Abstract Background and Aims Primary sclerosing cholangitis (PSC) increases the risk of developing cholangiocarcinoma (CCA), which is the leading cause of premature death in these patients. Current diagnostic methods for early detection of either condition remain largely suboptimal. This study investigates serum metabolites as potential non-invasive diagnostic biomarkers. Methods This multicenter international study analyzed 459 serum samples from 13 international centers, including patients with PSC (n = 216), PSC who developed CCA during follow-up (PSC to CCA; n = 24), PSC with concomitant CCA (PSC-CCA; n = 88), ulcerative colitis (UC; n = 14), and healthy individuals (n = 79). Serum metabolomics was evaluated by ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) and the accuracy of the single candidate metabolite biomarkers was further assessed. Machine learning was used to generate the best diagnostic and predictive algorithms. Results Fifty-two serum metabolites were identified as markers of PSC, independent of age, biological sex, cirrhosis, UC, or ursodeoxycholic acid (UDCA) treatment. A diagnostic model based on only six lipids distinguished PSC patients from healthy controls with high accuracy (AUC 0.952 discovery, 0.918 validation), independently of the levels of transaminases or cholestasis markers (i.e., GGT, AP, bilirubin) in blood. Subgroup analyses were performed in PSC patients with and without inflammatory bowel disease (IBD), confirming the model’s consistent performance across both subgroups when compared with their respective control groups – UC and healthy controls (AUC 0.959 and 0.939, respectively). The PSC model also confirmed its diagnostic performance in patients with established PSC-CCA or progression to CCA within 2 years, when compared to UC and healthy controls, showing AUC values ranging from 0.911 to 0.976. Additionally, 20 metabolites were significantly altered in PSC patients with CCA (PSC-CCA), regardless of demographics, cirrhosis, UDCA treatment, IBD status, or CCA subtype, and independently of serum transaminases and cholestatic markers levels. A model incorporating six lipids accurately identified PSC-CCA cases compared to PSC patients (AUC = 0.912 discovery; 0.896 validation), showing excellent performance in early-stage tumor detection (AUC 0.921) and outperforming CA19-9 (AUC 0.708). Importantly, the model retained high accuracy even in CCA patients with low CA19-9 levels (AUC 0.905) and in PSC patients who developed CCA within a year of clinical diagnosis (AUC 0.792). Conclusion The PSC and PSC-CCA blood tests provide a non-invasive approach for diagnosing PSC and PSC-CCA, addressing an important need in this population. Their integration into clinical practice could enhance risk stratification, early detection, personalized surveillance, and treatment decision-making in PSC patients.

Background & Aims: Primary sclerosing cholangitis (PSC) shows highly variable progression toward advanced fibrosis and cirrhosis, contributing to its poor prognosis and making accurate disease staging challenging. Growing evidence indicates that metabolic alterations play a key role in PSC pathogenesis and progression. This study aimed to characterize serum metabolomic profiles associated with cirrhosis in PSC and assess whether the same metabolomic signature could predict adverse outcomes—liver transplantation (LT)—in patients without advanced disease.Methods: In this international multicenter study, serum samples were collected from 241 PSC patients (9 centers, 7 countries), who were randomly assigned in a 60:40 ratio to a discovery cohort (n=144; 22% with cirrhosis) and a validation cohort (n=97; 25% with cirrhosis). Metabolites measured by ultra-high-performance liquid chromatography–mass spectrometry (UHPLC-MS) were evaluated individually for diagnostic performance and through multivariate logistic regression to identify those independently associated with cirrhosis. A metabolite-based diagnostic model was developed using LASSO regression in the discovery cohort and subsequently validated. The ability of the model to predict LT was examined in a subgroup of non-cirrhotic PSC patients with available survival data and compared with established prognostic tools, including the Amsterdam Oxford Model (AOM) and the revised Mayo Risk Score (rMRS).Results: Among the 484 metabolites identified, 115 were significantly associated with cirrhosis (50 increased, 65 decreased) and 16 remained independently associated after adjustment for age, sex, IBD, UDCA treatment, ALP and bilirubin levels. A model incorporating 11 metabolites accurately distinguished cirrhotic from non-cirrhotic patients in the discovery cohort (AUROC 0.88; Se 68%; Sp 93%; PPV 72%; NPV 91%). In the validation cohort the model achieved an AUROC of 0.73 (Se 42%; Sp 88%; PPV 53%; NPV 82%). In a subgroup of non-cirrhotic patients (n=85), 21 underwent LT, with a median time-to-LT of 34 (11–51) months. In this group, the metabolomic signature was independently associated with LT risk (HR 2.62, p=0.001). The model showed strong prognostic discrimination, with a C-statistic of 0.82 and time-dependent AUCs ranging from 0.81 to 0.88 across 12–60 months. Overall, the metabolomic model outperformed both AOM (C-statistic 0.76) and rMRS (C-statistic 0.75).Conclusion: Serum metabolomic profiles vary across PSC disease stages, indicating that metabolomics may serve as a valuable complementary tool for both disease staging and cirrhosis detection. In an exploratory analysis of non-cirrhotic patients with varying degrees of fibrosis, the metabolomic model was associated with future LT risk, suggesting that it may capture underlying disease severity linked to fibrosis even in earlier stages. These findings support the integration of metabolomic data into future patient stratification strategies.

Ursodeoxycholic acid (UDCA) is widely used to treat cholestatic liver diseases such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), yet its molecular mechanisms remain unclear. This study investigated the impact of long-term UDCA therapy on circulating levels of the microRNAs miR-34a and miR-506, which are implicated in PBC pathogenesis, and explored associated changes in inflammatory markers and signaling pathways. Serum samples from patients with PBC and PSC were collected before and after UDCA treatment and analyzed for miRNA expression as well as levels of TREM-2 and sCD163. In vitro studies using human cholangiocytes and lipopolysaccharide (LPS) stimulation assessed changes in the expression of miR-34a, TREM-2, and ADAM17. The results showed that the baseline levels of miR-34a and miR-506 were significantly elevated in PBC patients compared to controls and were significantly reduced after UDCA therapy in PBC but not in PSC. UDCA also decreased serum levels of TREM-2 and sCD163. In vitro, it suppressed the LPS-induced expression of miR-34a and ADAM17 while enhancing TREM-2 expression. Single-cell RNA sequencing of liver tissue and immunofluorescence staining confirmed TREM-2 expression in cholangiocytes. These findings suggest that UDCA modulates key inflammatory pathways and miRNAs in PBC, providing mechanistic insights into its therapeutic effect

INTRODUCTION:Primary sclerosing cholangitis (PSC) is a biliary disorder associated with a high risk of end‑stage liver disease and cholangiocarcinoma (CCA). Currently, prediction of unfavorable outcomes is hindered by a lack of valuable prognostic biomarkers. OBJECTIVES:The aim of the study was to assess the prevalence of autoantibodies in PSC and define their potential use as the predictors of the disease progress and CCA in a large, prospective cohort of PSC patients. PATIENTS AND METHODS:Enzyme‑linked immunosorbent assay was applied for detection of serum immunoglobulin (Ig) A anti‑gliadin antibodies at a concentration equal to or above 30 U and anti‑F‑actin antibodies at a concentration equal to or above 20 U in 624 patients with PSC and 305 healthy controls. Poor PSC outcomes were defined as liver transplantation or / and liver disease‑related death, that is, transplantation‑free survival, and CCA. RESULTS:Anti‑gliadin and anti‑F‑actin IgA antibodies were more frequent in the PSC patients than in the healthy controls (P <0.001 for both). The autoantibodies were associated with laboratory indices of the liver disease severity, including the model of end‑stage liver disease score, and anti‑F‑actin was associated with cirrhosis (P <0.001). During a median (interquartile range) follow‑up of 18.5 (8-33) months, 17.2% patients were transplanted, 4.6% died due to their liver disease, and 5.2% were diagnosed with CCA. Associations between anti‑F‑actin and anti‑gliadin and a shorter transplantation‑free survival (P <0.001 for both) were found. In the multivariable Cox proportional hazards regression analysis anti‑gliadin was an independent predictor of poor survival. No association between the analyzed antibodies and the incidence of CCA was detected. CONCLUSIONS:Anti‑gliadin and anti‑F‑actin IgA identify the subgroup of PSC patients with a more severe disease and at a risk of shortened transplantation‑free survival.

BACKGROUND:Primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) are immune-mediated cholestatic disorders characterized by progressive biliary inflammation and fibrosis, for which treatment options remain limited, underscoring the need for novel therapeutic targets. The leukemia inhibitory factor (LIF) is an IL-6-related cytokine that dysregulates the communication between epithelial cells and extracellular matrices by binding a heterodimeric complex formed by LIF receptor (LIFR) and gp130. The role of the LIF/LIFR system in PSC and PBC and its potential as a therapeutic target remain unclear. METHODS:We investigated LIF/LIFR system alteration in PSC and PBC and assessed the therapeutic potential of LIFR antagonism in a genetic mouse model of PSC (Abcb4-/- mice). Single-cell transcriptomics analyses were performed to evaluate LIF and LIFR expression in human liver samples. Whole liver RNA-seq and immunostaining were used to assess LIF/LIFR levels and correlation with fibrotic and immune markers. The effects of LIFR antagonism were evaluated in vitro using LRI-310, a steroidal LIFR antagonist, on human cholangiocytes, HSCs, endothelial cells, and macrophages. In vivo, LRI-310 was administered to Abcb4-/- mice, and effects on liver injury, cholestasis, fibrosis, leukocyte infiltration, and gene expression were assessed. RESULTS:LIF expression s enriched in human cholangiocytes, while LIFR is predominantly expressed by HSCs, endothelial cells, and macrophages. Whole liver RNAseq analysis and liver sections immunostaining demonstarted that increased LIF expression correlates with expression of markers of hepatic fibrosis and immune activation in PSC and PBC patients. LRI-310, a steroidal LIFR antagonist, attenuated human cholangiocytes activation and expression of inflammatory mediators, as well as the activation of liver sinusoidal cells and hepatic fibroblasts. In Abcb4-/- mice administration of LRI-310 mitigated liver injury, cholestasis, liver leukocytes infiltration and reduced the expression of biomarkers associated with fibrosis, inflammation, and bile acid dismetabolism. CONCLUSION:Cholangiocyte-derived LIF promotes the formation of a pro-inflammatory and pro-fibrotic niche centred on damaged cholangiocytes. LIFR antagonism reverses fibrosis and immune dysregulation in Abcb4-/- mice, supporting the development of anti-LIFR therapies in human cholangiopathies.

Primary sclerosing cholangitis (PSC) is a cholestatic liver disease with male predominance. This study investigated the role of microRNA-125b in PSC-related liver fibrosis, focusing on its interaction with transforming growth factor beta (TGF-β), androgen receptors (ARs), and apelin. Elevated serum and hepatic levels of miR-125b were observed in PSC patients, particularly in males and those with advanced fibrosis, and correlated with increased liver injury markers and FibroScan stiffness. miR-125b expression negatively correlated with apelin and TGF-β levels, while it positively correlated with AR expression. In vitro, miR-125b overexpression induced ARs and suppressed p53 and apelin, whereas lipopolysaccharide stimulation reduced miR-125b and enhanced pro-inflammatory genes, including TNF-α and TGF-β. Notably, ursodeoxycholic acid therapy significantly decreased serum miR-125b levels. These findings suggest that miR-125b contributes to inflammation and fibrogenesis in PSC, partly through the modulation of TGF-β, ARs, and apelin signalling. Moreover, the observed sex-based differences in miR-125b expression underscore the influence of androgens in PSC pathogenesis.

BACKGROUND AND AIMS:Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterised by progressive biliary inflammation and fibrosis, leading to liver cirrhosis and cholangiocarcinoma. GPBAR1 (TGR5) is a G protein-coupled receptor for secondary bile acids. In this study, we have examined the therapeutic potential of BAR501, a selective GPBAR1 agonist in a PSC model. METHODS:Single-cell analysis of healthy human liver samples and gene expression analysis of PSC liver samples were conducted. In vitro studies on a human cholangiocyte cell line (NHC), U937 and human hepatic stellate cells (hSteCs) were performed. Additionally, Abcb4-/- mice were treated with BAR501 for 12-24 weeks. RESULTS:Single-cell analysis demonstrated that GPBAR1 is expressed by macrophages, NK cells, sinusoidal cells and to a lesser extent by cholangiocytes. Total liver expression of GPBAR1 increases in PSC patients compared to that in healthy controls and positively correlates with markers for monocytes and NK cells and cytokeratin 19. In vitro treatment of NHCs with BAR501 reversed the acquisition of a pro-inflammatory phenotype and the downregulation of GPBAR1 expression promoted by LPS in an NF-κB-dependent manner. Treating Abcb4-/- mice reduced bile duct inflammation and liver fibrosis and prevented the downregulation of GPBAR1 expression. Treating mice with BAR501 also modulated the bile acid pool composition and reduced the dysbiosis-associated gut permeability, and intestinal and systemic inflammation. Ex vivo experiments using conditioned media from BAR501-treated cholangiocytes mitigated the activation of macrophages. CONCLUSIONS:Our study provides evidence for the therapeutic potential of selective GPBAR1 agonists in intestinal inflammation-associated cholestasis, warranting the evaluation of BAR501 in PSC patients.

INTRODUCTION Autoimmune hepatitis (AIH) is a chronic, progressive liver disease that, in most cases, may require lifelong immunosuppression. Hepatitis E virus (HEV) is a leading cause of acute, typically self-limited hepatitis worldwide, although immunocompromised patients may develop chronic hepatitis. OBJECTIVES We aimed to evaluate the impact of HEV seropositivity on the clinical course of AIH. PATIENTS AND METHODS The study involved a group of 374 adult patients with AIH (68% women; median [interquartile range] age, 34 [18-83] years; 38% with liver cirrhosis). Serum anti-HEV immunoglobulin (Ig) G and IgM antibodies were measured by enzyme-linked immunosorbent assay, liver fibrosis was assessed by liver stiffness measurement (LSM), and liver cirrhosis was confirmed with liver histology or LSM. RESULTS Fifty-five patients (15%) with AIH were anti-HEV IgG -positive. These patients were older ( P <0.001), had higher body mass index, and higher value of LSM (both P <0.05). In a multivariable model including the levels of alanine aminotransferase and IgG, the HEV seropositive status was associated with an increased risk of advanced liver fibrosis with odds ratio of 3.69 (95% CI, 1.26-10.77; P = 0.02), as reflected by liver stiffness equal to or above 10.5 kPa. Anti-HEV IgG seropositivity was, however, not linked with the type of treatment or worse AIH outcome. Seroprevalence of anti-HEV IgGin the patients with AIH was lower than in the general population of Polish blood donors (43%). CONCLUSIONS Previous HEV infection in patients with AIH is associated with more advanced liver fibrosis. However, the overall seroprevalence of anti-HEV IgG is lower in patients with AIH than in blood donors in Poland.

5-aminosalicylic acid (5-ASA) is a first-line treatment for maintaining colitis remission. It is a highly effective, safe, and well-tolerated drug with anti-inflammatory and chemo-preventive properties. While patients with primary sclerosing cholangitis (PSC) with concomitant ulcerative colitis are treated with 5-ASA, the molecular mechanisms underlying the drug’s chemo-preventive effects are not entirely understood. We previously reported that bile acids and lipopolysaccharide-induced miR-155 expression was associated with downregulating mismatch repair (MMR) proteins in CACO-2 cell lines. Therefore, in this investigation, a set of in vitro functional studies was performed to show the possible mechanisms behind the epigenetic relationship between miR-155 and 5-ASA’s prevention of high microsatellite instability (MSI-H). In transient transfection with miR-155Mimic, which behaves like endogenous miRNA, we confirmed the relationships between miR-155 and its target MMR in three human intestinal epithelial cell lines: CACO-2, NCM460D and HT-29. We have shown, for the first time, that 5-ASA modulates MLH1, MSH2, MSH6 in miR-155 transfected cells. These findings underline that chemoprotective 5-ASA therapy can effectively attenuate the expression of miR-155 and potentially prevent a development of MSI-H in a subset of colorectal cancers associated with PSC.

Primary sclerosing cholangitis (PSC) is characterised by the co-occurrence of inflammatory bowel diseases, particularly ulcerative colitis (UC). We investigated how the interaction of miR-125b with the sphingosine-1-phosphate (S1P)/ceramide axis may predispose patients with PSC, PSC/UC, and UC to carcinogenesis in the ascending and sigmoid colons. The overexpression of miR-125b was accompanied by the upregulation of S1P, ceramide synthases, ceramide kinases, and the downregulation of AT-rich interaction domain 2 in the ascending colon of PSC/UC, which contributed to the progression of high microsatellite instability (MSI-H) colorectal carcinoma. We also showed that the overexpression of sphingosine kinase 2 (SPHK2) and the genes involved in the glycolytic pathway in the sigmoid colon of UC led to the upregulation of Interleukin 17 (IL-17). In vitro stimulation of human intestinal epithelial cells (Caco-2, HT-29, and NCM460D) with lipopolysaccharide suppressed miR-125b and increased proinflammatory cytokines, whereas the induction of miR-125b activity by either a miR-125b mimetic or lithocholic acid resulted in the inhibition of miR-125b targets. In summary, miR-125b overexpression was associated with an imbalance in the S1P/ceramide axis that can lead to MSI-H cancer progression in PSC/UC. Furthermore, SPHK2 overexpression and a change in the cellular metabolic flux are important players in inflammation-associated colon cancer in UC.