclusions: GS‐986 is an adjuvant that stimulates pDCs to induce Tfh differentiation and antigen‐specific B‐cell production. This immune profile may be beneficial for therapeutic application as an immune modulator in CHB patients. [ABSTRACT FROM AUTHOR]

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

Epithelial plasticity, or epithelial‐to‐mesenchymal transition (EMT), is a well‐recognized form of cellular plasticity, which endows tumor cells with invasive properties and alters their sensitivity to various agents, thus representing a major challenge to cancer therapy. It is increasingly accepted that carcinoma cells exist along a continuum of hybrid epithelial–mesenchymal (E‐M) states and that cells exhibiting such partial EMT (P‐EMT) states have greater metastatic competence than those characterized by either extreme (E or M). We described recently a P‐EMT program operating in vivo by which carcinoma cells lose their epithelial state through post‐translational programs. Here, we investigate the underlying mechanisms and report that prolonged calcium signaling induces a P‐EMT characterized by the internalization of membrane‐associated E‐cadherin (ECAD) and other epithelial proteins as well as an increase in cellular migration and invasion. Signaling through Gαq‐associated G‐protein‐coupled receptors (GPCRs) recapitulates these effects, which operate through the downstream activation of calmodulin‐Camk2b signaling. These results implicate calcium signaling as a trigger for the acquisition of hybrid/partial epithelial–mesenchymal states in carcinoma cells. Synopsis: Cancer cells exist along a continuum of hybrid or partial EMT states. This is underpinned by a calcium‐dependent molecular mechanism in various carcinomas, resulting in the internalization of epithelial proteins. Calcium signaling induces a partial EMT (P‐EMT) phenotype characterized by the internalization of membranous E‐cadherin.Calcium induced P‐EMT tumor cells exhibit increased migration and invasion.P‐EMT is activated by Gαq associated GPCRs and downstream activation of calmodulin‐Camk2b signaling. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 cm2/m2, and the median body mass index was 23.4 kg/m2. Eighty‐two (35.0%) patients had sarcopenia (L3 skeletal muscle mass index: men <42, women <38 cm2/m2). 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cell replacement therapy for diabetes mellitus requires cost-effective generation of high-quality, insulin-producing, pancreatic β cells from pluripotent stem cells. Development of this technique has been hampered by a lack of knowledge of the molecular mechanisms underlying β-cell differentiation. The present study identified reserpine and tetrabenazine (TBZ), both vesicular monoamine transporter 2 (VMAT2) inhibitors, as promoters of late-stage differentiation of Pdx1-positive pancreatic progenitor cells into Neurog3 (referred to henceforth as Ngn3)-positive endocrine precursors. VMAT2-controlled monoamines, such as dopamine, histamine and serotonin, negatively regulated β-cell differentiation. Reserpine or TBZ acted additively with dibutyryl adenosine 3',5'-cyclic AMP, a cell-permeable cAMP analog, to potentiate differentiation of embryonic stem (ES) cells into β cells that exhibited glucose-stimulated insulin secretion. When ES cell-derived β cells were transplanted into AKITA diabetic mice, the cells reversed hyperglycemia. Our protocol provides a basis for the understanding of β-cell differentiation and its application to a cost-effective production of functional β cells for cell therapy. [ABSTRACT FROM AUTHOR]

The endoderm-inducing effect of the mesoderm-derived supportive cell line M15 on embryonic stem (ES) cells is partly mediated through the extracellular matrix, of which laminin a5 is a crucial component. Mouse ES or induced pluripotent stem cells cultured on a synthesized basement membrane (sBM) substratum, using an HEK293 cell line (rLN10-293 cell) stably expressing laminin-511, could differentiate into definitive endoderm and subsequently into pancreatic lineages. In this study, we investigated the differentiation on sBM of mouse and human ES cells into hepatic lineages. The results indicated that the BM components played an important role in supporting the regional-specific differentiation of ES cells into hepatic endoderm. We show here that knockdown of integrin b1 (Itgb1) in ES cells reduced their differentiation into hepatic lineages and that this is mediated through Akt signaling activation. Moreover, under optimal conditions, human ES cells differentiated to express mature hepatocyte markers and secreted high levels of albumin. This novel procedure for inducing hepatic differentiation will be useful for elucidating the molecular mechanisms controlling lineage-specific fates during gut regionalization. It could also represent an attractive approach to providing a surrogate cell source, not only for regenerative medicine, but also for pharmaceutical and toxicologic studies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]