Gastroenterology

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.

BackgroundShort bowel syndrome (SBS) resulting from extensive small bowel resection is characterized by severe malabsorption and represents the leading cause of intestinal failure. Although spontaneous intestinal adaptation can partially restore nutrient absorption, the temporal coordination and hierarchy of the adaptive mechanisms involved--particularly those linking the gut microbiota, enteroendocrine function, hyperphagia, and intestinal remodeling-- remain incompletely understood. MethodsWe investigated the kinetics of spontaneous intestinal adaptation in a rat model mimicking type 2 SBS over a 28-day postoperative period. Body weight, food intake, gastrointestinal transit, fecal losses, intestinal morphology, enteroendocrine hormone secretion, hypothalamic neuropeptide expression, and gut microbiota composition were assessed longitudinally in SBS and SHAM-operated rats. ResultsExtensive small bowel resection induced marked early weight loss, accelerated intestinal transit, diarrhea, and increased fecal energy losses that persisted throughout the follow-up. Profound gut microbiota remodeling occurred as early as day 7, remained largely stable thereafter, and was characterized by reduced diversity and enrichment in Lactobacillaceae and Enterobacteriaceae. Early elongation of remaining colon and epithelial remodeling were observed, preceding the jejunal hyperplasia, which became evident from day 14 onward. Enteroendocrine adaptation was marked by an early increase in plasma peptide YY levels, whereas glucagon-like peptide-1 showed a modest response. Food intake was increased in SBS rats from day 7 onward, and hyperphagia developed gradually and reached a plateau by the end of the third postoperative week, in parallel with increased hypothalamic AgRP levels and reduced POMC levels. No significant improvement of intestinal transit and fecal energy losses was observed during the study period. ConclusionIntestinal adaptation to extensive resection follows a time-dependent sequence in which early gut microbiota remodeling and colonic adaptation precede hyperphagia and small intestinal remodeling. These findings highlight the gut microbiota and the colon as central components of the early post-resection adaptation and potential therapeutic targets in SBS.

Background: Gastric cancer surveillance in CDH1 pathogenic variant carriers is challenging, as predictors of localized (stage T1a) and advanced (stage >T1a) signet ring cell carcinoma (SRCC) are not well defined. We established the Group of investigAtors STriving toward Research In CDH1 (GASTRIC) consortium to identify clinicopathological factors associated with localized and advanced SRCC. Methods: A retrospective observational study (1998-2025) of CDH1 carriers across twelve academic centers was performed. Clinical, endoscopic, and pathological data were compared between carriers with and without SRCC on endoscopy, and between those with advanced versus localized or no cancer on gastrectomy specimens. Results: Overall, 390 CDH1 carriers from 235 families were included. Presence of SRCCs on endoscopy was significantly associated with thickened folds, nodularity, masses, and intestinal metaplasia, while gastritis was negatively associated. Of 196 carriers (52.4%) undergoing gastrectomy, 11 (5.6%) had advanced cancers, 10(90.9%) of which showed endoscopic abnormalities. Identification of SRCC on baseline endoscopy was the most sensitive feature for advanced disease (0.81) but had moderate specificity (0.74), whereas masses and thickened folds were highly specific (0.99 and 0.96, respectively) but less sensitive. Negative predictive values were high (0.94-1.0), while positive predictive values were modest (0.13-0.66). On multivariate analysis, masses and SRCC foci on baseline endoscopy were independent predictors of advanced disease. Conclusion: Among CDH1 carriers, absence of endoscopic findings was reassuring, whereas significance of detected endoscopic and pathological abnormalities was less certain. Advanced cancer occurred in a small number of carriers, with endoscopic abnormalities in nearly all cases. Endoscopic surveillance might be an alternative to surgery in carriers without worrisome mucosal findings.

Pancreatic ductal adenocarcinoma (PDAC) frequently metastasizes to the liver, which drives patient mortality. CA19-9 is elevated in most PDAC tumors and is widely used as a clinical biomarker. Elevated serum levels are associated with poor outcomes. However, whether CA19-9 functionally contributes to metastatic progression has not been fully defined, in part because mice lack endogenous CA19-9 expression. Here, using syngeneic murine PDAC cells engineered to express CA19-9, we investigated its functional role in liver metastasis. In splenic injection models, CA19-9 expression markedly increased liver metastatic burden by promoting both metastatic seeding and subsequent metastatic outgrowth. In vitro, CA19-9 enhanced tumor cell adhesion to endothelial cells through interaction with E-selectin. Metastatic seeding of CA19-9-expressing cells was reduced by genetic deletion of E-selectin or antibody neutralization of either CA19-9 or E-selectin in vivo. Therapeutic targeting of CA19-9 with a neutralizing antibody markedly reduced liver metastatic burden after metastatic seeding. CA19-9 expression increased AKT signaling in PDAC cells and liver metastases, and CA19-9 levels correlated with AKT activation in human PDAC tissues. These findings show that CA19-9 promotes PDAC liver metastasis through E-selectin-dependent metastatic seeding and AKT-associated metastatic outgrowth, highlighting CA19-9 as a functional mediator of PDAC metastasis and a potential therapeutic target.

Background and AimsArtificial intelligence (AI) is increasingly applied to histological assessment in inflammatory bowel disease (IBD), but most approaches quantify features in isolation and ignore their anatomical location within the mucosa. We developed and validated PAIR-IBD (Perspectum AI Reading in IBD), an AI system that quantifies inflammatory cell populations, crypt injury, and epithelial damage within defined mucosal compartments to distinguish active disease, remission, and equivocal cases in ulcerative colitis (UC). MethodsA deep learning ensemble was trained on three IBD biopsy datasets to identify lymphocytes, neutrophils, eosinophils, and plasma cells, and to segment crypts, lamina propria (LP), and muscularis mucosae. Inflammatory cell densities and crypt injury metrics (mucin depletion, solidity, roughness, branching, and abscess formation) were quantified. PAIR-IBD outputs were compared between histologically active and remissive UC, evaluated in inconclusive cases, and correlated with manual pathology grading. ResultsNeutrophil density increased 3.5-fold in the LP and 15-fold within crypts in active UC (p<0.0001). Eosinophil density doubled and LP lymphocytes increased 1.4-fold. Active UC showed increased mucin depletion, crypt branching, and crypt abscesses, with reduced crypt solidity (p<0.0001 for all). PAIR-IBD metrics correlated with manual inflammatory and crypt injury scores (rs=0.23-0.72) and global indices (rs=0.27-0.65). Up to 89% of inconclusive cases aligned with remission-like profiles based on multiple independent AI metrics. ConclusionPAIR-IBD provides spatially resolved, quantitative assessment of inflammation and epithelial injury in UC, improving disease stratification and resolution of equivocal histology, with potential to support scoring consensus and improve accuracy of histological endpoints in clinical trials.

Colorectal cancer (CRC) is the leading cause of cancer mortality in young adults, with chemoresistance and metastatic progression severely limiting treatment options. MUC4, a membrane-bound mucin normally confined to the apical surface of epithelial cells, becomes aberrantly overexpressed across the tumor cell membrane in CRC and other cancer types, promoting cancer survival, immune evasion, and metastasis. Therefore, MUC4 is a promising candidate for the development of targeted therapies. We detected MUC4 cell surface expression in a panel of human CRC cell lines. We engineered MUC4-specific chimeric antigen receptor (CAR) T cells and evaluated their activity against methotrexate-resistant MUC4-expressing human CRC cell lines HT29-MTX and T84. MUC4 CAR-T cells efficiently eliminated HT29-MTX and T84 cells in vitro and delayed HT29-MTX subcutaneous tumor growth in vivo. Importantly, MUC4 CAR-T cell treatment significantly reduced tumor burden and improved survival in a highly aggressive and lethal CRC model, intraperitoneal HT29-MTX CRC metastatic dissemination in NSG mice. Mouse necropsies revealed no off-target in vivo toxicities associated with MUC4 CAR-T cell therapy. Altogether, our findings establish MUC4 as an important and clinically relevant CAR-T cell target and demonstrate therapeutic efficacy of MUC4 CAR-T cell therapy for invasive CRC, a setting where chemotherapy typically fails. MUC4-targeted CAR-T cell therapy has the potential to fill a critical treatment gap for patients with chemoresistant and invasive CRC and may extend to other MUC4-expressing solid tumors. Statement of PriorityColorectal cancer is increasingly diagnosed in younger individuals, a trend accompanied by a higher prevalence of aggressive and chemotherapy-resistant disease. Despite advances in systemic therapy, outcomes for patients with refractory or metastatic colorectal cancer remain poor, underscoring a critical unmet clinical need. CAR-T cell therapy offers a mechanism to selectively target tumor cells that evade conventional treatments and may thus be particularly valuable in mucinous, chemotherapy-resistant subsets. Developing effective CAR-T strategies for new targets in colorectal carcinoma, such as MUC4, is an urgent priority in this era of rising early-onset disease.

BackgroundEndoscopic resection is the standard treatment for rectal neuroendocrine tumors (r-NETs) [&le;]10 mm, yet the optimal technique remains controversial. Modified endoscopic mucosal resection (m-EMR) has emerged as a potential alternative compared to endoscopic submucosal dissection (ESD), but existing evidence is largely retrospective and the results of recent randomized controlled trials (RCTs) are inconclusive. AimsTo compare the efficacy and safety of m-EMR versus ESD for r-NETs [&le;]10 mm. MethodsWe systematically searched CENTRAL, PubMed, Embase, and WanFang from January 1st, 1970 to December 23, 2025 for RCTs comparing m-EMR with ESD in r-NETs [&le;]10 mm. The GRADE framework assessed evidence certainty, while trial sequential analysis (TSA) controlled random errors and evaluated conclusion validity. ResultsSix RCTs involving 440 patients were analyzed. No significant difference between m-EMR and ESD was found in histologic complete resection (RR = 1.00, 95% CI 0.97-1.03; I2 = 0%), en bloc resection rates (P = 0.75) and procedure-related complications (P = 0.94). And m-EMR was associated with a significantly shorter procedure time (P<0.00001) and lower hospitalization cost (P<0.00001). The evidence was of moderate certainty; TSA confirmed its reliability, and both cumulative and sensitivity analyses supported the robustness. ConclusionsModerate-certainty evidence indicates m-EMR achieves oncologic outcomes comparable to ESD while offering clear advantages in procedural efficiency and cost for r-NETs [&le;]10 mm, supporting m-EMR possibly as a preferred endoscopic strategy in clinical practice.

ObjectivesTo characterize ultra-processed food (UPF) circulating metabolic signatures associated with Crohns disease (CD) and to localize key metabolic mediators linking UPF intake to CD risk. DesignProspective cohort study. SettingTwo large multi-center cohorts (UK Biobank [UKB] and Whitehall II [WHII] study) across the UK and an Eastern multi-center cohort ONE-IBD Study from China. ParticipantsUK Biobank discovery cohort (n=10,229) for signature derivation, internal validation cohort (n=91,306), external validation cohort Whitehall-II (n=7,893), and three additional cohorts (two Western and ONE-IBD) for validation of key metabolic drivers. Main outcome measuresPrimary outcomes were UPF-related circulating metabolic signatures and their associations with CD risk; secondary outcomes included evidence supporting causal roles of candidate metabolites and genetic pathways assessed by Mendelian randomization, colocalization, and gene-environment analysis. ResultsA UPF metabolic signature of 73 metabolites was constructed and validated across cohorts (Spearman {rho}: 0.20-0.25). More pronounced UPF metabolic signature was associated with increased CD risk (HRper SD=2.65, 95% CI 1.57-4.48). WGCNA revealed a cluster enriched in fatty acids. Within this cluster, docosahexaenoic acid (DHA) emerged as the strongest, which mediated 17.1% of the UPF-CD association. External validation in ONE-IBD supported DHA as the strongest associated metabolite with UPF and CD. Mendelian randomization supported a causal protective effect of DHA on CD (OR=0.72, 95% CI 0.61- 0.83; P<0.001), with colocalization implicating rs174546 in the FADS1 gene. ConclusionThe adverse effects of UPF on CD risk may be driven by a relative deficiency of protective metabolites such as DHA, apart from additive harm to metabolic depletion. This reframes UPF-related risk and highlighting potential targets for precision nutrition in CD prevention.

Eosinophilic esophagitis (EoE) is a leading cause of chronic esophageal dysfunction driven by immune-mediated inflammation. Peak eosinophil count (PEC) in esophageal biopsies is routinely used to assess disease activity, and its associated molecular mechanisms have been well studied. However, PEC only partially captures overall disease severity, which is comprehensively captured by the Index of Severity for Eosinophilic Esophagitis (I-SEE). In contrast to PEC, the molecular and cellular programs associated with the I-SEE-defined disease severity, particularly in children, remain poorly understood. We integrated bulk transcriptomic profiling of pediatric esophageal biopsies with clinical severity metrics and a matched single-cell transcriptomic reference. Increasing severity was associated with a shift from type 2 inflammatory activation toward epithelial stress, cytoskeletal and junctional disruption, metabolic dysfunction, and extracellular matrix remodeling. Single-cell-informed analyses identified that proliferating and transitional epithelial cell states were strongly associated with higher I-SEE scores and exhibited impaired differentiation, heightened metabolic and oxidative stress responses, and structural remodeling programs not captured by bulk transcriptomic analyses alone. These findings reposition epithelial remodeling, rather than eosinophil burden alone, as a central molecular correlate of disease severity in pediatric EoE and provide a framework for improved disease stratification and therapeutic intervention.

Background and AimsPancreatic ductal adenocarcinoma (PDAC) is characterized by profound immune exclusion and resistance to immunotherapy. Although WNT signaling has been implicated in PDAC, its cellular source within the tumor microenvironment and its contribution to immune suppression remain poorly defined. This study investigated whether myeloid-derived WNT signaling promotes PDAC progression. MethodsTranscriptomic data from human PDAC cohorts, including The Cancer Genome Atlas (TCGA), and published single-cell RNA sequencing datasets were analyzed. Macrophage-associated WNT5A expression in human PDAC biopsies was assessed using in situ hybridization and immunofluorescence. Macrophage-derived WNT secretion was genetically disrupted using macrophage-specific Porcn knockout mice in orthotopic and subcutaneous KPC tumor models. Lineage-resolved spatial organization of macrophage subsets was characterized using Ms4a3 fate-mapping double-reporter mice with immunofluorescence and imaging mass cytometry. Macrophages-CD8 T cells interactions were assessed using tumor-educated macrophage conditioned media, pharmacologic ARG1 inhibition, and in vivo CD8 T cell depletion. ResultsPDAC tumors with high macrophage signatures showed enrichment of noncanonical WNT signaling, and macrophage-associated WNT5A was detected in human biopsies. Disruption of macrophage-derived WNT secretion suppressed tumor growth, reversed immune exclusion, and enhanced cytotoxic CD8 T cell infiltration. Spatial lineage-resolved analysis demonstrated progressive accumulation of Hexb tissue-resident macrophages that dominated advanced lesions and formed a WNT-rich niche closely associated with Trem2Arg1 monocyte-derived macrophages. Mechanistically, macrophage-derived noncanonical WNT activated a JNK/c-Jun-ARG1 axis that inhibited CD8 T cell proliferation, an effect abolished by myeloid WNT loss. ConclusionsMyeloid-derived noncanonical WNT establishes a lineage-structured macrophage niche that enforces immune exclusion in PDAC. Targeting macrophage-restricted WNT signaling represents a promising strategy to reprogram the PDAC immune microenvironment.

The chemokine CCL20 is implicated in inflammation and cancer but has proven challenging to target therapeutically. In this study, we precisely define what cells produce CCL20 in pancreatic inflammation and cancer. Through analysis of single cell RNA data, mutation and copy number signatures, gene methylation, and in vitro studies, we show that CCL20 and other NF-{kappa}B driven chemokine production is largely dependent on oncogenic KRAS in the malignant pancreas. Blockade of CCL20-CCR6 signaling in vivo using a novel partial agonist inhibitor, CCL20LD, increased recruitment of antigen presenting cells without significantly impinging tumor growth. Lastly, resistance to pan-RAS or allele-specific KRAS inhibitors decreased CCL20-dependent immune recruitment in culture. These results suggest that oncogenic KRAS activates NF-{kappa}B signaling in human pancreas cancer, resulting in pharmacologically reversible changes to chemokine production that may participate in immune suppression or immune evasion within the pancreas cancer microenvironment.

ObjectivesFree fatty acid receptors 2 and 3 (FFA2 and FFA3) are activated by nutrient-derived metabolites such as short-chain fatty acids (SCFAs) and ketone bodies, produced by the gut microbiota and host, respectively. This study aimed to investigate the intracellular signaling pathways recruited in glucagon-like peptide-1 (GLP-1) releasing enteroendocrine cells following activation of FFA2 and FFA3 to resolve the impact of nutrient status on enteroendocrine cell function. MethodsExperiments were performed using primary mouse colonic cultures and the mouse enteroendocrine cell line, GLUTag cells. Expression analysis by bulk RNA sequencing was used to determine expression of FFA2 and FFA3 in GLP-1 releasing cells. Measurement of GLP-1 secretion by sandwich ELISA was used to assess enteroendocrine cell function. Live-cell measurements of intracellular calcium and cAMP levels were performed to assess canonical second messenger signaling pathways. ResultsA SCFA mixture stimulated GLP-1 secretion from both primary mouse colonic cultures and GLUTag cells. In GLUTag cells, the FFA2 ligand 4-CMTB inhibited GLP-1 release independent of Gaq- and Gai-signaling as neither YM-254890 (Gaq inhibitor) nor pertussis toxin (Gai- uncoupler) altered its effect. However, 4-CMTB did elevate cAMP levels, suggesting an indirect mechanism for the increase in cAMP production. Stimulation of FFA2 with the Gai-biased ligand AZ1729 or the ketone body acetoacetate inhibited GLP-1 release and cAMP accumulation. AZ1729 was insensitive to pertussis toxin and OZITX, supporting atypical FFA2 signaling. Stimulation of FFA3 with AR420626 or the ketone body {beta}-hydroxybutyrate increased GLP-1 secretion from GLUTag cells, an effect that was not mediated by cAMP production. AR420626, but not {beta}-hydroxybutyrate increased intracellular calcium levels. ConclusionsOverall, activation of FFA2 inhibited secretory function in GLP-1-releasing enteroendocrine cells, whereas activation of FFA3 stimulated GLP-1 secretion via distinct intracellular signaling mechanisms. O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/708924v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@175dbaforg.highwire.dtl.DTLVardef@a9cc3eorg.highwire.dtl.DTLVardef@1a026e5org.highwire.dtl.DTLVardef@15997af_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG HighlightsO_LIExposure to physiological levels of SCFAs stimulates GLP-1 secretion from colonic EECs C_LIO_LIFFA2 and FFA3 regulate GLP-1 release via non-canonical signaling pathways C_LIO_LIKetone bodies activate SCFA receptors to differentially modulate GLP-1 levels C_LIO_LILigand bias enables nutrient-dependent tuning of EEC gut hormone secretion C_LI

Amatoxin-induced acute liver failure complicates misidentified foraged mushroom ingestion worldwide; abrupt multisystem collapse punctuates apparent improvement. Our prospective single-arm clinical trial investigated proactive toxicokinetic-based management to preserve elimination capacity: sustained enhanced hydration to maintain renal clearance; fasting plus octreotide to suppress meal-driven enterohepatic circulation; and intravenous silibinin to inhibit OATP1B3-mediated hepatic uptake, enabling safe passage and elimination of gallbladder-confined amatoxin-laden bile. Safety population (N=99) transplant-free recovery (TFR): 88.0% (87 recoveries, 6 transplants, 6 deaths). Protocol-adherent Efficacy population (n=86) TFR: 98.8% (85 recoveries, 1 transplant, 0 deaths). Multivariable analysis identified uninterrupted hydration as strongest TFR predictor (P<0.001), followed by earlier silibinin initiation (P=0.003); octreotide shortened INR recovery by 11 hours (P=0.033). These findings support a toxin elimination model in which preserved renal clearance and biliary sequestration are central recovery determinants. The kinetic balance between renal clearance and hepatic uptake governs both recovery and collapse.

BackgroundCurrent British Society of Gastroenterology (BSG) guidelines misclassify metachronous lesion risk after polypectomy in approximately 40% of patients. Building on evidence that immune exclusion drives progression of adenomas to colorectal cancer, this study examined immune profiles in screen-detected adenomas as a predictive biomarker for metachronous lesion risk. MethodsPatients undergoing polypectomy within the Scottish Bowel Screening Programme, with surveillance colonoscopy between 6 months and 6 years were included. Chromogenic immunohistochemistry (IHC; n=2642), 6-plex multiplex immunofluorescence (mIF; n=334), and spatially resolved 6000-plex single cell transcriptomics (n=7) were applied to adenoma microarrays. Cell density and location were measured using QuPath. Hierarchical then K-means clustering was used to define immune cell density-based clusters, which were compared to future lesion events using Kaplan-Meier curves and the log rank test. ResultsAfter adjustment for age, sex, site, size and dysplasia, adenoma CD3+ T cell density was significantly associated with future colorectal neoplasia (HR 1.43, 95% CI 1.19-1.71, p<0.001). Using mIF three immune cell density clusters were identified; 1) high T cell density, low macrophage density, 2) low T cell density, low macrophage density, and 3) high T cell, macrophage and SMA density, with significant differences in future lesion risk (Cluster 1: 22%, Cluster 2: 41%, Cluster 3: 36%, p=0.032). Bulk RNAseq and spatial transcriptomic analysis revealed significant variation in T cell and macrophage co-location and gene expression profiles between clusters. ConclusionAdenoma immune contexture emerges as a determinant of future metachronous lesion risk, offering a novel biomarker to refine surveillance and reduce disease burden. SummaryWhat is already known on this topic: O_LIPost-polypectomy surveillance is currently recommended to patients with high-risk pathological features to detect metachronous lesions and cancer. However current guidelines misclassify risk in a proportion of patients, leading to unnecessary surveillance for some, whilst falsely reassuring others. C_LI What this study adds: O_LIAnalysis of this large post-polypectomy surveillance cohort reveals that adaptive immune responses within removed index adenomas predicts low risk of metachronous lesions, while an immune excluded phenotype signals higher risk, independent of pathological characteristics, and patient risk factors. C_LI How this study might affect research, practice or policy: O_LIDefining immune cell spatial distributions and interactions that drive future adenoma and cancer risk will enable more precise risk stratification for surveillance, informing surveillance guidelines and shaping targeted colorectal cancer prevention strategies. C_LI

BackgroundPreoperative biliary stenting alters biliary colonization and may reduce the effectiveness of perioperative antibiotic prophylaxis in pancreatoduodenectomy. Although broader-spectrum regimens have been associated with improved infectious outcomes, their microbiological adequacy in routine clinical practice remains poorly defined. We therefore evaluated the real-world adequacy of a prolonged ampicillin-sulbactam protocol, its association with infectious outcomes and survival, and the potential impact of a universal piperacillin-tazobactam strategy. MethodsWe analyzed all consecutive patients who underwent elective pancreatoduodenectomy from 2002 to 2023 at our tertiary center. Demographic, operative, microbiological, and outcome data were retrieved from a prospectively maintained database. Patients were stratified by stent status. Adequacy of prophylaxis was defined as the full in vitro susceptibility of all bile isolates. The outcomes included 30-day infectious morbidity, clinically relevant POPF, PPH, DGE, reoperation, 30- and 90-day mortality and long-term survival. A coverage simulation was performed to compare ampicillin-sulbactam with a hypothetical universal piperacillin-tazobactam. Statistical methods included chi-square/Fishers exact tests, Mann-Whitney U tests, Cox models, McNemars test and Poisson regression. ResultsOf 956 patients, 424 (44%) had a biliary stent. Technical complications were comparable between groups, and rates of POPF and PPH were not increased. However, infectious morbidity was higher in stented patients, including sepsis (RR 1.62, 95% CI 1.05-2.51) and postoperative cholangitis (RR 2.20, 95% CI 1.36-3.56). Thirty- and 90-day mortality were increased (RR 2.88 and 2.73) but lost significance after adjustment. Bile cultures predominantly yielded Enterococcus and Enterobacterales with low ampicillin-sulbactam susceptibility. Overall adequacy was 21.7%. Among patients with bile cultures (n = 474), ampicillin-sulbactam covered 43.7% (207/474) versus 81.2% (385/474) with piperacillin-tazobactam; in stented patients with cultures (n = 397), coverage increased from 41.8% to 78.1%. Adequate ampicillin-sulbactam coverage was not associated with reduced infectious outcomes in Poisson models. ConclusionPreoperative stenting creates a polymicrobial, partially resistant biliary niche that ampicillin-sulbactam does not sufficiently cover. Our data shows that a piperacillin-tazobactam strategy substantially improves coverage and was therefore implemented at our center. Core message- Stented patients exhibit a distinct infectious risk profile characterized by Enterococcus-and Enterobacterales-dominated bile colonization rather than increased rates of technical complications. - In stented patients, real-world microbiological coverage of ampicillin-sulbactam was limited, and in vitro susceptibility did not independently translate into reduced postoperative infectious morbidity. - Broader prophylaxis, such as piperacillin/tazobactam, aligns with the actual flora and nearly doubles theoretical coverage, addressing the mismatch between stent-associated biofilms and narrow regimens.

Our understanding of human mucosal T cell clonotype distribution in health and disease has centered on immunodominant antigens. We performed single cell T cell receptor (TCR) and RNA sequencing as an untargeted approach to define distributions of T cell clonal groups in health and ulcerative colitis (UC) across 333,088 T cells in colon and peripheral blood. Healthy donor-specific TCR repertoires had limited blood-colon clonal sharing, which was highest in cytotoxic T effector memory (Tem) populations and lowest in regulatory T cells (Tregs), reflecting tissue-based compartmentalization. Within healthy colon, TCR repertoires showed high T cell clonal sharing independent of anatomic distance, associated with high intra-clonal phenotypic diversity. Colon cytotoxic and Th17 populations showed high dispersion across sites, while Tregs were compartmentalized. Clonal lineages dispersed across blood and colon upregulated trafficking markers, suggesting active movement between tissues, while those dispersed across colon sites upregulated residency markers, suggesting intra-colon repertoire sharing is mediated by long-term, slow moving clonal groups. In UC, Tregs were expanded across inflamed sites, and increased CD8 Tem clonal groups showed increased dispersion regardless of inflammation. These findings reveal principles of T cell clonal organization in the human colon during health and disease, identifying opposing patterns of clonal dispersion among Treg and Th17 clonal groups, high phenotypic diversity within dispersed clonal groups, and elevated cross-colon dispersion of CD8 Tem clonotypes in UC.

Type I interferons (IFN-Is) play a critical role in innate immunity, modulating the host response. While dysregulated IFN-I signaling has been implicated in autoimmune and infectious disorders, its role in inflammatory bowel disease (IBD) remains unclear. In this study, we extensively assessed the function of IFN-I signaling in human IBD and murine models of colitis. Expression of IFN-I signature genes was elevated in patients with active ulcerative colitis as well as multiple murine models of colitis. Single cell RNA sequencing revealed that upregulated IFN-I signature genes were enriched in myeloid cells, which exhibited increased expression of IFN receptors during mucosal inflammation. Mice carrying gain-of-function alleles of Ifnar1, a subunit of IFN-I receptor, showed heightened IFN-I signaling and altered colonic immune homeostasis at baseline, and were more susceptible to experimental colitis. In contrast, postnatal inhibition of IFNAR1, using either an inducible transgenic mouse model or an anti-IFNAR1 blocking antibody, protected against experimental colitis. Taken together, our findings reveal a previously under-recognized pathogenic role of IFN-I in IBD and provide a rationale for therapeutic intervention targeting this pathway.

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

Gallbladder cancer (GBC) is a highly lethal malignancy with limited experimental models to study disease biology or evaluate therapeutic responses. Although canonical Wnt activation is commonly used for patient-derived organoid (PDO) development and expansion, gallbladder PDOs has also been generated under Wnt-inhibitory conditions. No comparative assessment has determined how Wnt pathway modulation influences gallbladder PDO development, phenotype or drug response. This study systematically compared the impact of canonical Wnt activation (WNTAct medium containing CHIR99021) versus inhibition (WNTInh medium containing DKK1) on the establishment, propagation, molecular features and therapeutic responses of PDOs generated from malignant or non-malignant gallbladder tissues derived from the same patient. Both media supported successful PDO generation with comparable efficiency, preserving biliary epithelial functions and marker expression. Transcriptomic profiling confirmed selective enrichment of canonical Wnt target genes in PDOs generated in WNTAct cultures. WNTAct conditions enabled markedly superior long-term propagation, whereas WNTInh cultures more consistently retained the dysplastic features in malignant samples. Gemcitabine response assays demonstrated significantly greater drug sensitivity in PDOs grown in WNTAct medium, a phenotype reversible upon media switching but requiring extended adaptation, indicating a dynamic and context-dependent influence of Wnt signaling on chemotherapeutic vulnerability. Collectively, the findings reveal a trade-off between long-term propagation and histological fidelity in gallbladder PDOs and show that Wnt signaling modulates gemcitabine sensitivity in a reversible manner. This comparative framework provides practical guidance for selecting culture conditions for gallbladder PDO based disease modelling and precision oncology applications.

Background and ObjectivesInflammatory bowel disease (IBD) involves dysregulated immune responses and chronic intestinal inflammation. The nod-like receptor pyrin domain-containing 3 (NLRP3) inflammasome plays a critical role in IBD pathogenesis, but regulatory mechanisms remain not fully understood. Mitsugumin 53 (MG53, also known as TRIM72), originally identified as a critical membrane repair protein, has emerged as a novel regulator of inflammatory processes. To investigate the protective role of MG53 in colitis and elucidate its mechanisms in regulating NLRP3 inflammasome activation in IBD. MethodsWe used dextran sodium sulfate (DSS)-induced colitis models comparing MG53 knockout (MG53-/-) and wild-type (WT) mice, assessing disease severity, MG53 tissue uptake, and therapeutic effects of recombinant human MG53 (rhMG53). In vitro studies examined rhMG53s effects on NLRP3 inflammasome activation, caspase-1 cleavage, interleukin-1{beta} (IL-1{beta}) secretion, and MG53-NLRP3 interactions. ResultsMG53-/- mice showed more severe colitis with increased weight loss, higher disease activity scores, shortened colons, and greater inflammation. DSS treatment induced the accumulation of circulating MG53 in inflamed colonic tissue. rhMG53 administration ameliorated colitis severity in MG53-/- mice and dose-dependently suppressed NLRP3 inflammasome activation in vitro. MG53 interacted with NLRP3 and reduced apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and NLRP3 oligomerization without affecting upstream signaling or NLRP3 stability. ConclusionMG53 is a physiological regulator of NLRP3 inflammasome activation that protects against colitis, suggesting therapeutic potential for IBD.