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With the increase in the number of patients with sustained virologic response (SVR) in whom hepatitis C virus (HCV) was eradicated by the anti-HCV therapy, there are now many individuals in whom serum HCV RNA is absent despite positive serum HCV antibodies. However, in general clinical practice, HCV infection remains usually screened by measurement of serum HCV antibodies and patients with SVR can be misunderstood regarding HCV infection status.In the multicenter study, we conducted interviews with administered questionnaires to SVR individuals who had regular hospital visits after SVR. The prevalence of experiencing an incorrect diagnosis of HCV infection after SVR was assessed. Individuals who experienced this misunderstanding were further asked where they experienced it and how it made them feel.In a survey of 2,246 SVR individuals, 197 individuals (8.8%) were misunderstood as having persistent HCV infection by medical doctors due to positive HCV antibody, despite the absence of HCV viremia. These misunderstandings occurred most prevalently at a private clinic (55.3%). More than half (53.3%) of these individuals felt anxious about their HCV infection with becoming unsure about their HCV eradication status.Misunderstanding HCV status is commonly occurred in SVR individuals. Specialists in hepatology and infectious diseases should broadly emphasize the fact that most patients with HCV antibodies are now HCV-free because of the use of anti-HCV therapy.

Protein kinase C delta (PKCδ) is a multifunctional PKC family member and has been implicated in many types of cancers, including liver cancer. Recently, we have reported that PKCδ is secreted from liver cancer cells, and involved in cell proliferation and tumor growth. However, it remains unclear whether the extracellular PKCδ directly regulates cell surface growth factor receptors. Here, we identify epidermal growth factor receptor (EGFR) as a novel interacting protein of the cell surface PKCδ in liver cancer cells. Imaging studies showed that secreted PKCδ interacted with EGFR-expressing cells in both autocrine and paracrine manners. Biochemical analysis revealed that PKCδ bound to the extracellular domain of EGFR. We further found that a part of the amino acid sequence on the C-terminal region of PKCδ was similar to the putative EGFR binding site of EGF. In this regard, the point mutant of PKCδ in the binding site lacked the ability to bind to the extracellular domain of EGFR. Upon an extracellular PKCδ-EGFR association, ERK1/2 activation, downstream of EGFR signaling, was apparently induced in liver cancer cells. This study indicates that extracellular PKCδ behaves as a growth factor and provides a molecular basis for extracellular PKCδ-targeting therapy for liver cancer.

Introduction Management of elderly patients with cancer has become a global issue. We investigated the safety and tolerability of lenvatinib in hepatocellular carcinoma (HCC) patients ≥80 years old. Methods We retrospectively evaluated 61 HCC patients and divided them into 2 groups: an elderly group (n = 13, ≥80 years old) and a younger group (n = 48, Results The discontinuation of lenvatinib due to AEs was more frequent in the elderly group (8/13, 61.5%) than in the younger group (10/48, 20.8%) (P = 0.0043). Fatigue and appetite loss accounted for half of the cases discontinued due to AEs in the elderly group. The elderly group had a significantly lower 8-week-delivered dose intensity/body surface area ratio (147.2) and 8-week-relative dose intensity (50.0%) than those in the younger group (267.4, 67%) (P = 0.003, 0.029). The objective response rate was significantly lower in the elderly group (15.4%) than in the younger group (61.5%) (P = 0.021). The PFS in the elderly group tended to be shorter than that in the younger group (P = 0.058, hazard ratio [HR] 1.98). The modified albumin-bilirubin (mALBI) grade (hepatic function) (HR, 2.60; P = 0.01) and objective response (HR, 0.41; P = 0.011) were independently associated with the PFS in the multivariate analysis. Conclusion The management of AEs is crucial for adherence and maintaining the dose intensity of lenvatinib in elderly HCC patients.

Background/Aim: We aimed to investigate the association between The Geriatric Nutritional Risk Index (GNRI) and the tolerability of lenvatinib in patients with hepatocellular carcinoma (HCC). Patients and Methods: We retrospectively evaluated 61 HCC patients treated with lenvatinib and compared those with low GNRI (≤98, n=26) to those with high GNRI (>98, n=35). Results: The discontinuation of lenvatinib due to adverse events was more frequent in the low GNRI group (46.2%) than in the high GNRI group (17.1%) (p=0.014). Multivariate analysis revealed that low GNRI (p=0.014), hypothyroidism (model 1 p=0.021, model 2 p=0.013), and advanced age (p=0.026) were independently associated with the discontinuation of lenvatinib. The progression-free survival in the low GNRI group was significantly shorter than that in the high GNRI group (p=0.047). Conclusion: The GNRI might be independently associated with the tolerability of lenvatinib in patients with HCC.

Supplementary Materials and Methods Supplementary References Figures S1 to S9 Table S1

Expression of human protein kinase C delta (PKCδ) protein has been linked to many types of cancers. PKCδ is known to be a multifunctional PKC family member and has been rigorously studied as an intracellular signaling molecule. Here we show that PKCδ is a secretory protein that regulates cell growth of liver cancer. Full-length PKCδ was secreted to the extracellular space in living liver cancer cells under normal cell culture conditions and in xenograft mouse models. Patients with liver cancer showed higher levels of serum PKCδ than patients with chronic hepatitis or liver cirrhosis or healthy individuals. In liver cancer cells, PKCδ secretion was executed in an endoplasmic reticulum (ER)-Golgi–independent manner, and the inactivation status of cytosolic PKCδ was required for its secretion. Furthermore, colocalization studies showed that extracellular PKCδ was anchored on the cell surface of liver cancer cells via association with glypican 3, a liver cancer–related heparan sulfate proteoglycan. Addition of exogenous PKCδ activated IGF-1 receptor (IGF1R) activation and subsequently enhanced activation of ERK1/2, which led to accelerated cell growth in liver cancer cells. Conversely, treatment with anti-PKCδ antibody attenuated activation of both IGF1R and ERK1/2 and reduced cell proliferation and spheroid formation of liver cancer cells and tumor growth in xenograft mouse models. This study demonstrates the presence of PKCδ at the extracellular space and the function of PKCδ as a growth factor and provides a rationale for the extracellular PKCδ-targeting therapy of liver cancer.Significance:PKCδ secretion from liver cancer cells behaves as a humoral growth factor that contributes to cell growth via activation of proliferative signaling molecules, which may be potential diagnostic or therapeutic targets.

Protein secretion in cancer cells defines tumor survival and progression by orchestrating the microenvironment. Studies suggest the occurrence of active secretion of cytosolic proteins in liver cancer and their involvement in tumorigenesis. Here, we investigated the identification of extended-synaptotagmin 1 (E-Syt1), an endoplasmic reticulum (ER)-bound protein, as a key mediator for cytosolic protein secretion at the ER-plasma membrane (PM) contact sites. Cytosolic proteins interacted with E-Syt1 on the ER, and then localized spatially inside SEC22B

Protein secretion in cancer cells defines tumor survival and progression by orchestrating the microenvironment. Studies suggest the occurrence of active secretion of cytosolic proteins in liver cancer and their involvement in tumorigenesis. Here, we investigated the identification of extended-synaptotagmin 1 (E-Syt1), an endoplasmic reticulum (ER)-bound protein, as a key mediator for cytosolic protein secretion at the ER–plasma membrane (PM) contact sites. Cytosolic proteins interacted with E-Syt1 on the ER, and then localized spatially inside SEC22B+vesicles of liver cancer cells. Consequently, SEC22B on the vesicle tethered to the PM via Q-SNAREs (SNAP23, SNX3, and SNX4) for their secretion. Furthermore, inhibiting the interaction of protein kinase Cδ (PKCδ), a liver cancer-specific secretory cytosolic protein, with E-Syt1 by a PKCδ antibody, decreased in both PKCδ secretion and tumorigenicity. Results reveal the role of ER–PM contact sites in cytosolic protein secretion and provide a basis for ER-targeting therapy for liver cancer.

BACKGROUND Centrilobular zonal necrosis (CZN) is advocated as a histological hallmark present in a small number of patients with autoimmune hepatitis (CZN-AIH). Moreover, CZN has been detected in the absence of significant interface hepatitis, one of the most important histological findings of AIH. The concept of CZN-AIH as a distinctive subtype of AIH remains controversial, due to the rarity of CZN-AIH and the ambiguous definition of CZN. To elucidate the clinicopathological and immunogenetic features of CZN-AIH, and to evaluate the significance of co-existent interface hepatitis in CZN-AIH. METHODS A total of 102 biopsy samples of AIH, obtained at The Jikei University Katsushika Medical Center and Jikei University Hospital, were reviewed. The 32 patients whose biopsies showed CZN were selected as the CZN-AIH group and the remaining 70 were grouped as the non-CZN-AIH controls. In the CZN-AIH group, interface hepatitis was histologically present in 37.5% (n=12; mixed-type) and absent in 62.5% (n=20; pure-type). Data on clinical, histopathologic and immunogenetic features were statistically compared between the CZN-AIH group and the non-CZH-AIH controls. Additionally, significance of interface hepatitis in CZN-AIH was determined by comparative analysis of the mixed-type and pure-type subgroups. RESULTS Cases of CZN-AIH were more frequently of acute-onset hepatitis (56.2% vs chronic: 32.9%, P=0.031), lower immunoglobulin G level (P

Background and Aim Sarcopenia frequently develops in patient with liver cirrhosis (LC). Ethanol reduces muscle protein synthesis and accelerates proteolysis. However, the relationship between heavy alcohol consumption and sarcopenia remains controversial. This study aimed to investigate the characteristics and prevalence of sarcopenia among patients with alcoholic LC (ALC) in real‐world clinical settings. Methods This cross‐sectional study included 181 patients with LC. Heavy alcohol consumption was defined as >60 g/day. Sarcopenia was diagnosed according to the Japan Society of Hepatology criteria. Results Among the 181 patients, 64 (35.4%) were diagnosed with ALC. Patients with ALC were younger (median, 61.5 vs 72.0 years; P, This study aimed to investigate the characteristics and prevalence of sarcopenia among 181 patients with alcoholic liver cirrhosis (ALC). Patients with ALC were younger and had a lower prevalence of sarcopenia, despite advanced disease stage/impaired liver function reserve, compared to those with non‐ALC.

Background and Aim Although tenofovir alafenamide (TAF), as well as entecavir (ETV), is widely used as first‐line treatment for patients with chronic hepatitis B, there are only a few studies comparing sequential therapy from ETV to TAF and continuous ETV monotherapy in patients with maintained virologic response to ETV. Methods In a retrospective multicenter study, we investigated the efficacy and safety of sequential therapy from ETV to TAF (ETV‐TAF group) and compared them with continuous ETV monotherapy (ETV group), using propensity score matching, in chronic hepatitis B patients. Results From 442 patients, we analyzed 142 patients from each group comprising 71 patients matched for several data, including age, HBV genotype, hepatitis B envelope antigen, cirrhosis, alanine aminotransferase, platelet count, prior ETV monotherapy period, and hepatitis B surface antigen (HBsAg) change during prior ETV monotherapy. In the ETV‐TAF group, HBsAg levels significantly decreased from baseline to 48 weeks after switching to TAF (−0.02 log IU/mL, P = 0.038). HBcrAg levels also significantly decreased after switching to TAF (−0.1 log IU/mL, P = 0.004). However, there were no significant differences in the reduction of HBsAg and HBcrAg levels between the ETV‐TAF and ETV groups. There was no significant difference in the change of estimated glomerular filtration rate levels from baseline to 48 weeks between the two groups. Conclusions The present study indicated that the efficacy, especially of the HBsAg‐reducing action, and safety of sequential therapy from ETV to TAF were similar to those of continuous ETV monotherapy among chronic hepatitis B patients with maintained virologic response to ETV., In a retrospective, multicenter study involving 17 institutions, we investigated the efficacy and safety of sequential therapy from entecavir (ETV) to tenofovir alafenamide (TAF) (ETV‐TAF group). The present study indicated that the efficacy, especially of the hepatitis B surface antigen‐reducing action, and safety of sequential therapy from ETV to TAF were similar to those of continuous ETV monotherapy.

Supplemental Digital Content is available in the text., Aim Bone disorders are serious complications in patients with primary biliary cholangitis (PBC), especially in postmenopausal female patients. Given that osteoporosis interrelates closely with sarcopenia, the concept of osteosarcopenia (coexistence of the two complications) has been established. This study aimed to investigate the relationship between osteoporosis, sarcopenia, vertebral fracture, and osteosarcopenia in PBC patients. Methods This study involved 117 consecutive PBC patients (21 males and 96 females). Bone mineral density (BMD) was measured with dual-energy X-ray absorptiometry. Sarcopenia was diagnosed according to the Japan Society of Hepatology assessment criteria. Results Of the 117 patients, 33 (28.2%), 27 (23.1%), 21 (17.9%), and 18 (15.4%) had osteoporosis, sarcopenia, vertebral fracture, and osteosarcopenia, respectively. Multivariate analysis identified sarcopenia as a significant, independent risk factor associated with osteoporosis in all and female patients [odds ratio (OR) = 4.126, P = 0.018; OR = 6.510, P = 0.001, respectively], and vice versa (OR = 3.420, P = 0.040; OR = 4.012, P = 0.026, respectively). The skeletal muscle mass index and handgrip strength were significantly correlated with the BMD of the lumbar spine, femoral neck, and total hip (r = 0.46–0.59, P < 0.001). Patients with osteosarcopenia had significantly higher prevalence of vertebral fracture (10/18; 55.6%) than those without both osteoporosis and sarcopenia (5/75; 6.7%). Conclusion We demonstrated the prevalence of osteoporosis, sarcopenia, vertebral fracture, and osteosarcopenia in PBC, and noted that these complications interrelated closely with each other. Comprehensive assessment and treatment strategies for bone and muscle disorders are essential for PBC patients.

Background: Rifaximin is commonly used for hepatic encephalopathy (HE). However, the effects of long-term treatment for Japanese people are limited. Therefore, this study aimed to investigate the effects and safety of long-term treatment with rifaximin on HE. Methods: A total of 215 patients with cirrhosis administered with rifaximin developed overt or covert HE, which was diagnosed by an attending physician for >12 months. Laboratory data were extracted at pretreatment and 3, 6, and 12 months after rifaximin administration. The long-term effect of rifaximin was evaluated, and the incidence of overt HE during 12 months and adverse events was extracted. Results: Ammonia levels were significantly improved after 3 months of rifaximin administration and were continued until 12 months. There were no serious adverse events after rifaximin administration. The number of overt HE incidents was 9, 14, and 27 patients within 3, 6, and 12 months, respectively. Liver enzymes, renal function, and electrolytes did not change after rifaximin administration. Prothrombin activity is a significant risk factor for the occurrence of overt HE. The serum albumin, prothrombin activity, and albumin–bilirubin (ALBI) scores were statistically improved after 3 and 6 months of rifaximin administration. Moreover, the same results were obtained in patients with Child–Pugh C. Conclusions: The long-term rifaximin treatment was effective and safe for patients with HE, including Child–Pugh C.

BACKGROUND AND AIM Primary biliary cholangitis (PBC) patients who are refractory to ursodeoxycholic acid (UDCA) are at risk for progression to cirrhosis and liver failure. Bezafibrate could be an alternative second-line therapeutic option in these patients. This study aimed to evaluate the long-term outcome(s) of combined UDCA and bezafibrate therapy in UDCA-refractory PBC patients and identify prognostic factors. METHODS Among 445 patients treated with UDCA, 150 patients inadequately responded to UDCA monotherapy and received long-term UDCA plus bezafibrate (median, 15 years). Data from these patients were used for this retrospective analysis. RESULTS Combination therapy resulted in significant improvements in serum biochemistry and liver transplantation risk estimated using the UK-PBC-risk and the GLOBE scores. The cumulative normalization rates of alkaline phosphatase, gamma-glutamyltransferase, and immunoglobulin M (IgM) were significantly higher in patients without cirrhosis-related symptoms or liver-related events than in those with them. Overall, IgM constantly emerged as a significant factor associated with cirrhosis-related symptoms and liver-related events at all time points. Cumulative survival rates were significantly lower in patients with IgM ≥ 240 mg/dL than in patients with IgM

Based on high efficacy and safety demonstrated in clinical trials, treatment with glecaprevir/pibrentasvir (G/P) for 8 weeks is recommended for hepatitis C virus (HCV)-infected patients who are direct-acting antiviral (DAA) naïve, genotype 1 or 2, and noncirrhotic. The aim of this study was to validate real-world experience with 8-week G/P treatment in Japan. We conducted a prospective observational cohort study in 554 patients who underwent 8-week treatment from among 1,022 patients who initiated G/P therapy. The majority (54.5%) were male, with a median age of 66 years, and HCV genotype distribution was genotype 1, 43.8%; genotype 2, 55.3%; and mixed subtype, 0.9%. Overall, the sustained virologic response rate at 12 weeks (SVR12) was 92.8% (530/571) in the intention-to-treat population and 99.3% (526/530) in the per-protocol population. The SVR12 rates by subgroups were as follows: subtype 1a, 100% (6/6); 1b, 100% (189/189); 2a, 99.3% (150/151); 2b, 99.0% (103/104); and mixed subtype, 50% (2/4). Among four patients with virologic failure following 8-week treatment with G/P, none had baseline polymorphisms or treatment-emergent amino acid substitutions in NS3. However, 2 of 4 patients with virologic failure had treatment-emergent amino acid substitutions in NS5A. Adverse events (AEs) were reported in 21.5% of patients and 1.2% of patients discontinued due to drug-related AEs. In conclusion, G/P treatment for 8 weeks was safe and effective for DAA-naïve noncirrhotic genotype 1 or 2 patients in a real-world clinical setting in Japan.

Liver cancer is highly aggressive and globally exhibits a poor prognosis. Therefore, the identification of novel molecules that can become targets for future therapies is urgently required. We have reported that dual-specificity tyrosine-regulated kinase 2 (DYRK2) functions as a tumor suppressor by regulating cell survival, differentiation, proliferation and apoptosis. However, the research into its clinical application as a molecular target has remained to be explored. Here we showed that DYRK2 knockdown enhanced tumor growth of liver cancer cells. Conversely and more importantly, adenovirus-mediated overexpression of DYRK2 resulted in inhibition of cell proliferation and tumor growth, and induction of apoptosis both in vitro and in vivo. Furthermore, we found that liver cancer patients with low DYRK2 expression had a significantly shorter overall survival. Given the findings that DYRK2 regulates proliferation and apoptosis of cancer cells, DYRK2 expression could be a promising predictive marker of the prognosis in liver cancer. Stabilized or forced expression of DYRK2 may become thus a potential target for novel gene therapy against liver cancer.

Current models of cell-intrinsic immunity to RNA viruses centre on virus-triggered inducible antiviral responses initiated by RIG-I-like receptors or Toll-like receptors that sense pathogen-associated molecular patterns, and signal downstream through interferon regulatory factors (IRFs), transcription factors that induce synthesis of type I and type III interferons1. RNA viruses have evolved sophisticated strategies to disrupt these signalling pathways and evade elimination by cells, attesting to their importance2. Less attention has been paid to how IRFs maintain basal levels of protection against viruses. Here, we depleted antiviral factors linked to RIG-I-like receptor and Toll-like receptor signalling to map critical host pathways restricting positive-strand RNA virus replication in immortalized hepatocytes and identified an unexpected role for IRF1. We show that constitutively expressed IRF1 acts independently of mitochondrial antiviral signalling (MAVS) protein, IRF3 and signal transducer and activator of transcription 1 (STAT1)-dependent signalling to provide intrinsic antiviral protection in actinomycin D-treated cells. IRF1 localizes to the nucleus, where it maintains the basal transcription of a suite of antiviral genes that protect against multiple pathogenic RNA viruses, including hepatitis A and C viruses, dengue virus and Zika virus. Our findings reveal an unappreciated layer of hepatocyte-intrinsic immunity to these positive-strand RNA viruses and identify previously unrecognized antiviral effector genes. Using a short-hairpin RNA-based depletion strategy coupled with high throughput RNA sequencing, the authors unravel a layer of intrinsic immunity to positive-strand RNA viruses in immortalized hepatocytes that relies on basal expression levels of interferon regulatory factor 1 maintaining the transcription of a suite of antiviral genes.

Aim Primary biliary cholangitis (PBC) is an autoimmune liver disease with unknown pathogenesis. In PBC, activation of T-cell receptor (TCR) signaling is associated with inflammatory cytokine production through N-Ras upregulation. Although the CD4+ T cell TCR repertoire could be associated with PBC pathogenesis, it has not been evaluated. Thus, we analyzed the PBC-CD4+ T cell TCR repertoire using next generation sequencing (NGS). Methods Four PBC patients (one treatment-naive and three receiving ursodeoxycholic acid) and three healthy individuals were enrolled. NRAS expression in CD4+ T cells was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). N-Ras dynamics in CD4+ T cells were assessed by qRT-PCR and GTP-N-Ras activation assay. The TCR α- (TRA) and β-chain (TRB) repertoires on CD4+ T cells were analyzed by NGS and profiled using hierarchical analysis. Motif analysis was undertaken to elucidate the structure of PBC-specific TCRs. Results NRAS was upregulated in PBC relative to control CD4+ T cells (P 2). Among them, TRAV29/J22, TRBV6-5/J2-6, and TRBV10-1/J2-1 were expressed in PBC but the expression was negligible in the controls, with more mature and longer forms observed in PBC-CD4+ T cells. Conclusions N-Ras was upregulated in PBC-CD4+ T cells, and it enhanced TCR activation, indicating that PBC-CD4+ T cells were activated by N-Ras upregulation with differentially expressed TCR repertoires on their surfaces.

We developed a small portable sensor device using a p-type semiconductor cuprous bromide (CuBr) thin film to measure breath ammonia in real time with highsensitivity and selectivity. Breath ammonia is reportedly associated with chronic liver disease (CLD). We aimed to assess the practical utility of the novel CuBr sensor device for exhaled breath ammonia and the correlation between breath and blood ammonia in CLD patients. This was a feasibility and pilot clinical study of 21 CLD patients and 18 healthy volunteers. Breath ammonia was directly and quickly measured using the novel CuBr sensor device and compared with blood ammonia measured at the same time. CLD patients had significantly higher breath ammonia levels than healthy subjects (p = 1.51 × 10−3), with the level of significance being similar to that for blood ammonia levels (p= 0.024). Significant differences were found in breath and blood ammonia between the healthy and cirrhosis groups (p = 2.97 × 10−3 and 3.76 × 10−3, respectively). Significant, positive correlations between breath and blood ammonia were noted in the CLD group (R = 0.747, p = 1.00 × 10−4), healthy/CLD group (R = 0.741, p = 6.75 × 10−8), and cirrhosis group (R = 0.744, p = 9.52 × 10−4). In conclusion, the newly developed, easy-to-use, and small portable CuBr sensor device was able to non-invasively measure breath ammonia in real time. Breath ammonia measured using the device was correlated with blood ammonia and the presence of liver cirrhosis, and might be an alternative surrogate biomarker to blood ammonia.

ObjectiveMicroRNAs (miRNAs) are implicated in the pathogenesis of autoimmune diseases and could be biomarkers of disease activity. This study aimed to identify highly expressed circulating miRNAs in patients with autoimmune hepatitis (AIH) and to evaluate their association with clinical characteristics.MethodsMicroarray analyses were performed, and miRNA expression profiling for AIH, primary biliary cholangitis (PBC) and overlap syndrome (OS) using the serum of patients and healthy individuals was done. Samples were divided into discovery and test sets to identify candidate miRNAs that could discriminate AIH from PBC; the former included 21 AIH and 23 PBC samples, while the latter included five AIH and eight PBC samples.ResultsAmong 11 candidate miRNAs extracted in the discovery set, 4 (miR-3196, miR-6125, miR-4725–3 p and miR-4634) were specifically and highly expressed in patients with AIH in the test set. These four miRNAs discriminated AIH from PBC with high sensitivity (0.80–1.00) and specificity (0.88–1.00). In situ hybridisation analysis revealed that these miRNAs were expressed in the cytoplasm of hepatocytes in patients with AIH. Their expression levels were highest in untreated patients with AIH, followed by those in untreated patients with OS. They drastically or moderately decreased after prednisolone treatment. Histological analysis demonstrated that the expression levels of miR-3196, miR-6125 and miR-4634 in patients with AIH and OS were correlated with severe hepatic necroinflammatory activity.ConclusionThese circulating miRNAs are suggested to reflect hepatic necroinflammatory activity and serve as AIH-related and treatment-responsive biomarkers. These miRNAs could be beneficial in developing new therapeutic strategies for AIH.

Low vitamin D status is related to frailty and/or sarcopenia in elderly individuals. However, these relationships are unclear in patients with chronic liver disease (CLD). This study aimed at exploring the relationship between serum 25-hydroxyvitamin D [25(OH)D] levels and frailty or sarcopenia in 231 patients with CLD. Frailty was determined based on five factors (weight loss, low physical activity, weakness, slowness, and exhaustion). Sarcopenia was diagnosed by applying the Japan Society of Hepatology criteria. The patients were classified into three groups according to baseline 25(OH)D levels: low (L), intermediate (I), and high (H) vitamin D (VD) groups. Of the 231 patients, 70 (30.3%) and 66 (28.6%) had frailty and sarcopenia, respectively. The prevalence rate of frailty and sarcopenia significantly increased stepwise with a decline in the vitamin D status. The L-VD group showed the highest prevalence rates of frailty and sarcopenia (49.1% (28/57), p &lt, 0.001 for both), whereas the H-VD group showed the lowest prevalence rates of frailty (15.3% (9/59)) and sarcopenia (18.6% (11/59)) (p &lt, 0.001 for both). Multivariate analysis identified serum 25(OH)D levels as a significant independent factor related to frailty and sarcopenia. Serum 25(OH)D levels significantly correlated with handgrip strength, skeletal muscle mass index, and gait speed. In conclusion, low serum vitamin D level, especially severe vitamin D deficient status, is closely related to frailty and sarcopenia in patients with CLD.

Branched-chain amino acid (BCAA) and insulin-like growth factor 1 (IGF-1) are essential for muscle protein synthesis. We investigated the association of serum BCAA and IGF-1 levels with sarcopenia and gait speed in 192 patients with liver cirrhosis (LC). Sarcopenia was diagnosed according to the Japan Society of Hepatology criteria. Slow gait speed was defined as &lt, 1.0 m/s. Subjects were divided into three groups based on baseline BCAA or IGF-1 levels: low (L), intermediate (I), and high (H) groups. The L-BCAA group had the highest prevalence of sarcopenia (60.4%, p &lt, 0.001) and slow gait speed (56.3%, p = 0.008), whereas the H-BCAA group had the lowest prevalence of sarcopenia (8.5%, p &lt, 0.001). The L-IGF-1 group showed the highest prevalence of sarcopenia (46.9%, p &lt, 0.001), whereas the H-IGF-1 group had the lowest prevalence of sarcopenia (10.0%, p &lt, 0.001) and slow gait speed (18.0%, p = 0.003). Using the optimal BCAA and IGF-1 cutoff values for predicting sarcopenia (372 &mu, mol/L and 48.5 ng/mL, respectively), the sensitivity and specificity were 0.709 and 0.759 for BCAA and 0.636 and 0.715 for IGF-1, respectively. Low serum BCAA and IGF-1 levels were associated with sarcopenia and slow gait speed in patients with LC.

Expression of human protein kinase C delta (PKCδ) protein has been linked to many types of cancers. PKCδ is known to be a multifunctional PKC family member and has been rigorously studied as an intracellular signaling molecule. Here we show that PKCδ is a secretory protein that regulates cell growth of liver cancer. Full-length PKCδ was secreted to the extracellular space in living liver cancer cells under normal cell culture conditions and in xenograft mouse models. Patients with liver cancer showed higher levels of serum PKCδ than patients with chronic hepatitis or liver cirrhosis or healthy individuals. In liver cancer cells, PKCδ secretion was executed in an endoplasmic reticulum (ER)-Golgi–independent manner, and the inactivation status of cytosolic PKCδ was required for its secretion. Furthermore, colocalization studies showed that extracellular PKCδ was anchored on the cell surface of liver cancer cells via association with glypican 3, a liver cancer–related heparan sulfate proteoglycan. Addition of exogenous PKCδ activated IGF-1 receptor (IGF1R) activation and subsequently enhanced activation of ERK1/2, which led to accelerated cell growth in liver cancer cells. Conversely, treatment with anti-PKCδ antibody attenuated activation of both IGF1R and ERK1/2 and reduced cell proliferation and spheroid formation of liver cancer cells and tumor growth in xenograft mouse models. This study demonstrates the presence of PKCδ at the extracellular space and the function of PKCδ as a growth factor and provides a rationale for the extracellular PKCδ-targeting therapy of liver cancer. Significance: PKCδ secretion from liver cancer cells behaves as a humoral growth factor that contributes to cell growth via activation of proliferative signaling molecules, which may be potential diagnostic or therapeutic targets.

BACKGROUND Effective treatment of osteoporosis is essential for improving morbidity and health-related quality of life in chronic liver disease (CLD) patients. Denosumab has been shown to increase bone mineral density (BMD) and decrease the risk of osteoporotic fracture in the general population. However, there are few reports evaluating the efficacy of denosumab in CLD patients. AIM To investigated the effects and safety of denosumab in CLD patients with osteoporosis. METHODS Sixty CLD patients with osteoporosis were subcutaneously administered denosumab once every 6 mo. The study period for evaluating efficacy and safety was 12 mo. Changes from baseline in BMD at the lumbar spine, femoral neck, and total hip were evaluated at 12 mo of denosumab treatment. Bone turnover and quality were assessed by measuring serum tartrate-resistant acid phosphatase-5b (bone resorption marker), serum total procollagen type I N-terminal propeptide (bone formation maker), and plasma pentosidine (bone quality marker). RESULTS Among the 405 CLD patients, 138 (34.1%) patients were diagnosed with osteoporosis; among these, 78 patients met the exclusion criteria and thus 60 patients were finally included in the present study. The median percentage changes from baseline to 12 mo of denosumab treatment in BMD at the lumbar spine, femoral neck, and total hip were +4.44%, +3.71%, and +4.03%, respectively. Denosumab significantly improved BMD, regardless of sex, patient age, and presence of liver cirrhosis. Serum tartrate-resistant acid phosphatase-5b and procollagen type I N-terminal propeptide levels constantly and significantly declined after denosumab treatment (P < 0.001). Plasma pentosidine levels were also significantly lower at 12 mo of treatment (P = 0.010). No patients experienced fractures and moderate-to-severe adverse events, except for transient hypocalcemia. CONCLUSION Denosumab treatment was safe and increased BMD, suppressed bone turnover, and improved bone quality marker levels in CLD patients with osteoporosis, irrespective of differences in baseline characteristics.

As the incidence of hepatocellular carcinoma (HCC) caused by infection with the hepatotropic viruses hepatitis B and hepatitis C decreases, greater attention has become focused on HCC caused by nonalcoholic steatohepatitis (NASH), an advanced form of nonalcoholic fatty liver disease which has shown increasing prevalence in correspondence with the overall increase in metabolic syndrome over the recent decades. Several clinical population studies have shown a positive relationship between NASH and HCC, while also providing initial insights into the underlying mechanisms of HCC development from NASH. Research into the pathological progression of NASH to HCC has advanced by use of several beneficial rodent models. In this review, we summarize the established mouse models for preclinical research of NASH-associated HCC and discuss the underlying hepatic mechanisms of NASH-related tumorigenesis identified to date that could lead to new targets for treatment and prevention.

AIM Serum Mac-2 binding protein (M2BP) and Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA+ -M2BP) are used to estimate the liver fibrosis stage in chronic liver diseases. However, few head-to-head studies have been carried out to compare the two biomarkers in non-alcoholic fatty liver disease (NAFLD). METHODS Serum M2BP and WFA+ -M2BP levels were compared against clinical characteristics and liver histological manifestations in the same samples collected from 213 biopsy-proven NAFLD patients. RESULTS Median levels (range) of M2BP and WFA+ -M2BP were 1.58 (0.70-7.75) pg/mL and 0.85 (0.22-11.32) cut-off index (COI), respectively. Fibrosis stages 1, 2, 3, and 4 were determined in 136, 37, 17, and 23 patients, respectively. Median levels of both biomarkers increased stepwise with fibrosis progression. The M2BP and WFA+ -M2BP levels showed a significant positive correlation (r = 0.643, P = 2.91 × 10-26 ), but a marked discrepancy between both biomarkers was noted in five stage 4 and three stage 1 patients, who had high WFA+ -M2BP but relatively low M2BP levels. Most of these outliers had findings suggestive of more advanced fibrosis. For diagnosing any fibrosis severity, WFA+ -M2BP had greater area under the receiver operating characteristic curve (AUC) and predictive accuracy than M2BP. Among eight fibrosis markers/indices, WFA+ -M2BP yielded the second highest AUC (0.832) and the highest predictive accuracy (82.2%) to diagnose cirrhosis. In addition, WFA+ -M2BP showed the second highest predictive accuracy to diagnose severe fibrosis (78.4%) and significant fibrosis (76.1%). CONCLUSION This head-to-head comparison suggests that WFA+ -M2BP is superior to M2BP for distinguishing liver fibrosis stages in NAFLD patients. A marked discrepancy between the two biomarkers may be indicative of advanced NAFLD (UMIN000023286).

We investigated the usefulness of the Fracture Risk Assessment tool (FRAX) for predicting sarcopenia in chronic liver disease (CLD). In this cross-sectional study, we evaluated 321 patients with CLD. The FRAX with and without bone mineral density (BMD) was employed to calculate the 10-year risks of major osteoporotic and hip fractures. The FRAX score for high fracture risk was defined as a 10-year major osteoporotic fracture probability of ≥20% or a 10-year hip fracture probability of ≥3%. The diagnosis of sarcopenia was based on the Japan Society of Hepatology criteria. According to the FRAX, with and without BMD, 134 (41.7%) and 193 (60.1%) patients had a high fracture risk, respectively. The high fracture risk group had a significantly higher frequency of sarcopenia than the non-high fracture risk group. FRAX scores of major osteoporotic and hip fractures were negatively correlated with handgrip strength and muscle mass. Using the FRAX with BMD, the cutoff scores of major osteoporotic and hip fractures for predicting sarcopenia were 8.55% (sensitivity/specificity, 0.847/0.568) and 3.35% (0.729/0.746), respectively. Using the FRAX without BMD, they were 18.5% (0.635/0.725) and 7.65% (0.729/0.758), respectively. The FRAX is a simple and convenient screening tool for predicting sarcopenia in patients with CLD.

Background Sarcopenia and osteoporosis reduce life quality and worsen prognosis in patients with liver cirrhosis (LC). When these two complications coexist, a diagnosis of osteosarcopenia is made. We aimed to investigate the actual situations of sarcopenia, osteoporosis, osteosarcopenia, and vertebral fracture, and to clarify the relationship among these events in patients with LC. Methods We describe a cross-sectional study of 142 patients with LC. Sarcopenia was defined according to the Japan Society of Hepatology (JSH) criteria, Asian Working Group for Sarcopenia (AWGS) criteria, and European Working Group on Sarcopenia in Older People (EWGSOP2) criteria. The skeletal muscle mass index (SMI) and handgrip strength were assessed using bioelectrical impedance analysis and a digital grip strength dynamometer, respectively. Bone mineral density (BMD) was measured using dual energy X-ray absorptiometry, and vertebral fracture was evaluated using spinal lateral X-rays. The severity of LC was assessed using the Child-Pugh classification. Results Among the 142 patients, the prevalence of sarcopenia was 33.8% (48/142) according to the JSH and AWGS criteria and 28.2% (40/142) according to the EWGSOP2 criteria. The number of patients with osteoporosis, osteosarcopenia, and vertebral fracture was 49 (34.5%), 31 (21.8%), and 41 (28.9%), respectively. Multivariate analysis revealed a close association between sarcopenia and osteoporosis. Osteoporosis was independently associated with sarcopenia [odds ratio (OR) = 3.923, P = 0.010]. Conversely, sarcopenia was independently associated with osteoporosis (OR = 5.722, P r = 0.55 and 0.51, respectively; P r = 0.67 and 0.62, respectively; P r = 0.67 and 0.61, respectively; P Conclusions Sarcopenia, osteoporosis, osteosarcopenia, and vertebral fracture were highly prevalent and closely associated with one another in patients with LC. Specifically, patients with osteosarcopenia had the highest risk of vertebral fractures. Early diagnosis of these complications is essential for treatment intervention.

The incidence of metabolic syndrome with fatty liver is spreading on a worldwide scale. Correspondingly, the number of patients with the hepatic phenotype of metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), and in its advanced states, non-alcoholic steatohepatitis (NASH), and the subsequent hepatocellular carcinoma (HCC) derived from NASH (NASH-HCC) is increasing remarkably. A large-scale epidemiological study revealed that obesity can be a risk factor of such cancers as HCC. Moreover, despite the ongoing trends of declining cancer incidence and mortality for most cancer types, HCC has experienced a markedly increased rate of both. Considering the differences in liver-related mortality among NAFLD patients, NASH, and NASH-HCC should be included in the objectives of initiatives to manage NAFLD patients and their progression to the advanced stages. Unfortunately, research has yet to make a crucial drug discovery for the effective treatment of NASH and NASH-HCC, although it is urgently needed. The latest widespread concept of the “multiple parallel hits hypothesis,” whereby multiple factors contribute concurrently to disease pathogenesis has led to advances in the elucidation of hepatic and systemic molecular mechanisms driving NASH and the subsequent NASH-HCC progression; the results are not only extensive but promising for therapeutics. Here, we have summarized the myriad landmark discoveries of recent research into the pathogenic processes underlying NASH-HCC development and with the greatest possibility for a new generation of pharmaceutical products for interference and treatment.