Hepatology

Background and ObjectivesBariatric surgery is a highly effective obesity treatment, yet it may predispose individuals to alcohol-related liver injury. While altered ethanol metabolism following procedures like Roux-en-Y gastric bypass (RYGB) is well described, the long-term hepatic consequences, particularly the risk of portal hypertension in patients who develop alcohol-related hepatitis (AH,) remain poorly defined. MethodsUsing the TriNetX US Collaborative Network, we identified adult patients diagnosed with AH or alcohol-related cirrhosis. We compared outcomes between patients with a history of RYGB or sleeve gastrectomy (SG) who subsequently developed AH (Bariatric+AH group) and those with AH and no history of bariatric surgery (AH-only group). Propensity score matching was performed on over 44 demographic, clinical, and laboratory variables. Cox proportional hazards models and Kaplan-Meier survival curves were used to estimate the risk of clinically significant portal hypertension (PH) events, liver transplantation, and all-cause mortality at three-, five-, and seven-year follow-ups. ResultsAfter matching, 772 patients were included in each cohort. At 7 years post-index event, the Bariatric + AH group exhibited a significantly higher risk of PH-related complications compared to the AH-only group (HR 1.519; 95% CI, 1.15-2.005; p = 0.003). No significant differences were observed in liver transplantation (HR 1.412; 95% CI, 0.850-2.346; p = 0.181) or all-cause mortality (HR 1.085; 95% CI, 0.904-1.303; p = 0.381). These findings were consistent across all follow-up intervals. ConclusionBariatric surgery is associated with an increased long-term risk of portal hypertension in patients who develop alcohol-related hepatitis despite similar mortality and transplantation rates. These findings underscore the need for targeted postoperative counseling, liver-focused surveillance strategies, and integration of hepatologic risk assessment into metabolic surgery care pathways.

Background and aimsPopulation screening for liver disease in high-risk groups is recommended. Community diagnosis of liver disease is a challenge due to the asymptomatic nature of disease until very advanced stages. Moreover, regional variation in testing availability can result in people with clinically significant liver disease being missed. Machine learning (ML) has been proposed as a method to reduce diagnostic error and automate screening. We present a novel machine learning derived algorithm (ID LIVER-ML) designed to predict the risk of clinically significant liver disease in a high-risk community population to identify those needing further investigations or specialist referral. MethodsUsing data from 2039 patients recruited to two UK cohorts, we created a parsimonious model using investigations that would be available in primary care using liver stiffness measurement as reference standard. The performance of ID LIVER-ML was compared against FIB-4 score in a second unseen hold out cohort (n=327). ResultsID LIVER-ML performed well at identifying patients at risk of clinically significant liver fibrosis (sensitivity 0.90, Specificity 0.43, PPV 0.54, NPV 0.86, AUC 0.83) and outperformed conventional risk scoring systems (FIB-4: AUC 0.65; NAFLD Fibrosis Score: AUC 0.66; APRI: AUC 0.53; BARD: AUC 0.58). ConclusionMachine learning derived algorithms can help screen high risk populations in a community setting for liver fibrosis. ClinicalTrials.gov ID: NCT04666402 Impact and ImplicationsThe prevalence of steatotic liver disease is rising globally and is an increasingly significant challenge for healthcare systems. Existing risk stratification scores are not validated in a real-world cohort where patients have risk factors for multiple aetiologies of liver disease. Our work shows that a machine learning model can predict the risk of clinically significant liver disease using routine primary care data, better than existing non-invasive risk stratification tools in a real-world cohort. This highlights a potential role for machine learning in the automation of fibrosis risk assessment in primary care. Highlights- Machine learning derived algorithms can predict the risk of clinically significant liver disease in an at risk community population with a mixed aetiology of liver diseases. - The performance of the ML algorithm (ID LIVER-ML) is not affected by metabolic, alcohol, or mixed aetiologies. - ID LIVER-ML outperforms traditional risk stratification scoring systems such as FIB-4 and NAFLD fibrosis scores. - Compared to the FIB-4 score, the use of Machine Learning can reduce the need for secondary care investigations by 59%.

Background and AimsHighly aggressive hepatobiliary tumours include gallbladder cancer (GBC), hepatocellular carcinoma (HCC), intrahepatic and extrahepatic cholangiocarcinoma (iCCA, eCCA) and ampulla of Vater cancer (AoV). We aimed to identify plasma biomarkers for the early diagnosis of hepatobiliary cancer by leveraging the metabolomic signatures of established clinical risk factors. MethodBased on 273,190 participants from the UK Biobank, we (1) identified metabolites associated with gallstone-related conditions (e.g. cholecystitis), primary sclerosing cholangitis (PSC) and metabolic liver diseases (e.g. cirrhosis), and (2) evaluated the relationship between the identified metabolites and the risk of GBC, HCC, iCCA, eCCA and AoV. Findings were validated in an independent group of 227,809 participants from the UK Biobank. We also derived metabolomic scores summarizing the three risk-factor signatures and evaluated their ability to stratify cancer risk. ResultsWe identified 27 metabolites associated with gallstone-related conditions, 11 with PSC, and 34 with metabolic liver diseases, some of which showed associations with inconsistent directions across risk factors, suggesting distinct pathogenic processes. Several metabolites were associated with cancer risk in both the discovery and validation datasets, independently of established risk factors, predominantly for HCC (16 signals) and for iCCA (4), with one for GBC and none for eCCA and AoV. Metabolomic scores clearly distinguished individuals at high risk for HCC and iCCA. ConclusionThe preselection of plasma metabolites associated with established risk factors facilitated the subsequent identification and validation of biomarkers for early cancer detection. The identified metabolites suggest specific pathogenic pathways for each type of hepatobiliary cancer. Wider replication is urgently needed to advance toward clinical implementation. What you need to knowO_ST_ABSBACKGROUND AND CONTEXTC_ST_ABSClinical risk factors for hepatobiliary cancers often progress silently, making early identification of high-risk individuals difficult and highlighting the need for biological markers detectable before clinical diagnosis. NEW FINDINGSRisk-factor-based serum metabolomic profiling identified circulating metabolites that predict specific hepatobiliary cancers years before diagnosis, with strongest and most consistent signals for hepatocellular and intrahepatic cholangiocarcinoma. LIMITATIONSClinical risk factors were assumed to be frequently underdiagnosed in UK Biobank, and event numbers were relatively small for some cancers, which may have reduced power and attenuated associations for less common endpoints. CLINICAL RESEARCH RELEVANCEThis study shows that serum metabolic profiles can identify individuals at increased risk for hepatobiliary cancers long before symptoms appear, particularly for hepatocellular and intrahepatic cholangiocarcinoma. These findings support the development of precision risk-stratification strategies that may ultimately enable earlier surveillance. BASIC RESEARCH RELEVANCEBy first identifying metabolites linked to specific liver and biliary clinical conditions, the study clarifies which metabolites are indirectly associated with hepatobiliary cancers through known disease pathways. Testing these metabolites again while adjusting for diagnoses of those conditions then reveals which ones also show direct, pathway-independent associations with individual hepatobiliary cancers, providing clearer insight into cancer-specific metabolic mechanisms.

BackgroundChronic hepatitis B virus (HBV) infection (CHB) affects nearly 300 million individuals globally and remains incurable with current antiviral therapies, which suppress viral replication but rarely achieve functional cure defined by sustained loss of hepatitis B surface antigen (HBsAg). CHB is characterized by profound virus-induced immune tolerance that limits the efficacy of conventional therapeutic vaccination strategies. ObjectiveTo evaluate the therapeutic efficacy and immunological mechanisms of HEPLISAV-B, a CpG-1018-adjuvanted HBsAg vaccine, in breaking immune tolerance and inducing functional cure-like responses in a murine model of CHB. DesignUsing the adeno-associated virus-HBV (AAV-HBV) mouse model, mice with high levels of persistent HBV viremia were vaccinated with two doses of HEPLISAV-B. Virological outcomes in the blood and liver, immune responses and mechanisms were assessed. ResultsHEPLISAV-B induced rapid and durable HBsAg clearance, markedly reduced circulating and intrahepatic HBV DNA and RNA, and suppressed viral replication without hepatocellular injury. Vaccination elicited robust, sustained anti-HBs IgG1 and IgA responses, enhanced HBsAg-specific T and B cell immunity, reduced CD4 regulatory T cells, and decreased PD-1 expression on CD4 T cells. Therapeutic efficacy was strictly dependent on CD4 T cells and the CD40/CD40L signaling pathway, but independent of CD8 T cells, indicating a CD4-driven, non-cytolytic antiviral mechanism critical for HEPLISAV-B induced HBV control. ConclusionHEPLISAV-B effectively breaks HBV-induced immune tolerance and restores coordinated antiviral immunity through a CD4 T cell-/CD40L-dependent pathway. The findings support its potential as a therapeutic vaccine in CHB patients. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSChronic HBV infection is marked by profound virus-induced immune tolerance, current antiviral therapies and vaccines fail to reliably induce HBsAg loss or restore effective antiviral immunity, highlighting the need for immune-based therapeutic strategies. What this study addsThis study demonstrates that the clinically approved vaccine HEPLISAV-B can break HBV immune tolerance in a chronic HBV mouse model, inducing durable HBsAg clearance and anti-HBs immunity, non-cytolytic depletion of intrahepatic HBV DNA, through a mechanism strictly dependent on CD4 T cells and CD40/CD40L signaling. How this study might affect research, practice or policyThese findings defined a CD4 T cell-CD40L/CD40 axis that is critical in CHB functional cure, and support testing HEPLISAV-B as a therapeutic vaccine in CHB patients. O_FIG O_LINKSMALLFIG WIDTH=165 HEIGHT=200 SRC="FIGDIR/small/711721v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1d76f4dorg.highwire.dtl.DTLVardef@cc41cborg.highwire.dtl.DTLVardef@1f39288org.highwire.dtl.DTLVardef@192d0e_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

Background & aimsConstitutive activation of the {beta}-catenin pathway is a determining feature in the pathogenesis of two primary liver cancers, namely HCC and hepatoblastoma (HB). Activating alterations in CTNNB1 gene and, to a lesser extent, inhibiting alterations in APC gene are observed in 30 to 40% of HCC cases and 80 to 90% of HB cases. For both tumours, therapeutic management is far from optimal. Therefore, relevant experimental models are needed to increase our knowledge and test new therapeutic approaches. MethodsOrganoids and tumouroids were established from APC{Delta}hep and {beta}cat{Delta}ex3 mouse models, which are clinically relevant models for {beta}-catenin-activated HCC and mesenchymal HB. We developed a new methodological approach based on a dynamic suspension culture in a rotating bioreactor. Morphological and molecular characteristics and sensitivity to WNTinib, a treatment already successfully tested on human HCC and HB tumouroids, were evaluated by histology, immunohistochemistry, immunofluorescence, and RT-qPCR. ResultsThis easy-to-implement methodology allows for the rapid generation of a large number of organoids and tumouroids that are uniform in size and show no signs of cell death in their core. The robustness of the methodology is illustrated by the maintenance of the histological architecture, cell diversity and gene expression in organoids and tumouroids in comparison with the native liver tissues. In addition, the value of the HCC-derived tumouroids for evaluating cancer treatment was assessed based on their responsiveness to the {beta}-catenin antagonist WNTinib. ConclusionsThe organoids and tumouroids that we present here are new reliable in vitro cancer models, recapitulating the main features of {beta}-catenin-driven HCC and mesenchymal HB. They can be integrated into an appropriate platform for drug screening and could enable the development of "a la carte" therapies that are urgently needed for these indications. Impact and implicationsThis study addresses the critical need for representative in vitro models to investigate {beta}-catenin-driven liver cancers. The organoids and tumouroids developed here are particularly valuable for researchers seeking robust, reproducible models that accurately reflect the cellular diversity and gene expression profiles of native liver tumours. These findings have practical applications in exploring cancer mechanisms, screening new drugs, optimizing personalized treatment strategies, and reducing reliance on animal models, which ultimately benefits patients. HighlightsO_LIEasy and rapid generation of mouse liver organoids and tumouroids from {beta}-catenin activated tumours using culture in a bioreactor C_LIO_LITumouroids preserve histology, cell diversity, and gene expression of native tissue C_LIO_LIHCC-derived tumouroids respond to {beta}-catenin inhibitor WNTinib C_LIO_LIThese reliable 3D models reduce reliance on animal experiments for drug testing C_LI

Hepatocellular carcinoma (HCC) represents the third leading cause of cancer-related death worldwide and has been increasing in developed nations.1,2 The MYC oncogene or its paralogs are frequently amplified or overexpressed in subtypes of cancer associated with stem cell-like features and worse clinical outcomes,3,4 including in liver cancer.5 Unfortunately, selective inhibitors that target MYC or its transcriptional program are not yet clinically available for therapy of HCC. Here, we identified methionine metabolism as a selective vulnerability for MYC but not RAS-driven liver cancers. MYC-driven liver cancer cells are methionine dependent, with markedly diminished tumor growth when mice are fed a methionine low diet. While RAS-driven liver cancer was resistant to a low methionine diet. S-adenosylmethionine (SAM), the predominant methyl donor, partially rescues cell proliferation following methionine depletion, suggesting that methylation processes are especially critical in the context of MYC high tumor cells. Heavy isotope methionine tracing in MYC high cells identified increased levels of m5C nucleotides. We found NOP2, an rRNA m5C-methyltransferase, was regulated by both MYC overexpression and methionine abundance linking the two processes. Methionine depletion reduced methylation of multiple 28S rRNA residues as did NOP2 knockdown. Depletion of NOP2 selectively inhibited MYC liver cancer cell proliferation and in vivo tumor growth. Thus, methionine catabolism is critical for MYC-driven liver tumorigenesis and the rRNA methyltransferase NOP2 may serve as a new therapeutic target in liver cancer.

Liver sinusoidal endothelial cells (LSECs) play essential roles in liver regeneration after injury, but the underlying mechanisms remain incompletely defined. Here we report that leukemia inhibitory factor (LIF), which is rapidly induced after liver injury, acts as a key regulator of LSECs-driven liver regeneration through interaction with LSECs-enriched LIF receptor (LIFR). LIF directly stimulates LSECs proliferation and induces hepatocyte growth factor (HGF) release in a dose-dependent manner via LIFR signaling in LSECs, thereby indirectly promoting hepatocyte proliferation. Systemic LIF neutralization or endothelial cells (ECs)-specific Lifr loss impairs liver regeneration, whereas low-titer AAV-mediated LIF expression increases vascular density, elevates circulating HGF, and improves early liver recovery after partial hepatectomy (PHx) in mice. Together, these findings establish LIF-LIFR as a previously unrecognized endothelial axis to promote hepatocyte proliferation and suggest potential therapeutic strategies to enhance liver repair in patients. HighlightsO_LILIF is upregulated after liver injury and LIF neutralization impairs liver recovery. C_LIO_LILIFR displays the highest expression in ECs; endothelial-specific Lifr deletion delays liver regeneration after injury. C_LIO_LILIF mediates a positive feedback loop including LSECs proliferation as well as HGF release via LIFR pathway. C_LIO_LILIF overexpression increases liver-to-body weight ratio in a dose-dependent manner and accelerates liver regeneration at early stage. C_LI Abstract Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/707802v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@13b42feorg.highwire.dtl.DTLVardef@1ab6390org.highwire.dtl.DTLVardef@115c157org.highwire.dtl.DTLVardef@1486993_HPS_FORMAT_FIGEXP M_FIG C_FIG

Bile represents a clinically accessible biological fluid that can mitigates major limitations associated with tissue-based sampling for the generation of organoid models to study hepatobiliary disease, including biliary tract cancers where tissue availability is often limited. Importantly, bile can also enable the generation of non-malignant cholangiocyte organoids that are otherwise difficult to obtain. Here, we describe an operator-oriented, step-by-step protocol to generate organoids from fresh bile collected during endoscopic retrograde cholangiopancreatography (ERCP), together with two complementary workflows for siRNA delivery in 3D cultures. We detail critical control points that are often under-reported, yet considerably influence success and reproducibility. The protocol was optimized and applied in a real-world cohort of 21 patients undergoing ERCP, including benign biliary obstruction due to choledocholithiasis (n=5) and malignant strictures (n=16: cholangiocarcinoma n=13, gallbladder adenocarcinoma n=1, ampullary tumors n=2). Expandable organoids were established in 17/21 cases (81%), with establishment rates of 60% for choledocholithiasis and 85-100% across malignant entities. Anticipated results include organoid outgrowth within [~]2-3 weeks and morphological heterogeneity in cultures derived from malignant strictures, where normal-like and tumor-like populations may initially coexist and can drift toward a cystic phenotype under routine expansion, motivating optional manual handpicking when tumor-enriched lines are required. As downstream readouts, we show feasibility of DNA-based profiling in selected paired bile-organoid samples (targeted sequencing and ULP-WGS copy-number analysis) and demonstrate proof-of-concept gene silencing via siRNA in both dissociated cells prior to re-embedding, and intact fully formed organoids while preserving 3D architecture. Collectively, this workflow provides a practical and reproducible framework to establish, expand, characterize and functionally perturb bile-derived organoids from routine clinical procedures, facilitating standardized implementation across laboratories.

Background/AimsIntrahepatic cholangiocarcinoma (iCCA) represents an unmet clinical need due to its increasing incidence, aggressive biology, and limited treatment options. The extremely low-response rates to current systemic regimens and the emergence of adaptive resistance to targeted therapies underscore the urgent need for alternative therapeutic strategies. Given that the lineage-defining transcription factors SOX9 and YAP1 are central regulators of cholangiocyte and iCCA identity, we investigated their functional roles as potential therapeutic vulnerabilities across multiple preclinical models. MethodsPatient tissue-microarray (TMA) analysis, Sleeping-Beauty hydrodynamic tail vein injection-based iCCA models, and Cre-mediated inducible gene deletion systems were used to investigate the roles of Sox9 and Yap1. Deep-learning-based prediction, RNA-seq, ChIP-seq and immunohistochemistry analyses were performed to delineate transcriptional networks and downstream effectors associated with SOX9/YAP1 signaling. ResultsDual deletion of Sox9 and Yap1 effectively eradicated advanced iCCA while preserving intrahepatic bile ducts, regardless of oncogenic drivers. Mechanistically, SOX9 and YAP1 transcriptionally compensated for each other when one was absent, and ILF2, MGAT5, and WWTR1 were identified as key downstream effectors mediating this compensatory mechanism. Loss of Ilf2, Mgat5, or Taz suppressed iCCA, whereas overexpression of Ilf2 or Taz following Sox9/Yap1 co-deletion restored tumor development, indicating that ILF2 or TAZ can functionally substitute for YAP1 and SOX9 in sustaining iCCA. ConclusionsCo-targeting SOX9 and YAP1 offers a promising and safe broad-spectrum preventive/therapeutic approach for iCCA, potentially overcoming resistance to YAP1 inhibition. The adaptive resistance mechanism identified may extend to other malignancies, providing insights for addressing the advanced resistant to YAP1-TEAD-directed therapies.

Cancers develop in humans over months to years, and tumor-specific CD8 T cells (TST) can interact with cancer cells throughout tumorigenesis. Nevertheless, the long-term population dynamics of TST, especially within progressing tumors, are not well understood. A paradigm first established in chronic viral infection and applied to tumors describes a population hierarchy among exhausted T cells. Progenitor/stem-like exhausted T cells, which express the transcription factor T cell factor 1 (TCF1), maintain the population through self-renewal and by giving rise to terminally differentiated TCF1lo progeny. This has led to a focus on TCF1hi T cells, and though TCF1lo CD8 T cells are the predominant tumor-infiltrating/tumor-reactive subtype in patients, they have been largely overlooked. We leveraged our autochthonous liver cancer model to analyze TST differentiation and proliferation throughout tumorigenesis. Dual EdU/BrdU labeling studies revealed that throughout tumorigenesis, a subset of TCF1lo TST in the liver stochastically entered and exited cell cycle, and at later time points there was no evidence of a TCF1hi progenitor-like population. Moreover, TCF1-knockout TST proliferated and persisted robustly in tumors. Using liver cancer and melanoma models, we showed that tumor-resident TCF1lo TST proliferate and persist autonomously, even when new TST influx into tumors is inhibited. The prevailing notion is that only TCF1hi TST self-renew but we now demonstrate, using a clinically relevant mouse cancer model, that TCF1lo TST stochastically proliferate to achieve long-term population maintenance. Future studies to understand and harness this mechanism to improve T cell persistence in tumors could lead to novel immunotherapies for patients with cancer. SYNOPSISWe show that tumor-specific T cells with little/no expression of TCF1, previously considered incapable of self-renewal, can proliferate stochastically and persist long-term. As TCF1lo CD8 T cells are often the predominant tumor-reactive T cells found in tumors, future studies should be aimed at reprogramming these proliferating T cells within tumors.

Regeneration depends on tightly coordinated transcriptional programs governed by a dynamic epigenetic landscape to regulate cell identity, proliferation, and tissue remodelling following injury. The livers highly regenerative due to the ability to rapidly upregulate genes that drive the cell cycle and other genes important for regeneration. Trimethylation of histone 3 lysine 27 (H3K27me3) is deposited by the polycomb repressive complex 2 (PRC2) and many genes occupied by H3K27me3 in their promoters in uninjured livers become induced following PH. Here we test the hypothesis that depleting H3K27me3 by hepatocyte-specific deletion of Embryonic Ectoderm Development (EedHepKO), a key component of PRC2, changes the regenerative response in the liver. We show that Eed eliminates H3K27me3 in hepatocytes, resulting in reduced liver size, increased hepatocyte death, proliferation and fibrosis associated with upregulation of cell cycle and fibrogenic genes. Though these mice are less likely to survive two-thirds partial hepatectomy than wildtype controls, those that do survive increase liver mass faster than WTs. Importantly the genes that are occupied by H3K27me3 in control uninjured livers are upregulated in EEDHepKO and become further induced following PH. These data show that modulation of PRC2 activity disrupts epigenetic patterning, induces liver injury, and alters regenerative outcomes, suggesting that precise control of PRC2 function could be harnessed to enhance regenerative capacity.

The use of decellularized diseased livers in regenerative medicine is a promising approach for eliminating organ shortages. Bioengineering studies have shown that ECM can impact cell physiology, inducing cell activation, function, and ECM deposition, which suggests that the ECM has a "memory" that is involved in the outcome after recellularization. However, the effect of diseased ECM memory on new cells in vitro and in vivo has not been thoroughly investigated. Since it has been increasingly recognized that liver ECM changes due to different factors, it is comprehensively that diseased ECM obtained from discarded organs will ensure a distinct environment and impact cell survival and physiology. Thus, we aimed at investigating the impact of the memory of diseased ECM obtained from metabolic dysfunction-associated steatohepatitis (MASH)-derived organs on steatohepatitis establishment. To address this aim, we explored decellularized ECM obtained from rats and humans with MASH in different contexts. First, MASH ECM was characterized and then submitted to transplantation to investigate whether a MASH-derived ECM could be used as a scaffold for transplantation and to promote steatohepatitis features in control animals. Histological analysis revealed that the MASH-ECM was completely recellularized after transplantation in both control and MASH recipient rats. However, steatosis and fibrosis were observed in MASH ECM after transplantation in both groups. Molecular analysis showed that MASH ECM stimulates de novo lipogenesis and fibrosis 30 days after transplantation. Untargeted metabolomic analysis revealed that cells grown on MASH ECM had a similar metabolic profile, even when transplanted into healthy or MASH recipient rats. In addition, we observed that MASH ECM promoted impaired lipid oxidation and mitochondrial dysfunction when transplanted into healthy recipients. Altered lipid turnover and inflammatory signaling were observed in MASH ECM transplanted in MASH recipients. In vitro analysis revealed that MASH ECM induced lipid accumulation in HepG2 cells after 10 days of culture. Calcium signalling experiments obtained from HepG2 cells cultured in MASH ECM showed a lower response to ATP, a reduced calcium signalling amplitude, and a distinct response profile than that observed in healthy ECM. On the other hand, a diseased human-derived ECM could still provide an environment that allows cell development. Taken together, our data showed that MASH ECM impacts cell metabolism, promoting steatohepatitis maintenance. In conclusion, our data confirm that diseased ECM memory can impact cell physiology contributing to disease progression.

Hepatic ischemia-reperfusion injury (IRI) results from interrupted perfusion to the liver and contributes to acute liver dysfunction such as following liver transplantation. Lytic cell death pathways are major drivers of IRI and the subsequent inflammatory response. The transmembrane protein ninjurin-1 (NINJ1) was identified as the key executor of terminal plasma membrane rupture across multiple lytic cell death pathways implicated in hepatic IRI. We hypothesized that NINJ1-mediated lytic cell death drives IRI and that its therapeutic inhibition would mitigate liver IRI. Using human liver specimens, we found that NINJ1 is highly expressed in human liver tissue and that its activation correlates with early allograft dysfunction in patients undergoing liver transplantation. Utilizing a segmental hepatic IRI model in mice and rats, Ninj1 genetic deletion or pharmacologic inhibition diminished acute liver injury. Mice with hepatocyte- or macrophage-specific Ninj1 knockout both had reduced hepatocellular injury following IRI, suggesting that NINJ1 within both populations contributes to the resulting liver injury. Mechanistically, we found that hepatocytes and Kupffer cells are highly susceptible to hypoxia-induced NINJ1-mediated plasma membrane rupture, which can be pharmacologically prevented. These data position NINJ1 as a potential new therapeutic target to limit hepatic IRI, with important implications for organ preservation during liver transplantation.

BackgroundHepatocellular carcinoma (HCC) arises from diverse etiologies, but the balance between conserved and specific transcriptomic programs remains unclear. MethodsHBV and HCV cohorts were analyzed using GSVA to quantify Hallmark shifts. Biology was distilled into proliferation (ProlifHub) and hepatocyte-loss (HepLoss) modules, forming a composite HCCStateScore. An HBV injury axis was adjusted for proliferative state (E2F/G2M). Validation was performed using GSE14520 and GEPIA3. ResultsHallmark analysis revealed conserved proliferative activation and hepatocyte function suppression across etiologies. In HBV-HCC, the injury axis remained significantly elevated after adjusting for proliferation (p{approx}0.0147), indicating an injury component independent of the cell cycle. HCCStateScore robustly separated tumor from non-tumor tissue (AUC{approx}0.986, p=0). GEPIA3 confirmed concordant expression and survival associations for module genes. ConclusionsHCC features conserved opposing proliferation and hepatocyte-loss programs. HBV-associated tumors retain a distinct injury-linked component not fully explained by cell division. This validated score provides a framework for cross-cohort analysis and mechanistic prioritization in liver cancer research.

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

Vascular plasticity is a crucial biological asset enabling our bodies to rapidly adapt to infections and acute inflammation. However, repeated insult during chronic disease can result in these vascular adaptations becoming irreversible, thereby driving disease progression and fibrosis. This study aimed to understand if phenotypic changes in endothelial cell (EC) identity could be indicative of progressive fibrosis and thereby offer new diagnostic and therapeutic opportunities for patients with metabolic dysfunction-associated steatotic liver disease (MASLD). Previous research has documented that a significant shift in EC transcriptomic signature occurs during liver fibrosis in both pre-clinical models and patients. However, the protein expression profile, phenotype and functional role of these new EC subpopulations that are induced during fibrogenesis is unclear. In this study, we integrate high-resolution imaging, proteomic and transcriptomic analysis which collectively highlight a central role for endothelial-to-mesenchymal transition (EndMT)-induced EC plasticity in the derivation of fibrosis-associated EC (FAEC). We demonstrate that: 1) full spectrum flow cytometry can provide new opportunities to categorize and phenotype EC subpopulations, 2) two distinct EndMT-derived FAEC subpopulations expand during fibrogenesis; THY1.2+ICAM1+ and TAGLN+MCAM+ EC that display unique immunomodulatory and metabolic phenotypes, 3) TAGLN+ FAEC are a conserved, pro-fibrotic cell type arising at early stages of MASLD, and 4) increased hepatic expression of TAGLN is significantly associated with detrimental patient outcomes at all stages of liver disease. This study will pave the way for the development of FAEC-specific diagnostic and therapeutic approaches to tackle progressive fibrotic disease.

Mast cells (MCs) are myeloid cells of the innate immune system. As a first line of defence they fulfill effector functions and immune modulatory properties. Upon activation they release pro-inflammatory mediators such as cytokines and proteases. It has been suggested that MCs may contribute to the development of liver fibrosis. However, investigating hepatic MC biology in mice is challenging due to low MC numbers and a lack of suitable detection techniques relying on MC proteins and their modifications. Here, we evaluated whether the expression strength of MC markers correlates with the degree of liver fibrosis in mice and aimed to determine the frequency and localization of hepatic MCs. We applied both a toxic (DEN/CCl4 treatment) and a genetic (Mdr2-/- mice) liver fibrosis model in C57BL/6 mice and found a significant correlation between fibrosis grade and the expression of several established mast cell markers. This correlation was further supported in patients with fibrosis and hepatocellular carcinoma (HCC) using publicly available transcriptomics datasets. We used FACS to purify and isolate MCs from fibrotic mouse livers and verified MC signatures by qPCR analysis of MC-specific gene expression. Hepatic MCs were predominantly negative for Mast-Cell-Protease 5 (Mcpt5) and occurred at a low frequency (approximately 1-2% of leukocytes). Using Molecular CartographyTM of fibrotic liver sections, we determined the spatial localization, expression signature, abundance (approximately 2 cells/mm2) and cellular environment of murine hepatic MCs. In summary, we demonstrated the existence of MCs in murine fibrotic livers and defined an MC expression signature that correlates with the strength of liver fibrosis. These findings will help to study MC biology in murine models of liver disease more effectively in the future.

Ductular Reactions (DRs) are dynamic and complex multicellular responses that occur as a result of various hepatic injuries. Precise identification and quantification of the extent of DRs is a cornerstone of pre-clinical modelling of liver disease, with links to inflammation, fibrosis, regeneration, and disease severity. Here, we apply a deep learning model, Deep Understanding Convolutional Kernel or DUCK-Net, to the automated detection and segmentation of DRs in whole-slide histopathological images of murine models of liver damage. Following annotation of a training dataset by a specialist liver histopathologist, we demonstrate accelerated performance and accurate detection, achieving a mean Dice coefficient (model-expert segmentation overlap) of 85.4% and a specificity of 98%, indicating minimal false positives. Evaluation of model validity and utility was achieved with a histological time course of cholestatic injury and recovery using 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine diet (DDC) in mice. When assessed against a multiple linear regression model incorporating core epithelial and stromal components of the DR as quantified using IHC, DUCK-Net predicted the spatiotemporal response to injury and repair/resolution with a coefficient of determination (R2) of 0.88. Moreover, DUCK-Net kinetics strongly correlated with published spatial transcriptomic (Stereo-seq) analysis of the DDC model, demonstrating that H&E-based segmentation captures molecular DR dynamics comparable to, or exceeding that of individual IHC markers without the need for immunostaining. DUCK-Net provides a novel and accessible platform for rapid, accurate histological quantification of liver injury reflective of the matrix-rich, multicellular regenerative niche observed in DRs.

Cirrhosis, the end stage of chronic liver disease marked by fibrosis and impaired liver function, is associated with cirrhosis-associated immune dysfunction, a condition in which systemic inflammation coexists with impaired host defense and increased susceptibility to infections. However, intestinal intraepithelial lymphocytes (IELs), key mediators of epithelial immune defense, remain poorly characterized in this context. Using high-dimensional profiling of paired duodenal biopsies and peripheral blood across disease stages, we define IEL alterations in cirrhosis. Contrary to prior reports of immune exhaustion, lymphocyte effector function was preserved, while disease progression was marked by systemic inflammatory remodeling and increased tumor necrosis factor (TNF) production by circulating T cells. The IEL compartment was markedly altered, with loss of CD8{beta} IELs, expansion of natural killer (NK) IELs, and reduced CCR9CD8{beta} IELs, suggesting altered gut homing. These findings refine cirrhosis-associated immune dysfunction as inflammatory immune reprogramming coupled to impaired epithelial immune surveillance. HighlightsPeripheral lymphocytes from cirrhosis patients retain effector capacity with enhanced inflammatory activity Cirrhosis reshapes the duodenal intraepithelial lymphocyte landscape Reduced frequency of CCR9+CD8{beta} IELs indicates altered gut-homing in cirrhosis

Background and AimsNovel antiviral approaches capable of permanently inactivating the intrahepatic HBV DNA reservoir, the covalently closed circular DNA (cccDNA) and HBV DNA integrated into the host genome, are urgently needed. This study evaluated adenine base editing as a strategy to disrupt HBV replication by introducing mutations in the overlapping HBs/polymerase open reading frame (ORF). MethodsAn adenine base editor (ABE) and 3 guide RNAs (gS1-gS3) were designed to introduce missense mutations within the HBs/polymerase ORF. ABE mRNA and individual gRNAs were co-transfected into HBV-infected HepG2-hNTCP cells and primary human hepatocytes. Antiviral efficacy was further assessed in HepG2.2.15 and PLC/PRF/5 cells harboring integrated HBV DNA. In vivo, lipid nanoparticles (LNP)-mediated delivery of ABE mRNA and gRNAs was evaluated in HBVcircle DNA-transduced mice and in HBV-infected human liver-chimeric mice. The impact of HBs editing on hepatitis D virus (HDV) release was assessed using PLC/PRF/5 and Huh7 cell-based HDV replication models. ResultsAdenine base editing efficiently reduced HBsAg production and HBV replication in vitro by targeting both cccDNA and integrated HBV DNA. A single LNP injection of ABE-gS2 resulted in undetectable HBsAg in HBVcircle mice, while two injections achieved a 90% reduction in serum HBsAg in HBV-infected human liver chimeric mice. HBV DNA replication was also inhibited in vivo. Furthermore, HBs ORF base editing markedly suppressed HDV release in vitro. ConclusionsAdenine base editing of the HBs ORF effectively impairs HBV replication and HBsAg production in vitro and in vivo and concomitantly inhibits HDV release, highlighting its therapeutic potential.