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Background Intestinal fibrosis is a common complication of inflammatory bowel disease (IBD) with limited diagnostic modalities to detect and determine the degree of fibrosis. Fibroblast activation protein alpha (FAP) is a protein with both dipeptidyl peptidase and endopeptidase properties which is virtually absent in healthy tissues and has increased expression in chronic inflammatory and fibrotic diseases. In this study, we investigated the presence of FAP in intestinal fibrosis caused by Crohn’s disease (CD) and ulcerative colitis (UC). Methods For this study, transmural surgical resection specimens were retrieved from the Tytgat institute IBD biobank located at Amsterdam University Medical Center. IBD patients undergoing an ileocecal resection for stricturing CD or a subtotal colectomy for therapy-refractory disease, and patients undergoing a right-sided hemicolectomy or ileocecal resection for dysplasia or non-metastasized colorectal cancer (CRC), were included. All patients had an established diagnosis of (CD, UC, dysplasia or CRC) by previous endoscopy and biopsies and had provided informed consent prior to surgery. FAP protein and gene expression levels were determined using immunohistochemistry staining and quantitative polymerase chain reaction (qPCR). Mass cytometry was used to identify FAP–expressing cells. Results In total, 59 ileal and 35 colonic samples were stained and quantified to asses FAP expression (figure 1). A 1.7-fold and 1.9-fold increase of FAP protein expression was seen in inflamed (mean 22.3, p=0.0069); and stenotic (mean 25.5 p=0.0002) CD ileum compared to healthy non-IBD tissue (mean 13.1). Inflamed UC colon (mean 26.8, p = 0.0406) showed a 1.4-fold increase compared to healthy colon (mean 19.2). For the gene expression, 66 ileal and 42 colonic samples were processed and used for qPCR analysis. A 5-fold and 6.4-fold increase of FAP was found in inflamed (mean 0.011, p=0.0475) and stenotic (mean 0.014, p=0.0042) CD ileum, and a 1.7-fold increase was seen in inflamed UC colon (mean 0.278, p=0.0195) compared to controls (means: ileum 0.002; colon 0.165). Conclusion This is the first study reporting the potential of FAP as a fibrotic biomarker not only in stricturing CD, but also in therapy-refractory UC. Significantly increased FAP protein and gene expression is found in inflamed and stenotic ileum in CD and inflamed colon in UC. Additionally, CD90+GP38+ cells have been identified as the most present FAP expressing cells.

Background Up to 30% of Crohn's Disease (CD) patients suffer from perianal fistula creating a high disease burden. Treating CD fistula is challenging compared to cryptoglandular fistula, which have a significantly better wound healing response. Previous studies suggested epithelial to mesenchymal transition as an inducer of fistula formation, as markers were observed in fistula tracts. However, most studies compared CD fistula to normal mucosa, thus disregarding whether any observed difference was in fact a sign of healing (as CD fistula will have more active healing than uninjured mucosa) or a sign of pathology (as healing is not occurring effectively). Therefore, in this study we evaluated CD fistula, as well as both healthy mucosa (no active healing) and cryptoglandular fistula (effective healing) in order to determine the role of differences found. Methods Biopsies from the internal opening (n=20) of perianal fistula of CD patients were collected and compared to two publicly available datasets obtained from un-inflamed rectum (RISK cohort and GSE83245). In addition, curettage material of CD (n=54) and cryptoglandular fistula (n=15) was obtained. Expression profiling was performed using RNASeq. Results Comparing the mucosa at the internal opening of CD fistula to publicly available datasets of healthy rectum, 770 genes were differentially expressed. Pathway analysis revealed upregulation of proliferative and inflammatory processes, consistent with expected immune and wound healing responses. Interestingly, strongest upregulation was seen for processes of cornification and keratinization, including expression of IVL (involucrin) and FLGR2 (fillagrin2), involved in keratinocyte differentiation. To evaluate whether this was contributing to active wound healing or rather to persistent disease, we analyzed the actual tract in CD and cryptoglandular fistula. Strikingly, cornification and keratinization processes were more pronounced in cryptoglandular fistula, suggesting a role in effective healing rather than the persistent pathology seen in CD. Similar data was observed for TGFb, previously detected in CD fistula and thus considered a pathological contributor. While we confirm increased TGFb activity in CD fistula compared to normal mucosa, activity in cryptoglandular fistula tracts was higher than in CD fistula tracts. Again, this would suggest TGFb signaling as part of the wound healing response rather than part of the pathogenic mechanism. Conclusion Perianal fistulas show upregulation of keratinization and cornification pathways compared to healthy mucosa. Comparing CD and cryptoglandular fistula, we saw more cornification in the better healing cryptoglandular fistula, suggesting involvement in effective healing rather than pathology.

Background Previous studies suggest that faecal calprotectin can be used to discriminate Crohn’s disease (CD) perianal fistulas from cryptoglandular (CG) perianal fistulas. The aim of this research was to prospectively validate these findings and further investigate local calprotectin production inside fistula tracts. The aim of the current study was to prospectively analyse the clinical value of calprotectin levels in both the faeces and the fistula tract. Methods In this prospective study, all consecutive patients with an active perianal fistula undergoing examination under anesthetics were included. Faecal and scraping calprotectin levels collected during surgery were measured. The primary objective was to differentiate between active CD perianal fistulas and CG perianal fistulas based on faecal calprotectin. Secondary outcome parameters were the levels of local calprotectin in fistula tracts and the correlation with fistula characteristics and clinical prognostic parameters like the Geldof Classification system. Results Sixty-three fistula patients were included in this study (CD n=45; CG n=18). Faecal calprotectin values were significantly higher in CD patients compared to CG patients (396.8 [61.9-1136.8] vs. 47.3 [14.6-233.6] ug/g, p=0.004). The Area Under the Curve (AUC) was 0.733 (95% CI [0.644 to 0.903] with an optimal FC cut-off value of 344ug/g. Faecal calprotectin was not related to calprotectin levels in fistula scrapings which was not significantly different in CD and cryptoglandular fistulas (p=0.893). Calprotectin in fistula scrapings was also not related to fistula characteristics like number of tracts, duration of disease or (previous) seton drainage. However, a significant correlation was found between scraping calprotectin and fistula disease severity and prognosis as defined by a new Geldof classification system: Patients in group 2a (amenable for fistula repair) had significantly lower calprotectin levels (140 [31.0-149.0])ug/g) when compared to class 2b (symptom control) (706 [198.5 – 1936]ug/g, p=0.054) and class 2c (gradually debilitating disease) (3464.0 [328.5-5692.0]ug/g, p=0.008). Patients in class 3 (exhausted perineum) had the highest calprotectin levels highest (5928.0 [2605.0-6000]ug/g). Conclusion CD and CG perianal fistulas can be distinguished by measuring faecal calprotectin levels. The difference can not be explained by local fistula calprotectin production as calprotectin in fistula scrapings is not predictive of CD. However, the results of this study suggest that scraping calprotectin levels may be used to classify fistula patients and predict outcome of fistula treatment.

Background Perianal Crohn’s Disease (pCD) is a frequent and debilitating complication of Crohn’s Disease (CD). Magnetic Resonance Imaging (MRI) is the imaging modality for diagnosis, classification, and treatment monitoring in pCD. To evaluate treatment response, a validated radiological index is mandatory. Recently, the MAGNIFI-CD index has been developed. The aim of our study was to validate the MAGNIFI-CD index; evaluating reliability, responsiveness and accuracy. Methods At two tertiary IBD referral centers, patients (≥16yrs) with complex pCD that had pelvic MRI before and 3-24 months after medical and/or surgical treatment were included. Two independent, blinded abdominal radiologists randomly scored total MAGNIFI-CD and its separate items. Two clinicians scored clinical outcomes (remission, response, non-response) using the “fistula drainage assessment” (FDA). Interobserver agreement (ICCs or weighted K), responsiveness (according to FDA; Wilcoxon signed rank test), and test accuracy (ROC analyses for the follow-up (FU) MAGNIFI-CD) were determined. Youden indices (YI) were used to choose optimal cut-off values for the FDA categories. Results Sixty-seven patients (median age 30 [IQR 23-47], 51% female) were eligible with mean CD and pCD duration of 7.0 yrs (SD 8.7) and 3.7 yrs (SD 4.3), respectively. 44% was biological naïve, 9% had defunctioning ostomy and 20% had proctitis. Baseline MAGNIFI-CD was 18 [IQR 9–20]. MAGNIFI-CD had an almost perfect ICC of 0.88 (95%CI 0.78-0.92); reliability of the separate items was moderate to substantial. Between baseline and FU MAGNIFI-CD a significant decrease was observed in responders and remitters (p Conclusion The MAGNIFI-CD index is a reliable radiological index supported by an almost perfect agreement. Given the moderate agreement, the item “dominant feature” would need additional training or better definitions. The index shows a robust responsiveness to clinical change and does not improve significantly in non-responders. The MAGNIFI-CD seems to be an acceptable to excellent test to accurately determine response or remission. We suggest a cut-off value of ≤14 for response and ≤10 for remission at follow-up MAGNIFI-CD. These results support that the MAGNIFI-CD is suitable for treatment monitoring in clinical trials.

Background Adequate scoring of endoscopic recurrence in Crohn’s disease patients is of utmost importance, as it is used to diagnose recurrent disease, to predict disease outcomes, to (re-)initiate or change therapy and to monitor its effect. This study aims to assess the variance in endoscopic recurrence in patients after ileocolic resection for Crohn's disease using the most common classification systems, the Rutgeerts (RS) and modified Rutgeerts (mRS) classification. Methods A systematic literature search using MEDLINE, Embase and the Cochrane Library was performed. All RCTs and cohort studies describing endoscopic recurrence after an ileocolic resection for Crohn's disease were included. Main outcome was the range of endoscopic recurrence rates within twelve months postoperatively, defined as RS≥i2 and/or mRS≥i2b. A proportional meta-analysis was performed. Results Seventy-six studies comprising 7751 patients were included. The weighted mean of the endoscopic recurrence rates, defined as either RS≥i2 and/or mRS≥i2b, was 43.99% (95 per cent CI 43.56 – 44.43), ranging from 5.0% – 93.0%, fig 1. For studies reporting endoscopic recurrence defined as ≥i2 and for studies reporting recurrence defined as ≥i2b the weighted means were 44.0% (range 5.0% – 93.0%) and 41.1% (range 19.8% – 62.9%) respectively. Within studies reporting both RS and mRS the weighted means for endoscopic recurrence were 61.3% (range 39.1% – 84.7%) and 40.6% (range 19.8% – 62.0%) respectively. Conclusion This study demonstrates a major variance in scoring endoscopic recurrence after ileocolic resection for Crohn's disease, with the greatest variance when using the original Rutgeerts classification. This suggests a high likelihood of inadequate diagnosis of disease recurrence, with major implications for quality of life and health care consumption. Therefore, there is an important need to improve endoscopic scoring of recurrent disease.

Background Appendectomy has been suggested to decrease the risk of disease flares in UC, but we recently showed that even in therapy refractory patients, appendectomy may improve symptoms in a subset of patients. Despite several hypotheses, the effector mechanism of this phenomenon remains unclear. In this study we characterized gene expression profiles of appendices from therapy refractory UC patients either responding or non-responding to appendectomy, with the aim to identify pathways involved in response as well as guide selection of patients who are optimal candidates for appendectomy. Methods Appendix specimen from responders (N=10) and non-responders (N=10) to appendectomy for therapy refractory UC were included (COSTA-study NCT03912714 or off-label appendectomy). Endoscopic response was defined as steroid-free remission at 12 months without intensified medical treatment. The control group consisted of patients with acute appendicitis(N=2). Post-operative biopsies were taken from the appendix and the coecal base and snap frozen. RNA was isolated and expression profiling was performed using RNAseq. Results Unsupervised clustering indicated two separate clusters, one containing a more stromal phenotype expressing ADH1B, IGFBP5, Desmin and FLNA and the other expressing a more intestinal/B cell phenotype (OLMF4, PIGR, CEACAM5 and IHGA2). Stainings for the presence of collagen (stromal) versus pancytokeratin (epithelial) confirmed this analysis on protein level. Both clusters contained subclusters of responders and non-responders. In both the ‘responder’ subclusters, markers of immune activation were increased, but the type of response differed between ‘stromal responders’ and ‘epithelial responders’. In ‘stromal responders’ increased expression of acute phase genes was present (FABP4, ADIPOQ, SAA1 and CXCL8). In the ‘epithelial responders’, immune activity was more characterized by increased mitochondrial activation/oxidative phosphorylation. In both subclusters, non-responders lacked these immune activation markers. We also evaluated the expression of the differentiating genes in biopsies of the coecal bottom. Indeed, the coecal samples also largerly clustered according to response based on the gene expression profile of the matching appendices. Conclusion Patients responding to appendectomy showed two distinct gene expression patterns, suggesting two separate mechanisms of action. Although in both cases it clearly identified the requirement of active immune responses for response, the identifying markers differed between the two groups. This finding has consequences for both further functional studies into the effector mechanism of appendectomy in UC, but also in future efforts to identify patients suitable for this surgery.

Background A common complication of Crohn’s disease(CD) are perianal fistulae, highly impacting the quality of life in these patients. The pathogenesis of these fistulas is incompletely understood. Treatment is often given based on knowledge of disease processes in the inflamed intestine itself, but whether the immune activity in the fistula tract is similar remains unknown. Full characterization of the intestinal mucosa versus the fistula tract itself may reveal immunological differences between the two, and generate leads for both development of new interventional options as well as allow better stratification of patients. Methods Matched biopsies were obtained from the rectal mucosa, the internal opening of the fistula and the tract itself. A total of 30 patients were included (20 CD; 10 non-IBD patients). Tissues were processed to cell suspensions and selected for live CD66b- cells. Single cell RNASeq analysis was performed using 10x Chromium. In total, 111.955 immune cells were annotated and used for analysis. Results T cells comprised the largest cluster of immune cells, which was subclustered into 19 clusters. Both the internal opening and the fistula tracts contained elevated proportions of effector memory and cycling T cells. Conversely, the rectum contained more central and tissue resident memory cells. T cells expressing IL-22, which were previously described in fistula were detected in considerable number in the rectum and internal opening, but were scarce in the fistula tract itself. These cells were clearly distinct from the IL17A producing subset, and IL17F expressing cells and mainly found in the fistula tracts. Only the tract contained a clear subset of CXCL13 expressing T cells, suggesting follicular helper T cells. Presence of these cells was strongly correlated to the presence of lymphoid inducer cells as well as IgG producing B cells rather than the IgA subset found in the rectum. Clusters of myeloid cells were largely derived from the fistula tracts. Subclustering into 10 clusters indicated a shift from anti-inflammatory to pro-inflammatory macrophages in the tracts compared to the rectal mucosa. Finally, a striking difference in NK cells subsets was observed, with significantly more CD56bright NK cells present in the rectal samples while the internal opening and the tracts were comparable. Conclusion Cellular composition and activity of the immune cell compartment differs considerably between the rectum, the internal opening of the fistula and the tract. Subsequently, therapy targeted at processes involved in mucosal inflammation may not actually target the fistula itself, which may explain the low success rate in fistula resolution. Careful analysis of the fistula may help to identify targets useful in the treatment.

Background Increasing evidence suggests a role for the vermiform appendix in onset and relapse of ulcerative colitis (UC). Consequently, appendectomy is increasingly being seen as a potential alternative treatment. As a recent study suggests that patients with histological appendiceal inflammation are most likely to respond, pre-operative identification of ulcerative appendicitis could be beneficial1. Intestinal ultrasound (IUS) is increasingly utilized in the diagnosis and follow-up of patients with UC. The objective of this study was to characterize the appendix by IUS in UC patients with active and quiescent disease and healthy controls. Methods In this cross-sectional prospective observational cohort study, we performed appendiceal IUS on UC patients with active (A) disease (FCP>250 and SCCAI>5) and quiescent (Q) disease (FCP1000 examinations). Primary outcomes were signs of appendicitis on IUS, defined as a Results Out of 95 (A n=35, Q n=30, H n=30) included patients, the majority (n=58, 61.1%) had a visible appendix on IUS, which was similar among cohorts (p=0.56). The appendiceal diameter measured >6.0mm in a significantly higher proportion of active UC patients (A: 45.5%, Q: 5.6%, H: 0.0%, p=0.01). An increased diameter occurred irrespective of disease extent (42.9% of E1/E2 endoscopic active patients). Although the appendiceal diameter was higher for UC patients (Q-H p= 0.02, A-H p< 0.01), no significant difference between A and Q patients was found (p=0.15, Fig. 1). A higher proportion of UC patients had incompressibility when compared to healthy controls (Fig. 2a, p= 0.02). Hypervascularity was present in 54.5% of active UC (Fig. 2b, p= 0.02). Fig 1. Box plot of appendiceal diameter (mm) among cohorts Fig 2 (a, b). Bar charts of incompressibility (a) and hypervascularity (b) among cohorts a b Conclusion Appendiceal IUS was feasible in UC patients. Characteristics of appendiceal inflammation of IUS were reported for UC patients, as both the transverse appendiceal diameter and incompressibility rates were significantly higher in UC patients when compared to healthy controls. Correlating appendiceal IUS findings to histological inflammation could contribute to identifying UC patients most likely to benefit from appendectomy.

Background Perianal fistulising Crohn’s disease (CD) is an aggressive disease phenotype that can have a significant impact on patients’ quality of life. Current biological understanding of perianal fistulising CD remains inadequate and previous classification systems have not provided clear guidance on therapy in clinical practice nor on defining patient cohorts within clinical trials. To counter this unmet need, we propose a new classification system for perianal fistulising CD. Methods The proposed classification system was developed through a modified nominal group technique expert consensus process involving open discussion and formal voting on previously defined statements. Consensus agreement was defined a priori as 80% voting “strongly agree” or “agree with minor reservation”. Participants included gastroenterologists, radiologists, surgeons active in a tertiary IBD centre and a patient representative. Results The classification identifies four groups of patients with perianal fistulising CD. Key elements include stratification according to disease severity as well as disease outcome; synchronisation of patient and clinician goals in decision making, with a proactive, combined medical and surgical approach, on a ‘treat to patient goal’ basis; and identification of indications for curative fistula treatment, diverting ostomy and proctectomy. The new classification retains an element of flexibility, in which patients can cycle through different classes over time. Furthermore, with each specific class comes a paired treatment strategy suggestion and description of clinical trial suitability. Conclusion The proposed classification system is the first of its kind and is an important step towards tailored standardisation of clinical practice and research in patients with perianal fistulising CD.

Background The reliability and reproducibility of scoring endoscopic recurrence is of utmost importance because it generally determines initiating postoperative medical therapy and it is used as outcome parameter in clinical trials. Variability in endoscopic scoring may result in inappropriate therapeutic decisions and study conclusions. It is hypothesised that different types of anastomoses have an influence on scoring endoscopic recurrence using the Rutgeerts’ score. The aim of this study is to analyse to what extent the ulcerations are determined by the type of anastomosis and how it interferes with the scoring of Crohn’s recurrence. Methods This is a retrospective review of endoscopy videos to re-assess endoscopic recurrence. All videotapes of the ileocolonoscopy were blinded and scored by a trained gastroenterologist (MD) and a surgeon (WB). Videos were excluded if the quality of the video was low or if the anastomosis was not visualised properly. The primary outcome is the presence of ulcerations six months after surgery, on individual parts of the anastomosis: inverting stapled anastomosis (side to side conjunction) and everting type of junction (transection ends). In the latter group, at least one site – colon, ileum or both – was scored. Results Of 99 available endoscopy videos, eighty-nine eligible patients were included in this retrospective study for analysis. The inverting and everting stapled anastomosis were scored individually using the SES-CD. A SES-CD of zero was found in 21/89 (23.6%) of the inverting anastomoses versus 74/75 (98.67%) in the everting anastomoses. Conclusion This study demonstrated a significant difference in wound healing of the inverting anastomosis versus the everting anastomosis. Inverting anastomoses show ulcerations and a circular scar long after surgery irrespective of the presence of ulcerations of terminal ileum. A normal wound healing phenomena might mistakenly be seen for recurrent Crohns’ disease. Everting anastomosis, on the contrary, heal without residue.

Background Approximately 10% of ulcerative colitis (UC) patients undergoing ileal pouch-anal anastomosis (IPAA) are believed to develop ‘de novo Crohn’s disease’ (CD) of the pouch (1). Clear diagnostic criteria are lacking, but the diagnosis is mostly based on the development of fistula, strictures, treatment refractory disease, pre-pouch ileitis, or typical endoscopic findings fitting with CD, such as ulcers and patchy inflammation (1, 2). However, the findings on which this diagnosis is established may be caused by (unrecognized) surgical complications such as silent anastomotic leakage. It is important to distinguish these complications from what is truly a ‘de novo CD’ as treatment strategies differ in both groups. We aimed to investigate incidence rates and reasons for diagnosing CD of the pouch. Methods In total, 483 consecutive UC patients who underwent a proctocolectomy and IPAA in a tertiary IBD referral centre from January 1990 until December 2017 were retrospectively reviewed. Patients with a diagnosis ‘CD of the pouch’ were identified based on medical records, and could be based on either clinical, endoscopic, imaging and/or histological findings. Results Median follow-up time was 20 years (IQR 10 – 24). In 46 out of 483 patients (10%), de novo CD of the pouch was diagnosed, which was mostly based on endoscopic findings (29/46, 63%). Presence of pre-pouch ileitis was the most common reason to diagnose CD of the pouch, which was present in 18/29 patients (62%), followed by ulcerations in 6/29 (21%). On MRE, 19/46 (41%) patients had a fistula or sinus, 4/46 patients (9%) had a stricture (all proximal of the pouch). Interestingly, 12/46 patients (26%) never underwent cross sectional imaging after IPAA construction. Only ten patients (22%) showed histologic findings with a preference for CD in biopsies, revised colectomy specimen, and/or pouch excision specimen. Only two patients had granulomas, others showed signs of transmural inflammation, ulcerations or fibrosis. Other histologic findings were non-specific. Conclusion In our centre, 10% of patients were ‘diagnosed’ with de novo CD of the pouch. However, as confirmatory histological signs of CD were present in only 22%, chronic surgical complications as underlying cause might be of greater importance. It is advised to perform cross sectional imaging in every patient after a year after pouch construction to rule out silent leaks that might cause symptoms at a later stage mimicking Crohn’s disease, avoiding a diagnostic dilemma.

Background The aim of this study was to evaluate degree of fibrosis and disease activity using the MAGNIFI-CD index at MRI as predictors of long-term clinical perianal fistula closure in Crohn’s patients who had anti-TNF therapy and/or surgical closure. Methods Perianal Crohn’s patients who had anti-TNF and/or surgery between 2013 and 2020 with a post-treatment MRI and long-term clinical follow-up data were retrospectively included. Two radiologists scored the degree of fibrosis and items of the MAGNIFI-CD index at the post-treatment MRI. Long-term clinical closure was assessed 12 months after the post-treatment MRI and was defined as no production on palpation and/or closed external openings and absence of re-interventions. The accuracy of findings at MRI in predicting long-term clinical closure was evaluated in terms of a receiver operating characteristics (ROC) analysis. An area under the curve (AUC) with 95% confidence interval (CI) was calculated. Results Forty-nine patients were included; 31 female, median age 33 years (IQR 26–46). Post-treatment MRI was performed at a median of 7 months (IQR 4–14) after treatment. At long-term clinical follow-up, 25 patients showed fistula closure and 24 did not. Fourteen patients showed a 100% fibrotic fistula at post-treatment MRI of which all had a closed fistula at long-term clinical follow-up (Figure 1). ROC analysis showed an AUC of 0.90 (95% CI: 0.81–0.99) for degree of fibrosis in predicting long-term clinical closure. The median MAGNIFI-CD index at post-treatment MRI was 0 (IQR 0–5) in patients with long-term clinical closure and 16 (IQR 10–20) in patients without (Figure 2). ROC analysis showed an AUC of 0.95 (95% CI: 0.89–1.00) for the MAGNIFI-CD index in predicting long-term clinical closure. Abstract DOP11 – Figure 1: Degree of fibrosis at the post-treatment MRI in relation to long-term clinical closure. Abstract DOP11 – Figure 2: MAGNIFI-CD index at the post-treatment MRI in relation to long-term clinical closure. Conclusion Degree of fibrosis and MAGNIFI-CD index at post-treatment MRI are accurate in predicting long-term clinical closure of perianal fistulas in Crohn’s patients and can be valuable parameters for radiological follow-up. A completely fibrotic tract at MRI seems a robust indicator for long-term fistula closure.

Background Myeloid cells are key players in the sustained inflammatory process in Crohn’s disease (CD), and are therefore therapeutic targets. Human mononuclear myeloid cells express the cell selective expression of carboxylesterase 1 (CES-1). Esterase-sensitive motif (ESM) technology has been developed to specifically target ESM tagged small molecules on human mononuclear myeloid cells, based on their CES-1 expression, creating a larger therapeutic window with less side effects. This study investigates the presence of CES-1 expressing cells in PBMCs, intestinal biopsies and fistula cells of CD patients, highlighting the potential of ESM-technology in CD. This could benefit CD treatment in non-responsive patients or patients with a fistulizing phenotype. Methods Intestinal biopsies were collected from CD patients who underwent endoscopy. PBMCs were collected from CD patients who were receiving Vedolizumab (VDZ) treatment and categorized as responders or non-responders. Criteria for response was based on clinical (HBI ≤4) and biochemical (fecal calprotectine ≤250 µg/g) and/or endoscopic assessment (≥50% reduction in SES-CD score). Fistula tissue was of CD patients was obtained during surgery. CES1-positive cells were phenotyped by mass cytometry with a panel of 32-markers, including the myeloid markers CES-1, CD14, CD16, HLA-DR, CD11c, CD206. Results We demonstrated the presence of CD14+HLADR+CD11a+CD44+CD11b+ myeloid cells exclusively expressing CES-1 in intestinal biopsies of CD patients (n=7). CES-1 expression did not differ between inflamed and uninflamed tissue. In PBMCs of VDZ responders (n=5) and non-responders (n=6), we were able to identify different subsets of myeloid cells such as classical (CD14++CD16-), intermediate(CD14+CD16+) and non-classical (CD14-CD16++) monocytes, DCs and CD2+ DC precursors all demonstrating CES-1 expression. Percentage of CES-1 positive cells in inflamed PBMCs (or non-responders) was higher than in uninflamed PBMCs (responders) in all of the above-mentioned subsets. Furthermore, we demonstrated different myeloid populations within fistula cells (n=13) such as pDCs (CD123+CD68+HLA-DR+), DCs (CD11c+CD11b+HLA-DR+ CD44+), CD141+DCs (CD141+CD49d+HLA-DR+),Macrophage (CD14+CD11c+CD11b+HLA-DR+CD44+) and M2 Macrophages (CD14+CD163+CD206+CD11c+CD11b+HLA-DR+CD44+), all expressing CES-1. Conclusion We demonstrated specific CES-1 positive myeloid cells within intestinal biopsies, PBMCs of non-responsive patients, and fistulae samples of CD patients. Targeting CES-1+ with ESM-technology could provide new therapeutic targets for CD.

Background The PISA-II trial showed that anti-TNF induction combined with surgical closure induces MRI healing more frequently than anti-TNF alone at 18 months follow-up (FU) and that this was associated with no recurrences at the time. The aim of this study was to compare long-term outcomes of both treatment arms. Methods In this FU study, data were collected from patients who participated in the PISA-II trial, a multicentre, international patient preference randomised controlled trial that compared anti-TNF induction combined with surgical closure to anti-TNF therapy alone as treatment for Crohn’s perianal fistulas. FU data were collected from the time of enrolment in the PISA-II trial until November 2021. Primary outcome was radiological healing at MRI and secondary outcomes included long-term clinical closure, recurrences, anti-TNF trough levels (suboptimal level was defined as infliximab Results Long-term FU data were collected from 88 out of 94 patients included in the PISA-II trial, 35/38 in the surgical closure arm and 53/56 in the anti-TNF treatment arm. Median FU was 5 years (IQR 4–7). During long-term FU radiological healing occurred in significantly more patients in the surgical closure arm (40% vs 17%; P=0.018). Long-term clinical closure occurred in 71% in the surgical closure arm and in 60% in the anti-TNF treatment arm (P=0.533). Recurrences occurred in 20% in the surgical closure arm and in 36% in the anti-TNF treatment arm (P=0.111). One patient with radiological healing developed a recurrence (4.3%) vs 12/57 (21.1%) patients with clinical closure. Trough levels were available in 32/53 patients treated with anti-TNF. Around the time of recurrence a suboptimal anti-TNF serum trough level occurred in 6/10. In the surgical closure arm, 6 patients had more and 6 patients had less incontinence problems after treatment, comparable to the 5 patient in the anti-TNF treatment arm with more and 8 with less incontinence problems. All patients in the surgical closure arm who completed the questionnaire (strongly) agreed that undergoing surgery was the right decision and 79% of the patients in the anti-TNF arm (strongly) agreed, 14% neither agreed nor disagreed and 7% disagreed that anti-TNF treatment was the right decision. Conclusion This study further supports the previous finding that anti-TNF induction combined with surgical closure should be considered in patients with Crohn’s perianal fistulas as long-term outcomes are favourable. Interestingly, surgical closure does not seem to be correlated to decreased continence, and all patients agreed that surgery was the right decision.

Background Studies on the colorectal cancer (CRC) risk in patients with ulcerative colitis (UC) show conflicting results. The aim of the current study was to assess if there is an indication shift for surgery in patients with UC from refractory disease to malignant degeneration over the last 3 decades. Methods From the nationwide Dutch Pathology Registry (PALGA), pathology excerpts from all patients with histologically confirmed UC who underwent a colorectal resection for all indications between January 1st, 1991 and November 12th, 2020 were included. The primary outcome was the CRC rate in the colon specimens. Outcomes were compared between 3 time periods (P1: 1991–2000, P2: 2001–2010, P3: 2011–2020). Results Overall, 6,094 UC patients were included of which 5,026 underwent a (procto)colectomy and 1,068 a segmental resection. In 1,031 (16.9%) patients, pathological examination demonstrated CRC after a median disease duration of 11 years [IQR 3.0–19.0]. The CRC rate in the colectomy specimen increased from 11.3% in P1, to 16.1% in P2, to 22.8% in P3 (p-trend Conclusion This nationwide pathology study demonstrated an increased surgical indication rate for CRC in UC patients over the last 3 decades. We hypothesize that the expanding therapeutic armamentarium for UC likely leads to exhausting medical options and hence postponed colectomy. This however, might be at the expense of increased risk of CRC in the long term. Despite the introduction of surveillance programs, the incidence of advanced colon carcinoma remains high.

Background Early in inflammation granulocytes migrate from the circulation into inflamed areas and rapidly accumulate. They form an important role in first defense as well as the orchestration of downstream immune responses. In the circulation, several different subsets of granulocytes have been described, for example immature, intermediate and mature granulocytes. Surprisingly, despite their abundance and prominent function, this cell type is rarely studied from a tissue perspective, most likely due to their short lifespan ex vivo. One example of gastrointestinal disease with an extremely high number of granulocytes is the occurrence of perianal fistula which contain a considerable amount of granulation tissue. As curettage of this tissue is part of the treatment, the fistula tract is good source to study tissue derived granulocyte subsets. The aim of this study was to characterize granulocyte subsets in the fistula tract. Methods Curettage material of perianal fistula tracts was obtained during surgical intervention from patients with CD (n=15) and cryptoglandular fistulas (n=5). From 2 CD patients venous blood was taken and a ficoll step was performed. Single-cell suspensions were stained with a 35-antibody panel, including CD66, CD14, CD33, CD16 and CD11b. Samples were analyzed using mass cytometry (CyTOF). Results Granulocytes, characterized as CD45+CD66a+ cells, were the main cellular component of the fistula tract (64%±24) compared to other immune cells. This was more pronounced in cryptoglandular fistula compared to CD fistula (81% vs. 67%; p=0.019). Within these cells we could differentiate between 4 subsets, mature granulocytes (CD33+CD11b+CD16+), intermediate (CD33intCD11b+CD16int), immature granulocytes (CD33intCD11b-CD16int) and eosinophils (CD33-CD11b-CD16-). Immature granulocytes were the most abundant in de fistula tract (31%±15), closely followed by the mature (30%±23) and intermediate (23±9). No significant difference was seen in the percentages of granulocyte subsets between CD and cryptoglandular fistula. Conclusion Here, we show the presence of different subtypes of granulocytes in fistula derived granulation tissue. We show a heterogeneous group of cells ranging from immature neutrophils to mature neutrophils. Due to their high number in active ulcerating tissue, further evaluation of granulocyte subsets and function could be an interesting target for medical treatment options.

BACKGROUND & AIMS: Vitamin D deficiency is common in Crohn's disease (CD). High-dose vitamin D had anti-inflammatory effects in preclinical studies and trials of patients with CD. We performed a randomized trial to determine whether high-dose vitamin D prevents postoperative recurrence of CD after ileocolonic resection. METHODS: Patients with CD after ileocolonic resection with ileocolonic anastomosis were assigned randomly to groups given weekly 25,000 IU oral vitamin D (n = 72) or placebo (n = 71) for 26 weeks, at 17 hospitals in The Netherlands and Belgium, from February 2014 through June 2017. Patients were assessed at baseline and at weeks 2, 6, 12, and 26 for laboratory and clinical parameters, and underwent ileocolonoscopy at 26 weeks. The primary end point was endoscopic recurrence (modified Rutgeerts score, ≥i2b, as assessed by blinded readers) at 26 weeks. Secondary end points included clinical recurrence (Crohn's disease activity index, ≥220), quality of life (measured by the 36-Item Short Form Health Survey, Inflammatory Bowel Disease Questionnaire, and EuroQol, a 5-dimension questionnaire), and outcomes associated with the baseline serum concentration of vitamin D. RESULTS: In the vitamin D group, serum levels of 25-hydroxy vitamin D increased from a median of 42 nmol/L at baseline to 81 nmol/L at week 26 (P < .00001), whereas levels did not change significantly in the placebo group and remained unchanged at 43 nmol/L. In the intention-to-treat analysis, the proportion of patients with endoscopic recurrence at 26 weeks did not differ significantly between the vitamin D vs the placebo group (58% vs 66%; P = .37). The cumulative rate of clinical recurrence did not differ significantly between the groups (18.1% in the vitamin D group vs 18.3% in the placebo group; P = .91). Quality of life improved slightly over time in both groups, but did not differ significantly between groups (P = .07). There were few adverse events in either group. CONCLUSIONS: High-dose vitamin D, compared with placebo, did not reduce the incidence of postoperative endoscopic or clinical recurrence of CD in patients who underwent ileocolonic resection with ileocolonic anastomosis. ClinicalTrials.gov no: NCT02010762. ispartof: CLINICAL GASTROENTEROLOGY AND HEPATOLOGY vol:19 issue:8 pages:1573-+ ispartof: location:United States status: published

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