JHEP Reports

Background and aimsAlcohol disturbs hepatic lipid synthesis and transport, but the role of lipid dysfunction in the severity of alcohol-related liver disease (ALD) is unclear. We therefore characterised the liver and plasma lipidome in a biopsy-controlled cohort of patients with early ALD. MethodsWe performed ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry for lipidomics of the liver and plasma from 315 patients, and of plasma from 51 healthy controls matched for age, gender and BMI. We correlated lipid levels with histological fibrosis, inflammation and steatosis, after correction for multiple testing and adjustment for age, gender, statin use, BMI, HbA1c, HOMA-IR, and ongoing drinking. Moreover, we investigated the mechanism of dysregulated sphingolipid metabolism by whole-blood transcriptomics and qPCR sequencing of miRNA. ResultsWe detected 198 lipids in the liver and 236 lipids in the circulation from 18 lipid classes. Nearly all ceramides, sphingomyelins and lyso-phosphocholines in plasma decreased as fibrosis progressed. This was paralleled by a comparable decrease in the liver. Circulating and liver sphingomyelins were also inversely associated with hepatic inflammation. The lipidomic signature of healthy controls was only comparable to ALD patients with no fibrosis. Three circulating miRNA, highly involved in sphingomyelin metabolism, were dysregulated together with the mRNA expression of enzymes in the sphingomyelin degradation pathway. Mendelian randomization in Finnish and UK population biobanks externally validated our findings, suggesting a causal relationship between genetic disposition to ALD and low sphingolipid levels. ConclusionsLiver fibrosis severity in alcohol-related liver disease is characterized by selective lipid depletion in blood and liver, indicating profound effects of progressive disease on the bioactive sphingolipids, already from early stages of fibrosis. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/21260429v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@ad651org.highwire.dtl.DTLVardef@17c7c95org.highwire.dtl.DTLVardef@1d742f7org.highwire.dtl.DTLVardef@16da292_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIThe lipidome in the liver and circulation in alcohol-related liver disease (ALD) C_LIO_LISphingolipid, phospholipid and triacylglycerol levels were lowered in fibrosis C_LIO_LIExpression and regulation of genes in the sphingolipid pathway were affected C_LIO_LIALD has a causal link to lowered sphingomyelin levels in blood C_LI Lay summaryAlcohol causes a dysfunctional fat metabolism in the liver. In this study, we detected 198 different types of lipids in the liver and 236 in the blood stream of patients with different severity of alcohol-related liver disease. We found that patients with more severe scarring (fibrosis) of the liver, and more severe liver inflammation, had lower levels of sphingolipids both in the circulation and the liver. Sphingolipids regulate cell survival and inflammation, so they may be involved in the mechanism of progressive alcohol-related liver disease.

Background and aimsAlcohol-associated hepatitis (AH) is the most life-threatening form of alcohol-associated liver disease (ALD). AH is characterized by severe inflammation attributed to increased levels of ethanol, microbes or microbial components, and damage-associated molecular pattern (DAMP) molecules in the liver. HSPB1 (Heat Shock Protein Family B (Small) Member 1; also known as Hsp25/27) is a DAMP that is rapidly increased in and released from cells experiencing stress, including hepatocytes. The goal of this study was to define the role of HSPB1 in AH pathophysiology. MethodsSerum HSPB1 was measured in a retrospective study of 184 heathy controls (HC), heavy alcohol consumers (HA), patients with alcohol-associated cirrhosis (AC), and patients with AH recruited from major hospital centers. HSPB1 was also retrospectively evaluated in liver tissue from 10 HC and AH patients and an existing liver RNA-seq dataset. Finally, HSPB1 was investigated in a murine Lieber-DeCarli diet model of early ALD as well as cellular models of ethanol stress in hepatocytes and hepatocyte-macrophage communication during ethanol stress. ResultsCirculating HSPB1 was significantly increased in AH patients and levels positively correlated with disease-severity scores. Likewise, HSPB1 was increased in the liver of patients with severe AH and in the liver of ethanol-fed mice. In vitro, ethanol-stressed hepatocytes released HSPB1, which then triggered TNF-mediated inflammation in macrophages. Anti-HSPB1 antibody prevented TNF release from macrophages exposed to media conditioned by ethanol-stressed hepatocytes. ConclusionsOur findings support investigation of HSPB1 as both a biomarker and therapeutic target in ALD. Furthermore, this work demonstrates that anti-HSPB1 antibody is a rational approach to targeting HSPB1 with the potential to block inflammation and protect hepatocytes, without inactivating host defense. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/24313193v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@141816eorg.highwire.dtl.DTLVardef@1985d66org.highwire.dtl.DTLVardef@1c0ed4org.highwire.dtl.DTLVardef@118ca9_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIHSPB1 is significantly increased in serum and liver of patients with alcohol-associated hepatitis. C_LIO_LIEthanol consumption leads to early increases in HSPB1 in the mouse liver. C_LIO_LIHepatocytes subjected to ethanol stress release HSPB1 into the extracellular environment where it activates TNF-mediated inflammation in macrophages. C_LIO_LIAnti-HSPB1 antibody blocks hepatocyte-triggered TNF in a model of hepatocyte-macrophage communication during ethanol stress. C_LI

BackgroundAlcohol overuse disrupts liver function and alters microbial gut communities, with alcohol-related liver disease (ALD) accounting for half of all liver-related deaths worldwide. Bile acids (BAs) regulate liver and gut function, but their metabolism becomes disrupted in ALD. While it is known that gut microbes transform primary to secondary BAs, which are subsequently reabsorbed via the enterohepatic circulation, BA metabolism during ALD progression remains poorly understood. MethodsWe investigated BA co-metabolism in a cross-sectional cohort of individuals with ALD (n=462) and healthy controls (n=148). We validated key findings in two independent ALD cohorts (n=34 and n=52). We integrated BA concentrations, measured by targeted mass spectrometry in feces and plasma, with liver proteomics, and gut microbiome profiles derived from metagenomic and metatranscriptomic sequencing of fecal samples. ResultsAdvanced fibrosis was associated with decreased hepatic BA synthesis and impaired BA transport. Despite this, disease progression corresponded with increased levels of primary and secondary BAs in plasma and feces. The abundance of microbial secondary BA dehydroxylation and epimerization pathways in the gut microbiome changed with disease severity. Genes encoding early steps in the multi-step dehydroxylation pathway increased, whereas those involved in later steps were depleted, indicating a community-level microbial imbalance. In ALD, we identified Eggerthella lenta as a key mediator of BA dehydroxylation, while Mediterraneibacter torques and Bacteroides thetaiotaomicron facilitated most of the BA epimerization as a detoxification mechanism. ConclusionFibrotic ALD is characterized by disrupted primary BA synthesis and transport, leading to BA accumulation in the gut and blood circulation. Altered microbial secondary BA metabolism reflects a compensatory mechanism that becomes impaired at advanced fibrosis stages. Our findings highlight the gut-liver axis as an important factor influencing ALD progression. Impact and ImplicationsO_LIWith the progression of alcohol-related liver disease (ALD), levels of bile acids (BA) in serum and feces increase, but BA production and transport are impaired in the liver. C_LIO_LISecondary microbial BA metabolism, particularly epimerization and dehydroxylation, increases in ALD. However, a key enzyme, baiN, is depleted, illustrating a microbial community-level metabolic dysbiosis. C_LIO_LIThe main contributing microbial species were, among others, Mediterraneibacter torques, Bacteroides thetaiotaomicron, and Eggerthella lenta, which could serve as potential targets for future microbial-targeted interventions. C_LI Lay summaryAlcohol-related liver disease (ALD) from long-term alcohol overuse affects how the liver and gut interact, especially in handling bile acids (BAs), which are molecules produced by the liver and transformed by gut bacteria. Our study found that in people with ALD, the liver produces fewer BAs, but BAs accumulate in the gut and blood because their transport is impaired. We also observed that bacterial transformations of these BA change as the disease progresses, most likely due to an imbalance in the gut microbiome. For the first time, we identify specific bacterial species that strongly influence BA levels in ALD. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/25332046v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@19966f0org.highwire.dtl.DTLVardef@352be6org.highwire.dtl.DTLVardef@d5146dorg.highwire.dtl.DTLVardef@1304022_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 0:C_FLOATNO Graphical Abstract C_FIG

Background and AimsSteatotic liver disease (SLD) is characterized by excessive lipid accumulation in hepatocytes, and alcohol consumption may modify the disease course, but the evidence is inclusive. This systematic review and meta-analysis aimed to holistically evaluate the impact of mild, moderate, and high levels of alcohol consumption on hepatic and extrahepatic outcomes in SLD. MethodsWe systematically searched EMBASE, MEDLINE, Web of Science, and the Cochrane Central Register of Controlled Trials for relevant studies. The study outcomes included liver related events, malignancy, mortality and cardiovascular disease among adults with SLD who consumed alcohol. ResultsOf 2228 records identified, twenty-six studies comprising 466611 adults with SLD were included. High alcohol consumption was associated with an increased risk of liver-related events compared with abstinence (2.97, 95% CI 1.61-5.50; p<0.001), and a similar association was observed among alcohol drinkers overall (HR 1.93, 95% CI 1.60-2.33; p<0.001). Moderate alcohol consumption was associated with a higher incidence of malignancy (HR 1.41, 95% CI 1.13-1.78; p=0.677). In contrast, mild alcohol consumption was associated with lower all-cause mortality compared with abstinence (HR 0.88, 95% CI 0.78-0.98; p=0.001). No association was observed between alcohol consumption and cardiovascular disease incidence or hepatocellular carcinoma ConclusionsAlcohol intake may increase the risk of liver-related complications and cancer risk in individuals with SLD. Mild alcohol consumption was associated with lower all-cause mortality, and alcohol intake showed no association with cardiovascular disease incidence. Further studies are needed to clarify the dose-dependent effects of alcohol on hepatic and extrahepatic outcomes in SLD.

Alterations in cellular metabolism represent an important response to proliferative signals in both normal and transformed tissues. The benign proliferative process of liver regeneration after partial hepatectomy offers insight into homeostatic mechanisms to control liver mass, which are disrupted in liver disease induced by viral factors, alcohol, or associated with obesity. Moreover, successful targeting of cancer depends on the identification of genes and pathways that are selectively activated in the transformed state. Here, we present a differential transcriptomic and metabolomic analysis of benign proliferative and transformed liver, including associated plasma metabolite and lipid species. Using partial hepatectomy-induced liver regeneration and diethylnitrosamine (DEN) induced carcinogenesis, we identify and analyze alterations specific to multiple regenerative and transformed tissue states. Transcriptomics and LC/MS based metabolite profiling reveal fatty acid import and storage are specifically rewired during liver regeneration in a time dependent manner, a phenomenon not observed in liver tumors. In contrast, liver tumors exhibit preferential activation of numerous metabolic pathways, including glycolysis, serine biosynthesis, and polyamine metabolism. Alterations in serine metabolism occur at the earliest detectable stages in tumorigenesis and promote survival upon serine restriction. These data demonstrate that transformation-induced alterations in metabolism are distinct from those observed in normal regenerative cell division, which may be used to identify transformation-specific liabilities.

BackgroundRecently, the new nomenclature metabolic dysfunction-associated steatohepatitis (MASH) was proposed to supersede non-alcoholic steatohepatitis (NASH). To optimize the management of these patients, it is crucial to comprehend the similarities and differences between individuals with NASH and MASH. MethodsWe analyzed data from 13,846 participants in the third National Health and Nutrition Examination Surveys, along with their linked mortality through 2019. NASH and MASH were defined based on respective criteria. Survey-weight adjusted multivariable Cox proportional model was used to examine mortality. ResultsThe overall prevalence of steatohepatitis, NASH and MASH was 5.7% (n=788), 4.1% (n=564) and 5.5% (n=763), respectively. Most individuals with NASH (96.8%) could be categorized as MASH, but only 69.7% individuals with MASH qualified as NASH. During a median follow-up of 27 years, individuals with MASH exhibited a 53% higher risk of all-cause mortality (adjusted hazard ratio [aHR] 1.53, 95% CI 1.24-1.89). But individuals with NASH didnt show an association with all-cause mortality after adjustment for metabolic risk factors (aHR 1.14, 95% CI 0.91-1.44). Notably, individuals who met the criteria for MASH but not NASH (NASH(-)/MASH(+)) had a higher risk of all-cause mortality (aHR 2.47, 95% CI 1.71-3.57) compared to those with NASH(+)/MASH(+) (aHR 1.22, 95% CI 0.97-1.55). Moreover, advanced fibrosis was only associated with an increased risk of all-cause mortality in individuals with MASH, not NASH. ConclusionsMASH, rather than NASH, was associated with an elevated risk of all-cause mortality after adjusting for metabolic risk factors. Well-designed prospective studies are needed to assess and validate our findings.

Background & AimsAlterations in the glycosylation of blood proteins affect protein functionality and have been linked to various diseases. Metabolic dysfunction- associated steatotic liver disease (MASLD) is a silent disease, of which progression to advanced disease stages including metabolic dysfunction-associated steatohepatitis (MASH), fibrosis and cirrhosis often goes unnoticed. As current non- invasive diagnostic tests lack specificity, the purpose of this work was to study total blood protein N-glycosylation in individuals with MASLD and various degrees of fibrosis as compared to healthy controls. MethodsIn two independent cross-sectional cohort studies, blood N-glycosylation analysis was performed by mass spectrometry on released glycans of overall 132 MASLD patients and 99 age- and sex-matched healthy controls. Relationships between glycosylation traits and the disease spectrum of MASLD including fibrotic MASLD were investigated in comparison to healthy controls. Furthermore, publicly available transcriptomics datasets were used to explore glycosyltransferase expression in patients with MASLD. ResultsGlobally lower 2,3-sialylation distinguished MASLD from healthy controls (OR [CI]=0.36; [0.18-0.67]; p-value=0.019, and 0.11 [0.04-0.24]; p-value<0.000001), as well as non-fibrotic MASLD from its fibrotic counterparts (OR: 0.13 [0.06-0.26]; p- value<0.0001), but showed no association with steatohepatitis activity. Hepatic ST3GAL6, a sialyltransferase responsible for N-glycan 2,3-sialylation, negatively associated with fibrosis progression, similar to the observed glycomic signature. Both glycomic and transcriptomic signatures were replicated in independent cohorts. ConclusionsFibrotic MASLD is characterized by a global decrease of blood protein 2,3-sialylation and according decrease in hepatic 2,3-sialyltransferase expression, associating with disease progression. These findings suggest alterations in the N- glycan biosynthetic pathway and are potentially useful in the early diagnosis of fibrosis in MASLD. Lay SummaryNon-invasive markers of fibrotic MASLD perform suboptimal. This research identified that changes in blood protein glycosylation coincide with fibrosis development, offering blood-based markers that could potentially replace a liver biopsy. What You Need to KnowO_ST_ABSBACKGROUND AND CONTEXTC_ST_ABSThe majority of the plasma glycoproteins is synthesized in the liver and changes to their glycosylation are known to affect their function and to associate with liver disorders. NEW FINDINGSMASLD patients exhibit lower 2,3-sialylation on the complete range of their blood protein N-glycans, which coincides with the histological appearance of fibrosis, mediated likely via downregulation of hepatic ST3GAL6. LIMITATIONSWhile the findings of this study has could have implications for diagnosing fibrotic MASLD, the identified glycomic signature needs to be confirmed in a larger, ideally prospective patient cohort. CLINICAL RESEARCH RELEVANCEBy identifying specific signatures in the blood protein N-glycome, this research offers potential non-invasive markers for early diagnosis and monitoring of fibrosis in MASLD. Non-invasive diagnosis could potentially lessen the need for liver biopsies, and allow for timely intervention and improved disease management, ultimately leading to improvement of patient outcomes and the reduction of liver-related morbidity and mortality. BASIC RESEARCH RELEVANCEThe observed glycomic and transcriptomic signatures offer molecular-level insights into fibrosis development in MASLD. This paves the way for future research at the intersection of glycoscience and hepatology, that will offer deeper insights into the pathophysiology of this liver disease.

BackgroundSince liver disease is often clinically unapparent, non-invasive biomarkers predicting future major adverse liver outcomes (MALO) are urgently needed. Therefore, we assessed the usefulness of a novel, proximity extension assay (PEA)-based high-throughput targeted proteomics method to predict MALOs. MethodsPEA plasma proteomic data (>2900 proteins) and clinical information were accessed from the population-based UK Biobank (UKB) cohort, including >53000 individuals with a median follow-up of >10 years and its subcohorts of obese (>12900) and diabetic (>1600) participants. The validation cohorts comprised 287 subjects with severe alpha1-antitrypsin deficiency (AATD), Pi*ZZ genotype, and 960 people living with HIV (PLHIV), who underwent liver stiffness measurement (LSM) via transient elastography. Selected PEA parameters were compared to routine measurements. Bayes-moderated linear models (age and sex as covariates) assessed the differential abundance. Logistic regression was used to identify and validate a novel prognostic score. ResultsRoutine gamma-glutamyltransferase (GGT) and aspartate aminotransferase (AST) levels strongly correlated with PEA-based measurements (r=0.91 and r=0.68, respectively). Similarly, PEA-based thrombospondin-2 levels strongly correlated with immunoassay-based values (r=0.85). Twenty proteins were consistently associated with future MALOs/increased LSM in all cohorts. UKB cohort was used to develop a novel five-component PEA score that demonstrated superior predictive power (AUROC=0.84) compared to established indices/scores, including AST-to-platelet-ratio index (APRI, AUROC=0.73) and Fibrosis-4 index (FIB4, AUROC=0.72) and its attractive predictive power was sustained in diabetic/obese subcohorts. In PLHIV and AATD validation cohorts, all five components showed gradual increases across fibrosis stages, and PEA score numerically outperformed APRI/FIB4 in predicting significant liver disease. ConclusionOur study identifies a new PEA score consisting of epithelial/hepatic stellate cell markers that demonstrates an attractive discriminative ability in several independent cohorts and different liver disease aetiologies.

Introduction: The overlap between chronic hepatitis B (CHB) and metabolic dysfunction-associated steatotic liver disease (MASLD) is an emerging global health challenge. We investigated the impact of MASLD and metabolic comorbidity in a diverse London viral hepatitis clinic. Methods: This retrospective cross-sectional study (May 2018-Feb 2024) included adults with CHB having controlled attenuation parameter (CAP) measurements. MASLD was defined as CAP >264 dB/m plus [&ge;]1 cardiometabolic factor (CMF). We used univariable and multivariable models to examine MASLD's relationship with liver stiffness and hepatitis B viral load (HBV VL). Results: Among 323 individuals (67% male, median age 36), most were from Black (35%) or non-white British/Irish (29%) backgrounds. Overall, 64% had [&ge;]1 CMF, and 20% had MASLD. The CHB/MASLD group was significantly older (median 43 vs 35 years, p<0.001) with higher median alanine transaminase (35 vs 30 IU/L, p=0.02) and liver stiffness (5.3 vs 4.7 kPa, p<0.001). Following adjustment for covariates, MASLD remained significantly associated with liver stiffness ({beta} = 0.48 kPa, p=0.03). While univariable analysis showed significantly lower HBV VL in people with MASLD (median 54 vs 417 IU/ml, p=0.004), adjusted multivariable analysis revealed no significant association between MASLD and log10 HBV VL (p=0.2). Conclusions: Although adjusted analysis does not support an independent association between MASLD and HBV VL, the data highlight a substantial cardiometabolic burden in this CHB population and clearly link MASLD to more severe liver disease. Holistic consideration of metabolic comorbidities is crucial in comprehensive CHB management.

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.

BackgroundDihydrosphingolipids are lipid molecules biosynthetically related to ceramides. An increase in ceramides is associated with enhanced fat storage in the liver and inhibition of their synthesis is reported to prevent the appearance of steatosis in animal models. However, the precise association of dihydrosphingolipids with non-alcoholic fatty liver disease (NAFLD) is yet to be established. We employed a diet-induced NAFLD mouse model to study the association between this class of compounds and disease progression. MethodsMice were fed a high-fat diet enriched in cholesterol and supplemented with glucose and fructose up to 40 weeks. A mouse subgroup was treated with carbon tetrachloride to accelerate fibrosis development. Animals were sacrificed at different time-points to reproduce the full spectrum of histological damage found in human disease, including steatosis (NAFL) and steatohepatitis (NASH) with and without significant fibrosis. Blood and liver tissue samples were obtained from patients (n=195) whose NAFLD severity was assessed histologically. Lipidomic analysis was performed using liquid chromatography-tandem mass spectrometry. ResultsTriglyceride, cholesterol ester and dihydrosphingolipid levels were increased in the liver of model mice in association with the degree of steatosis. Dihydroceramide concentrations increased with the histological severity of the disease in liver samples of mice (0.024 {+/-} 0.003 vs 0.049 {+/-} 0.005, non-NAFLD vs NASH-fibrosis, p<0.0001) and patients (0.105 {+/-} 0.011 vs 0.165 {+/-} 0.021, p=0.0221). Several dihydroceramide and dihydrosphingomyelin species were increased in plasma of NAFLD patients and correlated with accumulation of liver triglycerides. ConclusionsDihydrosphingolipids accumulate in the liver in response to increased free fatty acid overload and are correlated with progressive histological damage in NAFLD. The increase in dihydrosphingolipids is related to upregulation of hepatic expression of enzymes involved in de novo synthesis of ceramides. HIGHLIGHTSO_LINeutral lipids and dihydrosphingolipids accumulate in liver in correlation with the histological severity of NAFLD in both mice and humans. C_LIO_LIThe ceramide pathway is stimulated to alleviate the free fatty acid excess in liver of NAFLD models. C_LIO_LIAppearance of significant fibrosis is associated with reduced concentrations of neutral lipids but not dihydrosphingolipids in a mouse model of NAFLD. C_LI

Background and AimsChronic hepatitis C virus (HCV) infection can lead to cirrhosis, development of hepatocellular carcinoma (HCC) and several extrahepatic manifestations. A sustained virological response (SVR) is achieved with direct-acting antivirals (DAA) in over 95% of the patients, but sequelae do not improve in all patients, suggesting permanent biological alterations induced by HCV infection. Therefore, we investigated the influence of chronic HCV infection, viral elimination and cirrhosis on inflammatory immune mediators. Approach and ResultsIn 102 chronic HCV patients, 46 with and 56 without cirrhosis, 92 soluble immune mediators (SIM) were measured in plasma samples at therapy start, end of treatment and long-term follow-up (median 96 weeks). 39 HBsAg positive persons with HBeAg negative infection served as controls. At baseline, 42 SIM were altered in chronic HCV patients (adj.p <0.05). Notably, patients with cirrhosis displayed a higher frequency and severity of alterations. At long-term follow-up, the SIM profile of the non-cirrhotic patients recovered to the level of the control group, while 41 SIM remained altered in cirrhotic patients. 33 of these SIM correlated with elastography, among them SIM linked to carcinogenesis as e.g. HGF, IL8 and IL6 (KEGG Pathways hsa05202, hsa05200). ConclusionsHCV-related changes in the inflammatory milieu can persist even after HCV elimination, specifically in cirrhotic patients. These changes are closely associated with liver damage and carcinogenesis. Our findings underscore the need for HCV elimination before extensive liver injury occurs and suggest further investigation of the relationship between persistent inflammatory milieu changes and long-term sequelae after HCV elimination. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/23297828v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@3a885borg.highwire.dtl.DTLVardef@ce3eb1org.highwire.dtl.DTLVardef@7601f6org.highwire.dtl.DTLVardef@17ad655_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background & AimsAlcohol-associated liver disease (ALD) is a major cause of liver disease worldwide with scarce therapeutic options. Animal models poorly recapitulate advanced ALD precluding the development of new treatments. Organoids have emerged as a powerful human-based preclinical tool. However, current patient-derived liver organoids fail to recapitulate the epithelial heterogeneity and its generation requires liver surgical resections, thus limiting personalized disease modeling. Here, we report the development of organoids from liver needle biopsies (b-Orgs) from patients with ALD. Methodsb-Orgs were generated from tru-cut biopsies from patients at early (n=28) and advanced (n=34) stages of ALD. b-Orgs were characterized by immunofluorescence, bulk and single cell RNA-sequencing and compared to parental tissues. b-Orgs were used to model ALD progression, identify pathogenic drivers, induce alcohol-associated hepatitis (AH) and evaluate response to prednisolone. ResultsPhenotypic and functional analysis of b-Orgs showed hepatocyte- enriched features. Single-cell RNA-sequencing revealed a heterogeneous cell composition comprising hepatocyte, biliary and progenitor populations, mirroring the epithelial landscape found in patients with advanced ALD. Moreover, b-Orgs preserved disease-stage features and allowed to identify the association of ELF3 with cell plasticity and disease progression. Finally, stimulation of b-Orgs with drivers of ALD induced pathophysiological features of alcohol-associated hepatitis, including ROS production, lipid accumulation, inflammation and decreased cell proliferation, which were mitigated in response to prednisolone. Conclusions Overall, we provide a human-based model that recapitulates epithelial complexity and patient specific features, allowing to identify drivers of cell plasticity and expanding organoid-based liver disease modeling for personalized medicine. Impact and implications Here, we describe the generation of biopsy-derived organoids (b-Orgs) from patients with liver disease. b-Orgs reproduce the liver epithelial cell composition found in patients liver tissue and are efficiently generated from different stages of the disease, providing a platform for patient- tailored disease modeling and drug testing.

Background and aims. The first direct-acting antivirals (DAAs) to treat the viral hepatitis C (HCV) became available in 2011. Despite numerous clinical studies of patient outcomes after treatment, few have evaluated changes in the liver microenvironment. Despite achieving sustained virologic response (SVR), patients may still experience adverse outcomes like cirrhosis and hepatocellular carcinoma. By comparing gene and protein expression in liver biopsies collected before and after treatment, we sought to determine whether specific signatures correlated with disease progression and adverse clinical outcomes. Methods. Biopsies were collected from 22 patients before and after DAA treatment. We measured [~]770 genes and used multispectral imaging with custom machine learning algorithms to analyze phenotypes of intrahepatic macrophages (CD68, CD14, CD16, MAC387, CD163) and T cells (CD3, CD4, CD8, CD45, FoxP3). Results. Before DAA treatment, patients showed two distinct gene expression patterns: one with high pro-inflammatory and antiviral gene expression and another with weaker expression. Patients with adverse outcomes exhibited significantly (p<0.05) more inflammatory activity and had more advanced fibrosis stages in their baseline biopsies than those with liver disease resolution. Patients who achieved SVR had significantly decreased liver enzymes, reduced inflammatory scores, and restored type 1 interferon pathways similar to controls. However, after DAA treatment, patients with persistently high gene expression (67%, pre-hot) still had significantly worse outcomes (p<0.049) despite achieving SVR. A persistent lymphocytic infiltrate was observed in a subset of these patients (76.5%). After therapy, anti-inflammatory macrophages (CD16+, CD16+CD163+, CD16+CD68+) increased, and T cell heterogeneity was more pronounced, showing a predominance of helper and memory T cells (CD3+CD45RO+, CD4+CD45RO+, CD3+CD4+CD45RO+). Conclusions. Patients who have more inflamed livers and more advanced fibrosis before DAA treatment should be closely followed for the development of adverse outcomes, even after achieving SVR. We can enhance patient risk stratification by integrating gene and protein expression profiles with clinical data. This could identify those who may benefit from more intensive monitoring or alternative therapeutic approaches, inspiring a new era of personalized patient care. Lay SummaryDirect-acting antiviral (DAA) therapy has dramatically improved the treatment of chronic HCV, making it curable for most people. This study determined gene and protein expression differences in the liver before and after treatment of HCV. These results will lead to a deeper understanding of the changes in the hepatic immune microenvironment with and without the virus present in the liver in hopes of improving patient surveillance, prognosis, and outcome in the future.

Background & AimsThe metabolic interplay between obesity and myopenia (MP) in chronic liver disease (CLD) remains poorly understood. We investigated whether progressive liver dysfunction mediates an obesity-MASLD (metabolic dysfunction{square}associated steatotic liver disease)-MP cascade and assessed the prognostic impact of MP and myopenic obesity (MO) in advanced CLD (ACLD). MethodsWe analyzed 859 CLD patients cross{square}sectionally and 169 obese patients longitudinally (median 38{square}months), using multimodal MRI to measure liver stiffness (LS), proton{square}density fat fraction (PDFF), and body composition. Temporal relationships between changes ({Delta}) in adiposity, muscle mass, and liver injury markers were assessed. Prognosis in ACLD (n=328) was evaluated using Cox regression. ResultsMP and MO were present in 29% and 8% of patients, respectively. In the longitudinal cohort, MO prevalence increased significantly from 15% to 23% (P{square}<{square}0.01). In fibrosis stages F0-2, {Delta}visceral adipose tissue significantly correlated with {Delta}PDFF, {Delta}ALT, and {Delta}LS (all P{square}<{square}0.01), whereas {Delta}muscle mass decreased, likely from weight loss. In F3-4, {Delta}ALBI score and {Delta}PDFF (hepatic fat "burning{square}out") independently correlated with {Delta}muscle mass (both P{square}<{square}0.01). In ACLD, MP--but not obesity itself--was an independent predictor of liver{square}related death (HR{square}2.27, 95%{square}CI{square}1.08-4.78, P{square}={square}0.025). ConclusionsOur findings suggest an obesity-MASLD-MP cascade driven by a liver-centered metabolic paradox: preserved hepatic function promotes harmful fat accumulation, whereas hepatic dysfunction leads to fat depletion (energy deficiency) and muscle loss. Recognition of this dynamic loop highlights the need for stage-specific strategies: fat reduction initially, followed by aggressive muscle preservation and energy repletion in ACLD.

Background and AimMASLD affects 30-38% of Indian adults, yet the contribution of genetic risk variants to disease susceptibility and fibrosis progression remains poorly characterised. We investigated the association of 12 candidate SNPs with MASLD susceptibility and fibrosis severity in North Indian patients, benchmarking allele frequencies against IndiGenomes and global populations. MethodsSixty-nine MASLD patients (75.4% male; median BMI 29.8 kg/m{superscript 2}) from a tertiary care liver clinic in New Delhi were genotyped for 12 SNPs using Illumina custom BeadChip array and Sanger sequencing. Patients were stratified by liver stiffness measurement (LSM): significant fibrosis ([&ge;]8 kPa, n=38) versus no significant fibrosis (<8 kPa, n=31). Allele frequencies were compared with IndiGenomes ([~]1,020 Indian individuals) and 1000 Genomes populations. ResultsPNPLA3 rs738409 G allele was the strongest within-cohort predictor of significant fibrosis (allelic OR 2.89, 95% CI 1.35-6.19, P=0.006; dominant model OR 3.94, P=0.008), with carriers demonstrating higher LSM (median 15.6 vs. 7.5 kPa, P=0.005). SAMM50 rs3761472 (OR 2.12, P=0.065) and FTO rs9939609 (OR 2.08, P=0.089) showed non-significant trends. In the population-level comparison, APOC3 rs2854116 T allele was the only variant significantly enriched after Bonferroni correction (64.0% vs. 47.9%; OR 1.93, 95% CI 1.35-2.77, P<0.001), followed by PNPLA3 (33.3% vs. 24.1%, OR 1.57, P=0.019) and SAMM50 (31.2% vs. 22.6%, OR 1.55, P=0.028). Notably, APOC3 showed no association with fibrosis (OR 0.96, P=1.000), suggesting a role in susceptibility rather than progression. All SNPs were in Hardy-Weinberg equilibrium. ConclusionsThis study reveals a dissociation between genetic determinants of MASLD susceptibility and fibrosis progression in North Indian patients. APOC3 rs2854116 predisposes to MASLD at the population level, while PNPLA3 rs738409 drives fibrosis severity within established disease, underscoring the need for ancestry-specific genetic risk stratification. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/26347059v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@a07808org.highwire.dtl.DTLVardef@12882adorg.highwire.dtl.DTLVardef@9b33a1org.highwire.dtl.DTLVardef@15aa5e8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background & aimsGenome-wide association studies in adults have identified variants in HSD17B13 and MARC1 as protective against NAFLD. It is not known if they are similarly protective in children and, more generally, whether the peri-portal inflammation of pediatric NAFLD and lobular inflammation seen in adults share common genetic influences. Therefore, we aimed to: establish if these variants are associated with NAFLD in children, and to investigate the function of these variants in hepatic metabolism using metabolomics. Methods960 children (590 with NAFLD, 394 with liver histology) were genotyped for rs72613567T>TA in HSD17B13, rs2642438G>A in MARC1. Genotype-histology associations were tested using ordinal regression. Untargeted hepatic proteomics and plasma lipidomics were performed in a subset of samples. In silico tools were used to model the effect of rs2642438G>A (p.Ala165Thr) on MARC1. Resultsrs72613567T>TA in HSD17B13 was associated with lower odds of NAFLD diagnosis (OR 0.7 (95%CI 0.6-0.9) and lower grade of portal inflammation (P<0.001) whilst rs2642438G>A in MARC1 was associated with lower grade of hepatic steatosis (P=0.02). Proteomics found reduced expression of HSD17B13 in carriers of the protective allele, whereas MARC1 levels were not affected by genotype. Both variants showed downregulation of hepatic fibrotic pathways, upregulation of retinol metabolism and perturbation of phospholipid species. Modelling suggests that p.Ala165Thr would disrupt the stability and metal-binding of MARC1. ConclusionsThere are shared genetic mechanisms between pediatric and adult NAFLD, despite their differences in histology. MARC1 and HSD17B13 are involved in phospholipid metabolism and suppress fibrosis in NAFLD.

Purpose Metabolic dysfunction-associated steatotic liver disease (MASLD) is a major cause of chronic liver disease and liver-related morbidity worldwide. Although dietary factors may influence MASLD progression, the long-term liver-specific implications of artificially sweetened beverage (ASB) intake remain unclear. We aimed to examine the association between ASB intake and the risk of liver-related adverse events and liver-related death among individuals with MASLD. Methods This prospective cohort study included 50,562 participants with MASLD from the UK Biobank. ASB intake was assessed using 24-hour dietary recalls and categorized as 0, >0-1, and >1 serving/day. Multivariable Cox proportional hazards models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for liver-related adverse events and liver-related death. Restricted cubic spline models were used to assess dose-response patterns, and competing-risk analyses were performed by treating liver-related death as a competing event for liver-related adverse events. Additional substitution, subgroup and sensitivity analyses were conducted to evaluate the robustness of the findings. Results During a median follow-up of 12.8 years, 292 liver-related adverse events and 91 liver-related deaths occurred. Compared with participants reporting no ASB intake, those consuming >1 serving/day had a higher risk of liver-related adverse events in the fully adjusted model (HR 1.40, 95% CI 1.02-1.93; P = 0.039), whereas the association for >0-1 serving/day was not statistically significant (HR 1.26, 95% CI 0.92-1.71; P = 0.149). The risk of liver-related adverse events increased across ASB intake categories (P for trend = 0.023). Restricted cubic spline analysis indicated a positive linear association between ASB intake and liver-related adverse events (P-overall <0.001; P-nonlinearity = 0.72). In competing-risk analysis, the association for >1 serving/day remained consistent after accounting for liver-related death as a competing event (sub-HR 1.40, 95% CI 1.02-1.93; P = 0.038; Gray test P = 0.006). The association was robust in sensitivity analyses. ASB intake was not significantly associated with liver-related death, and beverage substitution analyses showed no significant associations. Conclusion Among individuals with MASLD, high ASB intake, particularly >1 serving/day, was associated with an increased risk of liver-related adverse events, but not liver-related death. This association was consistent across dose-response, competing-risk, and sensitivity analyses, suggesting that high ASB intake may represent a potential dietary risk marker for adverse liver outcomes in MASLD.

Background: Metabolic adaptation is a recognized feature of therapeutic resistance in hepatocellular carcinoma (HCC), but it is unclear whether transcriptional states exposed during acquired resistance are restricted to drug adaptation or reflect broader aggressive tumor biology. We tested whether metabolic programs derived from a lenvatinib-resistance model identify a clinically adverse transcriptional state in an independent HCC patient cohort. Methods: The discovery framework was based on GSE186191, comprising parental and acquired lenvatinib-resistant Hep3B and Huh7 cells. A pre-specified 33-gene lipid-source ledger served as a biological anchor, and three discovery-derived programs, MYC Targets V2, mTORC1 Signaling, and Fatty Acid Metabolism, were frozen before patient-level evaluation. In TCGA-LIHC, single-sample enrichment scores for the three programs were population-standardized and summed to generate an integrated metabolic score. Overall survival was assessed by Kaplan-Meier and Cox analyses. Whole-transcriptome differences between high- and low-score tumors were characterized by preranked gene set enrichment analysis (GSEA). Results: The survival cohort comprised 282 patients (118 deaths), with 141 patients in each median-defined score group. High-score patients had shorter overall survival (log-rank P=0.000419). The continuous score was associated with mortality in univariable analysis (HR 1.86, 95% CI 1.33-2.61; P=0.000293) and in the frozen model adjusted for age, sex, and stage indicators (HR 1.93, 95% CI 1.35-2.76; P=0.000350; n=277). In 327 primary tumors, Fatty Acid Metabolism was strongly depleted in high-score tumors (NES -2.06; FDR<0.001). MYC Targets V2 (NES 1.18; FDR=0.232) and mTORC1 Signaling (NES 1.11; FDR=0.229) showed positive directional enrichment without FDR significance. Conclusions: A lenvatinib-resistance-derived transcriptional program is associated with an adverse-survival state in HCC. The strongest patient-level pathway feature is depletion of canonical fatty-acid metabolism, accompanied by directional MYC/mTORC1 features rather than statistically established pathway activation. These findings support a testable model of metabolic identity remodeling but do not establish causality or clinical prediction of lenvatinib response.

Understanding the genetic and immune heterogeneity in hepatocellular carcinoma (HCC) is crucial as treatments advance towards personalisation. This study characterises the genetic and immune heterogeneity within a population of 56 tumours from 28 patients, 10 of whom have multifocal disease, obtained from patients undergoing surgery at the Leeds Teaching Hospital NHS Trust. These samples represent the most common aetiological groups found in the UK: alcohol-related liver disease, non-alcoholic fatty liver disease and hepatitis C. Whole exome sequencing was performed on DNA from these tumour alongside matched background liver samples to determine tumour-specific variants. Tissue sections were stained for both proliferation and immune cell markers. Tumours across aetiological groups showed significant levels of variation, with high inter-individual variation. Analyses of multifocal tumours revealed significant discordance in genetic and immune cell profiles, both between multi-centric primary and metastatic tumours. This work emphasises the genetic and immuneheterogeneity in HCC across HCC subtypes, between and within individuals, highlighting mechansisms for therapeutic resistance and the need for personalised medicine.