Melanie Lingaya

A mechanistic multicentre, parallel group, randomised placebo-controlled trial of mesalazine for the treatment of IBS with diarrhoea (IBS-D)

Introduction Immune activation has been reported in the mucosa of IBS patients with diarrhoea (IBS-D), and some small studies have suggested that mesalazine may reduce symptoms. We performed a double-blind, randomised placebo-controlled trial of 2 g mesalazine twice daily versus placebo for 3 months in patients with Rome III criteria IBS-D. Primary outcome was daily average stool frequency during weeks 11–12; secondary outcomes were abdominal pain, stool consistency, urgency and satisfactory relief of IBS symptoms. Methods Participants were randomised after a 2-week baseline stool diary. All participants completed a 12-week stool diary and at the end of each week recorded the presence of ‘satisfactory relief of IBS symptoms’. Results 136 patients with IBS-D (82 women, 54 men) were randomised, 10 patients withdrew from each group. Analysis by intention to treat showed the daily average stool frequency during weeks 11 and 12 were mean (SD), 2.8 (1.2) in mesalazine and 2.7 (1.9) in the placebo group with no significant group difference, (95% CI) 0.1 (−0.33 to 0.53), p=0.66. Mesalazine did not improve abdominal pain, stool consistency nor percentage with satisfactory relief compared with placebo during the last two-weeks follow-up. Conclusions This study does not support any clinically meaningful benefit or harm of mesalazine compared with placebo in unselected patients with IBS-D. More precise subtyping based on underlying disease mechanisms is needed to allow more effective targeting of treatment in IBS. Trial registration number NCT01316718.

Characterisation of faecal protease activity in irritable bowel syndrome with diarrhoea: origin and effect of gut transit

Objectives Faecal serine proteases (FSPs) may play a role in irritable bowel syndrome with diarrhoea (IBS-D), but their origin is unclear. We aimed to structurally characterise them and define the impact of colonic cleansing and transit time. Design Faecal samples were obtained from 30 healthy volunteers (HV) and 79 patients with IBS-D participating in a trial of ondansetron versus placebo. Colonic transit was measured using radio-opaque markers. Samples were also obtained from 24 HV before and after colonic cleansing with the osmotic laxative MoviPrep. FSPs were purified from faecal extracts using benzamidine-Sepharose affinity chromatography. SDS-PAGE profiled components were identified using trypsinolysis and tandem mass spectrometry. Functional protease activity in faecal extracts was measured using a colorimetric assay based on the proteolysis of azo-casein. Results Protein analysis identified the most abundant FSPs as being of human origin and probably derived from pancreatic juice. Functional assays showed increased faecal protease (FP) and amylase in patients with IBS-D compared with HV. Those with higher amylase had significantly higher FP and greater anxiety. FP activity correlated negatively with whole gut transit in patients with IBS-D (Spearman r=−0.32, p=0.005) and HV (r=−0.55, p=0.014). Colon cleansing caused a significant rise in FP activity in HV from a baseline of median (IQR) 253 (140–426) to 1031 (435–2296), levels similar to those seen in patients with IBS-D. FSP activity correlated positively with days/week with urgency. Conclusions The most abundant FSPs are of human origin. Rapid transit through the colon and/or decreased (possibly bacterial) proteolytic degradation increases their faecal concentration and could contribute to visceral hypersensitivity in patients with IBS-D. ClinicalTrials.gov NCT00745004.

A mechanistic multi-centre, parallel group, randomised placebo controlled trial of Mesalazine for treatment of irritable bowel syndrome with diarrhoea (IBS-D)

Introduction Immune activation has been reported in the mucosa of IBS patients with diarrhoea (IBS-D), and some small studies have suggested that mesalazine may reduce symptoms. We performed a double-blind, randomised placebo-controlled trial of 2 g mesalazine twice daily versus placebo for 3 months in patients with Rome III criteria IBS-D. Primary outcome was daily average stool frequency during weeks 11–12; secondary outcomes were abdominal pain, stool consistency, urgency and satisfactory relief of IBS symptoms. Methods Participants were randomised after a 2-week baseline stool diary. All participants completed a 12-week stool diary and at the end of each week recorded the presence of ‘satisfactory relief of IBS symptoms’. Results 136 patients with IBS-D (82 women, 54 men) were randomised, 10 patients withdrew from each group. Analysis by intention to treat showed the daily average stool frequency during weeks 11 and 12 were mean (SD), 2.8 (1.2) in mesalazine and 2.7 (1.9) in the placebo group with no significant group difference, (95% CI) 0.1 (−0.33 to 0.53), p=0.66. Mesalazine did not improve abdominal pain, stool consistency nor percentage with satisfactory relief compared with placebo during the last two-weeks follow-up. Conclusions This study does not support any clinically meaningful benefit or harm of mesalazine compared with placebo in unselected patients with IBS-D. More precise subtyping based on underlying disease mechanisms is needed to allow more effective targeting of treatment in IBS. Trial registration number NCT01316718.

Effect of experimental stress on the small bowel and colon in healthy humans.

Symptoms of irritable bowel syndrome (IBS) are frequently reported to be exacerbated by stress. Animal studies suggest that corticotrophin releasing hormone (CRH) mediates the effect of stress on the bowel. We have shown that stressed IBS patients with diarrhea have constricted small bowels. We hypothesized that we could mimic this effect by applying experimental stress in the form of either hand immersion in ice water or CRH injection in healthy volunteers (HV).

Increased liver fat and glycogen stores after consumption of high versus low glycaemic index food: A randomized crossover study

To investigate the acute and longer‐term effects of low (LGI) versus high glycaemic index (HGI) diets on hepatic fat and glycogen accumulation and related blood measures in healthy volunteers.

Corticotropin-releasing factor increases ascending colon volume after a fructose test meal in healthy humans: a randomized controlled trial

BACKGROUND Poorly absorbed fermentable carbohydrates can provoke irritable bowel syndrome (IBS) symptoms by escaping absorption in the small bowel and being rapidly fermented in the colon in some susceptible subjects. IBS patients often are anxious and stressed, and stress accelerates small bowel transit, which may exacerbate malabsorption. OBJECTIVE In this study we investigated the effect of an intravenous injection of corticotropin-releasing factor (CRF) on fructose malabsorption and the resulting volume of water in the small bowel. DESIGN We performed a randomized, placebo-controlled crossover study of CRF compared with saline injection in 11 male and 10 female healthy subjects, examining the effect on the malabsorption of a 40-g fructose test meal and its transit through the gut, which was assessed by serial MRI and breath hydrogen measurement. Orocecal transit was assessed with the use of the lactose [(13)C]ureide breath test and the adrenal response to CRF was assessed by serial salivary cortisol measurements. RESULTS CRF injection caused a significant increase in salivary cortisol, which lasted for 135 min. Small bowel water content (SBWC) rose from baseline, peaking at 45 min after fructose ingestion, whereas breath hydrogen peaked later, at 75 min. The area under the curve for SBWC from -15 min to 135 min was significantly lower after CRF compared with saline [mean difference: 5911 mL · min (95% CI: 18.4, 11,803 mL · min), P = 0.049]. Considering all subjects, the percentage change in ascending colon volume rose significantly after CRF. This increase was significant for male (P = 0.026), but not female, volunteers. CONCLUSIONS CRF constricts the small bowel and increases fructose malabsorption, as shown by increased ascending colon volumes. This mechanism may help to explain the increased sensitivity of some stressed individuals to fructose malabsorption. This trial was registered at clinicaltrials.gov as NCT01763281.

Origin of increased fecal serine protease in patients with irritable bowel syndrome and diarrhoea (IBS-D)

Introduction Patients with IBS-D have been reported to have increased faecal serine protease activity which could induce visceral hypersensitivity and increase gut permeability by activating proteinase activated receptor-2 (PAR-2). These proteases could be either endogenous (pancreatic or mast cell) or bacterial in origin. Aim To define the type and origin of proteases using a proteomic method. Methods Study 1: Serine protease activity in stool supernatant from 36 patients meeting the Rome III criteria for IBS-D and 9 healthy controls was evaluated using a previous published method.1 Patients completed a one week stool diary and a hospital anxiety and depression score. Study 2: Serine proteases from a subset of 8 pooled patient samples were purified using a combination of affinity chromatography and SDS-PAGE. Protease identification using proteolysis and mass-spectrometry was performed. Study 3: Assessment of stool amylase and elastase was performed using two commercially available ELISAs. Results Study 1: Serine protease activity was significantly increased in patients, 621 ± 94 versus 211 ± 46 units of trypsin/mg protein in controls, p = 0.04. Serine protease activity was significantly correlated with anxiety r = 0.5 p = 0.002. Study 2: The predominant proteases were pancreatic in origin including trypsin, elastase and carboxypeptidase-B. Additionally there was abundant pancreatic amylase. Study 3: Stool amylase but not elastase was significantly increased in IBS-D 183 ± 35 103 units/ml versus controls 78 ± 44. Amylase also correlated with serine protease activity, r = 0.33, p < 0.05. Conclusion We confirm previous reports of increased serine proteases in IBS-D but these appear to be of pancreatic origin and associated with increased anxiety. Anxiety may cause rapid transit which reduces degradation by colonic bacteria and increases fecal protease.

PTU-047 High prevalence of clostridium difficile ribotype 078 in IBD outpatients

Background and Objectives Point prevalence studies have reported higher carriage rates of C. difficile in IBD patients compared with the general population, but longitudinal prospective data are lacking. The objectives of this observational study were to investigate and molecularly characterise isolates of C. difficile, collected prospectively on a monthly basis over a one-year period among IBD outpatients and healthy controls (HC). Methods At enrolment, recruited participants had established diagnoses of UC (n=16) and Crohn’s disease (n=6) and reported no recent hospitalisation or exposure to antibiotics. PCR ribotype and toxin status (cytotoxigenic culture) were determined for all +ive stool cultures. All participants underwent a monthly telephone interview to identify potential risk factors for C. difficile acquisition (changes in medications, exposure to antibiotics, clinic attendances, hospitalisation) and to assess for disease activity (Harvey-Bradshaw Index and Simple Clinical Activity Colitis Index). Results Two patients underwent physician-initiated laboratory testing of C. difficile during the sample collection phase, although no participants developed or were treated for C. difficile infection. C. difficile was cultured from 29/223 samples (13%) representing 16/22 patients and 1 of 5 HC with concurrent antibiotic exposure in 6/29 visits (20%). Of the toxin +ive isolates (n=25; 078, 005, 302 and 015), 72% (n=21) were PCR ribotype 078. Toxigenic negative ribotypes included 023, 026 and 656. Of those toxin +ive isolates, 9 samples (36%) were associated with relapsing IBD of which 7/9 were ribotype 078. Multiple stool specimens also tested +ive for different ribotypes in 3 patients with UC, all of whom were taking regular immunosuppressants. WGS studies of the 078 isolates revealed marked genetic similarity, with only 3 of the 21 isolates varying by 1 or more nucleotides when compared to the 078 reference genome, suggesting there may have been a common source for cross-transmission. Conclusions The high prevalence of PCR ribotype 078 in this IBD outpatient cohort is consistent with the recent emergence of this strain in the community. These results reinforce the importance of testing all in-and outpatients with an apparent flare or relapsing IBD for carriage of toxigenic C. difficile to inform optimal management strategies. Future research is needed to understand the predominance of 078 isolates in IBD, particularly in the context of clinical relapse.

OC-019 Iron Deficiency Anaemia and Hepcidin Levels in H. Pylori Infected Patients

Introduction IDA affects up to 5% of the adult population in the developing world. There is an association between H. pylori (Hp) infection and incidence of unexplained IDA, but the mechanisms remain unclear. In children it has been suggested that Hp disturbs the iron regulatory mechanism via hepicidin. This peptide hormone induces internalisation and degradation of the iron transporter protein ferroportin thus limiting iron absorption and release. Hepcidin expression is induced by inflammation, but how this relates to Hp and IDA has not been fully elucidated, particularly in adults. This pilot study aimed to characterise local and systemic iron transporter expression in IDA patients with and without Hp infection, in comparison to Hp negative dyspepsia controls. Methods Patients undergoing routine endoscopy for IDA (HpIDA and IDA groups, n = 18 and 40 respectively) or without IDA (control group, n = 18) donated blood and biopsy samples with informed consent and ethical approval. Hp status was assessed by three biopsy based tests and by serology. Duodenal and gastric biopsies were evaluated by immunohistochemistry for ferroportin and hepcidin, in addition to H&E staining for inflammation and atrophy grading. All scoring was carried out by experienced blinded histopathologists. A commercial ELISA assay was used to quantify serum Hepcidin-25. Results As expected, anaemia parameters were significantly lower in the HpIDA and IDA groups compared to the controls (P < 0.0001). Surprisingly, serum hepcidin concentrations were significantly reduced in the HpIDA and IDA groups compared to the controls (9 fold, P = 0.009 and 5 fold, P < 0.0001 respectively). Hp infection was associated with gastric expression of hepcidin, particularly in the corpus when compared to controls. This corresponded with the cytoplasmic relocalisation of ferroportin (n = 12; 67%) in the duodenal enterocytes of patients with HpIDA compared to controls, where the ferroportin was actively expressed. Hepcidin was also found to be expressed in the duodenum of both controls and HpIDA. Significant atrophy was observed in both IDA groups. Conclusion IDA was associated with significantly lower levels of serum hepcidin in contrast to previous studies. Local or systemic factors such as inflammatory mediators could be driving this response as more severe atrophy was observed in both IDA groups. We now aim to perform quantitative analysis of hepcidin in the gastric specimens using RT-qPCR to further evaluate whether local Hp transcriptionally upregulated hepcidin expression might cause these effects on iron transport. Disclosure of Interest None Declared

OC-091 Ondansetron slows transit and improves stool consistency in patients with diarrhoea predominant irritable bowel syndrome

Introduction 5-Hydroxytryptamine three receptor antagonists (5HT3RA) are effective in diarrhoea predominant irritable bowel syndrome (IBS-D), with a number needed to treat (NNT) for Alosetron of 7.1 Due to safety concerns (constipation [25%] and ischaemic colitis [0.1%]) Alosetron is not licensed in the UK. Ondansetron (OND) is a 5HT3 RA, widely and safely used for nausea, with constipation as a side effect. Methods We recruited 125 patients meeting the Rome III criteria from primary and secondary care to a two centre randomised, double-blind, placebo-controlled, crossover trial. Screening comprised of blood tests, rectal biopsy and a 1-week baseline Bristol stool form diary before randomisation to placebo (PLA) or OND for 5 weeks. Efficacy was optimised by dose titration during weeks 1–3 starting at 4 mg daily, increasing (max 8 mg three time a day) or decreasing as required. A 2-week washout preceded 5 weeks of the opposite therapy to that received in treatment 1. Symptom diaries including stool form, frequency, pain, bloating and urgency were completed daily. Transit using the Metcalf method was measured after each treatment period.2 The primary endpoint was the difference in average stool form between baseline and the last 2 weeks of each treatment. Stool consistency “responders” were defined as experiencing >50% reduction in the days/week with stool form 6 or 7. Analysis is presented by intention to treat. Results 80 women and 28 men completed the study (mean age 40.8, range 18–72, and 41.5, range 25–60). The mode dose of OND was <4 mg a day. Stool form improved significantly in the OND arm, mean change 1.4 (95% CI 1.2 to 1.6), vs 0.5 (95% CI 0.3 to 0.7) compared to PLA, p=<0.0001. Stool frequency improved on OND, mean 0.86 (95% CI 0.8 to 1.4), vs 0.44 (95% CI 0.4 to 0.89) on PLA, p=<0.01, as did urgency score 0.6 (95% CI 0.8 to 0.4) vs 0.3 (95% CI 0.5 to 0.2) p=<0.001. There were no significant improvements in pain or bloating. 74% of patients preferred OND while 26% preferred PLA or had no preference, χ2 p=<0.0001. 70% were “stool consistency responders” while taking OND compared with 33% taking PLA giving a NNT of 2.7. Whole gut transit slowed significantly while taking OND, 25 (13.5–47.5) h compared to 16 (7–29) h with PLA p=<0.001. 9% receiving OND complained of constipation compared to 2% on PLA, all but one responded to dose reduction alone. 2% withdrew because of constipation. There was no case of ischaemic colitis. Conclusion Using dose titration Ondansetron acts to slow whole gut transit and is highly effective in IBS-D with a low incidence of constipation. Competing interests None declared. References 1. Cremonini F. Neurogastroenterol Motil 2003. 2. Metcalf A. Gastroenterology 1987.