Hepatology Communications

BackgroundChronic infection with hepatitis C virus (HCV) is a leading cause of liver-related mortality. Direct-acting antivirals (DAAs) have revolutionised treatment by offering profound improvements in sustained viral clearance (SVR) and tolerability resulting in rapid expansion of treatment for individuals for whom HCV treatment had previously been less feasible, such as those with advanced liver disease or with drug and/or alcohol-related substance use. Given these clinical policy shifts, the primary objective of this study was to assess the impact of SVR on liver-related death among important clinical groups and the secondary objective was to explore changes in predictors of liver-related death by treatment era using real-world data from a large population-based cohort. MethodsWe conducted a population-based, linked cohort study of all Ontario residents with HCV viremia between January 1st, 1999, and December 31st, 2018, with follow up to 31st May 2021 (N=73,411). Population-level health administrative, clinical, and demographic data were accessed at ICES. Cause-specific hazard models were used to explore the impact of SVR on liver-related death and to identify factors associated with the rate of liver-related death in the DAA and pre-DAA treatment eras. The moderating effects of liver disease severity and substance-use disorder on the relationship between SVR and liver-related-mortality was explored by stratification. ResultsAmong Ontario residents diagnosed with living with HCV, the achievement of SVR was associated with a significant reduction in liver related mortality (adjusted hazard ratio [aHR] 0.22, 95%CI: 0.20-0.24 vs. no SVR). This was also observed across progressive liver disease severity levels (aHR 0.13, 95%CI: 0.10-0.17 for individuals without cirrhosis; aHR 0.11, 95%CI: 0.06-0.17 for those with compensated cirrhosis, and aHR 0.24, 95%CI: 0.22-0.27 for those with advanced liver disease vs. no SVR) and by substance use status (aHR 0.24, 95%CI: 0.21-0.27 for those with a history of substance use disorder; and aHR 0.21, 95%CI: 0.18-0.24 for those without vs. no SVR). Additionally, factors such as age at diagnosis, sex, liver disease severity, immigration status, birth year, substance use, HBV-coinfection, viral genotype, and markers of social marginalisation were independent predictors of liver-related mortality. However, sex, and viral genotype no longer displayed significant associations with liver-related death in the DAA era as was observed in the earlier treatment era. ConclusionsThis study provides real-world evidence showing profound impact of SVR on liver-related mortality in a population-based sample of individuals with CHC and highlights the importance of early diagnosis and treatment. This study further demonstrates significant mortality benefits of SVR regardless of substance use status highlighting the importance of supporting marginalised individuals in treatment access.

Background/AimsCholestatic liver diseases (CLD) are the leading indication for pediatric liver transplantation. Increased intrahepatic bile acid concentrations cause endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) is activated to maintain homeostasis. UPR dysregulation, including the inositol-requiring enzyme 1/X-box protein 1 (IRE1/XBP1) pathway, is associated with several adult liver diseases. We evaluated hepatic UPR expression in pediatric patients with end-stage CLD and hypothesize that an inability to appropriately activate the hepatic IRE1/XBP1 pathway is associated with the pathogenesis of CLD. MethodsWe evaluated 34 human liver explants. Cohorts included: pediatric CLD (Alagille, ALGS, and progressive familial intrahepatic cholestasis, PFIC), pediatric non-cholestatic liver disease controls (autoimmune hepatitis, AIH), adult CLD, and normal controls. We performed RNA-seq, quantitative PCR, and western blotting to measure expression differences of the hepatic UPR and other signaling pathways. ResultsMetascape pathway analysis demonstrated that the KEGG protein processing in ER pathway was downregulated in pediatric CLD compared to normal controls. Pediatric CLD had decreased hepatic IRE1/XBP1 pathway gene expression and decreased protein expression of p-IRE1 compared to normal controls. These CLD changes were not disease-specific to ALGS or PFIC. IRE1/XBP1 pathway gene expression was decreased in pediatric CLD compared to AIH disease controls. ConclusionPediatric CLD explants have decreased gene and protein expression of the protective IRE1/XBP1 pathway and down-regulated KEGG protein processing in the ER pathways. IRE1/XBP1 pathway expression differences occur when compared to both normal and non-cholestatic disease controls. Attenuated expression of the IRE1/XBP1 pathway is associated with cholestatic diseases and could be targeted to treat pediatric CLD.

IntroductionLiver fibrosis can progress to cirrhosis, liver failure, or hepatocellular carcinoma, which often requires transplantation and burdens healthcare systems around the world. Advances in single-cell RNA sequencing and machine learning have enhanced the understanding of immune responses in many liver diseases particularly alcohol liver cirrhosis (ALC) and non-alcoholic steatohepatitis (NASH). This study aims to identify key biomarkers involved in these conditions and assess their potential as non-invasive diagnostic tools. MethodsTwo gene expression profiles GSE136103 and GSE115469 were used to conduct differential gene expression (DEG) analysis. Using the results from DEG analysis, we then applied two machine learning network-based algorithms, master regulator analysis (MRA) and weighted key driver analysis (wKDA), to identify potential biomarker genes for NASH and ALC. ResultsA total of 1,435 and 5,074 DEGs were identified for ALC and NASH compared to healthy controls, including 1,077 shared DEGs between the two diseases. The MRA showed HLA-DPA1, HLA-DRB1, IFI44L, ISG15, and CD74 as the potential master regulators of ALC and HLA-DPB1, HLA-DQB1, HLA-DRB5, PFN1, and TMSB4X as the potential master regulators of NASH. In addition, wKDA analysis indicated CD300A, FCGR2A, RGS1, HLA-DMB, and C1QA as the key drivers of ALC and INPP5D, NCKAP1L, RAC2, PTPRC, and TYROBP as key drivers of NASH. ConclusionThis study presented a comprehensive framework for analyzing single-cell RNA-seq data, demonstrating the potential of combining advanced network-based machine-learning techniques with conventional DEG analysis to uncover actionable prognostic markers for ALC and NASH with potential use as target biomarkers in drug development.

Background & AimsA cholestatic pattern of liver enzymes is associated with progressive liver disease and major adverse liver-related outcomes (MALO) among patients with Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD). We aimed to authenticate the efficacy of a newly formulated liver function test (LFT) score for distinguishing patients with cholestatic vs. hepatocellular patterns and to evaluate its prognostic utility in MASLD patients. MethodsA retrospective longitudinal study on a dataset of over 250,000 individuals diagnosed with MASLD and/or obesity with cardiovascular risk factors. Patients were categorized into cholestatic (C), mixed (M), or hepatocellular (H) patterns according to the LFT score, or the well-known R score. Long-term MALO, major adverse cardiovascular events (MACE), and all-cause mortality were tracked. ResultsThe LFT score excelled in differentiating patients into C, M, or H groups accurately. While about two-thirds of our cohort initially showed a low FIB4 (<1.3), patients in the C category experienced a higher incidence of MALO and MACE compared to those in the H category (0.5% vs. 0.2% and 7.1% vs. 3.6%, respectively) over the span of 10 years post-diagnosis. Additionally, the 15-year overall survival rate was notably lower for C patients compared to their H counterparts (63% vs. 77%, p<0.0001). The LFT score was more effective than the R score in distinguishing between H and C patients for prognostic purposes, and a baseline cholestatic pattern indicates poorer outcomes regardless of subsequent LFT changes. ConclusionsThe LFT score accurately categorizes cholestatic MASLD patients and may serve as a useful prognostic tool.

Induced pluripotent stem cells (iPSCs) require further maturation before they can substitute primary human cells. Induced pluripotent stem cell hepatocyte-like cells (iHLCs) exhibit significantly lower hepatic functions than primary human hepatocytes (PHHs). Maturation of iHLCs has relied unsuccessfully on administering chemical cocktails in widely differing temporal patterns that are 106-fold higher in concentration than in vivo. Hence, there is no reproducible approach for iHLC maturation. We report the assembly of a multicellular 3D human liver organoid that recapitulates the in vivo hepatic microenvironment. Intra-and intercellular signaling between human hepatic cells and iHLCs results in their maturation. Within seven-days, iHLCs in organoids expressed markers of hepatocyte maturation that were statistically similar to PHHs including alphafetoprotein (AFP), hepatic nuclear factor (HNF)-4, and albumin. Ki67+ iHLCs decreased by 2-fold from Days 1 to 14. Expression of two cytochrome P450 (CYP) enzymes, CYP3A4 and CYP2E1, in iHLCs were statistically similar to PHHs by Days 7 and 14, respectively. Biotransformation of acetaminophen and ethanol were statistically similar to PHHs by Day 14. On Day 1, the concentration of endogenously secreted prostaglandin E2 (PGE2) was identical to values reported in adult humans. Over the 14-day culture, the concentrations of endogenously secreted hepatocyte growth factor (HGF) and Oncostatin M (OSM) increased until they were 26-36% of in vivo values. The organoids are secreting critically important maturation molecules that are similar to levels reported in healthy humans. These trends demonstrate how closely the multi-cellular organoids are emulating in vivo-like behavior while undergoing further maturation.

BackgroundHBsAg can be derived from intrahepatic cccDNA and integrated HBV DNA (iDNA). We examined the iDNA from liver tissues of 24 HBeAg(+) and 32 HBeAg(-) treatment-naive CHB participants. MethodsLiver tissues were obtained from the North American Hepatitis B Research Network (HBRN). For cccDNA analysis, DNA was heat-denatured and digested by plasmid-safe ATP-dependent DNase to remove rcDNA and iDNA prior to qPCR. For iDNA detection, total DNA was subjected to HBV hybridization-targeted next generation sequencing (HBV-NGS) assay. The HBV-host junction sequences were identified by ChimericSeq. Comparison of HBV cccDNA and iDNA with serum and intrahepatic virological parameters were assessed. ResultsIntrahepatic cccDNA, serum HBV DNA, HBV RNA, HBcrAg and qHBsAg were higher among the HBeAg(+) participants. Among the HBeAg(+) samples, 87% had positive intrahepatic HBcAg staining compared to 13% of HBeAg(-) samples (p<0.0001). HBsAg staining, in contrast, was present in over 85% of both HBeAg(+) and (-) livers. 23 (95.8%) HBeAg(+) participants had [&le;]50% iDNA of total HBV DNA whereas 25 (78.1%) HBeAg(-) participants had >50% iDNA in their livers. The iDNA junction-breakpoint distributions for the HBeAg(+) group were random with 15.9% localized to the DR2-DR1 region. In contrast, 52.4% of the iDNA were clustered at DR2-DR1 region among the HBeAg(-) participants. Microhomology-mediated end joining (MMEJ) patterns of dslDNA HBV integration was more frequent in HBeAg (+) livers. ConclusionSerum RNA and HBcrAg reflect the intrahepatic cccDNA concentrations. HBeAg(-) CHB participants had high levels of intrahepatic iDNA and HBsAg despite lower cccDNA levels suggesting that iDNA is the primary source of HBsAg in HBeAg(-) CHB.

Background & AimsCell-free DNA (cfDNA) has advanced cancer genetic profiling through liquid biopsy. While plasma is traditionally the primary source, emerging evidence highlights urinary cfDNA as a novel and noninvasive alternative. This study aimed to comprehensively assess transrenal DNA (trDNA) as a novel noninvasive biomarker source in HCC patients, compared to blood-based liquid biopsy. Approach & ResultsHBV DNA was used as a biomarker for trDNA. HBV-targeted and HCC-focused next generation sequencing (NGS) and whole genome sequencing (WGS) were used to compare fragment insert-sizes, the genome coverage, and germline genotyping accuracy. Urinary cfDNA overall exhibited a predominantly mononucelosomal pattern similar to plasma cfDNA, but with shorter fragments, broader size distribution and a more pronounced 10-bp periodicity. In contrast, trDNA were shorter and more variable among all patients. In HCC patients, trDNA was even shorter, with distinct 4-mer end motifs, compared to non-HCC trDNA. Higher concentrations of HCC-distinctive 4-mer end motif and TP53 mutations were found in urine compared to plasma. The overall genome coverage breadth by WGS was similar between urine and plasma cfDNA, with a higher fraction of covered cancer-associated mutation hotspots in urine cfDNA. In 101 HCC patients, there was a 78% overall concordance of HCC-associated mutations (TP53, CTNNB1, and hTERT) and in select 15 patients, 97% overall position-level concordance by targeted NGS between plasma and urine cfDNA. ConclusionUrine cfDNA has comparable features with distinct characteristics to plasma cfDNA and is a promising tool for liver cancer studies.

The heterogeneity of liver non-parenchymal cells (NPCs) is essential for liver structure and function. However, the current understanding of liver NPCs, especially in different liver diseases, remains incompletely elucidated. Here, a single-cell transcriptome atlas of 171,814 NPCs from healthy and 5 typical liver disease mouse models, including alcoholic liver disease, nonalcoholic steatohepatitis (NASH), drug-induced liver injury, cholestatic, and ischemia-reperfusion liver injury is constructed. The inter- and intra-group heterogeneity of 12 types (and numerous subtypes) of NPCs involving endothelial cells, hepatic stellate cells (HSCs), neutrophils, T cells, and mononuclear phagocytes (MPs) are summarized. A protective subtype of neutrophils characterized by Chil3high is validated and found significantly increasing only in drug-induced and cholestatic liver injury models. Transcriptional regulatory network analysis reveals disease-specific transcriptional reprogramming. Metabolic activity analysis indicates that fibrosis is accompanied by increases in glycolysis and retinol metabolism in activated HSCs and MPs. Moreover, we found that cell-cell interactions between cholangiocytes and immune cells contribute more to cholestatic liver fibrosis compared with NASH, while HSCs are more important for NASH fibrosis. Our atlas, together with an interactive website provides a systematic view of highly heterogeneous NPCs and a valuable resource to better understand pathological mechanisms underlying liver diseases.

Background & AimsMitochondrial dysfunction has been implicated in aging and various cancer development. As highly dynamic organelles, mitochondria constantly undergo fission, mediated by dynamin-related protein 1 (DRP1, gene name Dnm1l), and fusion, regulated by mitofusin 1 (MFN1), MFN2, and optic atrophy 1 (OPA1). However, whether and how dysregulation of mitochondria dynamics would be involved in liver pathogenesis and tumorigenesis is unknown. MethodsDnm1l Flox/Flox (Dnm1lF/F), Mfn1F/F and Mfn2F/F mice were crossed with albumin-Cre mice to generate liver-specific Dnm1l knockout (L-Dnm1l KO), L-Mfn1 KO, L-Mfn2 KO, L-Mfn1, Mfn2 double KO (DKO), and L-Mfn1, Mfn2, Dnm1l triple KO (TKO) mice. These mice were housed for various periods up to 18 months. Some mice also received hydrodynamic tail vein injections of a Sleeping Beauty transposon-transposase plasmid system with c-MYC and YAP. Blood and liver tissues were harvested for biochemical and histological analysis. ResultsL-Dnm1l KO mice had elevated serum alanine aminotransferase levels and increased hepatic fibrosis as early as two months of age. By 12 to 18 months, male L-Dnm1l KO mice developed spontaneous liver tumors, primarily hepatocellular adenomas. While female L-Dnm1l KO mice also developed liver tumors, their incidence was much lower. In contrast, neither L-Mfn1 KO nor L-Mfn2 KO mice had notable liver injury or tumorigenesis. However, a small portion of DKO mice developed tumors at 15-18 month-old. Increased DNA damage, senescence and compensatory proliferation were observed in L-Dnm1l KO mice but were less evident in L-Mfn1 KO, L-Mfn2 KO or DKO mice, indicating that mitochondrial fission is more important to maintain hepatocyte homeostasis and prevent liver tumorigenesis. Interestingly, further deletion of Mfn1 and Mfn2 in L-Dnm1l KO mice markedly abolished liver injury, fibrosis, and both spontaneous and oncogene-induced tumorigenesis. RNA sequencing and metabolomics analysis revealed significant activation of the cGAS-STING-interferon pathway and alterations in the tumor microenvironment pathways, alongside increased pyrimidine synthesis and metabolism in the livers of L-Dnm1l KO mice. Notably, the changes in gene expression and pyrimidine metabolism were considerably corrected in the TKO mice. ConclusionsMitochondrial dynamics and stability are essential for maintaining hepatic mitochondrial homeostasis and hepatocyte functions. Loss of hepatic DRP1 promotes liver tumorigenesis by increasing pyrimidine metabolism and activating the cGAS-STING-mediated innate immune response.

BackgroundA detailed understanding of the dynamic fate changes of hepatocytes, hepatic stellate cells (HSC), Kupffer cells (KC), and liver sinusoidal endothelial cells (LSEC) is critical for studying liver (patho)physiology during disease progression. Current isolation methods often focus on single cell types, limiting utility in comprehensive research. AimTo develop a novel, semi-automated protocol for the simultaneous isolation of hepatocytes and non-parenchymal cells (NPCs), including HSC, KC, and LSEC, from mouse and human, with high yield, purity, and viability from healthy and diseased livers. MethodThe protocol employs a two-step EGTA and collagenase II perfusion for tissue digestion. Hepatocytes were isolated by low-speed centrifugation and a Percoll gradient. Subsequently, magnetic-activated cell separation, using CD271 as a selective surface marker for HSC, CD11b for KC and CD146 for LSEC) was performed. Validation was achieved with immunofluorescence staining, flow cytometry, RT-PCR, and UV fluorescence, whereby yield, purity, and viability were assessed. ResultsWith our method, yield of hepatocytes, HSC, KC, and LSEC, is 33.4{+/-}5.5x10, 5.2{+/-}6.3x10, 12.4{+/-}4.8x10 and 18.2{+/-}8.9x10 cells per healthy mouse liver, respectively, with cell viabilities exceeding 89%, and purity surpassing 90%. CD271 was validated as an effective marker for purifying HSC in healthy and diseased human (n=4-6) and mouse livers. Compared to microfluidic and organ-on-a-chip approaches, with our protocol, we achieve higher yield and purity values while enabling the simultaneous isolation of multiple cell types from a single sample. ConclusionOur semi-automated protocol offers a scalable, reliable, and versatile solution for isolating main liver cell types with high yield, purity, and viability from both healthy and diseased tissues, advancing liver research and facilitating downstream investigations. Impact and implicationsO_LIBroad applicability: The CD271-based method efficiently isolates key liver cell types (Hepatocyte, HSC, KC, LSEC) simultaneously. C_LIO_LIVersatility in disease models: Effective for studying healthy, fibrotic, and damaged liver tissues. C_LIO_LIRobust across variability: Works reliably across different mouse strains, age groups, and conditions. C_LIO_LIHuman research potential: Scalable for high-purity isolation from human liver tissue, enabling translational studies. C_LIO_LIHigh-quality results: Ensures >85% viability and >90% purity, supporting reproducible liver research applications. C_LI

BackgroundGut microbiota alteration is implicated in the pathogenesis of alcoholic liver disease (ALD) and HCC. No study has characterized the dysbiosis associated with ALD by microbial culturomics, an approach that certifies viability and allows the characterization of pathobiont strain candidates. MethodsA case-control study was conducted on patients with ALD without HCC (ALD-NoHCC) (n=16), ALD with HCC (ALD-HCC) (n=19), and controls (n=24). 16S rRNA amplicon sequencing and microbial culturomics were used as complementary methods for gut microbiome profiling. ResultsBy microbial culturomics, Thomasclavelia ramosa was the most enriched and detected in all ALD samples (100%), while it was cultivated in only a small proportion of controls (20%, p < 0.001). By 16S rRNA amplicon sequencing and 3-groups linear discriminant analysis, T. ramosa was increased explicitly in the ALD-HCC group (LDA-score > 5, p < 0.05). ConclusionsT. ramosa, identified by culturomics and 16 rRNA sequencing, is associated with ALD and ALD-HCC. Alongside the recently reported in vitro genotoxicity of this species in colorectal cancer, this species has been identified as a candidate oncobiont in ALD-HCC. HighlightsO_LIThe gut microbiota signature of ALD and ALD-HCC was explored by microbial culturomics and 16S amplicon sequencing C_LIO_LIBy culturomics, T. ramosa was the most enriched and cultured from all included ALD patients, but in only 20% of controls (p < 0.05). C_LIO_LIT. ramosa was significantly associated with alcohol-related HCC by 16S sequencing. C_LIO_LIT. ramosa is identified as a putative oncobiont associated with ALD-HCC, thus opening new avenues for diagnosis and treatment. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/24312231v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@7db628org.highwire.dtl.DTLVardef@1420dc8org.highwire.dtl.DTLVardef@196fc7borg.highwire.dtl.DTLVardef@19a0ed4_HPS_FORMAT_FIGEXP M_FIG C_FIG

Alpha-1 antitrypsin deficiency (AATD) is a rare genetic disorder caused by accumulation of misfolded -1 antitrypsin within hepatocytes. AATD patients are prone to develop liver disease that remains undiagnosed until the late stages of the disease. Due to challenges in manipulating the -1 antitrypsin genes in mice, determining a true loss of function of -1 antitrypsin in previous AATD mouse models has been challenging. Here, we report generation and liver characterization of a new humanized transgenic mouse model for AATD with a background of a CRISPR-Cas9 generated SERPINA1-null mouse. Male and female transgenic mice for normal (Pi*M) and mutant (Pi*Z) variants of human -1 antitrypsin at 4-6 months of age were subjected to this study. The accumulation of human -1 antitrypsin in the hepatocytes and fibrotic features of the liver were monitored by performing an in vivo study. We demonstrate a strong liver phenotype satisfying clinically relevant manifestations of liver pathology associated with AATD, including hepatic accumulation of human -1 antitrypsin globules, liver deposition of extracellular matrix proteins, hepatic ER stress, and liver fibrosis in Pi*Z mice, in addition to mild systemic inflammation. In addition to major phenotypic criteria of AATD-associated liver fibrosis, accompanying single-nucleus RNA-seq data demonstrate activation of pathways associated with liver metabolic changes, inflammation, and regeneration. Data from this study suggest our humanized transgenic AATD mouse model could provide a suitable model to study -1 antitrypsin loss of function, replicate the pathophysiology of AATD associated liver disease, and evaluate therapeutic reagents against this disease. NEW & NOTEWORTHYWe have characterized a new humanized transgenic mouse model of -1 antitrypsin deficiency with a SERPINA1-null background that shows strong manifestations of liver disease. Our data explores the altered phenotype of -1 antitrypsin deficient hepatocytes and suggests a relationship between liver cell types during disease progression. This model may become a useful tool for investigating -1 antitrypsin loss of function, pathogenic mechanisms, and for drug discovery aimed at both prevention and treatment of the disease.

Background and AimsThe newly proposed nomenclature for steatotic liver diseases (SLD) aims to reduce the stigma associated with "non-alcoholic fatty liver disease" (NAFLD), increase awareness, and provide a framework for delineating pathogenic pathways. Approach and ResultsWe projected the new nomenclatures diagnostic scheme onto National Health and Nutrition Examination Survey (NHANES) data and determined SLD prevalence, fibrosis risk factors, subtypes, and consistency with previous classifications. Steatosis grade and fibrosis stage were estimated from vibration controlled transient elastography (VCTE). At a threshold of 240 dB/m, 62.1% [95% confidence interval (CI), 59.8-64.3%] of adults ([&ge;] 20 years) and 30.5% (95% CI, 27.1-34.0%) of adolescents (12-19 years) had SLD. By American Gastroenterological Association criteria, 19.3 million (95% CI, 15.8-22.8) adults with SLD qualify for hepatology referral. Over 98% of adults but only 85% of adolescents with NAFLD met criteria for definite MASLD. Significant fibrosis ([&ge;] 8.6 kPa) occurred in 13.5 million (95% CI, 10.9-16.2) adults with MASLD; risk factors varied by race and ethnicity. Significant fibrosis occurred in over 1.5 million adults without any identified LD and was associated with lead (Pb) exposure, odds ratio = 3.89 (95% CI, 2.00-7.56). ConclusionsThe overarching term, SLD, changes the diagnostic algorithm and creates an umbrella classification that highlights the extraordinary prevalence of liver steatosis. The more precise nomenclature establishes a valuable patient-centric platform for research and clinical care, clarifying risk groups and risk factors, including adolescents with NAFLD but without definite MASLD and adults without SLD in whom toxic exposures may increase fibrosis risk.

Background & AimsLiver cirrhosis is a major cause of death worldwide, and its prevalence is growing rapidly due to the growth of obesity and diabetes population with non-alcoholic fatty liver disease (NAFLD). Yet, no effective therapeutics have been developed to treat NAFLD or its more advanced stage, non-alcoholic steatohepatitis (NASH). This has raised great concern for a representative liver model to be developed so that novel drugs could be screened, identified and developed. Presently, we aim to develop a liver organoid entirely from human induced pluripotent stem cells (hiPSC) to model liver fibrogenesis and NAFLD. MethodsHepatoblasts (HBs), mesenchymal stem cells (MSCs), hepatic stellate cell (HSCs) and endothelial cells (ECs) were derived from hiPSCs, allowed to self-organized and differentiated into liver organoids. Liver functions, transcriptomic and protein expression of liver organoids were characterized and validated. Liver organoids were exposed to thioacetamide (TAA) and free fatty acids (FFA) to be induced into liver disease model. ResultsThe liver organoids we fabricated were highly vascularized, exhibited liver-specific functions and hepatic cellular spatial organization. The presence of liver specific ECs, macrophages and cholangiocytes were found within our organoids. TAA induced fibrosis in our liver organoids that exhibited diminished liver functions, elevated pro-inflammatory cytokines and fibrosis-related gene expression, as well as extensive collagen deposit. Organoids treated with FFA developed steatosis, inflammation and fibrosis. ConclusionsWe generated a novel method, that is Matrigel-independent and size-controllable, for making human liver organoids. These organoids can potentially be utilized as tissue-mimetic in vitro model for high throughput screening to identify drugs that can be used to treat liver fibrosis and NAFLD.

Hepatitis B surface antigen (HBsAg) loss or functional cure (FC), is considered the desirable therapeutic outcome for chronic hepatitis B (CHB) patients. However, the immuno-pathological biomarkers and underlying mechanisms remain unclear. Here we present a comprehensive single cell-transcriptomic atlas together with immune-phenotyping of disease-associated cell states (DACS) isolated from intra-hepatic tissue and matched PBMCs of either CHB or FC patients. We find that the intra-hepatic environment displays specific cell identities and molecular signatures that are distinct from PBMCs. FC is associated with emergence of an altered adaptive immune response marked by CD4 cytotoxic T lymphocytes (CD4-CTLs), and an activated innate response represented by liver-resident natural killer (LR-NK) cells. Overall, these findings provide novel insights into immuno-pathological cell states associated with FC that could serve as prognostic biomarkers.

Abstract/KeywordsO_ST_ABSBackground and AimsC_ST_ABSPrimary biliary cholangitis (PBC) is a chronic inflammatory autoimmune disease of the biliary epithelial cells, causing slow progression of cholestasis and fibrosis. The aim of the study is to summarize the imaging features of PBC and examine the correlation between its clinicopathological and radiologic features, elucidating the specific clinicopathological and radiologic differences between male and female PBC patients, as well as between AMA-positive and AMA-negative PBC patients. MethodsDemographic, laboratory, radiologic and survival data were collected retrospectively for patients diagnosed and treated for PBC at the Mount Sinai Health System between 2016 and 2020 with at least one ultrasound, CT, or MRI of abdomen available for assessment. Biochemical and radiologic data were compared between male v.s. female groups, and AMA-positive v.s. AMA-negative group. ResultsA total of 273 patients diagnosed with PBC were included. Non-specific hepatic parenchymal disease, cirrhosis, cholelithiasis, splenomegaly, lymphadenopathy, benign liver masses/cysts were the most common features on abdominal images. 24% of PBC patients had biliary tree abnormalities on MRI. LAD was reported in 38-47% of PBC patients. There was no significant difference in age, race, or ethnicity between the sexes of patients with PBC, however imaging characteristics of portal hypertension were more frequently seen in men. AMA-negative PBC patients had similar distributions of age, race/ethnicity and survival to AMA-positive PBC patients. ConclusionOur study summarized the clinicopathological and imaging features of PBC and distinct subgroups. This would assist in the diagnosis of PBC and avoid over-testing in real-world practice.

Background and aimsRare, pathogenic variants can cause severe liver disease, requiring transplantation in childhood, but it is unclear how common variants in the same genes affect adults. Here, we aimed to establish population-level genetic evidence for whether monogenic diseases are associated with liver injury in adulthood. MethodsWe identified 99 genes where pathological mutations cause significant liver disease in children. For each, we used data from over 1.8 million adults to identify associations with biomarkers of liver injury. Observations were validated in multiple cohorts of adults with clinical liver disease and transcriptomics. Finally, we illustrated the importance of the JAG1-NOTCH pathway on the ductular reaction using immunohistochemistry. ResultsMost genes (56% (55/99)) had at least moderate evidence of association with liver-related traits at a population level. We identified 82 genome-wide (p<5x10-8) associations with markers of liver injury in 41% (41/99) of genes. Loss of function variants in these genes had a ten-fold greater effect on liver enzymes and well-established variants in PNPLA3 had a three-fold greater effect. Variants in ABCC2, ASL, BCS1L, HFE, and SERPINA1 were linked with presence of clinical liver disease in adults. Aggregated effects of 35 variants as polygenic risk score (PRS) was associated with 0.6% lower prevalence of MASLD between highest and lowest PRS groups. Transcriptional expression of 30% of genes was associated with severity of MASLD. Expression of JAG1-NOTCH2 pathway was associated with severity of PSC. JAG1 and NOTCH2 were expressed in injured bile ducts but not adjacent unaffected ducts. ConclusionsOnset and severity of liver disease in adulthood is influenced by genes that also cause severe monogenic liver disease in children.

Background and AimsThe xenobiotic nuclear receptor Constitutive Androstane Receptor (CAR) is essential for xenobiotic tumor promotion in mouse models. In these models, {beta}-catenin is genetically activated in approximately 80% of tumors. Chronic Hepatitis B Virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC), and {beta}-catenin activation is also frequently activated in HBV-associated HCCs. The goal of this research was to determine whether activation of CAR in a mouse model of chronic HBV infection would result in tumor formation and whether these tumors would display increased {beta}-catenin activation. Approach and ResultsWe treated transgenic mice expressing the HBV X protein (HBx) in hepatocytes with a single dose of the potent CAR agonist TCPOBOP. After 10 months, these mice developed large liver tumors that are characterized by {beta}-catenin nuclear localization and upregulation of {beta}-catenin targets. The {beta}-catenin regulator FoxM1 and the oxidative stress master regulator Nrf2, both of which are CAR gene targets, were also overactivated in tumors. The CAR/HBx tumors share a conserved gene signature with HBV-related human hepatocellular carcinoma. ConclusionsActivation of CAR in the presence of HBx results in tumors with strong {beta}-catenin activation. The mouse model we have described reflects the gene expression patterns seen in human HBV-associated HCC and presents an attractive basis for future studies.

Background and aimsThe prevalence and etiology of liver fibrosis vary over time and impact racial/ethnic groups unevenly. This study measured time-trends and identified factors associated with advanced liver fibrosis in the U.S. MethodsStandardized methods were used to analyze data on 47,422 participants ([&ge;] 20 years) in the National Health and Nutrition Examination Survey (1999-2018). Advanced liver fibrosis was defined as Fibrosis-4 [&ge;]2.67 and/or Forns Index [&ge;]6.9 and elevated ALT. ResultsThe estimated number of people with advanced liver fibrosis increased from 1.3 million (95% CI, 0.8-1.9) to 3.5 million (95% CI, 2.8-4.2), a nearly 3-fold increase. Prevalence was higher in non-Hispanic Black and Mexican American persons than in non-Hispanic White persons. In multivariable logistic regression analysis, cadmium was an independent risk factor in all racial/ethnic groups. Smoking and current excessive alcohol use were risk factors in most. Importantly, non-Hispanic Black persons had a distinctive set of risk factors compared to non-Hispanic White persons that included poverty (OR = 2.09; 95%CI, 1.44-3.03), and susceptibility to lead exposure (OR = 3.25; 95%CI, 1.95-5.43), but did not include diabetes (OR = 0.88; 95% CI, 0.61-1.27, P =0.52). Non-Hispanic Black persons were more likely to have high exposure to lead, cadmium, polychlorinated biphenyls, and poverty than Non-Hispanic White persons. ConclusionsThe number of people with advanced liver fibrosis has increased, creating a need to expand the liver care workforce. The risk factors for advanced fibrosis varied by racial/ethnicity. These variations provide useful information for the design of screening programs. Poverty and toxic exposures were associated with the high prevalence of advanced liver fibrosis in non-Hispanic Black persons and need to be addressed. Lay summaryBecause liver disease often produces few warning signs, simple and inexpensive screening tests that can be performed by non-specialists are needed to allow timely detection and linkage to care. This study shows that non-Hispanic Black persons have a distinctive set of risk factors that need to be taken into account when designing liver disease screening tests. Exposure to exogenous toxins may be especially important risk factors for advanced liver fibrosis in non-Hispanic Black persons.

Background & AimsEthanol affects lipid metabolism through multiple pathways, leading to fatty liver development in most ALD patients. Recent studies have highlighted the role of calpain, a calcium-dependent protease, in liver inflammation and fibrosis. Calpain activity is regulated by its essential subunit, Capns1, (calpain-4, Capn4), which stabilizes and modulates the activity of its catalytic isoforms, calpain-1 and calpain-2. This study investigated calpains impact on lipid metabolism in ALD. Approach & ResultsSix-week-old C57Bl6/J mice were injected with rAAV8 vectors encoding Capn4 shRNA or control vectors. After four weeks, mice underwent a 10-day period of ad libitum ethanol consumption, followed by a single gavaged ethanol administration on day 11. Following Capn4 knockdown, microvesicular steatosis was attenuated. While triglycerides and free fatty acids levels showed no significant changes, cholesterol levels were significantly reduced in the ethanol (EtOH) group with Capn4 knockdown. Cpt1a expression increased significantly in the EtOH group with Capn4 knockdown. Western blot analysis revealed increased Cleaved-HMGCR to Pro-HMGCR ratio in Capn4 knockdown mice, suggesting reduced HMGCR activity and suppressed cholesterol biosynthesis. LXR expression was mainly increased in the cytoplasm in the EtOH group, and following Capn4 knockdown, it was relocalized to the nucleus via its activation. In addition, RNA sequencing analysis suggests that Capn4 knockdown contributes to the reprogramming of ethanol-induced disruptions in metabolic and homeostatic pathways, primarily those involving cholesterol metabolism. ConclusionsFurther investigation into the relationship between Capn4 and cholesterol biosynthesis proteins may provide insights into using calpain inhibitors as a therapeutic approach for alcohol-related hepatitis.