Vulnerability to recurrent episodes of acute decompensation/acute-on-chronic liver failure characterizes those triggered by indeterminate precipitants in patients with liver cirrhosis.

BACKGROUND: Acute decompensation (AD) of liver cirrhosis (LC) and subsequent acute-on-chronic liver failure (ACLF) are fatal and impair quality of life. Insufficient knowledge of the highly heterogeneous natural history of LC, including decompensation, re-compensation, and possible recurrent decompensation, hinders the development and application of novel therapeutics. Approximately 10%-50% of AD/ACLF is reported to be precipitated by any indeterminate (unidentifiable, cryptogenic, or unknown) acute insults; however, its clinical characteristics are unclear. METHODS: We conducted a single-center observational study of 2165 consecutively admitted patients with LC from January 2012 to December 2019. A total of 466 episodes of AD/ACLF in 285 patients, including their 285 first indexed AD/ACLF, were extracted for analysis. Stratified analyses of different acute precipitants, classified as indeterminate (AD/ACLFIND), bacterial infection (AD/ACLFBAC), gastrointestinal bleeding, active alcoholism, and miscellaneous, were performed. RESULTS: AD/ACLFIND was the leading acute precipitant (28%), followed by AD/ACLFBAC (23%). AD/ACLFIND showed better survival outcomes than AD/ACLFBAC (P = 0.03); however, hyperbilirubinemia, hyponatremia, or leukocytosis significantly and uniquely characterized subgroups of AD/ACLFIND with comparable or even worse survival outcomes than those of AD/ACLFBAC. Patients with subsequent AD/ACLF significantly tended to suffer from AD/ACLF with any organ failure in AD/ACLFIND but not in AD/ACLFBAC (P = 0.004, for trend). In competing risk analysis, patients with AD/ACLFIND were significantly more vulnerable to suffer from recurrent episodes of AD/ACLF within 180 days, compared to those triggered by other precipitants (P = 0.04). CONCLUSIONS: AD/ACLFIND, the leading acute precipitant, also plays a role in subsequent AD/ACLF. An abruptly exacerbating, remitting, and relapsing nature of systemic inflammation underlying AD/ACLF may also be useful for risk estimation.

Establishment and Long-Term Culture of Organoids Derived from Human Biliary Tract Carcinoma.

This protocol is a procedure for establishment and culture of cancer and non-cancer organoids using tissues from biliary tract carcinoma (BTC) patients. These BTC organoids can be used for various biological analyses and drug screening. One challenge in establishing and culturing BTC organoids is non-cancer cells contaminating surgically resected tumor tissues form organoids concurrently with cancer organoids. Careful validation that the established organoids are cancer-derived is important. For complete details on the use and generation of this protocol, please refer to Saito et al. (2019) in the journal Cell Reports.

Late-onset acute liver failure due to Wilson's disease managed by plasmapheresis and hemodiafiltration successfully serving as a bridge for deceased donor liver transplantation: a case report and literature review.

Late-onset acute liver failure due to Wilson's disease (WD-ALF) is rare. A 44-year-old female patient presenting acute hepatic decompensation with extreme coagulopathy was transferred to our hospital for evaluation for liver transplantation (LT). Alveolar hemorrhage and Coombs-negative acute hemolysis occurred during workup. Mechanical ventilation, plasmapheresis, and hemodiafiltration with zinc and chelation were started immediately before placing the patient on the waitlist for deceased donor LT (DDLT), with a tentative diagnosis of WD-ALF using the Leipzig score and quick diagnostic criteria suggested by the Acute Liver Failure Study Group Registry. The peak MELD score was 40, and the revised version of King's score for WD was 13. Serum free copper levels and the patient's overall general condition were stabilized with artificial support systems, although triphasic wave on electroencephalogram and liver atrophy were noted. She successfully underwent emergent DDLT approximately 2 weeks after suffering from acute hemolysis and survived. The genetic tests confirmed mutations at 2 loci in the ATP7B gene and, therefore, the diagnosis of WD. This is the first and oldest patient reported in Japan to present late-onset WD-ALF that was successfully treated with emergent DDLT.

CLIF-C Organ Failure Score and Liver Volume Predict Prognosis in Steroid-Treated Severe Acute Autoimmune Hepatitis.

Controversies and debates remain regarding the best management of severe acute-onset autoimmune hepatitis (SA-AIH) due to the lack of useful outcome or complication prediction systems. We conducted this clinical practice-based observational study to clarify whether Chronic Liver Failure Consortium Organ Failure scores (CLIF-C OFs) and the computed tomography-derived liver volume to standard liver volume (CTLV/SLV) ratio at admission to a tertiary transplant center can predict outcomes and complications due to infection. Thirty-four consecutive corticosteroid-treated patients with SA-AIH from 2007 to 2018 were included. Severe hepatitis was defined as an international normalized ratio (of prothrombin time) over 1.3 any time before admission. Of the 34 corticosteroid-treated patients with SA-AIH inclusive of 25 (73.5%) acute liver failure cases, transplant-free survival was observed in 24 patients (70.6%). Any infection was noticed in 10 patients (29.4%). CLIF-C OFs, at the cutoff of 9, significantly predicted survival (P = 0.0002, log-rank test), outperformed the Model for End-stage Liver Disease system in predicting outcome (P = 0.0325), and significantly discriminated between liver transplant and death in a competing risk analysis. SA-AIH was characterized as having decreased CTLV/SLV, which was also predictive of survival (P < 0.0001). Interestingly, CLIF-C OFs, especially the subscores for respiratory dysfunction, also predicted infection (P = 0.007). Conclusion: In corticosteroid-treated patients with SA-AIH, CLIF-C OFs and CTLV/SLV ratios predicted both survival outcome and complications due to infection. Further investigation is warranted to determine whether making decisions based on CLIF-C OFs or CTLV/SLV ratios is useful.

Clinical implications with tolvaptan on monitored bioimpedance-defined fluid status in patients with cirrhotic ascites: an observational study.

BACKGROUND: Prognostic value or clinical implications of fluid status monitoring in liver cirrhosis are not fully elucidated. Tolvaptan, an orally available, selective vasopressin V2-receptor antagonist approved for hyponatremia in the United States and European Union. It is also used for cirrhotic ascites at a relatively low dose (3.75 mg to 7.5 mg) in Japan, exerts its diuretic function by excreting electrolyte-free water. We hypothesized that bioimpedance-defined dynamic changes in fluid status allow prediction of response of V2 antagonism and survival in cirrhotic patients. METHODS: In this prospective observational study, 30 patients with decompensated liver cirrhosis who were unresponsive to conventional diuretics were enrolled. Detailed serial changes of body composition that were assessed by using non-invasive bioimpedance analysis (BIA) devices, along with biochemical studies, were monitored at 5 time points. RESULTS: Sixteen patients were classified as short-term responders (53%). Rapid and early decrease of BIA-defined intracellular water, as soon as 6 h after the first dose (ΔICWBIA%-6 h), significantly discriminated responders from non-responders (AUC = 0.97, P < 0.0001). ΔICWBIA%-6 h was highly correlated with the change of BIA-derived phase angle of trunk, e.g. reduced body reactance operated at 50 kHz after 24 h of the first dose of tolvaptan. Lower baseline blood urea nitrogen and lower serum aldosterone were predictive of a rapid and early decrease of ICWBIA. A rapid and early decrease of ICWBIA in response to tolvaptan was also predictive of a better transplant-free survival. CONCLUSIONS: BIA-defined water compartment monitoring may help predict short-term efficacy and survival in decompensated cirrhotic patients treated with tolvaptan.

A prospective trial of vaccine to prevent hepatitis B virus reactivation after hematopoietic stem cell transplantation.

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

The Effects of Continuous and Withdrawal Voluntary Wheel Running Exercise on the Expression of Senescence-Related Genes in the Visceral Adipose Tissue of Young Mice.

Obesity has become a global medical problem. The upregulation of senescence-related markers in adipose tissue may cause impairment of adipose tissue and disorders of systemic metabolism. Weight control through diet has been found to ameliorate senescence in the adipose tissue. Exercise is also important in maintaining a healthy lifestyle, however, very few researchers have examined the relationship between senescence-related markers in adipose tissue. Dietary restriction is also reported to have a legacy effect, wherein the effects are maintained for some periods after the termination of the intervention. However, very few researchers have examined the relationship between exercise and senescence-related markers in adipose tissue. Besides, there is no study on the long-term effects of exercise. Hence, we investigated whether the exercise could change the expression of senescence-related genes in the visceral adipose tissue of young mice and whether there was a legacy effect of exercise for 10 weeks after the termination of exercise. Four-week-old male ICR mice were assigned to one of the three groups: 20 weeks of sedentary condition, 20 weeks of voluntary wheel running exercise, or 10 weeks of exercise followed by 10 weeks of sedentary condition. The mice showed decreased expression in genes related to senescence and senescence-associated secretory phenotype, such as p53, p16, and IL-6, in the visceral adipose tissue in response to exercise. These effects were maintained for 10 weeks after the mice stopped exercising. Our study is the first report that exercise reduces the expression of senescence-related genes in the visceral adipose tissue of young mice, and that exercise causes the legacy effect.

Glucose Depletion Enhances the Stem Cell Phenotype and Gemcitabine Resistance of Cholangiocarcinoma Organoids through AKT Phosphorylation and Reactive Oxygen Species.

Cancer cells are strongly dependent on the glycolytic pathway for generation of energy even under aerobic condition through a phenomenon known as the Warburg effect. Rapid proliferation of cancer cells is often accompanied by high glucose consumption and abnormal angiogenesis, which may lead to glucose depletion. In the present study, we investigated how cholangiocarcinoma cells adapt to glucose depletion using a 3D organoid culture system. We cultured organoids derived from cholangiocarcinoma under glucose-free condition and investigated cell proliferation, expression of stem cell markers and resistance to gemcitabine. Cholangiocarcinoma organoids cultured under glucose-free condition showed reduced proliferation but were able to survive. We also observed an increase in the expression of stem cell markers including LGR5 and enhancement of stem cell phenotypic characteristics such as resistance to gemcitabine through AKT phosphorylation and reactive oxygen species. These findings indicate that cholangiocarcinoma cells are able to adapt to glucose depletion through enhancement of their stem cell phenotype in response to changes in microenvironmental conditions.

Generation of functional human hepatocytes in vitro: current status and future prospects.

Liver and hepatocyte transplantation are the only effective therapies for late-stage liver diseases, in which the liver loses its regenerative capacity. However, there is a shortage of donors. As a potential alternative approach, functional hepatocytes were recently generated from various cell sources. Analysis of drug metabolism in the human liver is important for drug development. Consequently, cells that metabolize drugs similar to human primary hepatocytes are required. This review discusses the current challenges and future perspectives concerning hepatocytes and hepatic progenitor cells that have been reprogrammed from various cell types, focusing on their functions in transplantation models and their ability to metabolize drugs.

Establishment of Patient-Derived Organoids and Drug Screening for Biliary Tract Carcinoma.

Biliary tract carcinomas (BTCs) are among the most aggressive malignancies and have a poor prognosis. Here, we successfully established organoid lines derived from intrahepatic cholangiocarcinoma, gallbladder cancer, and neuroendocrine carcinoma of the ampulla of Vater. These organoids derived from BTCs were cultured stably for >1 year and closely recapitulated the histopathology, gene expression, and genetic alterations evident in the primary tumors. Gene expression profiling of the organoids revealed that SOX2 could be a potential prognostic biomarker for patients with BTC. We screened a compound library consisting of drugs used clinically for their ability to suppress organoids derived from BTCs and found that the antifungal drugs amorolfine and fenticonazole significantly suppressed the growth of organoids derived from BTCs with minimal toxicity to normal biliary epithelial cells. Patient-derived organoids may be a powerful research tool for the clarification of molecular pathogenesis and the discovery of biomarkers and therapeutic drugs for refractory cancers.

Erratum: Publisher Correction: Epigenetic silencing of Lgr5 induces senescence of intestinal epithelial organoids during the process of aging.

[This corrects the article DOI: 10.1038/s41514-018-0031-5.].

Vonoprazan-Based Third-Line Therapy Has a Higher Eradication Rate against Sitafloxacin-Resistant Helicobacter pylori.

Eradication of Helicobacter pylori (H. pylori) is an effective strategy for preventing various gastrointestinal diseases such as gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma. However, the eradication success rate is decreasing because of a recent increase in drug-resistant strains of H. pylori. Here, we evaluated the success rate of eradication therapy with vonoprazan (VPZ), a new potassium-competitive acid blocker, against drug-resistant H. pylori. In total, 793 patients who received H. pylori eradication therapy were investigated retrospectively. All underwent esomeprazole (EPZ)-based triple therapy (n = 386) or VPZ-based triple therapy (n = 407) for first-, second- and third-line H. pylori eradication for 7 days. The overall success rates of first- and third-line H. pylori eradication were significantly higher for VPZ-based triple therapy (88.4% and 93.0%, respectively, per protocol (PP)) than for EPZ-based triple therapy (69.5% and 56.5%, respectively, PP). Moreover, the success rates of first- and third-line eradication of clarithromycin (CLR)- and sitafloxacin (STFX)-resistant H. pylori were significantly higher for VPZ-based triple therapy (72.0% and 91.7%, PP) than for EPZ-based triple therapy (38.5% and 20.0%, PP). In addition, patient age did not affect the eradication rate of VPZ-based first-line therapy, whereas the success rate of EPZ-based therapy was lower in patients under 65 years of age. Our results clearly demonstrated that VPZ-based therapy achieved a higher eradication rate even against CLR- and STFX-resistant H. pylori, and that patient age did not affect the eradication rate of VPZ-based therapy. These findings suggest that dual therapy using VPZ and amoxicillin may be sufficient for standard H. pylori eradication, and may thus also be beneficial for avoiding antibiotic misuse.

Epigenetic silencing of Lgr5 induces senescence of intestinal epithelial organoids during the process of aging.

To understand the molecular features underlying stem cell aging, we established intestinal epithelial organoids derived from both young and aged mice and investigated alterations in their senescence and epigenetic status. Senescence-related changes including accumulation of senescence-associated ß-galactosidase and up-regulation of Cdkn1a (p21) by DNA demethylation were observed in intestinal epithelial organoids derived from aged mice. We also demonstrated that the important stem cell marker Lgr5 was epigenetically silenced by trimethylation of histone H3 lysine 27, inducing suppression of Wnt signaling and a decrease of cell proliferation in organoids from aged mice. We further treated intestinal epithelial organoids from aged mice with nicotinamide mononucleotide (NMN), a key NAD+ intermediate. As a result, the organoids showed a higher NAD+ level, increased cell proliferative ability, activation of Lgr5 and suppression of senescence-associated genes, indicating that treatment with NMN could ameliorate senescence-related changes in intestinal epithelia. These findings suggest that organoids derived from aged animals could be a powerful research tool for investigating the molecular mechanisms underlying stem cell aging and for development of some form of anti-aging intervention, thus contributing to prolongation of healthy life expectancy.

Liver Fibrosis Markers Improve Prediction of Outcome in Non-Acetaminophen-Associated Acute Liver Failure.

A prognostic system for acute liver failure (ALF) with a higher predictive value is urgently needed. The role of extracellular matrix (ECM) remodeling in ALF has not been fully elucidated. We hypothesized that serologic fibrosis markers, which reflect ECM remodeling, are predictive of ALF outcome at first presentation. This observational study included 110 patients with acute liver dysfunction, of which 73 had non-acetaminophen-associated ALF (NAA-ALF). We evaluated serum levels of hyaluronic acid, 7S domain of type IV collagen (4COL7S), and Wisteria floribunda agglutinin-positive Mac-2-binding protein at first presentation to a tertiary center. Serologic fibrosis markers were significantly higher in NAA-ALF compared with acute hepatitis. Elevated hyaluronic acid and 4COL7S levels at first presentation correlated significantly with worse clinical outcomes. 4COL7S, along with age, ammonia, and the Model for End-Stage Liver Disease (MELD) score, was a significant prognostic factor in multivariate analysis; 4COL7S correlated significantly with coagulopathy, decreased hepatic synthetic functions, advanced hepatic encephalopathy, and liver atrophy and also predicted 180-day transplant-free survival. Cox regression models incorporating 4COL7S with the MELD system had profoundly improved predictive values that significantly surpassed the MELD system alone. Conclusion: Elevation of serologic fibrosis markers reflecting ECM remodeling in NAA-ALF predicted a worse clinical outcome. Incorporation of 4COL7S at first presentation to a transplant center improves the specificity while retaining the sensitivity of the MELD system. External validation of a fibrosis marker as part of a clinical prediction tool in ALF warrants further investigation.

Nrf2-mediated anti-oxidant effects contribute to suppression of non-alcoholic steatohepatitis-associated hepatocellular carcinoma in murine model.

The exact mechanisms of hepatocellular carcinoma development in non-alcoholic steatohepatitis remain unclear. In this study, we used a new class of high-fat diet, which could induce hepatocellular carcinoma development without the use of general chemical carcinogens or knockout mice. We investigated the correlation between hepatocellular carcinoma and oxidative stress/anti-oxidant effects after depletion of the gut microbiota by treatment with antibiotics. Mice fed with the steatohepatitis-inducing high-fat diet (STHD-01) for 41 weeks developed hepatocellular carcinoma. Antibiotic-treatment in mice fed with STHD-01 significantly depleted the gut microbiota and significantly ameliorated liver injury/histology. The tumor numbers of hepatocellular carcinoma were dramatically decreased by the antibiotics-treatment. We analyzed the factors involved in oxidative stress and anti-oxidant effects. Oxidative stress was elevated in mice fed with STHD-01, whereas some anti-oxidant factors were significantly elevated after antibiotics treatment. These results suggest that the gut microbiota is a key factor in improving oxidative stress induced by STHD-01 feeding.

Dual effects of the Nrf2 inhibitor for inhibition of hepatitis C virus and hepatic cancer cells.

BACKGROUND: We previously showed that knockdown of nuclear factor E2-related factor 2 (Nrf2) resulted in suppression of hepatitis C virus (HCV) infection. In this study, whether brusatol, an Nrf2 inhibitor, has dual anti-HCV and anticancer effects was explored. METHODS: The anti-HCV effect of brusatol was investigated by analyzing HCV RNA and proteins in a hepatic cell line persistently-infected with HCV, HPI cells, and by analyzing HCV replication in a replicon-replicating hepatic cell line, OR6 cells. Then, dual anti-HCV and anticancer effects of brusatol and enhancement of the effects by the combination of brusatol with anticancer drugs including sorafenib, which has been reported to have the dual effects, were then investigated. RESULTS: Brusatol suppressed the persistent HCV infection at both the RNA and protein levels in association with a reduction in Nrf2 protein in the HPI cells. Analysis of the OR6 cells treated with brusatol indicated that brusatol inhibited HCV persistence by inhibiting HCV replication. Combination of brusatol with an anticancer drug not only enhanced the anticancer effect but also, in the case of the combination with sorafenib, strongly suppressed HCV infection. CONCLUSIONS: Brusatol has dual anti-HCV and anticancer effects and can enhance the comparable effects of sorafenib. There is therefore the potential for combination therapy of brusatol and sorafenib for HCV-related hepatocellular carcinoma.

Bile acid metabolism regulated by the gut microbiota promotes non-alcoholic steatohepatitis-associated hepatocellular carcinoma in mice.

Gut microbiota plays a significant role in the development of hepatocellular carcinoma (HCC) in non-alcoholic steatohepatitis (NASH). However, understanding of the precise mechanism of this process remains incomplete. A new class steatohepatitis-inducing high-fat diet (HFD), namely STHD-01, can promote the development of HCC without the administration of chemical carcinogens. Using this diet, we comprehensively analyzed changes in the gut microbiota and its metabolic functions during the development of HCC in NASH. Mice fed the STHD-01 developed NASH within 9 weeks. NASH further progressed into HCC by 41 weeks. Treatment with antibiotics significantly attenuated liver pathology and suppressed tumor development, indicating the critical role of the gut microbiota in tumor development in this model. Accumulation of cholesterol and bile acids in the liver and feces increased after feeding the mice with STHD-01. Treatment with antibiotics did not reverse these phenotypes. In contrast, accumulation of secondary bile acids was dramatically reduced after the treatment with antibiotics, suggesting the critical role of the gut microbiota in the conversion of primary bile acids to secondary bile acids. Secondary bile acids such as deoxycholic acid activated the mTOR, pathway in hepatocytes. Activation of mTOR was observed in the liver of mice fed STHD-01, and the activation was reduced when mice were treated with antibiotics. Collectively, bile acid metabolism by the gut microbiota promotes HCC development in STHD-01-induced NASH.

Induction of differentiation of intrahepatic cholangiocarcinoma cells to functional hepatocytes using an organoid culture system.

Intrahepatic cholangiocarcinoma (IHCC) is a highly aggressive malignancy with a poor prognosis. It is thought to originate from cholangiocytes, which are the component cells of intrahepatic bile ducts. However, as patients with viral hepatitis often develop IHCC, it has been suggested that transformed hepatocytes may play a role in IHCC development. To investigate whether IHCC cells can be converted to functional hepatocytes, we established organoids derived from human IHCC and cultured them under conditions suitable for hepatocyte differentiation. IHCC organoids after hepatocyte differentiation acquired functions of mature hepatocytes such as albumin secretion, bile acid production and increased CYP3A4 activity. Studies using a mouse model of IHCC indicate that Wnt3a derived from macrophages recruited upon inflammation in the liver may promote the malignant transformation of hepatocytes to IHCC cells. The results of the present study support the recently proposed hypothesis that IHCC cells are derived from hepatocytes.