BACKGROUND & AIMS:Primary sclerosing cholangitis (PSC) is an immune-mediated liver disease with an incompletely understood pathogenesis. Dysregulation of monocytes and macrophages has been implicated in disease development, although their functional relevance remains insufficiently understood. The main aim of this study was to identify peripheral myeloid cell populations involved in PSC pathogenesis and to assess the therapeutic potential of targeting associated inflammatory signalling pathways. Additionally, we sought to enhance translational relevance through the application of a humanized immune system (HIS) mouse model. METHODS:Peripheral blood mononuclear cells from patients with PSC, patients with ulcerative colitis, and healthy controls were profiled using CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing). Inflammatory signalling via the IL-8:CXCR1/2 axis was further evaluated in both conventional and HIS mouse models of cholestatic liver disease. RESULTS:Patients with PSC exhibit a population of circulatory CXCL8+CD14+ monocytes with upregulated proinflammatory signalling. Serum concentrations of the proinflammatory cytokine IL-8, encoded by CXCL8, were increased in patients with PSC (p <0.001) and associated with poor prognosis. Patients with high IL-8 concentrations (≥27.8 pg/ml) at baseline had significantly worse transplant-free survival than those with lower IL-8 concentrations (log-rank hazard ratio 6.12, 95% CI 1.68-22.33). Intrahepatic IL-8+ macrophages were expanded during disease progression (p <0.05). Pharmacological Cxcr1 antagonism in conventional mouse models and antibody-mediated IL-8 neutralization in HIS mice reduced cholestatic liver injury. CONCLUSIONS:A circulating CXCL8+CD14+ monocyte subset and intrahepatic IL-8+ macrophages were identified as potential contributors to PSC pathogenesis. HIS mouse models could mark a significant advance in translational research on human immune responses. Targeting the IL-8:CXCR1/2 axis represents a promising therapeutic strategy that warrants further investigation. IMPACT AND IMPLICATIONS:The immune mechanisms underlying primary sclerosing cholangitis (PSC) remain incompletely understood, limiting the development of effective targeted therapies. We identified a circulating CXCL8+CD14+ monocyte subset and expanded intrahepatic IL-8+ macrophages associated with inflammatory activation in PSC, while elevated serum IL-8 concentrations identified patients with poorer transplant-free survival. Therapeutic inhibition of the IL-8:CXCR1/2 axis reduced cholestatic liver injury in both conventional and humanized immune system mouse models, supporting its relevance across experimental systems. These findings implicate IL-8-driven myeloid inflammation in PSC pathogenesis, identify IL-8 as a potential prognostic biomarker, and support further evaluation of IL-8-directed therapies in PSC.

Over the past 75 years, fluorinated contaminants present in aquatic, dietary, and environmental matrices have increasingly raised scientific and public health concerns. Among these substances, ultrashort perfluoroalkyl substances (US-PFAS) demonstrate substantial environmental persistence and mobility, comparable to that of their long-chain counterparts. Reported human serum concentrations of individual US-PFAS reach up to 27.7 ng/mL, substantially exceeding established thresholds for total PFAS levels. Nevertheless, data on human toxicity of US-PFAS remain limited. The present study aimed to investigate the impact of US-PFAS on cholestatic features and to elucidate the underlying mechanisms using an adverse outcome pathway network as a mechanistic compass. Human HepaRG liver cell cultures were exposed to 4 US-PFAS at various concentrations for 1 to 72 h. Transcriptomic profiles were assessed by RNA sequencing analysis. Functional alterations in early and key events associated with cholestatic pathology were evaluated using fluorescently labeled probes, while bile canaliculi dynamics were examined through in situ immunostaining and phase-contrast imaging. This revealed significant perturbations of molecular pathways involved in both early and late events relevant for cholestatic liver injury. US-PFAS did not significantly affect hepatocellular transporter activity or bile canalicular integrity at early time points (≤6 h). In contrast, prolonged exposure (24-72 h) to several US-PFAS elicited hepatocellular stress responses, such as oxidative stress and endoplasmic reticulum stress, and promoted inflammatory cell adhesion. Collectively, these findings demonstrate that US-PFAS have the capacity to affect biliary homeostasis and trigger downstream cholestatic events, providing mechanistic insight relevant for hazard identification.

In human athletes, training-induced improvements in insulin sensitivity typically coincide with increased GLUT4 expression and a glucose-centric fuel strategy. In horses, recent studies show the opposite: training is associated with reduced total GLUT4/GLUT12 abundance, challenging the assumption that enhanced glucose transport underpins improved insulin sensitivity. This study examined how insulin-dependent glucose transporters relate to insulin sensitivity in trained horses. After eight weeks of standardized aerobic harness training, horses displayed a more efficient insulin economy during an oral glucose tolerance test, with reduced peak insulin, lower insulin AUC, and a delayed time to peak. Intravenous glucose tolerance indices did not show corresponding improvements. Total GLUT4 and GLUT12 decreased with training, most clearly in acute post-exercise samples. Acute GLUT12 correlated positively with peak and total insulin responses, and its training-induced decline correlated with increased insulin time-to-peak. These data indicate that horses improve insulin sensitivity without upregulating or even downregulating GLUT4/GLUT12. This supports the emerging concept that equine training shifts insulin’s primary role toward non-glucose substrates (e.g., amino-acid–supported anaplerosis, lipid oxidation, microbiome-derived fuels). OGTT-type tests may be more sensitive to training adaptations than intravenous tests, and nutrition should support this shifted insulin profile with adequate amino acids, forage-first feeding, and moderated starch.

Abstract Background and aims Therapeutic outcomes for advanced hepatocellular carcinoma remain inadequate, despite recent advances using immunotherapy. Long-term effectiveness of systemic therapies, including second-line multi-tyrosine kinase inhibitor sorafenib, is limited by resistance mechanisms and adverse effects. Upregulated deubiquitinase UCH-L1 is frequently correlated with poor prognosis in cancers. Here, we investigated the therapeutic potential of combining pharmacological UCH-L1-inhibition with sorafenib in HCC. Methods UCH-L1 expression was analysed in TCGA-LIHC data and patient-derived HCC tissues. Sorafenib and LDN57444 effects were evaluated in vitro in cytotoxicity and invasion assays. Gene and protein expression were examined by RT-qPCR, Western blotting and immunohistochemistry. In vivo efficacy of drug synergy was assessed in an orthotopic xenograft mouse HCC model. Results In silico data-analysis revealed significantly higher UCH-L1 levels in patient HCC tumours versus non-tumour, associated with reduced overall survival. Low-dose sorafenib upregulated UCH-L1 in HCC cell line Hep3B. Paradoxically, this also promoted invasiveness and sustained MEK1/2-ERK1/2-pathway activation. Combining low-dose sorafenib with LDN57444 produced strong synergistic cytotoxicity in vitro , reverted MAPK-activation and suppressed invasion. Consistently, at low sorafenib dose co-treatment with LDN57444 completely inhibited tumour growth of Hep3B xenografts and enhanced sorafenib efficacy. Conclusion LDN57444 sensitises HCC cells to low-dose sorafenib by reverting drug-induced pro-oncogenic signalling and thereby strongly synergises with sorafenib to enhance anti-tumour efficacy in a HCC mouse model. This presents UCH-L1 as a player in treatment-induced adaptive response and supports further exploring UCH-L1-targeting in combination with sorafenib as therapeutic avenue for advanced HCC. Lay summary This study explores a new treatment approach for hepatocellular carcinoma (HCC) by combining two drugs: LDN57444, which blocks the enzyme UCH-L1, and sorafenib, a FDA-approved multi-tyrosine kinase inhibitor. We evaluated the effect of this drug combination in vitro using a HCC cell line and in an mouse HCC-model. The drug combination displayed strong, synergy in lowering HCC cell viability, and greatly reduced invasiveness and in vivo tumour growth. LDN57444 sensitised HCC cells to low doses of sorafenib by preventing UCH-L1-mediated activation of pro-oncogenic signalling. These findings highlight the potential of this new drug combination for treating advanced HCC thereby potentially reducing side-effects and countering drug resistance. Impact and implications Our preclinical research introduces a novel combination strategy against advanced HCC that holds potential to improve existing therapies, particularly the second-line multi-tyrosine kinase inhibitor sorafenib. The proposed combination of sorafenib with an inhibitor of the deubiquitinase UCH-L1 not only enhances sorafenib efficacy but present promise to also counter resistance mechanisms. Moreover, because effective responses are achieved at lower drug doses, this may in addition reduce therapy-associated adverse effects further increasing potential impact. While sorafenib is FDA-approved, the UCH-L1 inhibitor LDN57444 needs further (clinical) development to bring our promising findings to full translational potential for HCC patients and physicians.

IntroductionNADPH oxidase 1 inhibition (NOX1i) has shown to alter the tumor microenvironment in conventional mouse models for hepatocellular carcinoma (HCC). However, clinical translation is hampered by low translatability of these models.MethodsWe developed two novel human immune system (HIS) mouse models, co-transplanted with orthotopic human HCC cells, to investigate NOX1i in the context of human HCC and human adaptive (T cell-HIS-HCC) or innate (Myeloid-HIS-HCC) immune responses. Mice received NOX1i or vehicle twice per week for 3 weeks. Results were validated in ex vivo patient-derived precision-cut tumor slices (PCTS).ResultsT cell-HIS-HCC mice were mainly reconstituted with human T cells. Interestingly, the expression of cytokines and markers involved in both cancer progression and anti-tumor immunity was significantly lower in tumors of NOX1i-treated mice. In the Myeloid-HIS-HCC model, humanization in livers and tumors was dominated by macrophages. Significantly lower human immune cells were observed in tumors of NOX1i-treated Myeloid-HIS-HCC mice, in line with reduced VCAM1 and ICAM1 expression. NOX1i-treated Myeloid-HIS-HCC mice showed a similar shift in tumor-promoting cytokines and immune checkpoints as NOX1i-treated T cell-HIS-HCC mice. Moreover, gene expression of tumor-associated macrophage, HCC and proliferation markers tended to be lower in tumors of NOX1i-treated Myeloid-HIS-HCC mice. Ex vivo patient-derived PCTS treated with NOX1i also demonstrated lower gene expression of pro-tumorigenic cytokines and tumor-promoting markers.ConclusionOur data show that NOX1i modulates the tumor-immune microenvironment in human immune system HCC models, and might hold potential in combination therapy to rebalance the dysregulated immune profile in HCC.

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies, which is associated with a low 5-year survival rate. The importance of effective disease monitoring and prognostic evaluation is undeniable. For the present study, a systematic review was performed using extensive searches in Medline, Embase, Web of Science and Scopus up to December 29, 2023. The aim of the present study was to examine whether N-glycomics could predict the risk of developing HCC in adults with chronic liver disease and, if HCC was present, predict overall survival. As a secondary outcome, the prediction capability of HCC recurrence was assessed. After deduplication, 3,904 studies were identified, of which 30 were included. Overall, the median size of the study cohort was 144 patients, with a median follow-up time of 63.6 months. Three studies explored N-glycomics in whole serum, whereas the rest focused on individual glycoproteins, with Mac-2 binding protein glycosylation isomer (M2BPGi) being the most commonly studied. Most articles investigated baseline M2BPGi values as predictors for the development of HCC and demonstrated a median area under the curve of 0.83 with a cut-off index value of 1.8. In conclusion, it was revaled that N-glycan changes exhibit added value in determining patient prognosis in terms of survival, monitoring HCC development and recurrence.

Background & Aims: Although maternal obesity is an independent risk factor for metabolic dysfunction-associated steatotic liver disease (MASLD), the pathogenesis remains unclear. We aimed to evaluate the effect and mechanisms of multigenerational maternal Western diet (WD) on MASLD progression, and test drug candidates. Methods: Female mice were fed WD from 8 weeks before breeding initiation with a normal chow (NC)-fed male, throughout pregnancy and lactation. Male offspring were weaned onto NC or WD and assessed at the age of 24 days, 10 weeks, and 16 weeks (n = 5–11 per group). Additionally, offspring from dams with hepatic insulin receptor knockout were evaluated (n = 9–12 per group). Serum fibroblast growth factor 21 (FGF21) and mitochondrial open reading frame of 12S rRNA-c (MOTS-c) were measured in adolescents with MASLD with or without a history of maternal obesity. The therapeutic efficacy of FGF21, semaglutide and an amylin analogue was assessed from 8 to 16 weeks of age (n = 8–12 per group). Results: Starting from weaning age, maternal WD feeding aggravated body weight gain, insulin resistance, steatosis, and inflammation. Fibrosis was only observed in offspring exposed to maternal WD. Mechanistically, the latter exhibited reduced OXPHOS activity. Isolated maternal hepatic insulin resistance partially recapitulated offspring inflammation and fibrosis. Notably, OXPHOS was also downregulated in a transcriptomic dataset of maternal WD feeding in non-human primates. Serum FGF21 and MOTS-c correlated with MASLD severity and maternal obesity in adolescents. Particularly FGF21 treatment ameliorated steatohepatitis and mitochondrial function. Conclusions: Maternal WD aggravates MASLD in male offspring starting from weaning age, with mitochondrial dysfunction contributing to disease severity. This was reversed by FGF21 agonism. Impact and implications: The underlying mechanisms of maternal obesity contributing to metabolic dysfunction-associated steatotic liver disease (MASLD) severity in the offspring are not completely understood. Our study characterises the impact of multigenerational maternal Western diet on offspring MASLD development and identifies mitochondrial dysfunction as a contributor to disease severity. In this setting, pharmacological compounds targeting mitochondrial dysfunction appear to have the greatest therapeutic potential.

Portal hypertension (PH) can cause severe complications in patients with advanced chronic liver disease (aCLD). The pan-peroxisome proliferator-activated receptor (pan-PPAR) agonist lanifibranor reduces portal pressure in preclinical models of aCLD. Since the effect on PH might be secondary to fibrosis improvement, we investigated the effect of lanifibranor on PH, hepatic and splanchnic angiogenesis in mouse models of fibrotic and prehepatic non-fibrotic PH. Mice with fibrotic PH (common bile duct ligation; CBDL) and prehepatic PH (partial portal vein ligation; PPVL) received daily lanifibranor/vehicle for 14 or 7 days, respectively. Hemodynamics, serum, hepatic and mesenteric histology, and hepatic, mesenteric and liver sinusoidal endothelial cells (LSEC) gene expression levels were analyzed. Vascular corrosion casts of the venous mesenteric and hepatic vasculature were analyzed using scanning electron microscopy and µCT. Portal pressure was increased in CBDL mice. Lanifibranor treatment demonstrated a dose-dependent trend towards decreasing the elevated portal pressure, and reduced fibrosis. Hepatic mRNA levels of inflammatory, fibrotic and angiogenic markers were significantly downregulated in lanifibranor-treated CBDL mice. LSEC dysfunction was improved by lanifibranor. Compared to CBDL mice, portal pressure was more extensively elevated in PPVL mice, which was significantly reduced by lanifibranor. Superior mesenteric artery blood flow, which was increased in vehicle-treated PPVL mice, tended to decrease by lanifibranor. The expansion of the mesenteric vasculature and mesenteric protein level of angiogenetic markers in PPVL mice were reduced after lanifibranor. In conclusion, lanifibranor improves PH, independently from fibrosis reduction, potentially through reducing the venous mesenteric vasculature expansion and intrahepatic angiogenesis, and ameliorating LSEC function.

Background & Aims:Hepatopulmonary syndrome (HPS) is a severe pulmonary vascular complication of liver disease. Liver transplantation (LT) is the only definitive treatment, yet a contemporary synthesis of LT outcomes in HPS, alongside persisting uncertainties, is lacking. Methods:We conducted a systematic review and meta-analysis of 22 studies to evaluate HPS prevalence, post-LT survival, and HPS resolution. Results:Pooled HPS prevalence was 17.2% (11.6-24.7%, n = 1,171 studied patients) among patients with cirrhosis evaluated for LT, 26.1% (18.5-35.5%, n = 629) in patients with portal hypertension evaluated for LT and 21.6% (11.2-37.4%, n = 737) in LT recipients. Under current model for end-stage liver disease (MELD) exception policies, waitlist mortality was 13.1% (0.6-78.5%, n = 1,240 studied patients). Post-LT survival in patients with HPS was comparable to that in patients without HPS and aligned with international benchmarks. Pooled 1-year post-LT survival rates were 85.5% in prospective studies from the pre- and post-MELD era (n = 240 studied patients), 93.9% in the post-MELD era (n = 99), and 83.8% in registry-based studies (n = 800). Pooled estimates for 5-year post-LT survival rates were 85% (n = 144 studied patients), 92.9% (n = 99), and 76% (n = 739), respectively. Lower pre-LT PaO2 correlated with poorer post-LT survival, especially in very severe HPS. HPS resolved after LT in 90.1% (71.7-97.0%, n = 80 studied patients) of patients at 6 months, with complete resolution within 1 year in all patients assessed. Key gaps remain regarding the influence of underlying liver disease on HPS progression, the optimal timing of LT, and factors affecting post-LT recovery. Conclusions:LT offers substantial survival and clinical benefits for patients with HPS, with high rates of HPS resolution within a year. Patients with very severe HPS warrant closer monitoring, and further research is needed to address unresolved questions regarding disease trajectory and post-transplant outcomes. Impact and implications:This systematic review and meta-analysis shows that liver transplantation (LT) is an effective treatment for hepatopulmonary syndrome (HPS), resulting in high rates of syndrome resolution and post-transplant survival comparable to patients without HPS. These findings support the continued use of MELD exception policies and underscore the importance of timely referral for LT. While outcomes are generally favorable, patients with very severe HPS may experience worse post-transplant outcomes, highlighting the need for more tailored clinical management. A key unmet need remains in understanding the role of underlying liver disease severity in the natural history of HPS, as well as the optimal timing for LT and factors influencing post-transplant recovery.

Cholestatic liver injury is a complex adversity leading to the toxic accumulation of.noxious bile salts in the liver and systemic circulation. Cholestasis can be instigated by a plethora of chemicals originating from several applicability domains. Current efforts fail to predict the cholestatic potential of chemicals due to, at least in part, gaps in the mechanistic understanding of this type of adversity. A recently introduced adverse outcome pathway (AOP) network on cholestatic liver injury generated using artificial intelligence pulls up transporter changes, bile canalicular changes and hepatocellular changes as molecular initiating events (MIEs). The present study used this AOP network as the mechanistic basis for the development of an in vitro test battery to predict MIEs of cholestatic hepatotoxicity, including assays to monitor transporter changes at the sinusoidal uptake, canalicular efflux and basolateral efflux pole as well as bile canalicular changes. For this purpose, human HepaRG cells were exposed to known cholestatic chemicals covering various MIEs, non-cholestatic hepatotoxic chemicals and non-hepatotoxic chemicals. Subsequent application of the MIE test battery shows great potential for identifying cholestatic chemicals, while correctly predicting all negative control chemicals. In conclusion, the established in vitro test battery shows potential for early prediction of cholestatic chemicals.

BACKGROUND:Metabolic and bariatric surgery (MBS) procedures with extended biliary limb length are gaining popularity to expedite weight loss but can induce liver failure. We aimed to investigate the underlying pathophysiology for this potentially fatal complication. MATERIALS AND METHODS:We compared mouse models of vertical sleeve plication, sleeve gastrectomy, Roux-en-Y gastric bypass (RYGB), and one-anastomosis gastric bypass with three biliary limb lengths (25% = Ω 1 , 50% = Ω 2 , 75% = Ω 3 ) by analyzing mortality, weight loss, metabolic and liver health, bacterial translocation, inflammation, and biliary and fecal microbiome. Gut decontamination with oral antibiotics (amoxicillin, vancomycin, neomycin, and metronidazole) was performed in a subset of Ω 3 mice. Liver histology from mice with different biliary limb lengths was compared to samples from human patients who developed liver failure following biliopancreatic diversion or RYGB. RESULTS:RYGB and Ω 1&2 significantly improved glucose intolerance and liver steatosis compared to sham surgery. However, extending the biliary limb (Ω 3 ) resulted in 100% mortality. The Ω 3 procedure induced bacterial translocation of Enterococcus genus to the spleen and biliary fluid, consistent with increased serum lipopolysaccharide levels and terminal ileum, biliary limb, and hepatic inflammation. Liver histology in Ω 3 mice was characterized by mediovesicular steatosis, closely resembling the histological picture observed in patients with liver failure after MBS. Oral gut decontamination significantly improved Ω 3 1-week-survival from 31.3% to 80.0%, prevented bacterial overgrowth in biliary fluid and spleen, and decreased liver damage. CONCLUSION:Mortality in longer biliary limb MBS surgery is caused by bacterial overgrowth, translocation, and gut-liver axis inflammation, which were reversed by oral gut decontamination with antibiotics.