Gastroenterology

Background and AimsStem cell-derived organoids are promising platforms for therapeutic screening in inflammatory bowel disease (IBD), but identifying functional organoid readouts with translational utility is challenging. Colon epithelial organoids from patients with ulcerative colitis (UC) overexpress chemokines CXCL1, CXCL11, CCL2, and CCL28, yet whether these inflammatory signatures correlate with disease activity and treatment response is unknown. This short report investigates whether organoid-retained chemokines correlate with disease activity and therapeutic outcomes. MethodsWe interrogated three bulk and two single-cell transcriptomic datasets from IBD clinical trials encompassing anti-TNF and anti-integrin therapies to determine whether epithelial chemokines retained in UC organoids track clinical response and distinguish treatment responders from non-responders to biologic therapy across multiple IBD patient cohorts. ResultsIn bulk transcriptomic data, CXCL1, CXCL11, and CCL2 were elevated in active UC and normalized only in patients achieving clinical remission, independent of therapy class, with persistent chemokine overexpression in non-responders. Single-cell analysis demonstrated widespread chemokine overexpression in UC epithelial clusters, with consistent normalization of CXCL1, CXCL11, and CCL28 in LGR5-positive stem compartment of patients who achieved clinical remission, but not in non-responders. In Crohns disease, the resolution of these epithelial chemokines was not associated with clinical response. ConclusionsEpithelial chemokines, particularly CXCL1, CXCL11, and CCL28, track clinical remission in UC and represent candidate biomarkers and functional endpoints for epithelial-directed therapeutic strategies using stem cell-derived UC organoid models.

Irreversible electroporation (IRE) has shown promise for treating pancreatic ductal adenocarcinoma (PDAC), but whether IRE can induce an abscopal effect is not established. We demonstrated that the combination of IRE and anti-PD-1 antibody could trigger robust abscopal effects in preclinical models of metastatic PDAC. Data from multiple in vivo models, RNA-seq, scRNA-seq, and spatial immunofluorescence provide compelling evidence that IRE induced mitochondrial dysfunction and cellular stress, which triggered activation of the cGAS-STING pathway and subsequent systemic antitumor effects. IRE also led to inflammatory response characterized by tumor infiltration of myeloid cells and their polarization toward M1 state, turning immunologically "cold" tumors into "hot" tumors. Moreover, the presence of T cell/B cell clusters in tumors from mice treated with IRE plus PD-1 and the lack of antitumor efficacy in B cell knockout mice bearing orthotopic murine PDAC tumors indicate that B cells play an important role in IRE-mediated systemic antitumor immunity. SignificanceThis study shows that IRE plus a checkpoint inhibitor represents a promising therapeutic strategy for PDAC and supports advancing this treatment toward clinical translation. Our data also support potential combination strategies with immunomodulatory agents that can recruit and reprogram B cells to support T cell activation and cytotoxic effector functions.

Background and AimsThe tumor microenvironment in colorectal cancer (CRC) is richly innervated, yet the contribution of the enteric nervous system (ENS) to CRC biology remains poorly defined. ENS neurons express proenkephalin (PENK), which can be processed by proprotein convertase 1/3 (PCSK1) to generate Methionine-enkephalin (M-ENK), a bioactive peptide with growth-regulatory potential. We hypothesized that an ENS-derived PCSK1-M-ENK axis restrains CRC proliferation through opioid growth factor receptor (OGFr) signaling and is modulated by stress-associated glucocorticoid receptor (GR) signaling and GLP1 receptor (GLP1R) activity. MethodsPublicly available human CRC single-cell RNA-sequencing datasets were analyzed for OGFr expression. PCSK1 and M-ENK expression in murine ENS and tumor-associated tissue was assessed by immunofluorescence. Functional studies were performed using murine CRC organoids, and primary murine ENS neurons in mono- and co-culture. CRC proliferation was quantified by EdU incorporation following treatment with recombinant M-ENK, recombinant PCSK1, OGFr synthetic ligand naloxone, or PCSK1 inhibitors. Effects of dexamethasone and liraglutide on PCSK1 expression in ENS-containing murine tissue were evaluated. ResultsOGFr was enriched in CRC cells and positively associated with KRAS gene expression. A subset of adult murine colonic myenteric neurons expressed PCSK1 and M-ENK. M-ENK dose-dependently suppressed proliferation of CRC organoid cells. ENS neurons also suppressed CRC proliferation in a PCSK1-dependent manner. Dexamethasone reduced, whereas liraglutide increased, PCSK1 expression. ConclusionsThese findings define a previously unrecognized ENS-derived neuro-oncologic pathway that is associated with reduced CRC cell proliferation and identify the GR/GLP1R-PCSK1-M-ENK axis as a potentially actionable therapeutic node. SummaryThis study identifies a neuronal PCSK1 - M-ENK pathway in the ENS that directly suppresses colorectal cancer growth through local OGFr activation, revealing a previously unrecognized neuropeptidergic mechanism of tumor control within the intestinal microenvironment.

Background & AimsBulevirtide, a viral entry inhibitor used to treat chronic hepatitis delta virus (HDV) infection, targets the hepatic bile salt transporter Na+-Taurocholate Co-transporting Polypeptide (NTCP). As Bulevirtide displays preclinical potential to mitigate cholestatic liver injury, NTCP inhibition is currently explored as treatment for primary sclerosing cholangitis (PSC), a condition frequently associated with colitis. Here, we investigated the immunomodulatory effects of Bulevirtide in lipopolysaccharide (LPS)-induced inflammation and dextran sodium sulfate (DSS)-induced colitis in mice. MethodsThe immunomodulatory properties of the bile salt taurochenodeoxycholic acid (TCDC) were investigated in LPS-challenged mouse bone marrow-derived macrophages (BMDM) and human BLaER1 macrophages. The therapeutic efficacy of Bulevirtide against LPS-induced inflammation and DSS-induced colitis was evaluated in Slco1a/1b-/- FVB and C57BL/6J mice, which recapitulate human bile salt dynamics. ResultsIn BMDMs, TCDC reduced pro-inflammatory tumor necrosis factor alpha (TNF), increased anti-inflammatory interleukin (IL)-10, and suppressed inflammasome activation, as evidenced by reduced IL-1{beta}, IL-18 and cleaved-IL-1{beta} levels. Consistently, TCDC also reduced TNF and IL1B expression in human BLaER1 macrophages. In both FVB and C57BL/6J Slco1a/1b-/- mice, Bulevirtide increased plasma bile salt levels at least 30-fold. This systemic elevation of bile salts reduced plasma TNF and increased IL-10 in LPS-treated mice. Moreover, Bulevirtide attenuated DSS-induced colitis, evidenced by reduced disease scores and reduced intestinal Tnf expression. ConclusionThese findings highlight the anti-inflammatory effects of bile salts in preclinical models of colitis and support NTCP inhibition as a future therapeutic strategy to ameliorate both cholestasis and colitis in PSC. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/719641v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@38efbeorg.highwire.dtl.DTLVardef@3e09borg.highwire.dtl.DTLVardef@8f1262org.highwire.dtl.DTLVardef@100179c_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisInhibition of the Na+-Taurocholate Co-transporting Polypeptide using Bulevirtide induces systemic bile salt elevation and mitigates acute inflammation and colitis in mice. These findings support clinical evaluation of Bulevirtide in primary sclerosing cholangitis with protective effects against cholestasis and colitis.

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 Recent mouse model data demonstrate that chronic colonization with toxigenic Clostridioides difficile promotes colonic tumorigenesis via intraluminal toxin B (TcdB), its main virulence factor. In a prior multisite hospital cohort, we found that history of positive tcdB stool testing was associated with increased CRC risk in a dose-dependent manner, though limited by small sample size. We aimed to validate this association in a larger cohort with extended follow-up and greater geographic distribution using the Veterans Health Administration (VHA) Corporate Data Warehouse (CDW). Methods We conducted a retrospective cohort study among adults receiving care through the VA from 2000-2025 who underwent C. difficile testing. Data collected from the VHA CDW and National Death Index (NDI) included demographics, comorbidities, medications, CRC risk factors, and cancer incidence and death. The first C. difficile test date defined cohort entry; individuals with prior CRC were excluded. Ever C. difficile positivity was defined by a positive PCR or EIA results. The number of positive tests (episodes) was also determined to define recurrent positivity. Follow-up time ended at the first occurrence of CRC incidence or mortality, death from other causes, or censor date. Follow-up time was split for individuals who converted from negative to positive, with follow-up time updated accordingly. Multivariable Cox proportional hazards models were used to estimate hazard ratios (HRs) for C. difficile exposure and CRC incidence and mortality after adjustment for confounders. Tests for linear trend and tests for interaction were conducted to assess effect modification by sex and IBD status, while time-lag intervals were evaluated for 1, 3, 5, and 10 years before the outcome. Results Among 806,844 veterans with C. difficile testing, those with positive tests were more likely to be older, male, to have diabetes, to use aspirin, and to have a lower BMI than those with negative tests. Race and IBD prevalence were similar between the groups. There was no overall association between ever C. difficile positivity and CRC incidence (HR = 0.99, 95% CI 0.93-1.05). However, recurrent C. difficile positivity was associated with increased risk in a dose-response manner [2-3 episodes HR = 1.30 (95% CI 1.16-1.47), and >3 episodes HR = 1.58 (95% CI 1.17-2.14) compared to negative tests; ptrend< 0.001]. Further, ever C. difficile positivity was associated with increased CRC mortality risk (HR = 1.21, 95% CI 1.13-1.30; p < 0.001). Recurrent C. difficile positivity was associated with increased mortality risk but was particularly strong for those with >3 episodes among individuals with IBD (HR=3.84, 95% CI 1.98-7.45). In sensitivity analyses, the increased risk of CRC incidence and mortality attenuated beyond 10 years. Conclusion Prior positive C. difficile testing was associated with increased CRC incidence and mortality in a dose-dependent manner, particularly among patients with IBD. These findings extend animal model evidence, epidemiologically establishing C. difficile presence as an independent risk factor for subsequent colorectal tumorigenesis and supporting investigation into recurrent CDI, especially among patients with IBD, as a potential modifiable CRC risk factor.

Cholangiocarcinoma (CCA) is a highly aggressive malignancy characterized by poor prognosis, limited therapeutic options, and a predominantly immunosuppressive tumor microenvironment. Protein arginine methyltransferase 1 (PRMT1), the major mediator of asymmetric arginine dimethylation, has been implicated in multiple oncogenic processes, although its role in CCA remains unknown. Here, we demonstrate that PRMT1 is frequently overexpressed in human CCA and is associated with aggressive molecular subtypes and immune-desert tumors. Genetic dependency analyses and pharmacological inhibition using type I PRMT inhibitors markedly impaired CCA cell proliferation, clonogenicity, and tumoroid growth. Transcriptomic profiling revealed that PRMT1 inhibition induces broad alterations in gene expression and alternative splicing, affecting pathways involved in proliferation, apoptosis, DNA damage response, metabolism, and immune signaling. Mechanistically, PRMT1 targeting promoted genomic stress, accumulation of cytosolic double-stranded DNA, and activation of the cGAS-STING-TBK1-IRF3 signaling axis, resulting in enhanced interferon signaling and increased expression of T cell-recruiting chemokines, including CXCL9 and CXCL10. PRMT1 inhibition also synergized with cisplatin, poly-ADP-ribose polymerase (PARP) inhibition, and PRMT5 blockade in vitro and in patient-derived tumoroids. Importantly, in an aggressive orthotopic murine model of intrahepatic CCA, combined treatment with the PRMT1 inhibitor GSK3368715 and anti-PD-1 antibodies significantly reduced tumor burden and increased CD4+ and CD8+ T-cell infiltration compared with monotherapies. Collectively, these findings identify PRMT1 as a critical regulator of CCA growth and immune evasion and support the therapeutic potential of PRMT1 inhibition, particularly in combination with immunotherapy.

Background & AimsPrimary sclerosing cholangitis (PSC) is a progressive cholangiopathy characterized by ductular remodeling, inflammation, and periportal fibrosis, for which effective medical therapies remain limited. The Hippo pathway effector TEAD1 has been implicated in liver regeneration and fibrogenesis; however, its role in cholestatic injury remains poorly defined. We investigated whether hepatocyte TEAD1 regulates injury-associated remodeling in a PSC-mimicking model and whether this mechanism is conserved in human PSC liver. MethodsHepatocyte-specific TEAD1 knockout mice (Alb-TEAD1-/-) and littermate controls were subjected to DDC-induced cholestatic injury. Ductular reaction, fibrosis, inflammation, and bile acid-related gene programs were assessed by histology, immunostaining, and gene expression analyses. Translational relevance was evaluated using bulk and single-cell transcriptomic datasets from human PSC liver. ResultsHepatocyte TEAD1 deletion attenuated DDC-induced fibrosis, ductular expansion, and inflammatory cell accumulation, while preserving hepatocyte proliferative responses. TEAD1-deficient livers exhibited reduced expression of profibrotic mediators, including Spp1, Ctgf, and Cyr61, with decreased extracellular matrix deposition. In contrast, canonical transcriptional adaptations to cholestatic stress, including suppression of bile acid uptake, induction of efflux pathways, and repression of bile acid synthesis genes, were preserved in the absence of TEAD1. Analysis of human PSC datasets demonstrated coordinated upregulation of TEAD1 and TEAD-associated target genes. Single-cell transcriptomic analysis further revealed hepatocyte-enriched TEAD1 expression and activation of a TEAD1 target gene program across all hepatic zones in PSC, with effect sizes exceeding those observed in non-parenchymal populations. TEAD1 activation was accompanied by co-expression of profibrotic mediators and downregulation of hepatocyte differentiation markers, consistent with a maladaptive hepatocyte state. ConclusionsHepatocyte TEAD1 drives ductular, inflammatory, and fibrogenic remodeling during cholestatic injury without disrupting bile acid metabolic adaptation. These findings identify TEAD1 as a hepatocyte-intrinsic regulator of epithelial-stromal crosstalk and establish conserved activation of this pathway in human PSC, supporting TEAD-directed signaling as a therapeutic target.

IntroductionBiliary fibrosis and inflammation are central to the pathogenesis of cholangiopathies such as primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC). Inflammatory and fibrogenic stimuli, such as transforming growth factor-{beta} (TGF{beta}) and lipopolysaccharide (LPS) signaling, drive these processes, but their underlying transcriptional mechanisms in cholangiocytes remain incompletely defined. We investigated the role of Runt-related transcription factor 1 (RUNX1) as a transcriptional co-regulator of fibroinflammatory signaling in cholangiocytes. MethodsHuman PSC-derived cholangiocytes (PSC-Cs) and mouse large biliary epithelial cells (MLEs) were subjected to RUNX1 knockdown or pharmacologic inhibition (Ro5-3335 or AI-10-104). Cytokine secretion was profiled by Luminex multiplexing; RUNX1 genomic binding and protein interactome were assessed by ChIP-qPCR, ChIP-seq, and LC-MS/MS. In vivo, Mdr2-/- mice received Ro5-3335, and cholangiocyte-selective Runx1 knockout mice (Krt19-CreERT) were challenged with a DDC diet, followed by evaluation of fibrosis and inflammation. ResultsRUNX1 expression was significantly increased in cholangiocytes from PSC and PBC patients, and Mdr2-/- mice. RUNX1 knockdown or inhibition reduced IL6, TNF, and other proinflammatory cytokines in PSC-Cs and attenuated TGF{beta}-, LPS-, and TNF-induced Il6 and Ccl2 expression in MLEs. ChIP-qPCR and ChIP-seq revealed TGF{beta}-induced RUNX1 binding to the Il6 promoter and 727 additional genomic sites enriched for fibrosis and inflammatory pathways; predicted upstream regulators included TGF{beta}, TNF, and NF{kappa}B signaling. Proteomic analysis identified TGF{beta}-induced RUNX1 interactions with SMAD2 and NF{kappa}B2. In vivo, Ro5-3335 treatment in Mdr2-/- mice reduced hepatic collagen, ECM gene expression, immune cell infiltration, and serum liver injury markers and bile acids. Similarly, cholangiocyte-specific Runx1 deletion mitigated fibrosis, inflammation, and liver injury in DDC-fed mice. ConclusionRUNX1 is a central transcriptional hub integrating TGF{beta} and inflammatory signals in cholangiocytes. Its inhibition attenuates biliary fibrosis and inflammation in cholestatic models, supporting RUNX1 as a potential therapeutic target in fibroinflammatory cholangiopathies.

Pain in pancreatic ductal adenocarcinoma (PDAC) is associated with poor survival, but whether altered pain processing carries prognostic significance is unknown. We analyzed a prospective cohort of 143 patients with PDAC who underwent pancreatic quantitative sensory testing (PQST) after diagnosis. Patients were classified as having normal pain processing (n=84), segmental hyperalgesia (n=30), or widespread hyperalgesia (n=29). Survival was measured from the date of P-QST assessment. During follow-up, 70 deaths occurred. Widespread hyperalgesia was associated with increased mortality in unadjusted Cox analysis (HR 1.96, 95% CI 1.14,3.35) and after adjustment for age, sex, tumor stage, comorbidity, opioid treatment, and body mass index (adjusted HR 2.33, 95% CI 1.30,4.15). Segmental hyperalgesia was not associated with mortality. Kaplan Meier analysis demonstrated lower survival probability in the widespread hyperalgesia group (log rank p=0.025). These findings suggest that widespread hyperalgesia, reflecting altered central pain processing, identifies a subgroup of PDAC patients at increased risk of mortality independent of conventional clinical factors.

The intestinal epithelium relies on rapid repair to maintain homeostasis after injury, and dysregulation of this process contributes to the pathogenesis of inflammatory bowel disease and colorectal cancer. Interleukin-11 (IL-11), a fibroblast-derived cytokine elevated in these diseases, has well-documented effects on stromal cells, but its direct action on intestinal epithelial cells remains poorly characterized. Here, we used mouse colon organoids as an isolated epithelial system to directly examine the effects of IL-11 on epithelial cells. IL-11 stimulation activated the canonical JAK/STAT3 pathway, as evidenced by increased STAT3 phosphorylation and Socs3 induction in a concentration-dependent manner. In a pipetting-based mechanical disruption model, IL-11 significantly increased the number of organoids recovered. Although mechanical disruption dominated the overall transcriptional landscape, RNA-seq analysis identified coordinated upregulation of STAT3 target genes and proliferation-related pathways specifically in response to IL-11. Pharmacological inhibition of STAT3 attenuated the IL-11-induced promotion of organoid recovery, indicating that STAT3 signaling mediates the epithelial response to IL-11 and maintains organoid size under basal conditions. Together, these findings demonstrate that IL-11 directly promotes intestinal epithelial repair after mechanical disruption through STAT3-dependent signaling, providing a mechanistic basis for its protective role in acute colonic injury.

Fibrostenotic complications represent a major cause of morbidity in Crohns disease (CD), yet the cellular mechanisms that drive intestinal fibrosis independent of active inflammation remain poorly understood. Here, we identify impaired fatty acid oxidation (FAO) as a defining metabolic feature of fibroblasts in fibrostenotic CD. Untargeted lipidomics of non-inflamed colonic tissue from CD patients demonstrated enrichment of triacylglycerols and long-chain acylcarnitines, suggesting altered lipid utilization. Across three independent RNA-sequencing cohorts, including treatment-naive pediatric ileal biopsies, FAO genes (CPT1A, CPT2, SLC25A20) were selectively downregulated in patients with or destined to develop fibrostenotic disease. Single-cell RNA-sequencing localized these transcriptional alterations specifically to fibroblasts within strictured ileum. Primary fibroblasts derived from fibrostenotic CD exhibited increased neutral lipid accumulation, impaired mitochondrial fatty acid trafficking, and diminished responsiveness to PPAR{gamma}-mediated suppression of TGF{beta}-induced myofibroblast activation. Together, these findings demonstrate that FAO impairment is a conserved, fibroblast-specific metabolic program associated with intestinal fibrosis in CD and suggest that metabolic modulation of stromal cells represents a potential therapeutic strategy for fibrostenotic disease.

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.

Background and Aims: Diet plays a critical role in managing Crohns disease (CD) inflammation. We assessed whether dietary replacement of animal protein (AnimalP) by soy-pea protein (SoyP) decreases the pro-inflammatory potential of gut microbiota and intestinal inflammation in CD patients. Design: In an open-label, randomized controlled feeding trial at University Hospitals Cleveland Medical Center, CD participants and healthy controls were randomized (1:1) to a soy-pea or animal protein diet for 7-days. Primary outcomes were the absolute difference (d7-d0) in; Crohns Disease Activity Index (CDAI) score and fecal myeloperoxidase (MPO). Secondary outcomes included fecal calprotectin (FC) and high-sensitivity C-reactive protein (hsCRP). Murine fecal transplantation experiments were performed to determine the inflammatory potential of diet-altered gut microbiota. Results: The study randomized 66 participants and 60 were included in the final analysis (n=31 CD, n=29 HC). After 7 days, CD-SoyP participants were more likely than CD-AnimalP to show reductions in HBI (RR=4.68, 95% CI: 1.22-17.98, P=0.009) and fecal MPO (RR=2.30, 95% CI: 1.04-4.85, P=0.032), with a similar directional trend for CDAI (RR=1.52, 95% CI: 0.89-2.58, P=0.135). No participants experienced worsening of CDAI. The rank-based composite CDAI-MPO score was lower in the CD-SoyP vs CD-AnimalP group (median [IQR]: 5 [4-6] vs 8 [7-9]; P=0.012). Stratified analyses showed significant reductions in fecal MPO among CD participants with lower baseline disease activity (CDAI <150; P<0.0001), but not in those with higher activity (P=0.799) Conclusion: Short-term addition of plant-based soy-pea protein within a controlled diet exerted a beneficial, anti-inflammatory effect in CD, with evidence of greater effects among participants with lower baseline disease activity. ClinicalTrials.gov, Number NCT04065048.

Patients with gastric intestinal metaplasia (GIM), a precancerous lesion, are at high risk for progressing to gastric cancer. Identifying these patients is critical to enable gastric cancer interception. Current approaches rely primarily on histologic evaluation of GIM severity and extent, which may be improved by incorporating molecular features that distinguish high-risk lesions. Our prior single-cell and spatial transcriptomics study identified differentially expressed genes associated with the highest-risk category of GIM. They included ANPEP expressed in enterocytes and CPS1 and OLFM4 expressed in intestinal stem-like or progenitor cells. We evaluated the protein expression and localization of these three markers to understand the cellular features associated with GIM risk and their spatial distribution within metaplastic tissues. Using multiplex immunofluorescence, whole slide image analysis and confocal microscopy, we examined protein expression from 100 tissue biopsies annotated for metaplasia severity using the Operative Link on Gastric Intestinal Metaplasia Assessment (OLGIM) system. Tissue samples included control gastric tissue, GIM, dysplasia and adenocarcinoma. Quantitative whole slide image analysis demonstrated that CPS1 expression had a modest association with disease severity. Although ANPEP was strongly associated with GIM severity, it was also frequently expressed in stromal regions outside epithelial glands. In contrast, OLFM4 expression was largely restricted to epithelial glands and showed a strong association with increased OLGIM severity. These OLFM4-positive epithelial cells were present in discrete glandular foci that expanded with increasing severity of metaplasia. Within individual metaplastic glands, OLFM4 expression was highest at the gland base with decreased expression toward the gland surface. Overall, these findings identified OLFM4 as a protein marker associated with high-risk GIM. The spatial organization of OLFM4-expressing cells at the base of metaplastic glands and their focal expansion within tissues suggest the presence of a stem cell-like epithelial compartment that may contribute to the progression of GIM towards gastric cancer.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease for which the vast majority of the approved therapies target the immune system. We aimed to identify consistently dysregulated genes and pathways across independent UC transcriptomic cohorts, distinguishing constitutive from inflammation-dependent changes. We performed a random-effects meta-analysis of 14 microarray datasets from the Gene Expression Omnibus (972 mucosal biopsies, 9 platforms), comparing inflamed UC, uninflamed UC, and inflamed Crohns disease (CD) to controls, as well as UC to CD directly. The inflamed UC analysis revealed an upregulated inflammatory transcriptomic profile in UC, providing a rationale for the use of all approved anti-inflammatory therapies. In parallel, the predominant downregulated signal was metabolic, driven by PPARGC1A, PPARGC1B, and ES-RRA, indicating a coordinated, inflammation-dependent collapse. The uninflamed UC analysis revealed a separate set of potentially constitutive vulnerabilities -- fully suppressed glucuronidation, upregulated translation, complement priming, and altered iron export -- that are not downstream of the energy collapse. The metabolic deficit was more severe in UC than in CD, while immune pathways were shared. These findings suggest a two-layer model of UC pathology: a constitutive impairment of metabolic pathways that is further exacerbated by inflammation. The inflammation analysis reveals new targets for immune suppression while the constitutive analysis identifies targets for proactive intervention between flares directed at the metabolic deficiency.

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.

Human genetic studies have identified defects in multiple mechanisms that predispose the risk of developing inflammatory bowel diseases (IBD), which include alterations in adaptive and innate immune responses, epithelial integrity and regulation of the intestinal mucus layer. Despite the importance of intestinal barrier integrity in the pathogenesis of IBD, essentially all current therapies modulate the immune responses. In this study, we determined the high resolution cryo-EM structure of human NXPE1, a IBD associated protein. Based on the structural homology, we identified NXPE1 as an O-acetyltransferase. Since NXPE1 is a pseudo gene in mouse, we generated knockout mouse model that lacked two of the mouse NXPE1 homologs, Nxpe2 and Nxpe4. The O-acetylation of sialic acid on red blood cells was abolished in the double knockout mice, confirming the sialic acid O-acetyltransferase function of NXPE1 family members. These findings underscore the potential of NXPE1 as a novel therapeutic target of the intestinal barrier functions for the treatment of IBD.

We previously reported that the murine lncRNA Epr is essential for maintaining colon mucosal integrity and permeability. Mice lacking Epr in the colon are more susceptible to colitis and tumor development. Additionally, we demonstrated that human EPR expression is reduced in ulcerative colitis and in a small cohort of colon adenocarcinoma patients. Here, we present evidence that human and mouse EPR share several key physiological features: preferential binding to the KH1 domain of their interacting protein, KSRP; specific expression in canonical and immature goblet cells of the large intestine; and a functional role in intestinal goblet cell development. The correlation between EPR levels and survival in large cohorts of metastatic colon adenocarcinoma patients, together with the capacity of human EPR to inhibit cell proliferation and induce apoptosis in two distinct human colon adenocarcinoma cell lines, suggests that EPR may serve as both a valuable prognostic marker for goblet cell-derived adenocarcinomas and a potential therapeutic target.