Ileal Biopsies Research Articles

A Novel Microbial Dysbiosis Index and Intestinal Microbiota-Associated Markers as Tools of Precision Medicine in Inflammatory Bowel Disease Paediatric Patients.

Recent evidence indicates that the gut microbiota (GM) has a significant impact on the inflammatory bowel disease (IBD) progression. Our aim was to investigate the GM profiles, the Microbial Dysbiosis Index (MDI) and the intestinal microbiota-associated markers in relation to IBD clinical characteristics and disease state. We performed 16S rRNA metataxonomy on both stools and ileal biopsies, metabolic dysbiosis tests on urine and intestinal permeability and mucosal immunity activation tests on the stools of 35 IBD paediatric patients. On the GM profile, we assigned the MDI to each patient. In the statistical analyses, the MDI was correlated with clinical parameters and intestinal microbial-associated markers. In IBD patients with high MDI, Gemellaceae and Enterobacteriaceae were increased in stools, and Fusobacterium, Haemophilus and Veillonella were increased in ileal biopsies. Ruminococcaceae and WAL_1855D were enriched in active disease condition; the last one was also positively correlated to MDI. Furthermore, the MDI results correlated with PUCAI and Matts scores in ulcerative colitis patients (UC). Finally, in our patients, we detected metabolic dysbiosis, intestinal permeability and mucosal immunity activation. In conclusion, the MDI showed a strong association with both severity and activity of IBD and a positive correlation with clinical scores, especially in UC. Thus, this evidence could be a useful tool for the diagnosis and prognosis of IBD.

Biological variability of human intraepithelial lymphocytes throughout the human gastrointestinal tract in health and coeliac disease.

Intraepithelial lymphocytes are the first line of defence of the human intestinal immune system. Besides, their composition is altered on patients with coeliac disease (CD), so they are considered as biomarkers with utility on their diagnose and/or monitoring. Our aim is to address their variability through the human gastrointestinal tract in health and characterized them in further depth in the coeliac duodenum. Intraepithelial lymphocytes were isolated from human gastric, duodenal, ileal and colonic biopsies, then stained with specific antibodies and acquired by flow cytometry. Our results confirmed that the profile of Intraepithelial lymphocytes change through the length of the human gastrointestinal tract. Besides and given the central role that Interleukin-15 (IL-15) elicits on CD pathogenesis; we also assessed the expression of its receptor revealing that there was virtually no functional IL-15 receptor on duodenal Intraepithelial lymphocytes. Nevertheless and contrary to our expectations, the active IL-15 receptor was not increased either on Intraepithelial lymphocytes from CD patients. IL-15 might require additional stimulus to activate intraepithelial lymphocytes. These findings may provide novel tools to aid on a CD diagnosis and/or monitoring, at the time that provide the bases to perform functional studies in order of getting a deeper insight in the specific function that Intraepithelial lymphocytes elicit on CD pathogenesis.

Paediatric Crohn’s disease: histologic findings at initial presentation

BackgroundDiagnosing paediatric Crohn’s disease (CD) based on histology can present challenges. We evaluate the histological spectrum of treatment-naïve biopsies from children with CD and assess these findings’ diagnostic and predictive...

Histologic Activity From Neoterminal Ileal Biopsies in Patients With Crohn's Disease in Endoscopic Remission is Associated With Postoperative Recurrence.

Following ileocolic resection (ICR), the clinical importance and prognostic implications of histologic activity on biopsies in Crohn's disease (CD) patients with endoscopic remission are not well defined. The aim of this study was to determine if histologic activity in patients with endoscopic remission is associated with future risk of endoscopic and/or radiologic postoperative recurrence (POR). In this multicenter retrospective cohort study, adult patients with CD who underwent ICR between 2009 and 2020 with endoscopic biopsies of ileal mucosa from Rutgeerts i0 on index colonoscopy were included. The composite rate of endoscopic (Rutgeerts score ≥i2b) and radiologic (active inflammation on imaging) recurrence was compared in patients with and without histologic activity using a Kaplan-Meier survival analysis. A multivariable Cox proportional hazard regression model including clinically relevant risk factors of POR, postoperative biologic prophylaxis, and histology activity was designed. A total of 113 patients with i0 disease on index colonoscopy after ICR were included. Of these, 42% had histologic activity. Time to POR was significantly earlier in the histologically active versus normal group ( P = 0.04). After adjusting for clinical risk factors of POR, histologic activity (HR 2.37, 95% CI 1.17-4.79; P = 0.02) and active smoking (HR 2.54, 95% CI 1.02-6.33; P = 0.05) were independently associated with subsequent composite POR risk. In patients with postoperative CD, histologic activity despite complete endoscopic remission is associated with composite, endoscopic, and radiographic recurrence. Further understanding of the role of histologic activity in patients with Rutgeerts i0 disease may provide a novel target to reduce disease recurrence in this population.

A172 A CLASSIC CASE OF WHIPPLE'S DISEASE

Abstract Background Whipple’s disease is a systemic disorder caused by the Gram-positive bacillus Tropheryma whippelii. It is classically defined by the four cardinal clinical manifestations of arthralgias, weight loss, diarrhea and abdominal pain. Classic Whipple’s disease is considered extremely rare, with an estimated global incidence of 30 cases per year, though this may be an underestimate. Aims To present a case of classic Whipple’s disease, so as to raise awareness and clinical suspicion for this rare condition. Methods We performed a retrospective chart review of a case of classic Whipple’s disease diagnosed in Saint John, NB, in 2020. Results A 55-year-old female presented to hospital with intractable weakness and diarrhea. Her history was notable for several years of right leg weakness, myalgias and low-grade fevers. More acutely, she presented to hospital due to worsening secretory diarrhea resulting in 15kg weight loss. Initial investigations were remarkable for an elevated C-reactive protein. The patient was admitted for coordinated evaluation for possible rheumatologic and infectious causes. As part of that workup, colonoscopy was performed. Colonoscopy revealed an abnormal ileum, with villi described as edematous with a mosaic pattern alternating between erythematous and “white tipped”. On histologic assessment, ileal biopsies showed macrophages with PAS-positive diastase resistant granules suspicious for Whipple’s disease. A follow-up esophagogastroduodenoscopy with duodenal biopsies was thus performed to gain further tissue for confirmatory testing. The duodenal mucosa was also noted to have a mosaic pattern with hypertrophic villi alternating between pale and erythematous in appearance. Duodenal biopsies were taken and sent for PCR testing. This testing was positive for T. whippelii, confirming the diagnosis of Whipple’s disease. The patient was initiated on long-term antibiotic therapy with ceftriaxone transitioning to trimethoprim-sulfamethoxazole prior to discharge. She unfortunately re-presented one month post-discharge with recurrent fevers, malaise and arthralgias. Given suspicion that her disease may be resistant to trimethoprim-sulfamethoxazole, she was transitioned to doxycycline and hydroxychloroquine as second-line therapy. She clinically improved on this regimen, and was able to be de-escalated to long-term doxycycline monotherapy after one year. At a recent follow-up, nearly three years from her initial diagnosis, she was doing well with essentially no ongoing symptoms of her Whipple’s disease. Conclusions Whipple’s disease is a rare infection presenting with intractable diarrhea and abdominal discomfort. Our case highlights the excellent clinical response that can be achieved with successful diagnosis and management of this otherwise debilitating disease. Funding Agencies None

Linking gene expression to clinical outcomes in pediatric Crohn’s disease using machine learning

Pediatric Crohn’s disease (CD) is characterized by a severe disease course with frequent complications. We sought to apply machine learning-based models to predict risk of developing future complications in pediatric CD using ileal and colonic gene expression. Gene expression data was generated from 101 formalin-fixed, paraffin-embedded (FFPE) ileal and colonic biopsies obtained from treatment-naïve CD patients and controls. Clinical outcomes including development of strictures or fistulas and progression to surgery were analyzed using differential expression and modeled using machine learning. Differential expression analysis revealed downregulation of pathways related to inflammation and extra-cellular matrix production in patients with strictures. Machine learning-based models were able to incorporate colonic gene expression and clinical characteristics to predict outcomes with high accuracy. Models showed an area under the receiver operating characteristic curve (AUROC) of 0.84 for strictures, 0.83 for remission, and 0.75 for surgery. Genes with potential prognostic importance for strictures (REG1A, MMP3, and DUOX2) were not identified in single gene differential analysis but were found to have strong contributions to predictive models. Our findings in FFPE tissue support the importance of colonic gene expression and the potential for machine learning-based models in predicting outcomes for pediatric CD.

MUCIN MRNA ISOFORM SIGNATURES AS POTENTIAL NOVEL BIOMARKERS TO EVALUATE DISEASE STATUS IN PATIENTS WITH INFLAMMATORY BOWEL DISEASES

Abstract Mucosal barrier dysfunction and aberrant mucin expression are major hallmarks in the pathophysiology of IBD. Mucins are highly polymorphic, and the presence of genetic differences can alter gene expression, resulting in several mRNA isoforms via alternative splicing. While most isoforms encode similar biological functions, others alter protein function, potentially resulting in progression towards disease. Currently, little attention has been given to the importance of mucin mRNA isoforms in IBD. The aim of our study is to investigate the potential of mucin mRNA isoforms as novel biomarkers for the evaluation of IBD activity and subtypes. To obtain this goal, RNA was extracted from colonic and terminal ileal biopsies of IBD patients that underwent an endoscopy at the Antwerp University Hospital (UZA). Additionally, patients without a history of IBD undergoing an endoscopy due to a positive iFOBT which show no endoscopic abnormalities, were included as controls. Library preparation was performed with the PacBio Iso-Seq multiplex protocol adapted for targeted transcriptome sequencing. Targeted capture was accomplished by using a custom-designed pool of probes, developed for the capture of all mucin gene transcripts. Samples were sequenced on the PacBio Sequel platform at the University of Antwerp. The data was analyzed by using the isoseq3-pipeline and additional filtering with SQANTI3 was performed. In total 106 biopsies were sequenced on the PacBio platform. The resulting intestinal mucin transcriptome was merged with the human reference transcriptome. On this combined mucin transcriptome Illumina bulk RNA sequencing data from over 2000 intestinal biopsies (GEO dataset GSE193677) were mapped to determine mucin isoform expression. An external dataset (GEO dataset GSE165512) was used for additional validation. A classification random forest was trained on this data to distinguish inflamed IBD from non-inflamed control patients based on the mucin isoform expression alone. The model performed well on train and test datasets but decreased in the external validation (Table 1). Dividing the samples based on disease phenotype greatly increases performance on the external validation dataset (Table 1). When only training on ileal biopsies, the model proved to be excellent in distinguishing Crohn’s disease patients from controls (Table 1). Classification of inflamed ulcerative colitis from control patients based on only the biopsies from the distal colon was similar to the latter (Table 1). Our machine learning model was able to distinguish Crohn’s disease from control patients and ulcerative colitis from control patients based on mucin mRNA isoform expression. In addition, our data suggests that the mucin isoform expression may vary between different regions within the colon. Table 1 Area under the receiver operator curve (AUCROC) from a random forest classifier trained on mucin isoform expression of inflamed biopsies of IBD and non-inflamed control patients.

GROUP 3 INNATE LYMPHOID CELLS RELY ON ER STRESS RESPONSE FOR CYTOKINE PRODUCTION IN IBD

Abstract BACKGROUNDS Group 3 innate lymphoid cells (ILC3s) recently emerged as important regulators and potential drug targets for IBD. However, the response of ILC3s to environmental stimuli during intestinal inflammation remains elusive. IRE1a-XBP1 serves as the regulatory hub of the endoplasmic reticulum stress response that plays a vital role in intestinal inflammation. METHODS We generated conditional knockout Ire1aflo/flox,Rorc-Cre (Ire1aΔRorc) and Rag-/-Ire1aΔRorc mice with Ire1a deleted in ILC3s. Intestinal infections were induced by Citrobacter rodentium and Clostridium difficile. ILC3s-derived cytokines including IL-22 is crucial for host defense in the acute phase of C. rodentium and C. difficile infection. Dextran sodium sulfate (DSS) was given in drinking water to induce acute colitis. We recruited patients with active Crohn’s disease (CD) and collected ileal biopsies during routine colonoscopy before starting ustekinumab, a non-selective anti-IL-23 antibody. Mucosal ILC3s were isolated for analysis of ILC3s by flow cytometry. RESULTS In murine intestinal ILC3s, the activity of IRE1a-XBP1 pathway follows a robust 24h circadian rhythm which parallels that of clock genes and cytokines including IL-22. XBP1s is activated in ILC3s ex vivo by vasoactive intestinal peptide, which was previous shown to orchestrate the circadian expression of IL-22 by ILC3s. IRE1a-XBP1 in intestinal ILC3s is further activated in response to IL-23 and colitis in mice, as well as in inflamed IBD tissues compared to normal tissues from healthy individuals. We further showed that IRE1a-XBP1 activation in stimulated ILC3s requires mitochondrial reactive oxygen species (mtROS) (Fig 1). Using the Ire1aΔRorc mice, we demonstrated that IRE1a-XBP1 is critical for cytokine expression by ILC3s. Ire1aΔRorc mice are highly susceptible to acute C. rodentium and C. difficile infection as well as acute DSS colitis (similar vulnerabilities were found in Rag-/-Ire1aΔRorc mice compared to Rag-/-Ire1aflox/flox littermates). Ire1aΔRorc mice exhibit reduced IL-22 and IL-17A in ILC3s and impaired epithelial barrier function with diminished expression of mucins and antimicrobial peptides. The susceptibilities of Ire1aΔRorc and Rag-/-Ire1aΔRorc mice to colitis were rescued by administration of recombinant IL-22 (Fig 2). Moreover, the frequency of XBP1s+ ILC3s in ileal mucosa from CD patients before initiation of ustekinumab positively correlates with response to therapy. CONCLUSIONS We demonstrate that a non-canonical mtROS-IRE1a-XBP1 pathway augments cytokine production by intestinal ILC3s. Loss of IRE1a-XBP1 in ILC3s impairs the optimal and rhythmic expression of protective cytokines and renders the mice more susceptible to intestinal infections and colitis. We also identify XBP1+ ILC3s as a potential biomarker for predicting responsiveness to anti-IL-23 therapies in IBD.

P900 A PROOF-OF-CONCEPT, PLACEBO-RANDOMIZED CONTROLLED TRIAL TARGETING ADHERENT AND INVASIVE ESCHERICHIA COLI (AIEC) WITH ANTIBIOTICS IN CROHN’S DISEASE: the TEOREM TRIAL

Abstract Background Adherent-invasive E. coli (AIEC) are found in the ileal mucosa of 21-63% of patients with ileal Crohn’s disease (CD) vs 0-16% of controls. This trial tested the efficacy of antibiotics targeting AIEC in CD patients with ileal involvement, colonized by AIEC. Methods Eligible patients had endoscopically active (CDEIS ≥8) ileal or ileocolonic CD and either a clinically inactive, or mild to moderately active disease that responded to budesonide or prednisolone. AIEC search was performed on ileal biopsies, by phenotypical assays. The primary endpoint was the endoscopic overall response at week 12, defined as segmental response in at least 1 segment, and < 20% increase of CDEIS in other segments. Segmental response was defined by decrease ≥ 50% of surface of ulcerations, or loss of deep ulcerations (if any). Secondary endpoints were ΔCDEIS ≥ 50% in at least 1 segment with CDEIS ≥ 8 at baseline, and remission (CDEIS < 6 in all segments). Endoscopic endpoints were assessed by a central, anonymous and blinded scoring of video recordings of colonoscopies at preinclusion and week 12. Patients were randomized 1/1 between oral Ciprofloxacin 500 mg bid + Rifaximin 800 mg bid and double placebo for 12 weeks. In this subgroup of patients selected for AIEC colonization, we were in search of a strong signal of efficacy. We hypothesized that 60% of patients in the experimental arm and 25% of those in the placebo arm would reach the primary endpoint. 31 patients in each arm were planned to demonstrate such a difference. Results Between May 2016 and June 2021, 155 patients were screened, of whom 91; 27 were AIEC positive and 24 patients (12 in each arm) were randomized in 10 centres. 17 patients had a clinically active disease. All AIEC strains of randomized patients were sensitive to ciprofloxacin and rifaximin. Clinical and endoscopic characteristics were similar in the 2 arms. 2 patients did not have colonoscopy at week 12 (1 tendinitis and 1 had no colonoscopy during the COVID pandemic). 7/10 patients cleared AIEC at week 12 in the experimental arm, vs 6/12 in the placebo arm. The 3 patients who received antibiotics and remained AIEC+ after antibiotics acquired resistance to ciprofloxacine. Results are displayed in the table. There was no association between AIEC clearance and endoscopic endpoints. A Bayesian, post hoc analysis showed that the probability to obtain a 35% difference in the primary endpoint between the two arms of the trial was of 25%. Conclusion Antibiotics were not statistically superior to placebo to achieve endoscopic response and remission in patients with ileal CD colonized with AIEC. There was no association between AIEC clearance and endoscopic endpoints. Differences were numerically in favour of antibiotics.

DOP23 The role of histology for the prediction of clinical relapse in Crohn’s Disease: A substudy of the STORI cohort

Abstract Background Deep remission has become the target in the management of Crohn’s disease (CD). The role of histology in the management of CD has not yet been established. The aim of this work was to study the impact of histological inflammation on the risk of clinical relapse in patients with CD in clinical and endoscopic remission and to study the correlation between histology and endoscopic scores and biomarkers. Methods All patients included in STORI (1) were eligible for inclusion. Fecal calproptectin and colonoscopy with CDEIS calculation were performed at inclusion. Two biopsies, taken either from the most inflammed macroscopic area or from an area with previous inflammation in case of mucosal healing (MH) were prospectively performed. MH was defined by the absence of ulcers. Different histological scores were calculated by 2 independent pathologists: Robart, Geboes, modified Geboes, Nancy, and DCA-IBD scores. Histological remission was defined by Nancy=0 ; Geboes ≤2 ; modified Geboes of grade 0 ; Robarts ≤3, IBD-DCA of C≤1 and A=0. Clinical relapse was prospectively evaluated in the STORI trial as CDAI>250 or CDAI between 150 and 250 with an increase of 70 points during 2 successive weeks compared to the inclusion visit. (1) Louis E et al .Gastroenterology 2012 Results Eighty biopsies (22 ileal and 58 colonic) were available from 76/115 patients included in STORI. Among the 45 patients with MH, 30 had colonic biopsies, 11 had ileal biopsies and 4 had biopsies in both locations. Among colonic biopsies 68% had histological remission according to Nancy and modified Geboes scores, 65% according to DCA-IBD and Geboes scores and 71% according to Robarts score. Among ileal biopsies 53% had histological remission according to Nancy score, 47% according to Modified Geboes, Geboes and DCA-IBD scores, and 67% according to the Robarts score. Thirty-five patients (46%) experienced a clinical relapse during the follow-up including 16/45 (36%) and 19/31 (61%) in the group with MH and without MH respectively. Histological characteristics and histological scores were not predictive of clinical relapse. In the total cohort (n=76), the CDEIS score was correlated to Nancy (p<0,001, r=0,43), Geboes (p<0,001, r=0,47) and Robarts (p<0,001, r=0,45) scores. Fecal calprotectin was correlated to Nancy (p<0,001, r=0,48), Geboes (p<0,001, r=0,45) and Robarts (p<0,001, r=0,46) scores. The histological scores of Nancy, Robarts and Geboes were highly correlated (p<0,0001 ; r > 0,9). Conclusion Although correlated with endoscopy and biomarkers, histology was not predictive of clinical relapse in CD patients in clinical and endoscopic remission. These findings do not support the use of histology to predict clinical relapse in CD in clinical practice.

DOP89 Unfolded protein response controls group 3 innate lymphoid cells in IBD

Abstract Background Group 3 innate lymphoid cells (ILC3s) have recently emerged as important regulators and potential drug targets for IBD. However, the response of ILC3s to environmental stimuli during intestinal inflammation remains elusive. IRE1a-XBP1 serves as the regulatory hub of endoplasmic reticulum stress and the unfolded protein response (UPR) that plays a vital role in intestinal inflammation. Methods We generated conditional knockout Ire1aflo/flox,Rorc-Cre (Ire1aΔRorc) and Rag-/-Ire1aΔRorc mice with Ire1a deleted in ILC3s. Intestinal infections were induced by Citrobacter rodentium and Clostridium difficile. ILC3s-derived cytokines including IL-22 is crucial for host defense in the acute phase of C. rodentium and C. difficile infection. Dextran sodium sulfate (DSS) was given in drinking water to induce acute colitis. We recruited patients with active Crohn’s disease (CD) and collected ileal biopsies during routine colonoscopy before starting ustekinumab, a non-selective anti-IL-23 antibody. Mucosal ILC3s were isolated for analysis of ILC3s by flow cytometry. Results In murine intestinal ILC3s, the activity of IRE1a-XBP1 pathway follows a robust 24h circadian rhythm which parallels that of clock genes and cytokines including IL-22. XBP1s is activated in ILC3s ex vivo by vasoactive intestinal peptide, which was previous shown to orchestrate the circadian expression of IL-22 by ILC3s. IRE1a-XBP1 in intestinal ILC3s is further activated in response to IL-23 and colitis in mice, as well as in inflamed IBD tissues compared to normal tissues from healthy individuals. We further showed that IRE1a-XBP1 activation in stimulated ILC3s requires mitochondrial reactive oxygen species (mtROS) (Fig 1). Using the Ire1aΔRorc mice, we demonstrated that IRE1a-XBP1 is critical for cytokine expression by ILC3s. Ire1aΔRorc mice are highly susceptible to acute C. rodentium and C. difficile infection as well as acute DSS colitis (similar vulnerabilities were found in Rag-/-Ire1aΔRorc mice compared to Rag-/-Ire1aflox/flox littermates). Ire1aΔRorc mice exhibit reduced IL-22 and IL-17A in ILC3s and impaired epithelial barrier function with diminished expression of mucins and antimicrobial peptides. The susceptibilities of Ire1aΔRorc and Rag-/-Ire1aΔRorc mice to colitis were rescued by administration of recombinant IL-22 (Fig 2). Moreover, the frequency of XBP1s+ ILC3s in ileal mucosa from CD patients before initiation of ustekinumab positively correlates with response to therapy. Conclusion We demonstrate that a non-canonical mtROS-IRE1a-XBP1 pathway augments cytokine production by intestinal ILC3s. We also identify XBP1+ ILC3s as a potential biomarker for predicting responsiveness to anti-IL-23 therapies in IBD.

Expression of the cobalamin transporters cubam and MRP1 in the canine ileum-Upregulation in chronic inflammatory enteropathy.

Chronic inflammatory enteropathy (CIE) in dogs, a spontaneous model of human inflammatory bowel disease (IBD), is associated with a high rate of cobalamin deficiency. The etiology of hypocobalaminemia in human IBD and canine CIE remains unknown, and compromised intestinal uptake of cobalamin resulting from ileal cobalamin receptor deficiency has been proposed as a possible cause. Here, we evaluated the intestinal expression of the cobalamin receptor subunits, amnionless (AMN) and cubilin (CUBN), and the basolateral efflux transporter multi-drug resistance protein 1 (MRP1) in 22 dogs with CIE in comparison to healthy dogs. Epithelial CUBN and AMN levels were quantified by confocal laser scanning microscopy using immunohistochemistry in endoscopic ileal biopsies from dogs with (i) CIE and normocobalaminemia, (ii) CIE and suboptimal serum cobalamin status, (iii) CIE and severe hypocobalaminemia, and (iv) healthy controls. CUBN and MRP1 expression was quantified by RT-qPCR. Receptor expression was evaluated for correlation with clinical patient data. Ileal mucosal protein levels of AMN and CUBN as well as mRNA levels of CUBN and MRP1 were significantly increased in dogs with CIE compared to healthy controls. Ileal cobalamin receptor expression was positively correlated with age, clinical disease activity index (CCECAI) score, and lacteal dilation in the ileum, inversely correlated with serum folate concentrations, but was not associated with serum cobalamin concentrations. Cobalamin receptor downregulation does not appear to be the primary cause of hypocobalaminemia in canine CIE. In dogs of older age with severe clinical signs and/or microscopic intestinal lesions, intestinal cobalamin receptor upregulation is proposed as a mechanism to compensate for CIE-associated hypocobalaminemia. These results support oral supplementation strategies in hypocobalaminemic CIE patients.

Mechanisms underlying distinct subcellular localization and regulation of epithelial long myosin light-chain kinase splice variants

Intestinal epithelia express two long myosin light-chain kinase (MLCK) splice variants, MLCK1 and MLCK2, which differ by the absence of a complete immunoglobulin (Ig)-like domain 3 within MLCK2. MLCK1 is preferentially associated with the perijunctional actomyosin ring at steady state, and this localization is enhanced by inflammatory stimuli including tumor necrosis factor (TNF). Here, we sought to identify MLCK1 domains that direct perijunctional MLCK1 localization and their relevance to disease. Ileal biopsies from Crohn's disease patients demonstrated preferential increases in MLCK1 expression and perijunctional localization relative to healthy controls. In contrast to MLCK1, MLCK2 expressed in intestinal epithelia is predominantly associated with basal stress fibers, and the two isoforms have distinct effects on epithelial migration and barrier regulation. MLCK1(Ig1-4) and MLCK1(Ig1-3), but not MLCK2(Ig1-4) or MLCK1(Ig3), directly bind to F-actin invitro and direct perijunctional recruitment in intestinal epithelial cells. Further study showed that Ig1 is unnecessary, but that, like Ig3, the unstructured linker between Ig1 and Ig2 (Ig1/2us) is essential for recruitment. Despite being unable to bind F-actin or direct recruitment independently, Ig3 does have dominant negative functions that allow it to displace perijunctional MLCK1, increase steady-state barrier function, prevent TNF-induced MLCK1 recruitment, and attenuate TNF-induced barrier loss. These data define the minimal domain required for MLCK1 localization and provide mechanistic insight into the MLCK1 recruitment process. Overall, the results create a foundation for development of molecularly targeted therapies that target key domains to prevent MLCK1 recruitment, restore barrier function, and limit inflammatory bowel disease progression.

The FOXP3+ Pro-Inflammatory T Cell: A Potential Therapeutic Target in Crohn’s Disease

Background and aimsThe incidence of Crohn’s disease (CD) continues to increase worldwide. The contribution of CD4+ cell populations remains to be elucidated. Here, we aim to provide an in-depth transcriptional assessment of CD4+ T cells driving chronic inflammation in CD. MethodsWe performed single cell RNA-sequencing in CD4+ T cells isolated from ileal biopsies of CD patients compared to healthy individuals. Cells underwent clustering analysis, followed by analysis of gene signalling networks. We overlapped our differentially expressed genes with publicly available microarray datasets and performed functional in vitro studies, including an in vitro suppression assay and organoid systems, to model gene expression changes observed in CD TREGs and to test predicted therapeutics. ResultsWe identified five distinct FOXP3+ regulatory T (Treg) subpopulations. Tregs isolated from healthy controls represent the origin of pseudotemporal development into inflammation-associated subtypes. These proinflammatory Tregs displayed a unique responsiveness to TNFα signaling with impaired suppressive activity in vitro and an elevated cytokine response in an organoid co-culture system. As predicted in silico, the histone deacetylase inhibitor Vorinostat normalized gene expression patterns, rescuing the suppressive function of FOXP3+ cells in vitro. ConclusionWe identified a novel, proinflammatory FOXP3+ T cell subpopulation in CD patients and developed a pipeline to specifically target these cells using the FDA-approved drug Vorinostat.

Fecal miR-223 is a noninvasive biomarker for estimating Crohn's disease activity.

MicroRNA-223 (miR-223) has emerged as a promising noninvasive biomarker for Crohn's disease (CD). However, it is unclear which tissue derived miRNA-223 can more accurately estimate CD disease activity. To collect serum, terminal ileal mucosa biopsy and fecal samples from CD patients and healthy controls. The CD Activity Index (CDAI) score, Montreal classification, maintenance medicines, peripheral blood inflammatory markers, fecal calprotectin (FC) and the Simple Endoscopic Score for CD (SES-CD) were recorded. To compare the expression of miR-223 in the serum, intestinal tissue, and feces. MiR-223 expression levels in the serum, intestinal tissue and feces of CD patients were significantly higher than those of controls. The level of miR-223 in the serum, intestinal tissue and feces increased significantly in active CD patients compared with that in inactive CD patients. The levels of serum, intestinal tissue and fecal miR-223 were correlated with the CDAI. Serum miR-223 was also correlated with C-reactive protein (CRP) and IL-6, tissue miR-223 correlated with IL-6 and FC, and fecal miR-223 correlated with FC. In terms of the association with FC, fecal miR-223 had a higher Spearman r value than tissue miR-223. The area under the curve (AUC) values of serum, tissue and fecal miR-223 to diagnose CD were similar to those of CRP and FC (AUC > 0.8). The AUC values of tissue and fecal miR-223 to evaluate CD disease activity were 0.832 and 0.818, respectively, and were higher than serum miR-223, CRP and FC. Fecal miR-223 had a higher specificity of 92.3%. Fecal miR-223 might be a novel, noninvasive biomarker for estimating the disease activity of CD patients.

Diagnostic Utility of Mucosal Biopsies Taken During Colonoscopy-Guided Colonic Manometry Catheter Placement.

The diagnostic utility of mucosal biopsies taken during colonoscopy-guided colonic manometry catheter placement is unknown. The aims of our study were to determine the frequency and histopathology results of mucosal biopsies during these procedures and to assess whether there were any associations between the histology or gross findings with manometry results. We performed a retrospective chart review of children who had a colonic manometry study completed between 2008 and 2020 at a quaternary children's hospital. We captured patient demographics, biopsy locations, histopathology results, gross endoscopy findings, and manometry results. The chi-squared test and when appropriate Fisher exact test was used to evaluate categorical associations. One hundred forty-eight patients were included. One hundred eighteen (80%) had colonic biopsy and 63 (43%) had ileal biopsy. Colonic histology findings, which patients could have multiple, included lymphonodular hyperplasia (34%), normal (27%), chronic inflammation (24%), melanosis coli (21%), colonic eosinophilia (10%), and acute inflammation (8%). Ileal histology findings included increased Peyer patches (44%), normal (44%), acute inflammation (11%), chronic inflammation (3%), eosinophilia (5%), and eosinophilic ileitis (3%). The majority of acute and chronic inflammation was graded as mild. There were no statistically significant associations of histology to gross endoscopy or manometry findings. Colonic biopsies are obtained in the majority of patients presenting for colonic manometry evaluation with ileal biopsies obtained less frequently. Histopathology findings are noted frequently, but the majority are the result of or did not impact clinical care. There were no associations between abnormal histopathology or abnormal gross endoscopy findings with colonic manometry results.

Diagnostic performance of sonographic activity scores for adult terminal ileal Crohn’s disease compared to magnetic resonance and histological reference standards: experience from the METRIC trial

ObjectivesThe simple ultrasound activity score for Crohn’s disease (SUS-CD) and bowel ultrasound score (BUSS) are promising intestinal ultrasound (IUS) indices of CD, but studied mainly in small settings with few sonographers. We compared SUS-CD and BUSS against histological and magnetic resonance enterography (MRE) reference standards in a post hoc analysis of a prospective multicentre, multireader trial.MethodsParticipants recruited to the METRIC trial (ISRCTN03982913) were studied, including those with available terminal ileal (TI) biopsies. Sensitivity and specificity of SUS-CD and BUSS for TI CD activity were calculated with 95% confidence intervals (CI), from the prospective observations of the original METRIC trial sonographers against the histological activity index (HAI) and the simplified magnetic resonance index of activity (sMARIA).ResultsWe included 284 patients (median 31.5 years, IQR 23–46) from 8 centres, who underwent IUS and MRE. Of these, 111 patients had available terminal ileal biopsies with HAI scoring. Against histology, sensitivity and specificity for active disease were 79% (95% CI 69–86%) and 50% (31–69%) for SUS-CD, and 66% (56–75%) and 68% (47–84%) for BUSS, respectively. Compared to sMARIA, the sensitivity and specificity for active CD were 81% (74–86%) and 75% (66–83%) for SUS-CD, and 68% (61–74%) and 85% (76–91%) for BUSS, respectively. The sensitivity of SUS-CD was significantly greater than that of BUSS against HAI and sMARIA (p < 0.001), but its specificity was significantly lower than of BUSS against the MRE reference standard (p = 0.003).ConclusionsParticularly when compared to MRE activity scoring, SUS-CD and BUSS are promising tools in a real-world clinical setting.Clinical relevance statementWhen tested using data from a multicentre, multireader diagnostic accuracy trial, the simple ultrasound activity score for Crohn’s disease (SUS-CD) and bowel ultrasound score (BUSS) were clinically viable intestinal ultrasound indices that were reasonably sensitive and specific for terminal ileal Crohn’s disease, especially when compared to a magnetic resonance reference standard.Key PointsThe simple ultrasound activity score for Crohn’s disease and bowel ultrasound score are promising intestinal ultrasound indices of Crohn’s disease but to date studied mainly in small settings with few sonographers.Compared to histology and the magnetic resonance reference standard in a multicentre, multireader setting, the sensitivity of simple ultrasound activity score for Crohn’s disease is significantly greater than that of bowel ultrasound score.The specificity of simple ultrasound activity score for Crohn’s disease was significantly lower than that of bowel ultrasound score compared to the magnetic resonance enterography reference standard. The specificity of both indices was numerically higher when the magnetic resonance enterography reference standard was adopted.

Collagen VI promotes recovery from colitis by inducing lymphangiogenesis and drainage of inflammatory cells.

Despite a number of studies providing evidence that the extracellular matrix (ECM) is an active player in the pathogenesis of intestinal inflammation, knowledge on the actual contribution of specific ECM molecules in the progression of inflammatory bowel disease (IBD) remains scant. Here, we investigated the role of a major ECM protein, collagen VI (ColVI), in gut homeostasis and elucidated the impact of its deregulation on the pathophysiology of IBD. To this end, we combined in vivo and ex vivo studies on wild type and ColVI-deficient (Col6a1-/- ) mice both under physiological conditions and during experimentally induced acute colitis and its subsequent recovery, by means of gut histology and immunostaining, gene expression, bone marrow transplantation, flow cytometry of immune cell subpopulations, and lymph flow assessment. We found that ColVI displayed dynamic expression and ECM deposition during the acute inflammatory and recovery phases of experimentally induced colitis, whereas the genetic ablation of ColVI in Col6a1 null mice impaired the functionality of lymphatic vessels, which in turn affected the resolution of inflammation during colitis. Based on these findings, we investigated ColVI expression and deposition in ileal specimens from two cohorts of patients affected by Crohn's disease (CD) and correlated ColVI abundance to clinical outcome. Our results show that high ColVI immunoreactivity in ileal biopsies of CD patients at diagnosis correlates with increased risk of surgery and that ColVI expression in biopsies taken at the resection margin during surgery, and showing inactive disease, predict disease recurrence. Our data unveil a key role for ColVI in the intestinal microenvironment, where it is involved in lymphangiogenesis and intestinal inflammation. Altogether, these findings point at the dysregulation of ColVI expression as a novel factor contributing to the onset and maintenance of inflammation in CD via mechanisms impinging on the modulation of inflammatory cell recruitment and function. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Diagnostic yield of random colon biopsy sampling in patients with chronic diarrhea and normal colonoscopy: a systematic review and meta-analysis

Background and AimsMost colonoscopy examinations in patients with chronic unexplained diarrhea do not reveal endoscopic abnormalities, and routine random colon biopsy specimens are taken for further diagnostic assessment. In this study, we aimed to perform a systematic review and meta-analysis to study the pooled diagnostic value of random colon biopsy sampling in this patient population.MethodsMultiple databases (MEDLINE, Scopus, Embase) were searched in May 2022 for studies evaluating the diagnostic yield of random biopsy sampling in patients with chronic diarrhea with normal colonoscopy examinations. Standard meta-analysis methods used the random-effects model, and heterogeneity was assessed using the I2 statistics.ResultsTwenty-one studies were included in the study (3322 patients). The pooled rate of normal colon biopsy sampling was 48% (95% confidence interval [CI], 31-65.2; I2 = 98%), and normal ileal biopsy sampling was 88.5% (95% CI, 51.3-98.3; I2 = 93%). The pooled rate of significant histopathologic change was 30% (95% CI, 20-41; I2 = 96%). The pooled rates of specific diagnoses based on random colon biopsy sampling were as follows: microscopic colitis, 15% (95% CI, 10-21; I2 = 92%); lymphocytic colitis, 9% (95% CI, 6 -13; I2 = 85%); collagenous colitis, 3.5% (95% CI, 2.4-5.7; I2 = 80%); nonspecific colitis, 20% (95% CI, 11-32.8; I2 = 97%); inflammatory bowel disease, 1.7% (95% CI, .8-3.5; I2 = 54%); and eosinophilic colitis, 2.3% (95% CI, 1.1-4.5; I2 = 69%).ConclusionsThis meta-analysis of 21 studies demonstrated significant histopathologic change in 30% and microscopic colitis in 15%. Normal colon biopsy sampling was noted in 48% and normal ileum biopsy sampling in 88.5%. Ileal biopsy data were based on limited data and studies (only 3 included). In studies with a mean patient age <49 years, 40% demonstrated significant histopathologic changes. Most colonoscopy examinations in patients with chronic unexplained diarrhea do not reveal endoscopic abnormalities, and routine random colon biopsy specimens are taken for further diagnostic assessment. In this study, we aimed to perform a systematic review and meta-analysis to study the pooled diagnostic value of random colon biopsy sampling in this patient population. Multiple databases (MEDLINE, Scopus, Embase) were searched in May 2022 for studies evaluating the diagnostic yield of random biopsy sampling in patients with chronic diarrhea with normal colonoscopy examinations. Standard meta-analysis methods used the random-effects model, and heterogeneity was assessed using the I2 statistics. Twenty-one studies were included in the study (3322 patients). The pooled rate of normal colon biopsy sampling was 48% (95% confidence interval [CI], 31-65.2; I2 = 98%), and normal ileal biopsy sampling was 88.5% (95% CI, 51.3-98.3; I2 = 93%). The pooled rate of significant histopathologic change was 30% (95% CI, 20-41; I2 = 96%). The pooled rates of specific diagnoses based on random colon biopsy sampling were as follows: microscopic colitis, 15% (95% CI, 10-21; I2 = 92%); lymphocytic colitis, 9% (95% CI, 6 -13; I2 = 85%); collagenous colitis, 3.5% (95% CI, 2.4-5.7; I2 = 80%); nonspecific colitis, 20% (95% CI, 11-32.8; I2 = 97%); inflammatory bowel disease, 1.7% (95% CI, .8-3.5; I2 = 54%); and eosinophilic colitis, 2.3% (95% CI, 1.1-4.5; I2 = 69%). This meta-analysis of 21 studies demonstrated significant histopathologic change in 30% and microscopic colitis in 15%. Normal colon biopsy sampling was noted in 48% and normal ileum biopsy sampling in 88.5%. Ileal biopsy data were based on limited data and studies (only 3 included). In studies with a mean patient age <49 years, 40% demonstrated significant histopathologic changes.

Multi-Omics Unravels Metabolic Alterations in the Ileal Mucosa of Neonatal Piglets Receiving Total Parenteral Nutrition.

Total parenteral nutrition (TPN) is life-saving therapy for the pediatric patients with intestinal failure (IF) who cannot tolerate enteral nutrition (EN). However, TPN-induced metabolic alterations are also a critical issue for the maintenance of intestinal homeostasis, and thus the global metabolomic signatures need to be addressed. In this study, ileal mucosal biopsies were collected from 12 neonatal Bama piglets receiving either EN or TPN for 14 days, and changes in the intestinal metabolism were examined by multi-omics (HM350 Metabolomics + Tandem Mass Tag (TMT)-based proteomics). As a result, a total of 240 compounds were identified by metabolomics, including 56 down-regulated and 9 up-regulated metabolites. Notably, tissue levels of fatty acyl-carnitines (decreased by 35-85%) and succinate (decreased by 89%) dramatically decreased in the TPN group, suggestive of disrupted processes of fatty acid oxidation (FAO) and the citrate cycle, respectively. Interestingly, however, no differences were found in the production of adenosine 5'-triphosphate (ATP) between groups, suggesting that these dysregulated metabolites may have mainly led to the loss of bioactive compounds rather than energy deficit. Additionally, 4813 proteins were identified by proteomics in total, including 179 down-regulated and 329 up-regulated proteins. The analysis of protein-protein interactions (PPI) indicated that most of the differentially expressed proteins were clustered into "lipid metabolism" and "innate immune responses". In summary, this work provided new findings in TPN-induced intestinal metabolic alterations, which would be useful to the improvement of nutritional management for IF patients.