Sigmoid Colon Biopsies Research Articles

Transcriptomic and Metabolomic Correlates of Increased Colonic Permeability in Postinfection Irritable Bowel Syndrome

Background and aimPost-infection irritable bowel syndrome (PI-IBS) is well-known epidemiologically; however, its physiological and molecular characteristics are not well studied. We aimed to determine the physiological phenotypes, colonic transcriptome, fecal microbiome, and metabolome in PI-IBS. MethodsFifty-one Rome III Campylobacter PI-IBS patients and 39 healthy volunteers (HV) were enrolled. Participants completed questionnaires, in vivo intestinal permeability, gastrointestinal transit, and rectal sensation. Fecal samples were collected for shotgun metagenomics, untargeted metabolomics, and sigmoid colonic biopsies for bulk RNAseq. Differential gene expression, differences in microbiota composition and metabolite abundance were determined. Gene and metabolite clusters were identified via weighted gene correlation network analysis and correlations with clinical and physiological parameters determined. ResultsPI-IBS (59% F, 46±2 yrs.) and HV (64% F, 42±2 yrs.) demographics were comparable. Mean IBS-symptom severity score was 227; 94% were non-constipation. 2-24h lactulose excretion was increased in PI-IBS, suggesting increased colonic permeability (4.4±0.5 mg vs. 2.6±0.3 mg, p=0.01). Colonic transit and sensory thresholds were similar between the two groups. Overall, expression of 2036 mucosal genes and 223 fecal metabolites were different, with changes more prominent in females. Fecal N-acetylputrescine was increased in PI-IBS and associated with colonic permeability, worse diarrhea, and negatively correlated with abundance of Collinsella aerofaciens. Histamine and N-acetyl histamine positively associated with 2-24 hr lactulose excretion. Eight weighted gene coexpression modules significantly correlated with phenotypes (sex, stool frequency, colonic permeability, transit). ConclusionsCampylobacter PI-IBS patients demonstrate higher colonic permeability which associated with changes in polyamine and histamine metabolites. Female patients demonstrated greater molecular changes.

Infliximab Tissue Concentrations in Patients With Stable Ulcerative Colitis Are Correlated With More Durable Infliximab-associated Disease Remission.

We aimed to determine the correlation between tissue and plasma infliximab concentrations in an outpatient ulcerative colitis (UC) cohort based on histologic disease activity in addition to their relationship with long-term clinical outcomes. We assessed intraparticipant variability in infliximab concentrations between adjacent intestinal samples and the correlation between disease activity and tumor necrosis factor-α (TNF-α). A prospective cohort study was conducted in participants with UC receiving infliximab. Blood and 2 sigmoid colon biopsies were obtained at the index colonoscopy for infliximab and TNF-α quantification. Histological disease activity was assessed. Participants were followed for 2 years for the occurrence of hospitalization, surgery, disease relapse, and infliximab discontinuation. A positive correlation was observed between mean plasma and uninflamed tissue infliximab concentrations only (Rs = 0.75, P = .0071). Lower mean tissue infliximab concentrations correlated with a shorter time to disease relapse vs those with higher mean tissue concentrations (Rs = 0.77, P = .032). This was not seen when using plasma infliximab concentrations. Additionally, no significant intraparticipant variability of infliximab concentrations was observed for all participants independent of disease activity. Neither plasma nor tissue TNF-α correlated with disease activity. These findings support data generated in patients with Crohn's disease: plasma infliximab concentrations are reflective of infliximab exposure in tissue in the UC patient in remission, but not for those with active disease. Increasing tissue concentrations in the noninflamed tissues may improve durability of infliximab. Neither plasma nor tissue TNF-α appear to correlate with UC disease activity. Larger follow-up studies would be of benefit.

Luminal Polyamines Disrupt Intestinal Barrier and Undergo Complex Regulation by Host and Commensal Microbiota

Background: Polyamines produced by host cells and intestinal microbiota and are critical to overall health. Studies have shown putrescine, a polyamine, can strengthen, or in some instances disrupt, gastrointestinal (GI) barrier integrity, yet the regulation of polyamine concentrations in the GI tract is unknown. Host colonocytes and microbiota both metabolize polyamines, and in GI diseases like irritable bowel syndrome (IBS) where intestinal microbiota is significantly altered, polyamine metabolism may be affected. Aim: Determine regulation of luminal polyamine concentration and its effects on intestinal barrier integrity. Hypothesis: Luminal polyamine concentration is suppressed by commensal intestinal microbiota and increasing concentration of putrescine results in intestinal barrier disruption. Methods: Microbiota effects on luminal polyamine concentrations were assessed by humanizing germ-free (GF) mice with a single oral gavage of commensal human microbiota. Polyamine effect on barrier integrity was assessed in vitro using Caco-2 cells and ex vivo on Ussing using mouse colonic tissue with 300Da fluorescein and 4kDa FITC-Dextran tracers. Changes in transepithelial resistance (TER) and rate of tracer flux was recorded. Untargeted metabolomics with shotgun metagenomics was completed on human fecal samples and sigmoid colonic biopsies were collected for bulk RNAseq. Bray-Curtis distance was determined at the species level using the PERMANOVA method and used to test relationships between beta-diversity and fecal polyamine concentration. Results: Humanization with human commensal microbiota (1.2% C. aerofaciens) lead to a 20-fold increase in cecal putrescine and a 20-fold decrease in fecal putrescine compared to GF controls. In vitro addition of putrescine (100mM) caused increased fluorescein flux (3.50±0.24 vs 1.44±0.04 mg/mL) and a rapid loss of transepithelial resistance compared to controls (% of Baseline TER, 36.6±8.2 vs 102.8±5.4, 3h postexposure). Ex vivo addition of putrescine caused a loss of TER (57% vs 89% of baseline by 3h) and an increased rate of 4kDa FITC-Dextran flux compared to untreated controls (1236±131 vs 562±70 ng/hr/cm2). Transcriptomic profiles show decreased expression of ornithine decarboxylase antienzyme (OAZ1, FDR=0.018) in IBS which inhibits the rate limiting enzyme for putrescine production. Increased lactulose excretion (2-24h), suggestive of increased distal colonic permeability, correlated with N-acetylputrescine concentration (p=0.07). N-acetylputrescine also correlated positively with increased stool frequency (p=0.009) and looser consistency (p=0.013). C. aerofaciens, a polyamine consumer, negatively correlated with n-acetylputrescine (FDR=0.02) suggesting a loss of specific taxa may lead to insuffcient polyamine metabolism. Conclusion: Polyamines, namely putrescine, disrupts intestinal barrier integrity, correlate with in vivo permeability, and are decreased in the colon by commensal microbiota. These observations identify putrescine as a negative effector of barrier function and suggest a novel role for the commensal microbiome in regulating barrier integrity through polyamine metabolism. Further work investigating host and microbiota mechanisms of polyamine metabolism, signaling and the specific taxa involved is needed to better understand the effects polyamines have on GI physiology. NIH DK 127998 (MG), support from Mayo Clinic Center for Individualized Medicine (CIM), and Mayo Clinic Center for Cell Signalling in Gastroenterology (C-Sig) P30DK084567. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

P626 JNJ-77242113, an oral peptide selectively targeting the IL-23 receptor, demonstrates pharmacodynamic activity in rat and human colon tissue explants following oral dosing

Abstract Background The interleukin (IL)-23 pathway is a pathogenic driver in psoriasis, psoriatic arthritis, and inflammatory bowel disease (IBD). There are currently no approved oral therapies selectively targeting the IL-23 pathway. JNJ-77242113 (JNJ-2113) is a targeted oral peptide that binds the IL-23 receptor with high affinity, and potently and selectively inhibits IL-23 proximal signaling and downstream cytokine production. Oral JNJ-2113 showed dose-dependent inhibition of IL-23–induced IL-17A production in rat blood and attenuated signs of disease in a rat colitis model. A Phase 1 study showed inhibition of IL-23–induced IFN-γ production in blood from participants dosed with JNJ-2113 (Fourie et al. ISID. 2023). Methods We evaluated IL-23–induced IL-22 expression in colon tissue after exposure to JNJ-2113 or JNJ-2100 (a tool molecule with similar affinity and potency). IL-22 mRNA was measured after IL-23 stimulation in rat colon biopsies treated with JNJ-2100 in vitro or in colon tissue from rats 6 h after oral JNJ-2100 dosing. Human colon explants from donors were treated in vitro with JNJ-2100 or JNJ-2113, stimulated with IL-23, and IL-22 mRNA was measured. Colon and rectum biopsies from healthy participants dosed for 10 days with oral JNJ-2113 or placebo in a Phase 1 study were obtained, stimulated with IL-23 ex vivo, and IL-22 mRNA was evaluated. Results Overall, inhibition of IL-23–induced IL-22 expression was observed in colon tissue exposed to JNJ-2100 or JNJ-2113. Dose-dependent inhibition of IL-22 expression was observed in rat colon biopsies exposed to JNJ-2100 in vitro. Colon explants from rats dosed with 0.3, 3, and 30 mg/kg oral JNJ-2100 showed dose-dependent inhibition of IL-22 expression (52%, 88% and 96%, respectively) compared with explants from rats treated with vehicle. Human colon explants exposed to JNJ-2100 or JNJ-2113 in vitro also showed dose-dependent inhibition of IL-23–induced IL-22 expression, with >90% inhibition at the highest doses. Importantly, in colon biopsies from Phase 1 participants dosed with oral JNJ-2113 versus placebo, significant inhibition of IL-23–induced IL-22 expression was seen (P<0.05), with an average of 65% and 66% inhibition observed in sigmoid colon and rectum biopsies, respectively. Conclusion These results demonstrate pharmacodynamic activity of JNJ-2100 in vitro and ex vivo in rat colon tissue, of JNJ-2100 and JNJ-2113 in vitro in human colon explants, and of JNJ-2113 in colon biopsies from healthy human volunteers. These data support development of JNJ-2113 for the treatment of IBD. Of note, a Phase 2b dose-ranging study has demonstrated clinical activity of JNJ-2113 in moderate-to-severe plaque psoriasis (NCT05223868).

The Mechanism and Potential Therapeutic Effects of Cyclosporin, Cyclophilin, Probiotics and Syndecan-1 in an Animal Model of Inflammatory Bowel Disease.

Background: Inflammatory bowel diseases (IBDs) have several treatment modalities including immunoregulators, like cyclosporine A, an immunosuppressant that interacts with cytoplasmic cyclophilin A, and probiotics. Aims: This study explored and compared the possible role of syndecan-1 in the IBD pathogenic process as well as the effectiveness of cyclophilin A, cyclosporine A, and their combination in the management of IBDs in the presence of probiotics. Methodology: IBD was induced in a total of 112 mice equally divided between syndecan-1 knock-out (KO) and Balb/c wild-type mice, using 2% dextran sulfate sodium (DSS) followed by intraperitoneal treatment with cyclosporine A, cyclophilin A, or a combination of both. In addition, a daily dose of probiotics was given in their drinking water. The animals were monitored for clinical signs and symptoms and checked for gross pathologies in the abdomen after 3 weeks. Descending and sigmoid colon biopsies were collected and fixed for routine microscopy or frozen for protein extraction and molecular testing for IL-6, CD3, CD147, and beta 1 integrins as well as pAkt expression. Results: The data showed that the induction of IBD in the syndecan-1 KO mice was more severe at the clinical, histological, and molecular levels than in the wild type. The combined CypA-CyA treatment showed no added inhibitory effect compared to single-drug treatment in both strains. Probiotics added to the combination was more effective in the wild type and, when used alone, its inhibition of IL-6 was the highest. As for the CD147 marker, there were more suppressions across the various groups in the KO mice except for the probiotics-alone group. Concerning CD3, it was significantly increased by the CypA-CyA complex, which led to more inflammation in the KO mice. Probiotics had little effect with the combination. In relation to beta 1 integrins, the CypA-CyA combination made no significant difference from CyA alone, and adding probiotics to the combination resulted in higher beta 1 integrin expression in the KO mice. As for pAkt, it was very well expressed and upregulated in both strains treated with DSS, but the effect was much larger in the KO mice. In brief, the CypA-CyA complex showed a decrease in the expression of pAkt, but there was no added effect of both drugs. Probiotics along with the complex had a similar reduction effects in both strains, with a greater effect in the wild-type mice, while probiotics alone led to a similar reduction in pAkt expressions in both strains. Conclusions: The differential effects of CyA, CypA, probiotics, and their combinations on the various inflammatory markers, as well as the histological alterations and clinical signs and symptoms, speak in favor of a clear role of syndecan-1 in reducing inflammation. However, probiotics need to be considered after more explorations into the mechanisms involved in the presence of CypA and CyA especially since pAkt is less active in their presence.

Highly Multiplexed Immunohistochemistry Can Predict Steroid Refractory Gastrointestinal (GI) Acute Gvhd at the Time of Endoscopy

Background: For patients with high-risk hematologic disease, hematopoietic cell transplant (HCT) often represents the only option for cure. However, HCT is fraught with complications, leading to morbidity and mortality. Acute GVHD (aGVHD), specifically steroid refractory (SR) GI aGVHD, is a major cause of early mortality. Two issues with SR GI aGVHD predominate: (1) We do not understand the mechanisms differentiating SR vs steroid sensitive (SS) GI aGVHD; (2) We do not have an accurate test to predict SR vs SS GI aGVHD at the time of endoscopic evaluation. Here, we used state-of-the-art multiplexed immunohistochemistry (IHC) to interrogate immune mediators of GI aGVHD, and to develop a robust assay capable of predicting SR vs SS disease from standard GI biopsies. Methods: We analyzed all HCT patients at Boston Children's Hospital (BCH) from 1/2014-12/2021 (n=551) who received a first lower GI endoscopy for suspected GI aGVHD (n=109). We tracked their SS vs SR status and their Overall Survival (OS) through 2 yr post-HCT. Classic histology was performed and in addition, each sigmoid colon biopsy (formalin-fixed) was analyzed with a series of novel multiplexed IHC panels (interrogating Pan-CK, DAPI, CD3, CD8, CD20, FoxP3, GITR, CD68, Granzymes B and K, CD103, Ki67, ECP, NCR1, BCMA, CD11c, and MPO, with quantitative image analysis performed using HALO Indica Labs Hyperplex module). Density/proportions of multiple cellular infiltrates and their ratios were assessed. Receiver Operator Characteristic (ROC) and K-nearest neighbors (KNN) classification were used to distinguish SS from SR aGVHD. Results: Of 109 biopsies, 60 had histologic evidence of lower GI aGVHD inflammation, 12 had other GI inflammation (eg infections, drug effects) and were not further analyzed, and 15 did not have inflammation and therefore were not diagnosed with histologic aGVHD (termed ‘No Inflammation’ (NI). In an additional 22 biopsies, histology was characterized as “Rare Apoptosis” (RA), a diagnosis for which there is no national consensus about severity or the need for steroid treatment. At BCH, the vast majority of patients with RA on endoscopy were treated as not having aGVHD (with only 3/22 receiving steroids, and all 3 being SS). We tracked OS and IHC characteristics for SS aGVHD, SR aGVHD, NI and RA. Fig 1 shows 2 yr OS, which confirms the inferior OS of SR vs SS aGVHD (SR: 58% vs SS: 93% p <0.0001). Importantly, Fig 1 demonstrates that even without steroids, the OS of RA was equivalent to no GI aGVHD (NI: 89% vs RA: 86% p = 0.74). Multiplexed IHC (performed on 68 biopsies from sequential cases collected from 1/2018-12/2021) was associated with several novel findings ( Fig 2): (1) By IHC, RA had fewer immune infiltrates vs aGVHD and NI: CD3 density: aGVHD (both SS and SR combined): 782 ± 133, NI: 386 ± 37, RA: 165 ± 26, p = 9.5x10 -5; CD8 density: aGVHD: 381 ± 83, NI: 123 ± 24, RA: 42 ± 26, p<0.001; CD4 conventional T cells (CD4 Tconv): aGVHD: 359 ± 47, NI: 237 ± 23, RA: 114 ± 19, p<0.001. Ratios of immune infiltrates also distinguished the 3 subtypes: the ratio of CD8:CD4 Tconv: aGVHD: 0.95 ± 0.14; NI: 0.59 ± 0.10 RA: 0.44 ± 0.29 p = 0.013; CD8:CD68: aGVHD: 0.35 ± 0.03, NI: 0.26 ± 0.04 RA: 0.11 ± 0.02, p<0.001. CD8:Epithelial cells (epi): aGVHD: 0.153 ± 0.04; NI: 0.045 ± 0.01; RA: 0.014 ± 0.003, p<0.001. These results underscore the immunologically benign nature of RA, predicting its superior OS. Importantly, multiplexed IHC could distinguish SR vs SS aGVHD at the time of endoscopy, a finding with major clinical implications. For example, amongst other distinguishing IHC characteristics, the ratio of CD8:CD4Tconv: SR: 1.3 ± 0.22 vs SS: 0.68 ± 0.15 p< 0.01; CD8:epi: SR: 0.220 ± 0.008 vs SS: 0.106 ± 0.042 p = 0.04). A KNN-based classifier identified thresholds with high accuracy (81%), specificity (69%), sensitivity (93%); AUC = 0.852 for SS vs SR aGVHD ( Fig 2). Conclusions: We used state-of-the-art multiplexed IHC to interrogate the immune cells associated with GI aGVHD, with 2 major findings: We find RA associated with superior OS without steroid treatment, and with minimal immune infiltration, confirming its benign nature. We also created a novel multiplexed IHC assay capable of accurately classifying SR vs SS aGVHD from standard-of-care biopsy specimens. If confirmed in a multicenter setting, this would be a major advance, allowing physicians to tailor appropriate therapies to patients at highest risk of developing SR GI aGVHD at the time of initial endoscopic evaluation.

The ClC-2 Chloride Channel Activator, Lubiprostone, Improves Intestinal Barrier Function in Biopsies from Crohn's Disease but Not Ulcerative Colitis Patients.

The prostone analog, lubiprostone, is approved to manage constipation-predominant irritable bowel syndrome. Lubiprostone also protects intestinal mucosal barrier function in animal models of colitis. The aim of this study was to determine if lubiprostone improves barrier properties in isolated colonic biopsies from Crohn's disease (CD) and ulcerative colitis (UC) patients. Sigmoid colon biopsies from healthy subjects, CD and UC patients in remission, and CD patients with active disease were mounted in Ussing chambers. Tissues were treated with lubiprostone or vehicle to determine the effects on transepithelial electrical resistance (TER), FITC-dextran 4kD (FD4) permeability, and electrogenic ion transport responses to forskolin and carbachol. Localization of the tight junction protein, occludin, was determined by immunofluorescence. Lubiprostone significantly increased ion transport across control, CD and UC remission biopsies but not active CD. Lubiprostone selectively improved TER in both CD remission and active disease biopsies but not in control or UC biopsies. The improved TER was associated with increased membrane localization of occludin. Lubiprostone selectively improved barrier properties of biopsies from CD patients vs. UC and independent of an ion transport response. These data indicate that lubiprostone has potential efficacy in improving mucosal integrity in Crohn's disease.

S149 Patient-Derived Enteroids and Colonoids for Measuring Enteroendocrine Function in Response to Luminal Contents: A Proof-of-Concept Study in Parkinson’s Disease

Introduction: In vitro studies of the gut-brain axis will require precise models of enteroendocrine function that replicate both patient-specific phenotype and intestinal milieu. Here we establish one such model for measuring the effects of stool and short-chain fatty acids (SCFA) on L-cell activity in Parkinson’s disease (PD). We have adapted a patient-derived organoid system which replicates intestinal epithelial function, immune responsiveness, and barrier integrity to respond directly to luminal contents including stool. Given the emerging importance of the enteroendocrine system in mediating microbiota-derived gut-brain signals in PD and other neurologic diseases, our system provides a needed platform for querying L-cells from a single stem-cell niche. Methods: Ileal and sigmoid colon biopsies were taken from PD patients and matched healthy volunteers between the ages of 40 and 80 years. 3D spherical organoids were generated from crypts isolated by EDTA, embedded in an extracellular matrix protein scaffold, and treated with growth factors to expand Lgr5+ stem cells through serial passages. Organoid polarity was inverted such that the apical surface faced outward. Enteroids and colonoids were then cocultured with stool supernatant or SCFA mixtures that resembled PD or control intestinal milieu. Glucagon-like peptide 1 (GLP-1) secreted by L-cells into the spheres was measured by ELISA after lysis and normalized to DNA content. Results: 3D epithelial organoids were successfully generated from ileal and colonic crypts from 7 PD and 7 control patients. L-cell presence was verified by immunohistochemistry for GLP-1 and chromogranin A. Spherical organoids were successfully inverted before coculture. GLP-1 secretion by control organoids was diminished in the presence of PD stool or SCFA relative to control milieu, and secretion by PD organoids was further diminished in either coculture condition. Conclusion: This proof-of-concept study establishes patient-derived organoids as a platform technology for measuring enteroendocrine responses to luminal contents, including stool and SCFA. Here, we use this technique to demonstrate that PD and healthy L-cells respond differently to SCFA in the stool. However, our novel system may be adapted to study a vast array of patient-specific enteroendocrine responses to stool microbiota, metabolites, and drugs.

S2072 Microscopic Colitis Induced by KRAS Targeted Therapy, Sotorasib

Introduction: Microscopic colitis is a chronic inflammatory condition in the colon where the patients usually present with chronic non-bloody, watery diarrhea. Colonoscopy usually shows normal mucosa and the diagnosis is established by colon biopsies that show the characteristic submucosal histologic changes. Microscopic colitis has been associated with smoking and different medications such as NSAIDs, PPIs, and SSRIs. We present a case of microscopic colitis that was found to be associated with the KRAS inhibitor, Sotorasib. Case Description/Methods: A 52-year-old non-smoker male with a history of stage IV poorly differentiated lung adenocarcinoma status post radiation and chemotherapy 6 months ago, clostridium difficile diarrhea 6 months ago, and pulmonary embolism on Rivaroxaban. The patient was then started on KRAS targeted therapy with Sotorasrib 960 mg daily given the progression of his carcinoma. One month later, he developed watery diarrhea. PCR stool, including clostridium difficile, was unremarkable. Sotorasib dose was decreased to 480 mg daily and then to 240 mg daily, however; he continued to have severe diarrhea and then was admitted to the hospital. He stated that he had been having 6-8 episodes of loose watery non-bloody stools associated with poor appetite and decreased oral intake. He denied any nausea or vomiting. The patient was started on IV fluids and Imodium while Sotorasib was held. CT abdomen with oral and IV contrast showed stable metastatic right iliopsoas mass and mild colonic wall thickening. Colonoscopy was done and showed grossly normal colonic and rectal mucosa. The cecum, ascending, and sigmoid colon biopsies showed inflammatory granulation tissues consistent with microscopic colitis. Diarrhea significantly improved after discontinuation of Sotorasib. The patient was discharged once his diarrhea resolved which did not recur afterward. Discussion: Sotorasib is an antineoplastic, KRAS inhibitor, which is typically used in advanced non-small-cell lung cancer. Diarrhea has been established as a common side effect (47%) of this medication that can limit its use in the indicated cases. This is the first case report in the literature, up to our knowledge, that shows an association between microscopic colitis and Sotorasib. Understanding the underlying pathophysiology of diarrhea as a limiting side effect of Sotorasib may help the patients to tolerate such medication.

Exercise effects on DNA methylation in EVL, CDKN2A (p14, ARF), and ESR1 in colon tissue from healthy men and women

ABSTRACT Physical activity reduces risk of colon cancer by 20–30%. Aberrant methylation patterns are common epigenetic alterations in colorectal adenomas, and cancers and play a role in cancer initiation and progression. Alterations identified in normal colon tissue represent apotential ‘field cancerization’ process, where normal colon is primed for carcinogenesis. Here, we investigate methylation patterns in three genes –Ena/VASP-like (EVL), (CDKN2A (p14, ARF)), and Oestrogen Receptor-1 (ESR1)– in normal colon tissue collected at baseline and 12 months from 202 sedentary men and women, 40–75 years, enrolled in a randomized controlled trial testing an exercise intervention vs. control (http://clinicaltrials.gov/show/NCT00668161). Participants were randomized to moderate-to-vigorous intensity exercise, 60 minutes/day, 6 days/week for 12 months, or usual lifestyle. Sigmoid colon biopsies were obtained at baseline and 12-months, DNA extracted, and bisulphite converted. Droplet digital methylation-specific PCR was performed for EVL, p14ARF, and ESR1. Generalized estimating equations modification of linear regression was used to model relationships between intervention effects and gene methylation levels, adjusting for possible confounders. There were no statistically significant differences between methylation patterns at 12-months between exercisers and controls. ESR1 methylation patterns differed by sex: women −10.58% (exercisers) +11.10% (controls); men +5.54% (exercisers), −8.16% (controls) (P=0.05), adjusting for BMI and age. There were no statistically significant changes in methylation patterns in any gene stratified by change in VO2max or minutes/week of exercise. While no statistically significant differences were found in gene methylation patterns comparing exercises vs. controls, 12-month exercise effects on ESR1 methylation differed by sex, warranting further study.

S2452 Two Cases of Ulcerative Colitis Flare Following SARS-CoV-2 mRNA Vaccination

Introduction: The advent of mRNA vaccines has been a major component of the vaccination effort against COVID-19. Immunization with mRNA vaccines has been associated with a systemic inflammatory response.1 We present two cases of patients with ulcerative colitis (UC) in long-term remission who experienced flare following vaccination. Case description/methods: Patient 1: A 50-year-old female with history of UC in remission of 4 years on mesalamine who received both doses of the mRNA-1273 mRNA vaccine, 28 days apart, presented with diarrhea and bloody stools that developed 1 week after dose 1. Patient had worsening diarrhea 1 week following vaccination that progressed to hematochezia. Abdominopelvic CT demonstrated contiguous colitis. She was treated with mesalamine and methylprednisolone. Her symptoms improved in 2 days, and she was discharged on a steroid taper. Patient 2: A 52-year-old female with history of UC in remission of 5 years on mesalamine who received both doses of the BNT162b2 mRNA vaccine, 21 days apart, presented with abdominal pain, diarrhea, and bloody stool that developed 1 day after dose 2. Abdominopelvic CT demonstrated diffuse colitis, and she underwent colonoscopy, which was aborted due to extensive colitis. Sigmoid colon biopsy confirmed crypt abscess formation architectural distortion and ulceration. She was treated with mesalamine and methylprednisolone. Her acute disease was complicated by bowel perforation, and she underwent total colectomy. Discussion: The presence of an environmental trigger is frequently linked to onset of disease flare in patients with UC.2 These two patients were in symptomatic remission on 5-ASA maintenance agents for several years prior to vaccination. The lack of clear alternate environmental changes in these patients suggests provocation of flare by the inflammatory response following vaccination. Existing literature evaluating SARS-CoV-2 mRNA vaccine adverse-events in patients with UC suggests that gastrointestinal side-effects are uncommon.3 In addition, biologic agent use may decrease rate of vaccine-related adverse events.3 Recommendations from an international expert consensus favors vaccination of patients with UC.4 In addition, recent literature demonstrates strong immunologic response following vaccination of patients with UC despite concurrent use of biologic agents.5 Consideration should be given to pre-treatment of patients with UC in remission to decrease risk of flare.Figure 1.: References.

S2445 When Recurring Infections Mask an Atypical Presentation of Inflammatory Bowel Disease

Introduction: Pyoderma gangrenosum (PG) is an uncommon and severe extraintestinal manifestation (EIM) of inflammatory bowel disease (IBD) with head or scalp involvement being exceeding rare. About one-third of presentations can be precipitated by skin trauma or infection, otherwise known as pathergy. These ulcers develop acutely, may not correlate with IBD activity, and can predate IBD diagnosis. Here we present an atypical presentation of PG in the setting of newly-diagnosed IBD. Case description/methods: A 35-year-old male with prior diagnosis of recurrent, diffuse soft tissue infections presented for management of presumed sepsis due to soft tissue infection. Two weeks prior to presentation, patient developed painful and purulent left lower extremity ulcer followed by worsening of facial and scalp ulcerations (Fig A-C). Patient reported prior diagnosis of facial folliculitis 1 year ago triggered by a stressor event that resolved but then had recurrence 3 months prior to this encoutner. Skin biopsy was unremarkable and was managed with IV antibiotics due to suspected infection. CT abdomen and pelvis during that admission showed circumferential sigmoid and rectal wall thickening concerning for colitis, but patient did not pursue further outpatient work up. Since being discharged from prior hospitalization, the patient experienced ongoing melenic stools. Prior to hospital transfer, patient was febrile to 39.4˚C, WBC count of 33k/ul and hemoglobin of 6.1g/dl for which endoscopy was performed showing diffuse erosive gastritis. Wound cultures grew staphylococcal aureus, and he was started on IV antibiotics. Initial concerns for IBD were based on imaging, ulcer concerning for PG, and three-year history of diarrhea elicited after admission. Laboratory work up is summarized in Table 1. Sigmoidoscopy showed cobblestoning with non-bleeding mucosal ulcerations and sigmoid colon biopsies demonstrated severely active colitis (Fig D,E). Skin biopsy was concerning for PG with superimposed infection. Patient was started on steroid therapy with improvement of diarrhea and inflammatory markers. Patient was discharged on oral steroids and planned follow up for initiation of biologic therapy. Discussion: This patient received a new diagnosis of IBD almost solely based on his presenting dermatological findings. This individual initially presented with recurrent skin wounds, however, eliciting further history of recurrent diarrhea, shifted the work up beyond infectious etiologies to earlier diagnosis and treatment of PG and IBD.Table 1.: Laboratory Data Depicting Patient Hospital Course.Figure 1.: Figure A and B: An extensive, necrolytic ulceration with an irregular, violaceous and undermined borders of the left lower extremity Figure C: Diffuse ulcerated lesions of the head and scalp Figure D and E: Endoscopy image of rectosigmoid mucosa with ulcerations, congestion, erythema, granularity and loss of vascularity.

Utilizing Deep Learning to Analyze Whole Slide Images of Colonic Biopsies for Associations Between Eosinophil Density and Clinicopathologic Features in Active Ulcerative Colitis.

Eosinophils have been implicated in the pathogenesis of ulcerative colitis and have been associated with disease course and therapeutic response. However, associations between eosinophil density, histologic activity, and clinical features have not been rigorously studied. A deep learning algorithm was trained to identify eosinophils in colonic biopsies and validated against pathologists' interpretations. The algorithm was applied to sigmoid colon biopsies from a cross-sectional cohort of 88 ulcerative colitis patients with histologically active disease as measured by the Geboes score and Robarts histopathology index (RHI). Associations between eosinophil density, histologic activity, and clinical features were determined. The eosinophil deep learning algorithm demonstrated almost perfect agreement with manual eosinophil counts determined by 4 pathologists (interclass correlation coefficients: 0.805-0.917). Eosinophil density varied widely across patients (median 113.5 cells per mm2, interquartile range 108.9). There was no association between eosinophil density and RHI (P = 0.5). Significant differences in eosinophil density were seen between patients with Montreal E3 vs E2 disease (146.2 cells per mm2 vs 88.2 cells per mm2, P = 0.005). Patients on corticosteroids had significantly lower eosinophil density (62.9 cells per mm2 vs 124.1 cells per mm2, P = 0.006). No association between eosinophil density and biologic use was observed (P = 0.5). We developed a deep learning algorithm to quantify eosinophils in colonic biopsies. Eosinophil density did not correlate with histologic activity but did correlate with disease extent and corticosteroid use. Future studies applying this algorithm in larger cohorts with longitudinal follow-up are needed to further elucidate the role of eosinophils in ulcerative colitis.

Overt IL-32 isoform expression at intestinal level during HIV-1 infection is negatively regulated by IL-17A.

Untreated HIV infection was previously associated with IL-32 overexpression in gut/intestinal epithelial cells (IEC). Here, we explored IL-32 isoform expression in the colon of people with HIV (PWH) receiving antiretroviral therapy (ART) and IL-32 triggers/modulators in IEC. Sigmoid colon biopsies (SCB) and blood were collected from ART-treated PWH (HIV + ART; n = 17; mean age: 56 years; CD4+ cell counts: 679 cells/μl; time on ART: 72 months) and age-matched HIV-uninfected controls (HIVneg; n = 5). The IEC line HT-29 was used for mechanistic studies. Cells from SCB and blood were isolated by enzymatic digestion and/or gradient centrifugation. HT-29 cells were exposed to TLR1-9 agonists, TNF-α, IL-17A and HIV. IL-32α/β/γ/D/ε/θ and IL-17A mRNA levels were quantified by real-time RT-PCR. IL-32 protein levels were quantified by ELISA. IL-32β/γ/ε isoform transcripts were detectable in the blood and SCB, with IL-32β mRNA levels being predominantly expressed in both compartments and at significantly higher levels in HIV + ART compared to HIVneg. IL-17A transcripts were only detectable in SCB, with increased IL-17A levels in HIVneg compared with HIV + ART and negatively correlated with IL-32β mRNA levels. IL-32β/γ/ε isoform mRNA were detected in HT-29 cells upon exposure to TNF-α, Poly I:C (TLR3 agonist), Flagellin (TLR-5 agonist) and HIV. IL-17A significantly decreased both IL-32 β/γ/ε mRNA and cell-associated IL-32 protein levels induced upon TNF-α and Poly I:C triggering. We document IL-32 isoforms abundant in the colon of ART-treated PWH and reveal the capacity of the Th17 hallmark cytokine IL-17A to attenuate IL-32 overexpression in a model of inflamed IEC.

DOP06 Histologic activity in Ulcerative Colitis patients achieving endoscopic healing is associated with higher rate of relapse and a distinct mucosal microbial and transcriptional profile

Abstract Background Histologic remission (HR) in ulcerative colitis (UC) has been associated with better clinical outcomes. However, this therapeutic target is hard to achieve with the available medications. We aim to assess the relapse-free survival (RFS) of patients with persistent histologic activity (HA) upon achieving endoscopic healing (EH) and the microbial and molecular signatures associated with this condition. Methods Left-sided and extensive UC patients and healthy controls (HC) were recruited at colonoscopy and sigmoid colon (SC) biopsy samples were obtained for HA evaluation, 16S rRNA sequencing and host RNA-seq. EH was defined as Mayo endoscopic subscore (MES) 0–1, and HA and histologic remission (HR) as the presence and absence of neutrophil infiltration, respectively. UC relapse was retrospectively evaluated and defined as symptomatic and endoscopic worsening. Cumulative RFS was assessed by log-rank test. Principal component analysis (PCA) was used for capturing cluster structure. For differential expressed genes (DEG) and enrichment pathway analyses, glmQLFTest in EdgeR and enrichKEGG in clusterProfiler were applied, respectively. Genes with log fold change > 2 and a log count per million > 1 were considered DEG. Dada2 algorithm in QIIME2 for amplicon sequence variant generation and ANCOM-BC package for differential abundance (DA) taxa analysis were applied. P-values of omics data were corrected by false discovery rate (FDR) method. Results Sixty-five UC patients had EH (37 MES 0 and 28 MES 1). Nine (20.9%) MES 0 and 17 (40.5%) MES 1 showed HA. Over a median follow-up of 4.2 years (IQR 2.6–6.1), UC patients with HA showed a significant lower RFS compared to HR (p < 0.05; Figure 1). This difference was not observed between MES 0 and MES 1 (p = 0.2). Transcriptomics data was available for 27 samples with HR and 9 samples with HA, as well as 43 HC and 13 MES 2–3 samples. PCA showed that HR and HA samples cluster with HC and MES 2–3 samples, respectively (Figure 2). Compared to HR, 163 DEG (118 up and 45 downregulated) were found in HA. Enrichment analysis showed significant upregulation of cytokine-cytokine interaction, Th17 and chemokine signaling pathways (Figure 3). Microbiome data was available for 43 and 19 samples with HR and HA, respectively. Three genera had significant DA in HA compared to HR (Figure 4). Conclusion HA in UC is better than MES at distinguishing a higher risk of relapse and has a transcriptomics profile closer to active endoscopic disease despite EH with significant upregulation of chemokine, cytokine and Th17 pathways. A different microbial signature is also observed in HA. These particular signatures could help to define strategies to reduce the risk of UC relapse in patients achieving EH.

Association between Brachyspira and irritable bowel syndrome with diarrhoea

ObjectiveThe incidence of IBS increases following enteric infections, suggesting a causative role for microbial imbalance. However, analyses of faecal microbiota have not demonstrated consistent alterations. Here, we used metaproteomics to...

S2812 A Rare Case of Small Bowel Obstruction Mimicking Crohn’s Disease

INTRODUCTION: Mesothelioma is an uncommon, but feared malignancy. Its peritoneal variant is an even rarer entity and is classified based on 3 histologic subtypes: sarcomatoid, epithelioid, and biphasic. The sarcomatoid variant carries the worst prognosis. CASE DESCRIPTION/METHODS: A 26-year old female presented with a 6-month history of recurrent abdominal pain, nausea, loose stools, and unintended weight loss. Her past medical history was significant for squamous cell carcinoma (SCC) of the tongue which was treated with surgical resection including lymph node dissection with plans for adjuvant chemotherapy/radiation after evaluation of her gastrointestinal symptoms. She underwent extensive workup that included labs, stool studies, EGD, colonoscopy, and MR enterography; resulting in a presumed diagnosis of Crohn's disease based on endoscopic, but not histologic, evidence of ileitis. She was referred to our inflammatory bowel disease center, and a plan to repeat colonoscopy and ileoscopy with biopsies was made after seeing our GI and surgical subspecialists. Before outpatient workup could be completed, the patient presented emergently with nausea, reduced flatus, and reduced stooling. CT showed distal small bowel obstruction due to abnormal thickening of 5–10 cm of the terminal ileum with mucosal enhancement and extensive wall edema and inflammation of the adjacent mesenteric fat. Inpatient colonoscopy with ileoscopy revealed mild erythema and edema of the terminal ileum and sigmoid colon. A non-traversable, smooth appearing stricture was encountered 5 cm proximal to the ileocecal valve. The endoscopic appearance was not consistent with Crohn's disease. Ileal biopsies revealed prominent lymphangiectasia and associated erosion of overlying mucosa, and sigmoid colon biopsies were normal. Due to worsening symptoms, a diagnostic laparoscopy was performed, revealing an extraluminal, 6–7 cm that compressed the terminal ileum and was adherent to the sigmoid colon. She underwent an ileocecal resection with primary anastomosis. Final pathology revealed metastatic malignant mesothelioma of the sarcomatoid variant. She recovered well from surgery and is undergoing oncologic care. DISCUSSION: Our case demonstrates a unique presentation of a rare, clinically challenging entity that mimicked a typical presentation of Crohn’s disease.Figure 1.: CT Abdomen/Pelvis.Figure 2.: Terminal Ileal Biopsy with dilated lymphatic spaces and overlying eroded mucosa- 4x.Figure 3.: Terminal Ileal Biopsy with dilated lymphatic spaces and overlying eroded mucosa- 10x.

S0248 Mucosal Association of Clostridioides difficile in the Colon May Play a Role in Infection Recurrence

INTRODUCTION: Clostridioides difficile infection (CDI) is increasing in prevalence and recurrent disease has increased by nearly 190%. Factors leading to recurrence remain poorly understood and may be related to C. difficile genetics and persistence within the gut after treatment. In this pilot study, we aim to clarify whether C. difficile colonizes the colonic mucosa to evade treatment and persist after initial standard antibiotic treatment and whether this may contribute to recurrence. METHODS: All human studies were approved by institutional IRB. Patients with recent CDI underwent flexible sigmoidoscopy to obtain sigmoid colon biopsies within 2 weeks of completing antibiotic therapy for CDI. Those already with recurrent CDI were excluded. Samples were fixed in methacarn solution and then stained with DAPI for DNA, and fluorescence in situ hybridization probes for bacteria (EUB388), mucin (MUC2) and a C. difficile 16S rRNA specific probe. Tissue permeability for fluorescein isothiocyanate (FITC 4kDa) was assessed in an Ussing chamber for 4 hours post procurement. Clinical information was obtained by review of electronic health records. Patients were contacted 8 weeks post CDI treatment to determine if recurrent CDI developed. RESULTS: 14 patients completed the study (mean age 55.8 years, 64.3% female). 4 patients (28.6%) developed recurrent CDI (3 confirmed by C. difficile stool testing, 1 based on recurrence of symptoms and favorable response to treatment) within 8 weeks of treatment. There were no significant differences in age, gender, recent hospitalization or recent antibiotic use between those with and without recurrence (Table 1). We assessed the presence of C. difficile in the colonic biopsy specimens using FISH and detected C. difficile on tissue staining in 2/4 (50.0%) patients who subsequently developed recurrent CDI, as compared to 3/10 (30.0%) without recurrence (Figure 1). C. difficile carrier status was assessed in biopsies from healthy patients with no CDI history and was found in 4/15 (26.7%). Colonic mucosal permeability was higher in patients who developed recurrent CDI compared to those who did not (P = 0.028 on mixed-model two way ANOVA with Sidak’s multiple comparison post hoc test; Figure 2).Table 1.: Characteristics of Post-CDI treatment patients.Figure 1.: Colonic mucosal biopsies showing presence of C. difficile (yellow arrows) and non-C. difficile bacteria (red arrows) in the mucus layer. Blue represents DNA (DAPI), green represents mucin (MUC2), red represents C. difficile specific probe.CONCLUSION: C. difficile can colonize the colonic mucus layer which may allow it to evade treatment and contribute to recurrent CDI. Preliminary data suggests that mucosal colonization of C. difficile post treatment and increased colonic mucosal permeability may predict recurrent CDI.Figure 2.: Colonic tissue permeability in relation to recurrent CDI status.

Parallel studies of mucosal immunity in the reproductive and gastrointestinal mucosa of HIV-infected women.

The effects of HIV on the gastrointestinal tract (GIT), including CD4 depletion, epithelial disruption, and collagen deposition, are well documented and only partially reversed by combination antiretroviral therapy (cART). However, the effects of HIV on the female reproductive tract (FRT) are poorly understood, and most studies have focused on ectocervix and vagina without assessing the upper tract. Here, we investigated CD4+ T-cell frequency, phenotype, and HIV-specific T-cell responses in the endocervix and endometrium of HIV-infected women, comparing these tissues to the GIT. Mucosal samples and blood were obtained from 18 women: four who were HIV-positive and not on cART for at least 3years prior to sampling, including two natural controllers (viral load [VL] undetectable and CD4 >350); nine women on cART with low to undetectable VL; and five HIV-uninfected women. Mucosal samples included terminal ileum, sigmoid colon, endocervical cytobrush, endocervical curettage, and endometrial biopsy. T-cell frequency, phenotypes, and HIV-specific T-cell responses were analyzed by multiparameter flow cytometry. T-cell activation, measured by CD38/HLA-DR co-expression, remained significantly elevated in endometrium following cART, but was lower in gastrointestinal tissues. HIV-specific CD8+ T-cell responses were detected in ileum, colon, and endometrial tissues of women both on and off cART, and were of higher magnitude on those not on cART. Our findings reveal differences in CD4+ T-cell frequencies, immune activation, and HIV-specific T-cell responses between the gastrointestinal and reproductive tracts, and highlight differences between HIV controllers and women on cART.

Sialylation and fucosylation modulate inflammasome-activating eIF2 Signaling and microbial translocation during HIV infection

An emerging paradigm suggests that gut glycosylation is a key force in maintaining the homeostatic relationship between the gut and its microbiota. Nevertheless, it is unclear how gut glycosylation contributes to the HIV-associated microbial translocation and inflammation that persist despite viral suppression and contribute to the development of several comorbidities. We examined terminal ileum, right colon, and sigmoid colon biopsies from HIV-infected virally-suppressed individuals and found that gut glycomic patterns are associated with distinct microbial compositions and differential levels of chronic inflammation and HIV persistence. In particular, high levels of the pro-inflammatory hypo-sialylated T-antigen glycans and low levels of the anti-inflammatory fucosylated glycans were associated with higher abundance of glycan-degrading microbial species (in particular, Bacteroides vulgatus), a less diverse microbiome, higher levels of inflammation, and higher levels of ileum-associated HIV DNA. These findings are linked to the activation of the inflammasome-mediating eIF2 signaling pathway. Our study thus provides the first proof-of-concept evidence that a previously unappreciated factor, gut glycosylation, is a force that may impact the vicious cycle between HIV infection, microbial translocation, and chronic inflammation.