Elucidation of pericholangitis and periductal fibrosis in cholestatic liver diseases via extracellular vesicles released by polarized biliary epithelial cells.

Cholestatic liver diseases causes inflammation and fibrosis around bile ducts. However, the pathological mechanism has not been elucidated. Extracellular vesicles (EVs) are released from both the basolateral and apical sides of polarized biliary epithelial cells. We aimed to investigate the possibility that EVs released from the basolateral sides of biliary epithelial cells by bile acid stimulation induce inflammatory cells and fibrosis around bile ducts, and they may be involved in the pathogenesis of cholestatic liver disease. Human biliary epithelial cells (H69) were grown on cell culture inserts and stimulated with chenodeoxycholic acid + IFN-γ. Human THP-1-derived M1-macrophages, LX-2 cells, and KMST-6 cells were treated with the extracted basolateral EVs, and inflammatory cytokines and fibrosis markers were detected by RT-PCR. Highly expressed proteins from stimulated EVs were identified, and M1-macrophages, LX-2, KMST-6 were treated with these recombinant proteins. Stimulated EVs increased the expression of TNF, IL-1ß, and IL-6 in M1-macrophages, TGF-ß in LX-2 and KMST-6 compared with the corresponding expression levels in unstimulated EVs. Nucleophosmin, nucleolin, and midkine levels were increased in EVs from stimulated cells compared with protein expression in EVs from unstimulated cells. Leukocyte cell-derived chemotaxin-2 (LECT2) is highly expressed only in EVs from stimulated cells. Stimulation of M1-macrophages with recombinant nucleophosmin, nucleolin, and midkine significantly increased the expression of inflammatory cytokines. Stimulation of LX-2 and KMST-6 with recombinant LECT2 significantly increased the expression of fibrotic markers. These results suggest that basolateral EVs are related to the development of pericholangitis and periductal fibrosis in cholestatic liver diseases.NEW & NOTEWORTHY Our research elucidated that the composition of basolateral EVs from the biliary epithelial cells changed under bile acid exposure and the basolateral EVs contained the novel inflammation-inducing proteins NPM, NCL, and MK and the fibrosis-inducing protein LECT2. We report that these new results are possible to lead to the potential therapeutic target of cholestatic liver diseases in the future.

A new model to estimate duration of survival in patients with hepatocellular carcinoma with BCLC intermediate stage.

It is difficult to determine whether an individual therapy contributes to the elongation of survival because of the difficulty of organizing clinical research in patients who receive multiple treatments in HCC. We aimed to establish a new model of survival prediction in patients with intermediate stage HCC to establish standards in the recent and coming multi-MTA era. This analysis was prepared using a data set of 753 patients diagnosed HCC prior to 2017. Multiple regression analysis showed age, naïve or recurrence, the size of the largest tumor nodule, the number of nodules, total bilirubin, albumin and α-fetoprotein as independent predictors of survival. A Weibull model had the best fit and, based on these predictors, we established a new predicted survival model. The survival duration can be predicted the proposed model; EXP (4.02580 + (- 0.0086253) × age + (- 0.34667) × (naïve/recurrence) + (- 0.034962) × (number of nodules) + (- 0.079447) × (the size of the largest nodule) + (- 0.21696) × (total bilirubin) + 0.27912 × (albumin) + (- 0.00014741) × (α-fetoprotein)) × (- natural logarithm(0.5))^0.67250. This model is useful for the planning and evaluating the efficacy of recent sequential therapies in multi-MTA era.

Role of Bile-Derived Extracellular Vesicles in Hepatocellular Proliferation after Partial Hepatectomy in Rats.

Although liver regeneration has been extensively studied, the effects of bile-derived extracellular vesicles (bile EVs) on hepatocytes has not been elucidated. We examined the influence of bile EVs, collected from a rat model of 70% partial hepatectomy (PH), on hepatocytes. We produced bile-duct-cannulated rats. Bile was collected over time through an extracorporeal bile duct cannulation tube. Bile EVs were extracted via size exclusion chromatography. The number of EVs released into the bile per liver weight 12 h after PH significantly increased. Bile EVs collected 12 and 24 h post-PH, and after sham surgery (PH12-EVs, PH24-EVs, sham-EVs) were added to the rat hepatocyte cell line, and 24 h later, RNA was extracted and transcriptome analysis performed. The analysis revealed that more upregulated/downregulated genes were observed in the group with PH24-EVs. Moreover, the gene ontology (GO) analysis focusing on the cell cycle revealed an upregulation of 28 types of genes in the PH-24 group, including genes that promote cell cycle progression, compared to the sham group. PH24-EVs induced hepatocyte proliferation in a dose-dependent manner in vitro, whereas sham-Evs showed no significant difference compared to the controls. This study revealed that post-PH bile Evs promote the proliferation of the hepatocytes, and genes promoting cell cycles are upregulated in hepatocytes.

Objective Response by mRECIST to Initial Lenvatinib Therapy Is an Independent Factor Contributing to Deep Response in Hepatocellular Carcinoma Treated with Lenvatinib-Transcatheter Arterial Chemoembolization Sequential Therapy.

Objective: There is limited information regarding the benefits of Lenvatinib-transcatheter arterial chemoembolization (LEN-TACE) sequential therapy for unresectable hepatocellular carcinoma (u-HCC). We compared the efficacy and safety of LEN-TACE sequential therapy to LEN monotherapy and investigated the factors contributing to the LEN-TACE sequential therapy deep response. Methods: We enrolled a multicenter cohort of 247 patients with u-HCC treated with LEN between 2018 and 2020. Propensity score matching identified 63 matching pairs of patients with well-balanced characteristics. We retrospectively compared the clinical outcomes, including overall survival (OS), progression-free survival (PFS), and incidence of adverse events (AEs), between the LEN-TACE and LEN monotherapy groups. Additionally, we evaluated the tumor response, change in albumin-bilirubin (ALBI) score, factors affecting PFS and OS, and independent predictors contributing to the LEN-TACE sequential therapy deep response. In this study, at eight weeks after resumption of LEN after initial TACE, "deep response" was defined as achieving complete response or partial response (PR) on modified Response Evaluation Criteria in Solid Tumors (mRECIST), and at least a 30% decrease in the sum of diameters of target lesions, taking the baseline sum diameters as the reference. Results: The OS and PFS in the LEN-TACE group were significantly higher than those in the LEN monotherapy group (p = 0.002 and p = 0.037, respectively). The incidence of AEs related to LEN was not significantly different between the two groups. In LEN-TACE sequential therapy, the objective response rate was 61.9%, and the disease control rate was 74.6%, according to the mRECIST criteria. No significant change in the ALBI score was observed during sequential LEN-TACE therapy. Multivariable analyses revealed that deep response was independently associated with the outcome of the initial response to LEN by mRECIST: PR (odds ratio: 5.176, 95% confidence interval: 1.528-17.537, p < 0.001). Conclusions: LEN-TACE sequential therapy may provide more clinical benefits than LEN monotherapy in u-HCC patients who responded to initial LEN treatment. Objective response according to mRECIST to initial LEN is an independent factor contributing to LEN-TACE sequential therapy deep response.

Influence of skeletal muscle volume loss during lenvatinib treatment on prognosis in unresectable hepatocellular carcinoma: a multicenter study in Tohoku, Japan.

Sarcopenia is associated with poor prognosis of patients with hepatocellular carcinoma (HCC). We investigated the association of skeletal muscle volume (SMV) and its change in HCC patients taking lenvatinib. In 130 HCC patients, psoas mass index (PMI) was calculated as the left-right sum of the major × minor axis of psoas muscle at the third lumbar vertebra, divided by height squared. Patients were classified into two groups (low and normal PMI) based on indices of < 6.0 cm2/m2 for man and < 3.4 cm2/m2 for women. Change in PMI per month during the lenvatinib administration period (ΔPMI/m) was calculated; and patients were classified into two groups (severe and mild atrophy) based on the ΔPMI/m rate, as ≥ 1% or < 1%, respectively. There was no significant difference in Overall survival (OS) between the low and normal PMI groups at the start of lenvatinib administration. OS was significantly lower in the severe atrophy group than in the mild atrophy group (median; 15.2 vs. 25.6 months, P = 0.005). Multivariate analysis revealed a significant association of severe atrophy with OS (hazard ratio 1.927, P = 0.031). Progressive loss of SMV is a strong predictor of poor prognosis in HCC patients taking lenvatinib.

Epidemiology and surveillance of autoimmune hepatitis in Asia.

Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease that mainly injures the hepatocytes. The autoimmune disease might be involved in its aetiology, but this remains to be confirmed. Recently epidemiological studies of AIH in Asia have been broadly conducted, revealing characteristics and management of AIH patients in Asia. In East Asia, most AIH patients are type 1, and type 2 AIH is very rare. However, type 2 AIH in South Asia is as frequent as in Europe and the USA. HLA-DR4 is associated with the characteristics of type 1 AIH in East Asia, whereas HLA-DR3 occurs in AIH patients from South Asia. AIH prevalence worldwide is increasing, and several studies have reported a prevalence of 19.44, 22.80 and 12.99 per 100 000 people in Europe, the USA and Asia respectively. A meta-analysis of studies on AIH showed similar annual incidence rates for all regions, with 1.31, 1.37 and 1.00 per 100 000 people in Asia, Europe and the USA respectively. The increase in the rates could be attributable to the increased awareness of disease concepts and diagnosis. In South Asia, most cases were diagnosed as AIH only after having progressed to cirrhosis, which may cause a higher mortality rate in South Asia than in East Asia. Therefore, the early diagnosis and treatment of AIH patients can improve the current situation in Asia.

Environmental factors, medical and family history, and comorbidities associated with primary biliary cholangitis in Japan: a multicenter case-control study.

BACKGROUND: Primary biliary cholangitis (PBC) is considered to be caused by the interaction between genetic background and environmental triggers. Previous case-control studies have indicated the associations of environmental factors (tobacco smoking, a history of urinary tract infection, and hair dye) use with PBC. Therefore, we conducted a multicenter case-control study to identify the environmental factors associated with the development of PBC in Japan. METHODS: From 21 participating centers in Japan, we prospectively enrolled 548 patients with PBC (male/female = 78/470, median age 66), and 548 age- and sex-matched controls. These participants completed a questionnaire comprising 121 items with respect to demographic, anthropometric, socioeconomic features, lifestyle, medical/familial history, and reproductive history in female individuals. The association was determined using conditional multivariate logistic regression analysis. RESULTS: The identified factors were vault toilet at home in childhood [odds ratio (OR), 1.63; 95% confidence interval (CI), 1.01-2.62], unpaved roads around the house in childhood (OR, 1.43; 95% CI, 1.07-1.92), ever smoking (OR, 1.70; 95% CI, 1.28-2.25), and hair dye use (OR, 1.57; 95% CI, 1.15-2.14) in the model for lifestyle factors, and a history of any type of autoimmune disease (OR, 8.74; 95% CI, 3.99-19.13), a history of Cesarean section (OR, 0.20; 95% CI, 0.077-0.53), and presence of PBC in first-degree relatives (OR, 21.1; 95% CI, 6.52-68.0) in the model for medical and familial factors. CONCLUSIONS: These results suggest that poor environmental hygiene in childhood (vault toilets and unpaved roads) and chronic exposure to chemicals (smoking and hair dye use) are likely to be risk factors for the development of PBC in Japan.

XIAP Knockdown in Alcohol-Associated Liver Disease Models Exhibits Divergent in vitro and in vivo Phenotypes Owing to a Potential Zonal Inhibitory Role of SMAC.

Alcohol-associated liver disease (ALD) has been recognized as the most common cause of advanced liver disease worldwide, though mechanisms of pathogenesis remain incompletely understood. The X-linked inhibitor of apoptosis (XIAP) protein was originally described as an anti-apoptotic protein that directly binds and inhibits caspases-3, 7, and 9. Here, we investigated the function of XIAP in hepatocytes in vitro using gain and loss-of-function approaches. We noted an XIAP-dependent increase in caspase activation as well as increased inflammatory markers and pro-inflammatory EV release from hepatocytes in vitro. Primary hepatocytes (PMH) from Xiap Alb.Cre and Xiap loxP mice exhibited higher cell death but surprisingly, lower expression of inflammation markers. Conditioned media from these isolated Xiap deleted PMH further decrease inflammation in bone marrow-derived macrophages. Also, interestingly, when administered an ethanol plus Fas-agonist-Jo2 model and an ethanol plus CCl4 model, these animals failed to develop an exacerbated disease phenotype in vivo. Of note, neither Xiap Alb . Cre nor Xiap AAV8.Cre mice presented with aggravated liver injury, hepatocyte apoptosis, liver steatosis, or fibrosis. Since therapeutics targeting XIAP are currently in clinical trials and caspase-induced death is very important for development of ALD, we sought to explore the potential basis of this unexpected lack of effect. We utilized scRNA-seq and spatially reconstructed hepatocyte transcriptome data from human liver tissue and observed that XIAP was significantly zonated, along with its endogenous inhibitor second mitochondria-derived activator of caspases (SMAC) in periportal region. This contrasted with pericentral zonation of other IAPs including cIAP1 and Apollon as well as caspases 3, 7, and 9. Thus providing a potential explanation for compensation of the effect of Xiap deletion by other IAPs. In conclusion, our findings implicate a potential zonallydependent role for SMAC that prevented development of a phenotype in XIAP knockout mice in ALD models. Targeting SMAC may also be important in addition to current efforts of targeting XIAP in treatment of ALD.

Intrahepatic distribution of nerve fibers and alterations due to fibrosis in diseased liver.

Autonomic nerve fibers in the liver are distributed along the portal tract, being involved in the regulation of blood flow, bile secretion and hepatic metabolism, thus contributing to systemic homeostasis. The present study investigated changes in hepatic nerve fibers in liver biopsy specimens from patients with normal liver, viral hepatitis and non-alcoholic steatohepatitis, in relation to clinical background. The areal ratio of nerve fibers to the total portal area was automatically calculated for each sample. The nerve fiber areal ratios (NFAR) for total nerve fibers and sympathetic nerve fibers were significantly lower in liver affected by chronic hepatitis, particularly viral hepatitis, and this was also the case for advanced liver fibrosis. However, the degree of inflammatory activity did not affect NFAR for either whole nerves or sympathetic nerves. Comparison of samples obtained before and after antiviral treatment for HCV demonstrated recovery of NFAR along with improvement of liver fibrosis.

A machine learning-based treatment prediction model using whole genome variants of hepatitis C virus.

In recent years, the development of diagnostics using artificial intelligence (AI) has been remarkable. AI algorithms can go beyond human reasoning and build diagnostic models from a number of complex combinations. Using next-generation sequencing technology, we identified hepatitis C virus (HCV) variants resistant to directing-acting antivirals (DAA) by whole genome sequencing of full-length HCV genomes, and applied these variants to various machine-learning algorithms to evaluate a preliminary predictive model. HCV genomic RNA was extracted from serum from 173 patients (109 with subsequent sustained virological response [SVR] and 64 without) before DAA treatment. HCV genomes from the 109 SVR and 64 non-SVR patients were randomly divided into a training data set (57 SVR and 29 non-SVR) and a validation-data set (52 SVR and 35 non-SVR). The training data set was subject to nine machine-learning algorithms selected to identify the optimized combination of functional variants in relation to SVR status following DAA therapy. Subsequently, the prediction model was tested by the validation-data set. The most accurate learning method was the support vector machine (SVM) algorithm (validation accuracy, 0.95; kappa statistic, 0.90; F-value, 0.94). The second-most accurate learning algorithm was Multi-layer perceptron. Unfortunately, Decision Tree, and Naive Bayes algorithms could not be fitted with our data set due to low accuracy (< 0.8). Conclusively, with an accuracy rate of 95.4% in the generalization performance evaluation, SVM was identified as the best algorithm. Analytical methods based on genomic analysis and the construction of a predictive model by machine-learning may be applicable to the selection of the optimal treatment for other viral infections and cancer.

IRE1A Stimulates Hepatocyte-Derived Extracellular Vesicles That Promote Inflammation in Mice With Steatohepatitis.

BACKGROUND & AIMS: Endoplasmic reticulum to nucleus signaling 1 (ERN1, also called IRE1A) is a sensor of the unfolded protein response that is activated in the livers of patients with nonalcoholic steatohepatitis (NASH). Hepatocytes release ceramide-enriched inflammatory extracellular vesicles (EVs) after activation of IRE1A. We studied the effects of inhibiting IRE1A on release of inflammatory EVs in mice with diet-induced steatohepatitis. METHODS: C57BL/6J mice and mice with hepatocyte-specific disruption of Ire1a (IRE1αΔhep) were fed a diet high in fat, fructose, and cholesterol to induce development of steatohepatitis or a standard chow diet (controls). Some mice were given intraperitoneal injections of the IRE1A inhibitor 4µ8C. Mouse liver and primary hepatocytes were transduced with adenovirus or adeno-associated virus that expressed IRE1A. Livers were collected from mice and analyzed by quantitative polymerase chain reaction and chromatin immunoprecipitation assays; plasma samples were analyzed by enzyme-linked immunosorbent assay. EVs were derived from hepatocytes and injected intravenously into mice. Plasma EVs were characterized by nanoparticle-tracking analysis, electron microscopy, immunoblots, and nanoscale flow cytometry; we used a membrane-tagged reporter mouse to detect hepatocyte-derived EVs. Plasma and liver tissues from patients with NASH and without NASH (controls) were analyzed for EV concentration and by RNAscope and gene expression analyses. RESULTS: Disruption of Ire1a in hepatocytes or inhibition of IRE1A reduced the release of EVs and liver injury, inflammation, and accumulation of macrophages in mice on the diet high in fat, fructose, and cholesterol. Activation of IRE1A, in the livers of mice, stimulated release of hepatocyte-derived EVs, and also from cultured primary hepatocytes. Mice given intravenous injections of IRE1A-stimulated, hepatocyte-derived EVs accumulated monocyte-derived macrophages in the liver. IRE1A-stimulated EVs were enriched in ceramides. Chromatin immunoprecipitation showed that IRE1A activated X-box binding protein 1 (XBP1) to increase transcription of serine palmitoyltransferase genes, which encode the rate-limiting enzyme for ceramide biosynthesis. Administration of a pharmacologic inhibitor of serine palmitoyltransferase to mice reduced the release of EVs. Levels of XBP1 and serine palmitoyltransferase were increased in liver tissues, and numbers of EVs were increased in plasma, from patients with NASH compared with control samples and correlated with the histologic features of inflammation. CONCLUSIONS: In mouse hepatocytes, activated IRE1A promotes transcription of serine palmitoyltransferase genes via XBP1, resulting in ceramide biosynthesis and release of EVs. The EVs recruit monocyte-derived macrophages to the liver, resulting in inflammation and injury in mice with diet-induced steatohepatitis. Levels of XBP1, serine palmitoyltransferase, and EVs are all increased in liver tissues from patients with NASH. Strategies to block this pathway might be developed to reduce liver inflammation in patients with NASH.

Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptor Deficiency Promotes the Ductular Reaction, Macrophage Accumulation, and Hepatic Fibrosis in the Abcb4-/- Mouse.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL; TNFSF10) receptor (TR) is a pro-apoptotic receptor whose contribution to chronic cholestatic liver disease is unclear. Herein, we examined TRAIL receptor signaling in a mouse model of cholestatic liver injury. TRAIL receptor-deficient (Tnsf10 or Tr-/-) mice were crossbred with ATP binding cassette subfamily B member 4-deficient (Abcb4-/-, alias Mdr2-/-) mice. Male and female wild-type, Tr-/-, Mdr2-/-, and Tr-/-Mdr2-/- mice were assessed for liver injury, fibrosis, and ductular reactive (DR) cells. Macrophage subsets were examined by high-dimensional mass cytometry (time-of-flight mass cytometry). Mdr2-/- and Tr-/-Mdr2-/- mice had elevated liver weights and serum alanine transferase values. However, fibrosis was primarily periductular in Mdr2-/- mice, compared with extensive bridging fibrosis in Tr-/-Mdr2-/- mice. DR cell population was greatly expanded in the Tr-/-Mdr2-/- versus Mdr2-/- mice. The expanded DR cell population in Tr-/-Mdr2-/- mice was due to decreased cell loss by apoptosis and not enhanced proliferation. As assessed by time-of-flight mass cytometry, total macrophages were more abundant in Tr-/-Mdr2-/- versus Mdr2-/- mice, suggesting the DR cell population promotes macrophage-associated hepatic inflammation. Inhibition of monocyte-derived recruited macrophages using the CCR2/CCR5 antagonist cenicriviroc in the Mdr2-/- mice resulted in further expansion of the DR cell population. In conclusion, genetic deletion of TRAIL receptor increased the DR cell population, macrophage accumulation, and hepatic fibrosis in the Mdr2-/- model of cholestasis.

Targeted Apoptosis of Ductular Reactive Cells Reduces Hepatic Fibrosis in a Mouse Model of Cholestasis.

BACKGROUND AND AIMS: In cholestatic liver diseases, ductular reactive (DR) cells extend into the hepatic parenchyma and promote inflammation and fibrosis. We have previously observed that multidrug-resistant 2 (Mdr2-/- ) double knockout (DKO) mice lacking tumor necrosis factor-related apoptosis-inducing ligand receptor (Tr-/- ) display a more extensive ductular reaction and hepatic fibrosis compared to Mdr2-/- mice. This observation suggests that the magnitude of the DR-cell population may be regulated by apoptosis. APPROACH AND RESULTS: To examine this concept, we cultured epithelial cell adhesion molecule-positive reactive cholangioids (ERCs) obtained from wild-type (WT), Tr-/- , Mdr2-/- and DKO mice. Single-cell transcriptomics and immunostaining of both WT and DKO ERCs confirmed their DR-cell phenotype. Moreover, DKO ERCs displayed a unique translational cluster with expression of chemokines, indicating a reactive state. Incubation with the myeloid cell leukemia 1 (MCL1) inhibitor S63845, a proapoptotic BH3-mimetic therapy, significantly decreased DKO and Mdr2-/- ERC viability compared to WT. Intravenous administration of S63845 significantly reduced the DR-cell population and markers of inflammation and liver fibrosis in Mdr2-/- and DKO mice. Furthermore, DKO mice treated with S63845 displayed a significant decrease in hepatic B lymphocytes compared to untreated mice as assessed by high-definition mass cytometry by time-of-flight. Coculture of bone marrow-derived macrophages with ERCs from DKO mouse livers up-regulated expression of the B cell-directed chemokine (C-C motif) ligand 5. Finally, DR cells were noted to be primed for apoptosis with Bcl-2 homologous antagonist/killer activation in vitro and in vivo in primary sclerosing cholangitis liver specimens. CONCLUSIONS: DR cells appear to play a key role in recruiting immune cells to the liver to actively create an inflammatory and profibrogenic microenvironment. Pharmacologic targeting of MCL1 in a mouse model of chronic cholestasis reduces DR-cell and B-cell populations and hepatic fibrosis.

Activated cholangiocytes release macrophage-polarizing extracellular vesicles bearing the DAMP S100A11.

In mouse models of biliary tract diseases, macrophages are recruited to the periductal milieu and promote injury and cholestasis. Although cell necrosis with release of biomolecules termed damage-associated molecular patterns (DAMPs) promotes recruitment and activation of macrophages, necrosis was not observed in these studies. Because extracellular vesicles (EVs) are important in cell-to-cell communication, we postulated that activated cholangiocytes may release EVs containing DAMPs as cargo. Both the human (NHC) and mouse cholangiocyte (603B) cell lines display constitutive activation with mRNA expression of chemokines. Proteomic analysis revealed that EVs from both cell lines contained the DAMP S100A11, a ligand for the receptor for advanced glycation end products (RAGE). Bone marrow-derived macrophages (BMDM) incubated with EVs derived from the mouse 603B cell line increased mRNA expression of proinflammatory cytokines. Genetic or pharmacologic inhibition of RAGE reduced BMDM expression of proinflammatory cytokines treated with EVs. RAGE signaling resulted in activation of the canonical NF-κB pathway, and consistently, proinflammatory cytokine expression was blunted by the IKKα/ß inhibitor TPCA-1 in BMDM incubated with EVs. We also demonstrated that primary mouse cholangiocyte-derived organoids express chemokines indicating cholangiocyte activation, release EVs containing S100A11, and stimulate proinflammatory cytokine expression in BMDM by a RAGE-dependent pathway. In conclusion, these observations identify a non-cell death mechanism for cellular release of DAMPs by activated cholangiocytes, namely by releasing DAMPs as EV cargo. These data also suggest RAGE inhibitors may be salutary in macrophage-associated inflammatory diseases of the bile ducts.

Incidence of development of hepatocellular carcinoma in Japanese patients infected with hepatitis B virus is equivalent between genotype B and C in long term.

Hepatitis B virus (HBV) genotypes B (HBV/B) and C (HBV/C) are the most prevalent genotypes among Japanese patients with hepatitis. Reportedly, HBV/C infection has been associated with more severe disease progression, manifesting as developing cirrhosis and hepatocellular carcinoma (HCC), than HBV/B infection. However, no long-term studies have examined the development of HCC in HBV/B-infected patients in Japan. The aims of our study were to compare the incidence of HCC in HBV/B- or HBV/C-infected patients. A total of 241 patients were followed up among 295 hepatitis B surface antigen (HBsAg)-positive carriers. Genotypes of HBV were A in 1% (4/295), B in 61% (179/295), C in 37% (110/295) and D in 1% (2/295) patients, and 96% of HBV/B were infected with subgenotype Bj. The mean age at HCC diagnosis was significantly higher in HBV/B than in HBV/C (67.0 ± 10.0 vs 57.7 ± 8.0 years, P < 0.001). The value of FIB-4 index was significantly higher in HBV/B than in HBV/C (P < 0.01). The rate of HCC was higher in HBV/C than in HBV/B, and a significant difference was observed until the 20-year observation period (P = 0.048). However, thereafter, HCC associated with HBV/B increased, and no significant difference was observed between HBV/B and HBV/C. HCC development was consistently observed even in HBV/B infection, especially among elderly patients with advanced fibrosis compared with HBV/C. HBV/B-infected patients developed HCC later in life, and in the long term, we found no differences in incidence of HCC development rates between these two genotypes.

MiR-139-5p is associated with inflammatory regulation through c-FOS suppression, and contributes to the progression of primary biliary cholangitis.

Primary biliary cholangitis (PBC) is a chronic cholestatic liver disease characterized pathologically by destruction of intrahepatic bile ducts. PBC is largely classified into three subtypes based on clinical course: (i) gradually progressive, (ii) portal hypertension, and (iii) hepatic failure. Previous studies have indicated that serum levels of the pro-inflammatory cytokine TNF-α, is elevated in PBC patients with fibrosis. Although the severity of cholangitis might also be related to the PBC subtype, its etiology has been unclear. Several studies have shown that microRNAs (miRNAs) demonstrate specific expression patterns in various diseases. In the present study, we evaluated miRNA expression patterns among the PBC subtypes using comprehensive deep sequencing. We also carried out histologic examination by laser capture microdissection and investigated how the identified miRNAs were involved in PBC clinical progression using the miRNA transfection method. On average, ~11 million 32-mer short RNA reads per sample were obtained, and we found that the expression levels of 97 miRNAs differed significantly among the four groups. Heat mapping demonstrated that the miRNA profiles from hepatic failure and portal hypertension type were clustered differently from those of the gradually progressive type and controls. Furthermore, we focused on miR-139-5p, which has an adequate number of total short reads. Quantitative reverse transcription PCR showed that miR-139-5p was significantly downregulated in clinically advanced PBC. Also, examination of liver tissues demonstrated that the expression of lymphocyte-derived miR-139-5p was significantly higher in hepatocytes. In vitro, the level of TNF-α was significantly elevated in supernatant of cells with upregulation of miR-139-5p. Furthermore, c-FOS gene transcription was repressed. Thus, we have demonstrated a novel inflammation-regulatory mechanism involving TNF-α and c-FOS transcription through miR-139-5p in the NF-κB signaling pathway. We conclude that the specific miRNA miR-139-5p might be involved in the pathogenesis of PBC, especially during clinical progression.

Serum metabolome profiles characterized by patients with hepatocellular carcinoma associated with hepatitis B and C.

AIM: To clarify the characteristics of metabolite profiles in virus-related hepatocellular carcinoma (HCC) patients using serum metabolome analysis. METHODS: The serum levels of low-molecular-weight metabolites in 68 patients with HCC were quantified using capillary electrophoresis chromatography and mass spectrometry. Thirty and 38 of the patients suffered from hepatitis B virus-related HCC (HCC-B) and hepatitis C virus-related HCC (HCC-C), respectively. RESULTS: The main metabolites characteristic of HCC were those associated with glutathione metabolism, notably 13 γ-glutamyl peptides, which are by-products of glutathione induction. Two major profiles, i.e., concentration patterns, of metabolites were identified in HCC patients, and these were classified into two groups: an HCC-B group and an HCC-C group including some of the HCC-B cases. The receiver operating characteristic curve for the multiple logistic regression model discriminating HCC-B from HCC-C incorporating the concentrations of glutamic acid, methionine and γ-glutamyl-glycine-glycine showed a highly significant area under the curve value of 0.94 (95%CI: 0.89-1.0, P < 0.0001). CONCLUSION: The serum levels of γ-glutamyl peptides, as well as their concentration patterns, contribute to the development of potential biomarkers for virus-related HCC. The difference in metabolite profiles between HCC-B and HCC-C may reflect the respective metabolic reactions that underlie the different pathogeneses of these two types of HCC.

Animal models of primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is characterized histologically by the presence of chronic non-suppurative destructive cholangitis of the small interlobular bile duct, leading to chronic progressive cholestasis. Most PBC patients are asymptomatic and have a reasonable prognosis, but a few develop esophageal varices or jaundice, rapidly leading to liver failure within a short period. As multiple factors appear to be involved in the onset of PBC, its clinical course may be complicated. Therefore, the use of an animal model would be valuable for clarifying the pathogenesis of PBC. Here, we review recent data of selected PBC models, particularly spontaneous models, xenobiotic immunized models, and infection-triggered models. There are a number of spontaneous models: the NOD.c3c4, dominant-negative TGF-ß receptor II, IL-2Rα-/-, Scurfy, and Ae2a,b-/- mice. These animal models manifest distinct clinical and immunological features similar, but also often different, from those of human PBC. It is clear that a combination of genetic predisposition, environmental factors, and immunological dysfunction contribute to the pathogenesis of PBC. The diverse clinical course and complexity of the immunological mechanisms of PBC cannot be fully recapitulated solely any single animal model. The challenge remains to develop a progressive PBC disease model that exhibits fibrosis, and ultimately hepatic failure.

Epiregulin promotes the emergence and proliferation of adult liver progenitor cells.

We have previously reported that epiregulin is a growth factor that seems to act on liver progenitor cells (LPCs) during liver regeneration. However, the relationship between epiregulin and LPCs has remained unclear. The aim of the present study was to clarify the role of epiregulin during liver regeneration. The serum levels of epiregulin in patients with acute liver failure were examined. A liver injury model was developed using mice fed a diet containing 0.1% 3.5-diethoxycarbonyl-1.4-dihydrocollidine (DDC) to induce LPCs. We then evaluated the expression of epiregulin and LPCs in these mice. The proliferation of epithelial cell adhesion molecule + LPCs cultured with epiregulin was examined in vitro, and finally epiregulin was overexpressed in mouse liver. In patients with acute liver failure, serum epiregulin levels were elevated significantly. In DDC mice, LPCs emerged around the portal area. Epiregulin was also detected around the portal area during the course of DDC-induced liver injury and was partially coexpressed with Thy1. Serum epiregulin levels in DDC mice were also significantly elevated. Recombinant epiregulin augmented the proliferative capacity of the LPCs in a dose-dependent manner. In mice showing overexpression of epiregulin, the expression of PCNA on hepatocytes was increased significantly. Finally, LPCs emerged around the portal area after epiregulin gene delivery. We concluded that epiregulin promotes the proliferation of LPCs and DNA synthesis by hepatocytes and is upregulated in the serum of patients with liver injury. Furthermore, induction of epiregulin leads to the appearance of LPCs. Epiregulin would be a useful biomarker of liver regeneration.