Assessing Disease Activity in Pediatric Crohn's Disease Using Ultrasound: The Pediatric Crohn Disease Intestinal Ultrasound Score.

OBJECTIVES: Currently, there is no consensus on how to score Crohn disease (CD) activity assessed by intestinal ultrasound (IUS) in children. This study aimed to design an easy-to-use IUS score for disease activity in pediatric CD. METHODS: Children undergoing ileo-colonoscopy for CD assessment underwent IUS the day before ileo-colonoscopy, assessed with simple endoscopic score for CD (SES-CD). IUS features were compared to the SES-CD on segmental level. Multiple regression analyses, separately for terminal ileum (TI) and colon, were done to assess predictors of disease activity and to develop a model. RESULTS: In 74 CD patients (median 15 years, 48% female), 67 TI and 364 colon segments were assessed. Based on receiver operating characteristics curves, bowel wall thickness (BWT) was categorized into low [1 point: 2-3 mm (TI) and 1.6-2 mm (colon)], medium [2 points: 3.0-3.7 mm (TI) and 2.0-2.7 mm (colon)], and high [3 points: >3.7 mm (TI) and >2.7 mm (colon)]. In TI, only BWT was retained in the model [high BWT: odds ratio (OR) 11.50, P < 0.001]. In colon, BWT (high BWT: OR 8.63, P < 0.001) and mesenteric fat (1 point: OR 3.02, P < 0.001) were independent predictors. A pediatric Crohn disease IUS score (PCD-US) cut-off of 1 resulted in a sensitivity of 82% (95% confidence interval, CI: 65%-93%) and 85% (95% CI: 80%-89%) and a cut-off of 3 in a specificity of 88% (72%-97%) and 92% (87%-96%) for TI and colon, respectively. Inter-observer agreement was moderate for TI and colon ( K : 0.42, K : 0.49, respectively). CONCLUSIONS: The PCD-US score is an easy-to-use and reliable score to detect or rule out CD activity on segmental level in children. External validation is needed before applying this score in clinical practice.

Intestinal Ultrasound to Assess Ulcerative Colitis Disease Activity in Children: External Validation and Comparison of 2 Intestinal Ultrasound Activity Indices.

BACKGROUND: There is currently no consensus on the definition of an abnormal intestinal ultrasound (IUS) for children with ulcerative colitis (UC). This cross-sectional study aimed to externally validate and compare 2 existing IUS indices in children with UC. METHODS: Children undergoing colonoscopy for UC assessment underwent IUS the day before colonoscopy, assessed with the Mayo endoscopic subscore. The UC-IUS index and the Civitelli index were compared with the Mayo endoscopic score in the ascending, transverse, and descending colon. The area under the receiver-operating characteristic curve for detecting a Mayo endoscopic score ≥2 of both scores was compared and sensitivity and specificity were calculated. RESULTS: A total of 35 UC patients were included (median age 15 years, 39% female). The area under the receiver-operating characteristic curve was higher for the UC-IUS index in the ascending colon (0.82 [95% confidence interval (CI), 0.67-0.97] vs 0.76 [95% CI, 0.59-0.93]; P = .046) and transverse colon (0.88 [95% CI, 0.76-1.00] vs 0.77 [95% CI, 0.60-0.93]; P = .01). In the descending colon, there was no difference (0.84 [95% CI, 0.70-0.99] vs 0.84 [95% CI, 0.70-0.98]). The optimal cutoff for the UC-IUS was <1 point to rule out a Mayo endoscopic score ≥2 (sensitivity: 88%, 100%, and 90% in the ascending, transverse, and descending colon, respectively) and a Mayo endoscopic score ≥2 could be detected using a cutoff of >1 (specificity: 84%, 83%, and 87%, respectively). For the Civitelli index, in our cohort, the optimal cutoff was <1 to rule out a Mayo endoscopic score ≥2 (sensitivity 75%, 65%, and 80%, respectively) and a cutoff >1 to detect a Mayo endoscopic score ≥2 (specificity 89%, 89%, and 93%, respectively). CONCLUSIONS: In this cohort, the UC-IUS index performed better than the Civitelli index. The UC-IUS index had both a high sensitivity and specificity in this cohort, when using 1 point as cutoff for a Mayo endoscopic score ≥2.

In this prospective study, we validated and compared 2 intestinal ultrasound indices to score pediatric ulcerative colitis: the UC-IUS index and the Civitelli index. In our cohort, the UC-IUS index was more accurate.

Intestinal Ultrasound Early on in Treatment Follow-up Predicts Endoscopic Response to Anti-TNFα Treatment in Crohn's Disease.

BACKGROUND: To assess treatment response, objective measures are superior to clinical improvement in Crohn's disease [CD]. Intestinal ultrasound [IUS] is an attractive, non-invasive alternative to endoscopy, demonstrating early transmural changes after treatment initiation. Therefore, we investigated IUS and contrast-enhanced ultrasound [CEUS] to predict [early] endoscopic treatment response. METHODS: Consecutive patients with endoscopically active CD, starting anti-TNFα therapy, were included. Clinical, biochemical, IUS, and CEUS parameters at baseline [T0], after 4-8 weeks [T1] and 12-34 weeks [T2] were collected. The most severely inflamed segment at endoscopy (highest segmental Simplified Endoscopic Score for Crohn's Disease [SES-CD]) and IUS (highest segmental bowel wall thickness [BWT]) was identified. At T2, endoscopic response [decrease in SES-CD ≥ 50%] and remission [SES-CD = 0] were scored. RESULTS: A total of 40 patients were included: 14 reached endoscopic remission and 17 endoscopic response. At T1 (3.1 mm [1.9-4.2] vs 5.3 mm [3.8-6.9], p = 0.005) and T2 (2.0 mm [1.8-3.1] vs 5.1 [3.0-6.3] mm, p = 0.002) BWT was lower in patients with endoscopic remission. At T1 and T2, 18% (area under the receiver operating curve [AUROC]: 0.77; odds ratio [OR]: 10.80, p = 0.012) and 29% [AUROC: 0.833; OR: 37.50, p = 0.006] BWT decrease predicted endoscopic response, respectively. To determine endoscopic remission, BWT 3.2 mm was most accurate [AUROC: 0.94; OR: 39.42, p < 0.0001] at T2. In addition, absence of colour Doppler signal [OR: 13.76, p = 0.03] and the CEUS parameter wash-out rate [OR: 0.76, p = 0.019] improved the prediction model. CONCLUSIONS: Reduction in BWT, already after 4-8 weeks of follow-up, predicted endoscopic response and remission. CEUS parameters were of limited value. Furthermore, we have provided accurate cut-offs for BWT reflecting endoscopic response and remission at different time points.

Point-of-care Intestinal Ultrasound in IBD Patients: Disease Management and Diagnostic Yield in a Real-world Cohort and Proposal of a Point-of-care Algorithm.

INTRODUCTION: Intestinal ultrasound [IUS] is useful for assessment of inflammation, complications, and treatment follow-up in inflammatory bowel disease [IBD] patients. We aimed to study outcomes and impact on disease management for point-of-care [POC] IUS in IBD patients. METHODS: Two patient cohorts undergoing POC IUS [January 2016-July 2018 and October 2019-December 2019] were included retrospectively. Disease management after IUS was analysed and IUS outcomes were compared with symptoms, biomarkers, and additional imaging within 8 weeks from IUS. To study differences in use of IUS over time, cohorts were compared. RESULTS: In total, 345 examinations (280 in Crohn's disease [CD]/65 in ulcerative colitis [UC]) were performed. Present inflammation on IUS was comparable between symptomatic and asymptomatic CD [67.6% vs 60.5%; p = 0.291]. In 60%, IUS had impact on disease management with change in medication in 47.8%. Additional endoscopy/magnetic resonance imaging [MRI] was planned after 32.8% examinations, showing good correlation with IUS in 86.3% [ρ = 0.70, p <0.0001] and 80.0% [ρ = 0.75, p <0.0001] of cases, respectively. Faecal calprotectin was higher in active versus inactive disease on IUS [664 µg/g vs 79 µg/g; p <0.001]. Over the years, IUS was performed more frequently to monitor treatment response and the use of MRI was reduced within the cohort. CONCLUSIONS: POC IUS affects clinical decision making and could detect preclinical relapse in CD patients, with potential to reduce additional endoscopy or MRI. In addition, the paradigm expands towards monitoring treatment and close follow-up for IUS. Based on our results, we propose a POC IUS algorithm for follow-up of IBD patients.

Hyperbaric oxygen therapy for the treatment of rectovaginal fistulas in patients with Crohn's disease: results of the HOT-REVA pilot study.

BACKGROUND: Positive effects of hyperbaric oxygen (HBO) on perianal fistulas in Crohn's disease (CD) have been described, but the effect on rectovaginal fistulas (RVFs) has not yet been studied. The aim was to investigate the efficacy, safety and feasibility of HBO in patients with RVF in CD. METHODS: In this prospective study, consecutive CD patients between November 2018 and February 2020 presenting with RVF at the outpatient fistula clinic of the Amsterdam University Medical Centre were included and selected to receive treatment with 30 daily HBO sessions, if fistulas were actively draining and any concomitant treatment regimen was stable at least 6 weeks prior to start of HBO. Patients with a stoma were excluded. The primary endpoint was clinical closure at 3-month follow-up, defined as cessation of complaints and/or closure of the external orifice if visible at baseline. Secondary outcomes were improvement of concomitant perianal fistulas as measured by the perianal disease activity index (PDAI) and fistula drainage assessment (FDA), as well as improvement in patient-reported outcomes (visual analogue scale (VAS), inflammatory bowel disease questionnaire (IBDQ), faecal incontinence quality of life scale (FIQL) and female sexual functioning index (FSFI)) at 3-month follow-up. RESULTS: Out of 14 eligible patients, nine patients (median age 50 years) were treated, all of whom had previously had one or more unsuccessful medical and/or surgical treatments for their RVF. Clinical closure occurred in none of the patients at 3-month follow-up. There was no improvement in PDAI and patient-reported outcomes (VAS, IBDQ, FIQL and FSFI). Two patients had concomitant perianal fistulas; using FDA, one patient had a clinical response and one patient was in clinical remission 3 months after HBO. There were two treatment-related adverse events during HBO concerning claustrophobia and fatigue. Furthermore, two patients had a surgical intervention due to RVF and two patients were treated with antibiotics for a urinary tract infection during follow-up. One patient had a dose reduction of ustekinumab because of decreased luminal complaints. CONCLUSION: Treatment with HBO was feasible, but in this therapy-refractory cohort without deviating ostomy no clinical closure of RVF or improvement in quality of life was seen 3 months after HBO. Treatment with HBO alone in this specific group of patients therefore appears to be ineffective.

Systematic Review: Histological Scoring of Strictures in Crohn's Disease.

BACKGROUND AND AIMS: In Crohn's disease, many patients develop a stricture, which can due to inflammation, fibrosis and muscular changes or all at the same time. Determining the predominant component has therapeutic consequences but remains challenging. To develop imaging techniques that assess the nature of a stricture, a gold standard is needed and histopathology is considered as such. This paper provides an overview of published histological scoring systems for strictures in Crohn's disease. METHODS: A systematic literature review according to PRISMA guidelines was performed of histological scoring indices that assessed whether a stricture was inflammation-predominant or fibrosis-predominant. Multiple libraries were searched from inception to December 2018. Two reviewers independently assessed abstracts and full-texts. RESULTS: Sixteen articles were identified as suitable for this systematic review. A large number of parameters were reported. Extent of neutrophil infiltration and extent of fibrosis in the bowel wall were most frequently described to reflect severity of inflammation and fibrosis, respectively. Among the 16 studies, only two described a numerical scoring system for the inflammatory and fibrotic component separately. Smooth muscle changes were scored in a minority of studies. CONCLUSIONS: Multiple scoring systems have been developed. There was large heterogeneity in scoring per parameter and construction of numerical scoring systems. Therefore, we feel that none of the systems is suitable to be used as gold standard. We offer an overview of histological parameters that could be incorporated in a future histological scoring index for strictures.

Bowel ultrasound measurements in healthy children - systematic review and meta-analysis.

BACKGROUND: Ultrasound (US) is a noninvasive method of assessing the bowel that can be used to screen for bowel pathology, such as Inflammatory Bowel Disease, in children. Knowledge about US findings of the bowel in healthy children is important for interpreting US results in cases where disease is suspected. OBJECTIVE: To assess the bowel wall thickness in different bowel segments in healthy children and to assess differences in bowel wall thickness among pediatric age categories. MATERIALS AND METHODS: We conducted a systematic search in the PubMed, Embase, Cochrane, and CINAHL databases for studies describing bowel wall thickness measured by transabdominal US in healthy children. We excluded studies using contrast agent. We calculated the pooled mean and standard deviation scores and assessed differences among age categories (0-4 years, 5-9 years, 10-14 years, 15-18 years), first with analysis of variance (ANOVA) and further with subsequent Student's t-tests for independent samples, corrected for multiple testing. RESULTS: We identified 191 studies and included 7 of these studies in the systematic review. Reported bowel wall thickness values ranged from 0.8 mm to 1.9 mm in the small bowel and from 1.0 mm to 1.9 mm in the colon. The mean colonic bowel wall thickness is larger in children ages 15-19 years compared to 0-4 years (range in difference: 0.3-0.5 mm [corrected P<0.02]). CONCLUSION: The reported upper limit of bowel wall thickness in healthy children is 1.9 mm in the small bowel and the colon, and mean thickness increases slightly with age in jejunum and colon. These values can be used as guidance when screening for bowel-related pathology in children.

Hyperbaric oxygen therapy for the treatment of perianal fistulas in Crohn's disease (HOT-TOPIC): study protocol of a prospective interventional cohort study with one-year follow-up.

Background: Perianal fistulizing Crohn's disease (pCD) has a significant impact on patients' health and quality of life. Current treatment options have a relatively low success rate and a high recurrence risk. Positive effects of hyperbaric oxygen (HBO2) therapy have been indicated in animal studies as well as in small case series. Methods/Design: This is a non-randomized, controlled pilot study. A total of 20 patients with pCD who have been refractory to standard therapy for at least six months will be included. Patients with a seton and stable treatment regimen will be included. Patients with anal strictures, rectovaginal fistulas, stoma or deep ulceration of the rectum will be excluded. Patients who are eligible but refuse HBO2 will be asked to serve as controls. Patients in the HBO2 group will be treated with 40 sessions of HBO2 therapy at 243-253 kPa, with the seton being removed after 30 sessions. Co-primary endpoints are changes in the perianal disease activity index and MRI-scores. Secondary outcomes are fistula drainage assessment, laboratory findings and patient-reported outcomes. Assessment will be done at baseline, 16 weeks, 34 weeks and 60 weeks after finishing HBO2. Discussion: The aim of this study is to investigate the feasibility and therapeutic effect of HBO2 on pCD. The one-year follow-up should provide information on the effect durability. A comparison between patients treated with HBO2 and patients who continue to receive standard care will be made. The risk of bias will be limited by using clearly defined inclusion and exclusion criteria, baseline characteristics and consecutive recruitment of patients through an outpatient fistula clinic. Trial registration: The HOT-TOPIC trial has been approved by the local Medical Ethical Committee of the Academic Medical Centre in Amsterdam, the Netherlands. The trial has been registered at the Netherlands Trial Register (www.trialregister.nl), registration number: NTR 6676. Protocol version: August 2017, version 3.0.

Ultrasound for Assessing Disease Activity in IBD Patients: A Systematic Review of Activity Scores.

BACKGROUND AND AIMS: Ultrasound [US] indices for assessing disease activity in IBD patients have never been critically reviewed. We aimed to systematically review the quality and reliability of available ultrasound [US] indices compared with reference standards for grading disease activity in IBD patients. METHODS: Pubmed, Embase and Medline were searched for relevant literature published within the period 1990 to June 2017. Relevant publications were identified through full text review after initial screening by two investigators. Data on methodology and index characteristics were collected. Study quality was assessed using a modified version of the Quadas-2 tool for risk of bias assessment. RESULTS: Of 20 studies with an US index, 11 studies met the inclusion criteria. Out of these 11 studies, 7 and 4 studied Crohn's disease [CD] and ulcerative colitis [UC0 activity indices, respectively. Parameters that were used in these indices included bowel wall thickness [BWT], Doppler signal [DS], wall layer stratification [WLS], compressibility, peristalsis, haustrations, fatty wrapping, contrast enhancement [CE], and strain pattern. Study quality was graded high in 5 studies, moderate in 3 studies and low in 3 studies. Ileocolonoscopy was used as the reference standard in 9 studies. In 1 study a combined index of ileocolonoscopy and barium contrast radiography and in 1 study histology was used as the reference standard. Only 5 studies used an established endoscopic index for comparison with US. CONCLUSIONS: Several US indices for assessing disease activity in IBD are available; however, the methodology for development was suboptimal in most studies. For the development of future indices, stringent methodological design is required.

Review article: pharmacological aspects of anti-TNF biosimilars in inflammatory bowel diseases.

BACKGROUND: Anti-tumour necrosis factor (anti-TNF) monoclonal antibodies have shown efficacy in inflammatory bowel diseases (IBD). As these therapies lose patent protection, biosimilar versions of the originator products are being developed, such as the infliximab biosimilar CT-P13; however, some uncertainty exists regarding their pharmacology in IBD. AIM: To review the literature on anti-TNF biosimilars focusing on pharmacokinetics, pharmacodynamic properties and comparative effectiveness, related to their use in IBD. METHODS: A PubMed literature search was performed using the following terms individually or in combination: 'biosimilars,' 'CT-P13,' 'Crohn's disease,' 'inflammatory bowel disease,' 'ulcerative colitis,' 'anti-TNFα therapy,' 'infliximab,' 'adalimumab,' 'pharmacokinetics,' 'immunogenicity.' RESULTS: Bioequivalence of CT-P13 and infliximab was shown in ankylosing spondylitis (AS) and therapeutic equivalence in rheumatoid arthritis (RA). Preliminary results of CT-P13 in IBD come from small post-marketing registries and case series with a relatively short-term follow-up period and suggest comparable efficacy and safety to infliximab. Inter- and intra-individual differences in exposure and response are well known for the original molecules but dosing regimens and concomitant medications are different for RA compared to IBD, limiting the ability to translate some of the pharmacology data in RA to IBD. Uncertainty exists about cross-reactivity of anti-drug antibodies and whether similar exposure-response relationships will be observed for biosimilars and efficacy thresholds for therapeutic drug monitoring can be used interchangeably. CONCLUSIONS: It is likely that biosimilars will be widely used for the treatment of IBD due to their cost savings and comparable efficacy. Nevertheless, robust post-marketing studies and pharmacovigilance are warranted in the coming years.

Proteinase-activated receptor-4 evoked colorectal analgesia in mice: an endogenously activated feed-back loop in visceral inflammatory pain.

BACKGROUND: Activation of proteinase-activated receptor-4 (PAR-4) from the colonic lumen has an antinociceptive effect to colorectal distension (CRD) in mice in basal conditions. We aimed to determine the functional localization of the responsible receptors and to test their role in two different hyperalgesia models. METHODS: Mice received PAR-4 activating peptide (PAR-4-AP, AYPGKF-NH(2)) or vehicle intraperitoneally (IP), and abdominal EMG response to CRD was measured. The next group received PAR-4-AP intracolonically (IC) with or without 2,4,6-triaminopyrimidine, a chemical tight junction blocker, before CRD. The SCID mice were used to test the role of lymphocytes in the antihyperalgesic effect. The effects of PAR-4-AP and PAR-4-antagonist (P4pal-10) were evaluated in water avoidance stress (WAS) model and low grade 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis. Spinal Fos protein expression was visualized by immunohistochemistry. KEY RESULTS: The antinociceptive effect of PAR-4-AP disappeared when was administrered IP, or with the blockade of colonic epithelial tight junctions, suggesting that PAR-4-AP needs to reach directly the nerve terminals in the colon. The CRD-induced spinal Fos overexpression was reduced by 43% by PAR-4-AP. The PAR-4-AP was antihyperalgesic in both hyperalgesia models and in mice with impaired lymphocytes. The PAR-4-antagonist significantly increased the TNBS, but not the WAS-induced colonic hyperalgesia. CONCLUSIONS & INFERENCES: The antinociceptive effect of PAR-4-AP depends on its penetration to the colonic mucosa. The PAR-4 activation is endogenously involved as a feedback loop to attenuate inflammatory colonic hyperalgesia to CRD.

Detailed esophageal function and morphological analysis shows high prevalence of gastroesophageal reflux disease and Barrett's esophagus in patients with cervical inlet patch.

Although the pathogenesis of cervical inlet patch (CIP) is not fully understood, most authors consider it as a congenital abnormality, whereas others surmise it to be related to gastroesophageal reflux disease (GERD). We aimed to evaluate esophageal function and the prevalence of GERD and Barrett's esophagus in patients with CIP. GERD is defined by the presence of erosive esophagitis or an abnormal pH monitoring. Seventy-one consecutive patients with endoscopic and histological evidence of CIP were prospectively evaluated. Esophageal symptom analysis, 24-hour simultaneous biliary reflux and double-channel pH-monitoring, and esophageal manometry were carried out in 65/71 (92%) patients and in 25 matched controls. Six patients were not suitable for testing and were, therefore, excluded. The histological evaluation of the heterotopic islands showed cardia and/or oxyntic mucosa in 64/65 (98%) patients and specialized intestinal metaplasia (SIM) in one patient (2%). The cardia and/or oxyntic mucosa was accompanied by focally appearing pancreatic acinar metaplasia and pancreatic ductal metaplasia in 7/64 (11%) and in 1/64 (2%), superficial mucous glands in 6/64 (9%), and SIM in 2/64 (3%) cases. In total, SIM was present in three patients (5%), and one of them had low-grade dysplasia. At the gastroesophageal junction, 28 (43%) patients had columnar metaplasia, including nine (14%) patients with SIM. Erosive esophagitis was present in 37 (57%) cases. Thirty-two patients (49%) had abnormal acid reflux in the distal and 25 (38%) in the proximal esophagus. Abnormal biliary reflux was present in 25 (38%) cases. On the basis of endoscopic and pH studies, GERD was established in 44/65 (68%) patients. Typical reflux symptoms were common (33/65, 51%). The combined 24-hour biliary and double-channel pH-monitoring detected significantly more significant acidic reflux at both measurement points and significantly longer bile exposure time in the distal esophagus in patients with CIP. Acid secretion in the CIP was detected in three (5%) cases. Esophageal manometry revealed decreased LES pressure and prolonged relaxation with decreased peristaltic wave amplitude, and an increased number of simultaneous contractions in the esophageal body. The detailed evaluation of the esophageal morphology and function in subjects with CIP showed a high prevalence of GERD and Barrett's esophagus. Further studies are needed to evaluate whether combined acidic and biliary reflux is able to promote similar histomorphological changes in the CIP, as it is shown distally in patients with Barrett's esophagus.

A renewed insight into Barrett's esophagus: comparative histopathological analysis of esophageal columnar metaplasia.

Specialized intestinal metaplasia (SIM) is considered as a premalignant condition of the esophagus, but other types of esophageal metaplasia are commonly neglected. A standardized histopathological analysis was focused not only on SIM but also on the presence of metaplastic processes typical of additional glands. A morphological study using standardized histopathological tests was carried out between 2004 and 2007, with biopsies taken from esophageal mucosa of 826 consecutive patients. Mean age and male : female ratio of patients were 55.6 ± 14.7 and 1.1 : 1, respectively. Only 4.1% (n = 34) of all cases proved to have SIM. The remainder of the cases (n = 615; 74.4%) contained cardiac-fundic mucosa without SIM. Some samples exhibited superficial mucous glands, pancreatic acinar metaplasia (PAM), and ciliated metaplasia accounting for 24% (n = 198), 14.9% (n = 123), and 0.2% (n = 2), respectively. SIM was colocalized with superficial mucous glands (103/198 superficial mucous gland cases; P < 0.001). Low-grade dysplasia (n = 51; 6.2%) and high-grade dysplasia (n = 9; 1.1%) were found mainly in SIM (37/51; 9/9; P = 0.071) with male preponderance (3 : 1 at low-grade and 2 : 1 at high-grade dysplasia). PAM was found mainly in cases without dysplasia (103 of 123 pancreatic metaplasias; P < 0.001). SIM alone in the esophagus is rare, and its frequent association with cardiac mucosa-type metaplasia testifies to transition of mucinous-goblet cell through pseudogoblet cells. PAM rather indicates absence of dysplasia, but superficial mucous glands predicts that SIM follows dysplasia.

Increased faecal serine protease activity in diarrhoeic IBS patients: a colonic lumenal factor impairing colonic permeability and sensitivity.

OBJECTIVES: Diarrhoea-predominant irritable bowel syndrome (IBS-D) is characterised by elevated colonic lumenal serine protease activity. The aims of this study were (1) to investigate the origin of this elevated serine protease activity, (2) to evaluate if it may be sufficient to trigger alterations in colonic paracellular permeability (CPP) and sensitivity, and (3) to examine the role of the proteinase-activated receptor-2 (PAR-2) activation and signalling cascade in this process. PATIENTS AND METHODS: Faecal enzymatic activities were assayed in healthy subjects and patients with IBS, ulcerative colitis and acute infectious diarrhoea. Following mucosal exposure to supernatants from control subjects and IBS-D patients, electromyographic response to colorectal balloon distension was recorded in wild-type and PAR-2(-/-) mice, and CPP was evaluated on colonic strips in Ussing chambers. Zonula occludens-1 (ZO-1) and phosphorylated myosin light chain were detected by immunohistochemistry. RESULTS: The threefold increase in faecal serine protease activity seen in IBS-D patients compared with constipation-predominant IBS (IBS-C) or infectious diarrhoea is of neither epithelial nor inflammatory cell origin, nor is it coupled with antiprotease activity of endogenous origin. Mucosal application of faecal supernatants from IBS-D patients in mice evoked allodynia and increased CPP by 92%, both of which effects were prevented by serine protease inhibitors and dependent on PAR-2 expression. In mice, colonic exposure to supernatants from IBS-D patients resulted in a rapid increase in the phosphorylation of myosin light chain and delayed redistribution of ZO-1 in colonocytes. CONCLUSIONS: Elevated colonic lumenal serine protease activity of IBS-D patients evokes a PAR-2-mediated colonic epithelial barrier dysfunction and subsequent allodynia in mice, suggesting a novel organic background in the pathogenesis of IBS.