Metabolic biomarkers significantly enhance the prediction of HBV-related ACLF occurrence and outcomes.

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) is a clinical syndrome associated with high short-term mortality in patients with chronic liver disease. Chronic hepatitis B is the main cause of ACLF (HBV-ACLF) in China and other Asian countries. To improve disease management and survival for patients with ACLF, we aimed to discover novel biomarkers to enhance HBV-ACLF diagnosis and prognostication. METHODS: We performed a metabolomics profiling of 1,024 plasma samples collected from patients with HBV-related chronic liver disease with acute exacerbation at hospital admission in a multi-year and multi-center prospective study (367 ACLF and 657 non-ACLF). The samples were randomly separated into equal halves as a discovery set and a validation set. We identified metabolites associated with 90-day mortality in the ACLF group and the progression to ACLF within 28 days in the non-ACLF group (pre-ACLF) using statistical analysis and machine learning. We developed diagnostic algorithms in the discovery set and used these to assess the findings in the validation set. RESULTS: ACLF significantly altered the plasma metabolome, particularly in membrane lipid metabolism, steroid hormones, oxidative stress pathways, and energy metabolism. Numerous metabolites were significantly associated with 90-day mortality in the ACLF group and/or pre-ACLF in the non-ACLF group. We developed algorithms for the prediction of 90-day mortality in patients with ACLF (area under the curve 0.87 and 0.83 for the discovery set and validation set, respectively) and the diagnosis of pre-ACLF (area under the curve 0.94 and 0.88 for the discovery set and validation set, respectively). To translate our discoveries into practical clinical tests, we developed targeted assays using liquid chromatography-mass spectrometry. CONCLUSIONS: Based on novel metabolite biomarkers, we established tests for HBV-related ACLF with higher accuracy than existing methods. CLINICAL TRIAL NUMBER: NCT02457637 and NCT03641872. IMPACT AND IMPLICATIONS: Acute-on-chronic liver failure (ACLF) is a clinical syndrome associated with high short-term mortality affecting 25% of patients hospitalized with cirrhosis. Chronic hepatitis B is the main etiology of ACLF in China and other Asian counties. There is currently no effective therapy. Early diagnosis and accurate prognostication are critical for improving clinical outcomes in patients with ACLF. Based on novel metabolite biomarkers, we developed liquid chromatography-mass spectrometry tests with improved accuracy for the early diagnosis and prognostication of HBV-related ACLF. The liquid chromatography-mass spectrometry tests can be implemented in clinical labs and used by physicians to triage patients with HBV-related ACLF to ensure optimized clinical management.

Endoplasmic reticulum stress promotes HBV production by enhancing use of the autophagosome/multivesicular body axis.

BACKGROUND AND AIMS: HBV infection has been reported to trigger endoplasmic reticulum (ER) stress and initiate autophagy. However, how ER stress and autophagy influence HBV production remains elusive. Here, we studied the effect of tunicamycin (TM), an N-glycosylation inhibitor and ER stress inducer, on HBV replication and secretion and examined the underlying mechanisms. APPROACH AND RESULTS: Protein disulfide isomerase (an ER marker), microtubule-associated protein 1 light chain 3 beta (an autophagosome [AP] marker), and sequestosome-1 (a typical cargo for autophagic degradation) expression were tested in liver tissues of patients with chronic HBV infection and hepatoma cell lines. The role of TM treatment in HBV production and trafficking was examined in hepatoma cell lines. TM treatment that mimics HBV infection triggered ER stress and increased AP formation, resulting in enhanced HBV replication and secretion of subviral particles (SVPs) and naked capsids. Additionally, TM reduced the number of early endosomes and HBsAg localization in this compartment, causing HBsAg/SVPs to accumulate in the ER. Thus, TM-induced AP formation serves as an alternative pathway for HBsAg/SVP trafficking. Importantly, TM inhibited AP-lysosome fusion, accompanied by enhanced AP/late endosome (LE)/multivesicular body fusion, to release HBsAg/SVPs through, or along with, exosome release. Notably, TM treatment inhibited HBsAg glycosylation, resulting in impairment of HBV virions' envelopment and secretion, but it was not critical for HBsAg/SVP trafficking in our cell systems. CONCLUSIONS: TM-induced ER stress and autophagic flux promoted HBV replication and the release of SVPs and naked capsids through the AP-LE/MVB axis.

Portal vein thrombosis compromises the performance of MELD and MELD-Na scores in patients with cirrhosis.

BACKGROUND AND AIMS: The accuracy of model for end-stage liver disease (MELD) and MELD with sodium (MELD-Na) scores in reflecting the clinical outcomes of patients with cirrhosis and portal vein thrombosis (PVT) remains unclear. This study aimed to evaluate the performance of scores in predicting 90-day mortality in patients with cirrhosis and PVT. METHODS: Post hoc analysis was performed in two prospective cohorts (NCT02457637 and NCT03641872). The correlation between the MELD/MELD-Na score and 90-day liver transplantation (LT)-free mortality was investigated in patients with cirrhosis with and without PVT. RESULTS: In this study, 2826 patients with cirrhosis were included, and 255 (9.02%) had PVT. The cumulative incidence of 90-day LT-free mortality did not significantly differ between patients with and without PVT (log-rank P = 0.0854). MELD [area under the receiver operating curve (AUROC), 0.649 vs. 0.842; P = 0.0036] and MELD-Na scores (AUROC, 0.691 vs. 0.851; P = 0.0108) were compared in patients with and without PVT, regarding the prediction of 90-day LT-free mortality. In MELD < 15 and MELD-Na < 20 subgroups, patients with PVT had a higher 90-day LT-free mortality than those without PVT (7.91% vs. 2.64%, log-rank P = 0.0011; 7.14% vs. 3.43%, log-rank P = 0.0223), whereas in MELD ≥ 15 and MELD-Na ≥ 20 subgroups, no significant difference was observed between patients with and without PVT. CONCLUSIONS: The performance of MELD and MELD-Na scores in predicting 90-day LT-free mortality of patients with cirrhosis was compromised by PVT. MELD < 15 or MELD-Na < 20 may underestimate the 90-day LT-free mortality in patients with PVT.

A novel prognostic nomogram for older patients with acute-on-chronic liver diseases (AoCLD): a nationwide, multicentre, prospective cohort study.

BACKGROUND: the incidence of acute-on-chronic liver disease (AoCLD) is increasing. OBJECTIVE: to investigate the clinical features and risk factors of AoCLD and construct an effective prognostic nomogram model for older patients with AoCLD. METHODS: data from 3,970 patients included in the CATCH-LIFE study were used, including 2,600 and 1,370 patients in the training and validation sets, respectively. Multivariate Cox regression analyses were performed to identify predictive risk factors in older individuals, and an easy-to-use nomogram was established. Performance was assessed using area under the curve, calibration plots and decision curve analysis (DCA). RESULTS: of the 3,949 patients with AoCLD, 809 were older with a higher proportion of autoimmune-related abnormalities, hepatitis C viral infection and schistosomiasis. In the older patient group, the incidence of cirrhosis, hepatic encephalopathy (HE), infection, ascites and gastrointestinal bleeding; neutrophil-to-lymphocyte ratio (NLR), aspartate-to-alanine transaminase ratio (AST/ALT), creatinine and blood urea nitrogen levels were higher, whereas incidence of acute-on-chronic liver failure, white blood cell, platelet and haemoglobin levels; albumin, total bilirubin (TB), AST and ALT levels; international normalised ratio (INR), estimated glomerular filtration rate and blood potassium levels were lower than in the younger group. The final nomogram was developed based on the multivariate Cox analysis in training cohort using six risk factors: ascites, HE grades, NLR, TB, INR and AST/ALT. Liver transplantation-free mortality predictions were comparable between the training and validation sets. DCA showed higher net benefit for the nomograph than the treat-all or treat-none strategies, with wider threshold probabilities ranges. CONCLUSIONS: our analysis will assist clinical predictions and prognoses in older patients with AoCLD.

Severe anemia is associated with increased short-term and long-term mortality in patients hospitalized with cirrhosis.

INTRODUCTION AND OBJECTIVES: The relationship between anemia and the outcome of patients with cirrhosis is not completely clear. Therefore, we performed this large-scale epidemiological study to investigate the prevalence and severity of anemia in patients with cirrhosis and acute decompensation or liver injury and how anemia impacts short-term and long-term outcomes. PATIENTS AND METHODS: Patients with cirrhosis and acute decompensation (AD) or acute liver injury (ALI) were enrolled in the Chinese AcuTe on CHronic LIver FailurE (CATCH-LIFE) studies, which consisted of two large, multicenter, prospective, observational cohorts between January 2015 and December 2016 and July 2018 and January 2019. We conducted data analysis on the prevalence of anemia and determined the relationship between anemia and prognosis. RESULTS: Among 1979 patients, 1389 (70.2%) had anemia, among whom 599 (41.3%) had mild anemia, 595 (15.8%) had moderate anemia and 195 (2.4%) had severe anemia. A linear association between hemoglobin level and 90-day or 1-year LT-free mortality was shown, and a 10 g/L decrease in hemoglobin level was associated with a 6.8% extra risk of 90-day death and a 5.7% extra risk of 1-year death. Severe anemia was an independent risk factor for 90-day [HR=1.649 (1.100, 2.473), p=0.016] and 1-year LT-free mortality [HR=1.610 (1.159, 2.238), p=0.005]. Multinomial logistic regression analysis further identified that severe anemia was significantly associated with post-28-day mortality but not within-28-day mortality. CONCLUSIONS: Anemia is common in patients with cirrhosis admitted for acute events. Severe anemia was associated with poor 90-day and 1-year prognoses in these patients.

MELD score < 18 rule out 28-day ACLF development among inpatients with hepatitis B-related previous compensated liver disease.

The acute-on-chronic liver failure (ACLF) development is highly dynamic. Currently, no satisfactory algorithm identifies patients with HBV at risk of this complication. The aim of the study was to characterize ACLF development in hospitalized HBV-related patients without previous decompensation and to test the performance of traditional prognostic models in ruling out ACLF development within 28 days on admission we conducted a cohort study. Two multi-center cohorts with hospitalized HBV-related previous compensated patients were analyzed. Performances of MELD, MELD-Na, CLIF-C AD, and CLIF-C ACLF-D in ruling out ACLF development within 28 days were compared and further validated by ROC analyses. In the derivation cohort (n = 892), there were 102 patients developed ACLF within 28 days, with profound systemic inflammatory levels and higher 28-day mortality rate (31.4% vs. 1.0%) than those without ACLF development. The MELD score (cut-off = 18) achieved acceptable missing rate (missed/total ACLF development) at 2.9%. In the validation cohort (n = 1656), the MELD score (<18) was able to rule out ACLF development within 28 days with missing rate at 3.0%. ACLF development within 28 days were both lower than 1% (0.6%, derivation cohort; 0.5%, validation cohort) in patients with MELD < 18. While in patients with MELD ≥ 18, 26.6% (99/372, derivation cohort) and 17.8% (130/732, validation cohort) developed into ACLF within 28 days, respectively. While MELD-Na score cut-off at 20 and CLIF-AD score cut-off at 42 did not have consistent performance in our two cohorts. MELD < 18 was able to safely rule out patients with ACLF development within 28 days in HBV-related patients without previous decompensation, which had a high 28-day mortality.

MHBSt167 induced autophagy promote cell proliferation and EMT by activating the immune response in L02 cells.

BACKGROUND: Hepatitis B virus can induce hepatocellular carcinoma (HCC) by inducing a host immune response against infected hepatocytes. C-terminally truncated middle surface protein (MHBSt) has been reported to contribute to HCC through transcriptional activation in epidemiology studies, while the underlying mechanism of MHBSt-induced HCC is unknown. METHODS: In this study, a premature stop at codon 167 in MHBS (MHBSt167) was investigated into eukaryotic expression plasmid pcDNA3.1(-). MHBSt167 expressed plasmid was transfected into the L02 cell line, cell proliferation was analyzed by CCK-8 and high-content screening assays, the cell cycle was analyzed by flow cytometry, and epithelial-to-mesenchymal transition and autophagy were analyzed by immunoblotting and immunofluorescence. NF-κB activation and the MHBSt167-induced immune response were analyzed by immunoblotting and immunofluorescence. IFN-α, IFN-ß and IL-1α expression were analyzed by qPCR. Autophagy inhibitors were used to analyze the relationship between the immune response and autophagy. RESULTS: The results showed that MHBSt167 promoted L02 cell proliferation, accelerated cell cycle progression from the S to G2 phase and promoted epithelial-to-mesenchymal transition through ER-stress, leading to autophagy and NF-κB activation and increased immune-related factor expression. The MHBSt167-induced acceleration of cell proliferation and the cell cycle was abolished by autophagy or NF-κB inhibitors. CONCLUSION: In summary, MHBSt167 could promote cell proliferation, accelerate cell cycle progression, induce EMT and activate autophagy through ER-stress to induce the host immune response, supporting a potential role of MHBSt167 in contributing to carcinogenesis.

Immunostimulatory siRNA with a uridine bulge leads to potent inhibition of HBV and activation of innate immunity.

BACKGROUND: Hepatitis B virus (HBV) infection is difficult to cure. HBV-specific immune tolerance plays a key role in HBV persistence, and enhancing cellular and humoral immunity will improve the control of HBV infection. The purpose of the study was to explore the anti-HBV and immunostimulatory effects of msiRNAs that introduce unpaired uridine bulges in the passenger strand. METHODS: msiRNAs targeting the HBV S and X genes were designed and named msiHBs and msiHBx, respectively. HepG2 cells were cotransfected with siRNA or msiRNA and the HBV replication-competent plasmid pHY106-wta or pHY106-X15. HepG2.215 cells were transfected with siRNA or msiRNA. The levels of HBsAg, HBeAg, and the cytokines TNF-α, IFN-α, IFN-ß, IL-1α, and IL-6 in the culture supernatant was detected by ELISA. The levels of intracellular HBV RNA, nuclear HBV replication intermediates, and HBV DNA in the supernatant were measured by quantitative RT-PCR and PCR. The levels of HBV replication intermediates were detected by Southern blotting. Peripheral blood mononuclear cells were transfected with siRNA or msiRNA, and the levels of secreted cytokines IFN-α and IFN-ß were detected by ELISA. The bioactivity of type I interferons in the supernatants was detected by the virus protection assay. RESULTS: msiHBx treatment led to a significant decrease in HBsAg (to a negative level) and HBV DNA (95.5%) in the supernatant and intrahepatocellular HBV replication intermediates (89.8%) in HepG2 cells with transient HBV replication and in HepG2.2.15 cells. There was no significant difference between msiHBx and siHBx in terms of the reduction in HBV proteins and HBV replication (P > 0.05). Compared with siHBx, msiHBx treatment of HepG2 cells transfected with the HBV replication-competent plasmid led to a significant increase in the levels of the antiviral cytokines TNF-α (3.3-fold), IFN-α (1.4-fold), and IFN-ß (2.5-fold) (P < 0.01), without upregulation of the proinflammatory cytokines IL-1α and IL-6. The virus protection assay results showed msiHBx-mediated type I interferons effectively protected L929 cells against ECMV infection. CONCLUSIONS: msiHBx could effectively inhibit HBV expression and replication and induce an antiviral innate immune response without proinflammatory activation. The dual RNAi and immunostimulatory activity of msiRNAs may play an important role in the control of HBV infection.

Prevalence and Clinical Significance of Portal Vein Thrombosis in Patients With Cirrhosis and Acute Decompensation.

BACKGROUND & AIMS: Portal vein thrombosis (PVT) is a common and serious complication in patients with cirrhosis. However, little is known about PVT in patients with cirrhosis and acute decompensation (AD). We investigated the prevalence and clinical significance of PVT in nonmalignant patients with cirrhosis and AD. METHODS: We performed a retrospective study of 2 cohorts of patients with acute exacerbation of chronic liver disease who participated in the Chinese AcuTe on CHronic LIver FailurE study, established by the Chinese Chronic Liver Failure Consortium, from January 2015 through December 2016 (n = 2600 patients) and July 2018 through January 2019 (n = 1370 patients). We analyzed data on the prevalence, clinical manifestations, and risk factors of PVT from 2826 patients with cirrhosis, with and without AD. RESULTS: The prevalence of PVT in patients with cirrhosis and AD was 9.36%, which was significantly higher than in patients with cirrhosis without AD (5.24%) (P = .04). Among patients with cirrhosis and AD, 63.37% developed PVT recently (the first detected PVT with no indication of chronic PVT). Compared with patients without PVT, a significantly higher proportion of patients with PVT had variceal bleeding (47.33% vs 19.63%; P < .001) and patients with PVT had a significantly higher median serum level of D-dimer (2.07 vs 1.25; P < .001). Splenectomy and endoscopic sclerotherapy were independent risk factors for PVT in patients with cirrhosis and AD. The 1-year mortality rate did not differ significantly between patients with vs without PVT. CONCLUSIONS: In an analysis of data from 2826 patients with cirrhosis, a significantly higher proportion of those with AD had PVT than those without AD. PVT was associated with increased variceal bleeding, which would increase the risk for AD. Strategies are needed to prevent PVT in patients with cirrhosis, through regular screening, to reduce portal hypertension. ClinicalTrials.gov no: NCT02457637 and NCT03641872.

Clinical features and outcomes of bacterascites in cirrhotic patients: A retrospective, multicentre study.

BACKGROUND & AIMS: Current guidelines on the management of bacterascites are limited. This multicentre, retrospective study investigated the clinical features and outcomes of cirrhosis patients with bacterascites. METHODS: Two series of cirrhosis patients were evaluated. The first included 418 patients with ascites-positive cultures at 11 hospitals during 2012-2018. Clinical characteristics and outcomes were recorded. The second included 208 patients with sterile ascites from a prospective cohort (NCT02457637). Clinical features and outcomes of cirrhotic patients with or without bacterascites were investigated. RESULTS: In the first series, bacterascites was diagnosed in 254/418 (60.8%) patients, and culture-positive spontaneous bacterial peritonitis (SBP) in 164/418 (39.2%) patients. Gram-positive bacteria were more prevalent in bacterascites patients than in culture-positive SBP patients (59.1% vs 22.0%; P < .001). For patients with acute-on-chronic liver failure (ACLF) in bacterascites and culture-positive SBP groups, the 28-day transplant-free mortality (41.3% vs 65.5%; P = .015) and the prevalence of in-hospital acute kidney injury (AKI) (84.8% vs 75%; P = .224). For patients without ACLF in the bacterascites (n = 208) and culture-positive SBP groups (n = 108), the 28-day transplant-free mortalities were 13% vs 13.9% (P = .822), the probabilities of progression to ACLF within 28 days were 10.1% vs 14.8% (P = .216) and the prevalences of in-hospital AKI were 14.4% vs 30.6% (P = .001). Bacterascites patients had higher 28-day mortality than those patients with sterile ascites, after propensity score matching (18.4% vs 8.6%; P = .010). CONCLUSION: Bacterascites patients had non-negligible poor clinical outcomes, including in-hospital AKI, progression to ACLF and 28-day mortality. Future studies are warranted to expedite the diagnosis of bacterascites and optimize antibiotic treatment.

Isoliquiritigenin inhibits TGF-ß1-induced fibrogenesis through activating autophagy via PI3K/AKT/mTOR pathway in MRC-5 cells.

Isoliquiritigenin (ISL), a natural flavonoid derived from the root of liquorice, has been reported to possess anti-inflammatory and antioxidant activities. Previous studies have found that ISL plays a crucial role in anti-fibrosis of adipose tissue and renal tissue; however, its effect on pulmonary fibrogenesis has not been demonstrated. In this study, we aimed to explore the roles and the underlying mechanisms of ISL in TGF-ß1-induced fibrogenesis using human lung fibroblast-derived MRC-5 cells. Cell proliferation and migration were determined by MTT and wound healing assay, respectively. The expression levels of alpha-smooth muscle actin (α-SMA), collagen type I alpha 1 (COLIA1) and fibronectin (FN), microtubule-associated protein light chain 3 (LC3) and related signaling molecules were detected by quantitative real-time PCR, western blot and immunofluorescence assay, correspondingly. EGFP-LC3 transfection was used for autophagy analysis. The results showed that ISL inhibited the TGF-ß1-induced proliferation and migration, and down-regulated the expressions of α-SMA, COLIA1 and FN. ISL treatment led to up-regulation of LC3 in TGF-ß1-treated MRC-5 cells, accompanied by significant decrease in the phosphorylation levels of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR). In addition, the inhibitory effects of ISL on TGF-ß1-induced fibrogenic features in MRC-5 cells were enhanced by pretreatment with autophagy activator Rapmycin and PI3K/AKT inhibitor LY294002 and reversed by autophagy inhibitor 3-methyladenine and PI3K/AKT activator IGF-1. Taken together, our results demonstrated that ISL could attenuate the fibrogenesis of TGF-ß1-treated MRC-5 cells by activating autophagy via suppressing the PI3K/AKT/mTOR pathway. Therefore, ISL holds a great potential to be developed as a novel therapeutic agent for the treatment of pulmonary fibrosis.

Acute-on-Chronic Liver Failure in China: Rationale for Developing a Patient Registry and Baseline Characteristics.

Definitions and descriptions of acute-on-chronic liver failure (ACLF) vary between Western and Eastern types, and alcoholism and hepatitis B virus (HBV) are, respectively, the main etiologies. To determine whether there are unified diagnostic criteria and common treatment programs for different etiologies of ACLF, a multicenter prospective cohort with the same inclusion criteria and disease indicators as those used in the European Consortium Acute-on-Chronic Liver Failure in Cirrhosis Study is urgently needed in Asia, where the prevalence of HBV is high. A multicenter prospective cohort of 2,600 patients was designed, drawing from 14 nationwide liver centers from tertiary university hospitals in China, and 2,600 hospitalized patients with chronic liver disease (both cirrhotic and noncirrhotic) of various etiologies with acute decompensation or acute hepatic injury were continuously recruited from January 2015 to December 2016. Data were collected during hospitalization, and follow-ups were performed once a month, with plans to follow all patients until 36 months after hospital discharge. Of these patients, 1,859 (71.5%) had HBV-related disease, 1,833 had cirrhotic disease, and 767 had noncirrhotic disease. The numbers and proportions of enrolled patients from each participating center and the baseline characteristics of the patients with or without cirrhosis are presented.

High serum resistin associates with intrahepatic inflammation and necrosis: an index of disease severity for patients with chronic HBV infection.

BACKGROUND: Studies have revealed that resistin plays a role as an intrahepatic cytokine with proinflammatory activities. This study investigated the association between serum resistin and fibrosis severity and the possible marker role of resistin in the inflammatory process of chronic hepatitis B. METHODS: In this study, 234 subjects with HBV infection were retrospectively selected, including 85 patients with chronic hepatitis B (CHB), 70 patients with HBV-related liver cirrhosis (LC-B), and 79 patients with HBV-related liver failure (LF-B). Serum levels of resistin, IL-1, IL-6, IL-17, IL-23, TNF-α, and TGF-ß1 were assayed by ELISA. Demographic and clinical characteristics of patients were extracted from clinical databases of Taihe Hospital, Hubei University of Medicine, including serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil), and liver stiffness (LS). RESULTS: All the selected patients with HBV infection showed significantly increased levels of serum resistin, which was rarely detectable in the healthy controls. Serum resistin levels in patients with CHB, LC-B, and LF-B were 4.119 ± 5.848 ng/mL, 6.370 ± 6.834 ng/mL, and 6.512 ± 6.076 ng/mL, respectively. Compared with the CHB group, patients with LC-B or LF-B presented with significantly higher serum levels of resistin (p < 0.01). On the other hand, all of the enrolled patients had high serum levels of IL-1, IL-6, IL-17, TNF-α, and TGF-ß1, but not IL-23. Interestingly, serum levels of resistin was significantly positively correlated with serum levels of TGF-ß1 in LC-B patients (R = 0.3090, p = 0.0290), with IL-17 in LC-B (R = 0.4022, p = 0.0038) and LF-B patients (R = 0.5466, p < 0.0001), and with AST (R = 0.4501, p = 0.0036) and LS (R = 0.3415, p = 0.0310) in CHB patients. CONCLUSIONS: High serum resistin associates with intrahepatic inflammation and necrosis and may be used as an index of disease severity for patients with chronic HBV infection.

Novel Woodchuck Hepatitis Virus (WHV) transgene mouse models show sex-dependent WHV replicative activity and development of spontaneous immune responses to WHV proteins.

The woodchuck model is an informative model for studies on hepadnaviral infection. In this study, woodchuck hepatitis virus (WHV) transgenic (Tg) mouse models based on C57BL/6 mice were established to study the pathogenesis associated with hepadnaviral infection. Two lineages of WHV Tg mice, harboring the WHV wild-type genome (lineage 1217) and a mutated WHV genome lacking surface antigen (lineage 1281), were generated. WHV replication intermediates were detected by Southern blotting. DNA vaccines against WHV proteins were applied by intramuscular injection. WHV-specific immune responses were analyzed by flow cytometry and enzyme-linked immunosorbent assays (ELISAs). The presence of WHV transgenes resulted in liver-specific but sex- and age-dependent WHV replication in Tg mice. Pathological changes in the liver, including hepatocellular dysplasia, were observed in aged Tg mice, suggesting that the presence of WHV transgenes may lead to liver diseases. Interestingly, Tg mice of lineage 1281 spontaneously developed T- and B-cell responses to WHV core protein (WHcAg). DNA vaccination induced specific immune responses to WHV proteins in WHV Tg mice, indicating a tolerance break. The magnitude of the induced WHcAg-specific immune responses was dependent on the effectiveness of different DNA vaccines and was associated with a decrease in WHV loads in mice. In conclusion, sex- and age-dependent viral replication, development of autoimmune responses to viral antigens, pathological changes in the liver in WHV Tg mice, and the possibility of breaking immune tolerance to WHV transgenes will allow future studies on pathogenesis related to hepadnaviral infection and therapeutic vaccines.

Emerging roles of RNA ac4C modification and NAT10 in mammalian development and human diseases.

RNA ac4C modification is a novel and rare chemical modification observed in mRNA. Traditional biochemical studies had primarily associated ac4C modification with tRNA and rRNA until in 2018, Arango D et al. first reported the presence of ac4C modification on mRNA and demonstrated its critical role in mRNA stability and translation regulation. Furthermore, they established that the ac4C modification on mRNA is mediated by the classical N-acetyltransferase NAT10. Subsequent studies have underscored the essential implications of NAT10 and mRNA ac4C modification across both physiological and pathological regulatory processes. In this review, we aimed to explore the discovery history of RNA ac4C modification, its detection methods, and its regulatory mechanisms in disease and physiological development. We offer a forward-looking examination and discourse concerning the employment of RNA ac4C modification as a prospective therapeutic strategy across diverse diseases. Furthermore, we comprehensively summarize the functions and mechanisms of NAT10 in gene expression regulation and pathogenesis independent of RNA ac4C modification.

Association of AST/ALT ratio with 90-day outcomes in patients with acute exacerbation of chronic liver disease: a prospective multicenter cohort study in China.

Background and aim: A high aspartate aminotransferase/alanine aminotransferase (AST/ALT) ratio is associated with liver injury in liver disease; however, no data exist regarding its relationship with 90-day prognosis in patients with acute exacerbation of chronic liver disease. Methods: In this study, 3,758 participants (955 with advanced fibrosis and 2,803 with cirrhosis) from the CATCH-LIFE cohort in China were included. The relationships between different AST/ALT ratios and the risk of adverse 90-day outcomes (death or liver transplantation) were determined in patients with cirrhosis or hepatitis B virus (HBV)-associated advanced fibrosis, respectively. Results: In the patients with HBV-associated advanced fibrosis, the risk of 90-day adverse outcomes increased with AST/ALT ratio; after adjusting for all confounding factors, the risk of adverse 90-day outcomes was the highest when AST/ALT ratio was more than 1.08 (OR = 6.91 [95% CI = 1.789-26.721], p = 0.005), and the AST/ALT ratio of >1.9 accelerated the development of adverse outcomes. In patients with cirrhosis, an AST/ALT ratio > 1.38 increased the risk of adverse 90-day outcomes in all univariables (OR = 1.551 [95% CI = 1.216-1.983], p < 0.001) and multivariable-adjusted analyses (OR = 1.847 [95% CI = 1.361-2.514], p < 0.001), and an elevated AST/ALT ratio (<2.65) accelerated the incidence of 90-day adverse outcomes. An AST/ALT ratio of >1.38 corresponded with a more than 20% incidence of adverse outcomes in patients with cirrhosis. Conclusion: The AST/ALT ratio is an independent risk factor for adverse 90-day outcomes in patients with cirrhosis and HBV-associated advanced fibrosis. The cutoff values of the AST/ALT ratio could help clinicians monitor the condition of patients when making clinical decisions.

sTREM-1 as promising prognostic biomarker for acute-on-chronic liver failure and mortality in patients with acute decompensation of cirrhosis.

BACKGROUND: Acute decompensation (AD) of cirrhosis is associated with high short-term mortality, mainly due to the development of acute-on-chronic liver failure (ACLF). Thus, there is a need for biomarkers for early and accurate identification of AD patients with high risk of development of ACLF and mortality. Soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) is released from activated innate immune cells and correlated with various inflammatory processes. AIM: To explore the prognostic value of sTREM-1 in patients with AD of cirrhosis. METHODS: A multicenter prospective cohort of 442 patients with cirrhosis hospitalized for AD was divided into a study cohort (n = 309) and validation cohort (n = 133). Demographic and clinical data were collected, and serum sTREM-1 was measured at admission. All enrolled patients were followed-up for at least 1 year. RESULTS: In patients with AD and cirrhosis, serum sTREM-1 was an independent prognosis predictor for 1-year survival and correlated with liver, coagulation, cerebral and kidney failure. A new prognostic model of AD (P-AD) incorporating sTREM-1, blood urea nitrogen (BUN), total bilirubin (TBil), international normalized ratio (INR) and hepatic encephalopathy grades was established and performed better than the model for end-stage liver disease (MELD), MELD-sodium (MELD-Na), chronic liver failure-consortium (CLIF-C) ACLF and CLIF-C AD scores. Additionally, sTREM-1 was increased in ACLF and predicted the development of ACLF during first 28-d follow-up. The ACLF risk score incorporating serum sTREM-1, BUN, INR, TBil and aspartate aminotransferase levels was established and significantly superior to MELD, MELD-Na, CLIF-C ACLF, CLIF-C AD and P-AD in predicting risk of ACLF development. CONCLUSION: Serum sTREM-1 is a promising prognostic biomarker for ACLF development and mortality in patients with AD of cirrhosis.

The clinical courses of HBV-related acute-on-chronic liver failure and a multi-state model to predict disease evolution.

BACKGROUND AND AIMS: Acute-on-chronic liver failure (ACLF) is a highly dynamic syndrome. The objective of this study was to delineate the clinical course of patients with HBV-ACLF and to develop a model to estimate the temporal evolution of disease severity. METHODS: We enrolled eligible patients from 2 large, multicenter prospective cohorts. The ACLF grade, organ failures, and outcomes were assessed at multiple time points (days 1/4/7/14/21/28). Probabilities for ACLF transitions between these disease states and to death within 28 days were calculated using a multi-state model that used baseline information and updated ACLF status. The model was validated in independent patients. RESULTS: Among all the 445 patients with HBV-ACLF, 76 represented disease progression, 195 had a stable or fluctuating course, 8 with improvement, and the remaining 166 with resolution within 28-day follow-up. New coagulation (63.64%) or renal failure (45.45%) was frequently observed during early progression. Patients with disease progression had a higher incidence of new episodes of ascites [10 (13.16%) vs. 22 (5.96%), p = 0.027] and HE [13(17.11%) vs. 21 (5.69%), p = 0.001], and a significant increase in white blood cell count. The multi-state model represented dynamic areas under the receiver operating characteristic curves ranging from 0.71 to 0.84 for predicting all ACLF states and death at 4, 7, 14, 21, and 28 days post-enrollment and from 0.73 to 0.94 for predicting death alone, performing better than traditional prognostic scores. CONCLUSIONS: HBV-ACLF is a highly dynamic syndrome with reversibility. The multi-state model is a tool to estimate the temporal evolution of disease severity, which may inform clinical decisions on treatment.

Characterization of acute-on-chronic liver diseases: A multicenter prospective cohort study.

BACKGROUND: Acute-on-chronic liver disease (AoCLD) accounts for the majority of patients hospitalized in the Department of Hepatology or Infectious Diseases. AIM: To explore the characterization of AoCLD to provide theoretical guidance for the accurate diagnosis and prognosis of AoCLD. METHODS: Patients with AoCLD from the Chinese Acute-on-Chronic Liver Failure (ACLF) study cohort were included in this study. The clinical characteristics and outcomes, and the 90-d survival rate associated with each clinical type of AoCLD were analyzed, using the Kaplan-Meier method and the log-rank test. RESULTS: A total of 3375 patients with AoCLD were enrolled, including 1679 (49.7%) patients with liver cirrhosis acute decompensation (LC-AD), 850 (25.2%) patients with ACLF, 577 (17.1%) patients with chronic hepatitis acute exacerbation (CHAE), and 269 (8.0%) patients with liver cirrhosis active phase (LC-A). The most common cause of chronic liver disease (CLD) was HBV infection (71.4%). The most common precipitants of AoCLD was bacterial infection (22.8%). The 90-d mortality rates of each clinical subtype of AoCLD were 43.4% (232/535) for type-C ACLF, 36.0% (36/100) for type-B ACLF, 27.0% (58/215) for type-A ACLF, 9.0% (151/1679) for LC-AD, 3.0% (8/269) for LC-A, and 1.2% (7/577) for CHAE. CONCLUSION: HBV infection is the main cause of CLD, and bacterial infection is the main precipitant of AoCLD. The most common clinical type of AoCLD is LC-AD. Early diagnosis and timely intervention are needed to reduce the mortality of patients with LC-AD or ACLF.