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

Predicting the response to Ondansetron, a 5HT3 receptor antagonist, in irritable bowel syndrome with diarrhoea: the utility of clinical features and objective biomarkers

Background: Patients with diarrhoea predominant irritable bowel syndrome (IBS-D) suffer from loose frequent stools with associated urgency and fear of incontinence. Relief from these symptoms is an important unmet need. The 5-HT3 receptor antagonist Alosetron has been shown to increase stool consistency, decrease urgency and reduce abdominal pain leading to a global increase in satisfaction with treatment [1]. Its use is restricted following an increased incidence of ischaemic colitis and this agent is not available in Europe. The serine proteases family of proteolytic enzymes have been identified as the source of increased faecal proteolytic activity in patients with IBS. These enzymes may be mechanistically important via their action on the Protease activated receptor (PAR) -2, inducing increases in permeability and hypersensitivity. Aims: To assess the efficacy of the commonly prescribed 5-HT3 antagonist Ondansetron, in patients with IBS-D and to identify biomarkers that might allow us to predict response defining an Ondansetron responsive endophenotype of IBS. To structurally characterise faecal serine proteases and define the impact of treatment with Ondansetron. Methods: 120 patients meeting Rome III criteria for IBS-D entered a randomised, double-blind, placebo controlled, cross-over study of 5 weeks of Ondansetron 4mg versus placebo with dose titration allowed up to two tablets thrice daily in the first 3 weeks. Patients completed daily bowel symptom diaries documenting stool consistency using the Bristol Stool Form Score (BSFS). Gut transit and small bowel water content were measured in the last week of each treatment. The primary endpoint was average stool consistency in the last 2 weeks of treatment. Faecal samples were obtained from 30 healthy volunteers (HV) and 79 IBS-D patients participating in a trial of Ondansetron versus placebo. Colonic transit was measured using radio-opaque markers. Faecal serine proteases (FSP) 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 colourimetric assay based upon the proteolysis of azo-casein. Results: Ondansetron significantly improved stool consistency In the intention to treat analysis n= 101, with a 1.39 (95% CI1.20-1.58)point decrease on the Bristol stool form scale whilst taking Ondansetron compared to a 0.51 (95% CI 0.32-0.72) point reduction whilst taking placebo p=<0.0001. Compared to placebo patients on Ondansetron experienced fewer days with urgency (p=0.01), lower urgency scores (P<0.001), reduced frequency of defecation (p=0.001) and less bloating (p=0. 25) although pain scores did not change significantly. Protein analysis identified the most abundant FSPs as being of human origin and likely pancreatic juice derived. Functional assays showed increased FSP and faecal amylase in IBS-D compared to HV. Those with higher amylase had significantly higher FSP and greater anxiety. FSP activity correlated negatively with whole gut transit in IBS-D (Spearman r=-0.32, p=0.005) and HV (r=-0.55, p=0.014), but was not affected by treatment with Ondansetron. Conclusions: Ondansetron is an effective and well tolerated treatment in patients with IBS-D with a low number of side effects. It slows whole gut transit, but without a demonstrable difference in small bowel water content. Clinical rather than biochemical indicators predicted responsiveness to Ondansetron best. Patients with less severe symptoms are more likely to respond well to Ondansetron which should prove a useful addition to the current rather limited therapies available for this important group of patients. Previous reports that FSP activity is elevated in some patients with IBS-D has been confirmed. We have increased our understanding of this phenomenon by characterising the proteins responsible for the serine protease activity, showing that most of this activity is likely due to human pancreatic enzymes

Increased fasting small bowel water content in untreated coeliac disease and scleroderma as assessed by MRI

Background and aims: The regular overnight migrating motor complex (MMC) ensures that the normal fasting small bowel water content (SBWC) is minimised. We have applied our recently validated non-invasive magnetic resonance technique to assess SBWC in newly diagnosed coeliac disease (CD), scleroderma (SCD) and irritable bowel syndrome (IBS) conditions, possibly associated with small intestinal bacterial overgrowth (SIBO).Methods: 20 CD and 15 SCD patients with gastrointestinal symptoms were compared to 20 healthy volunteers (HV) and 26 IBS with diarrhoea (IBS-D) patients as previously reported. All underwent a fasting, magnetic resonance imaging (MRI) scan on a 1.5 T Philips Achieva MRI scanner to assess fasting SBWC and colonic volumes. Stool and symptom diaries were completed for 1 week.Results: Median (Interquartile range, IQR) Compared to healthy volunteers, all the patients had significantly increased stool frequency and Bristol stool form score. SBWC was significantly increased in CD 109(53-224) vs. 53(31-98) mL in HV,

Predicting the response to Ondansetron, a 5HT3 receptor antagonist, in irritable bowel syndrome with diarrhoea: the utility of clinical features and objective biomarkers

Background: Patients with diarrhoea predominant irritable bowel syndrome (IBS-D) suffer from loose frequent stools with associated urgency and fear of incontinence. Relief from these symptoms is an important unmet need. The 5-HT3 receptor antagonist Alosetron has been shown to increase stool consistency, decrease urgency and reduce abdominal pain leading to a global increase in satisfaction with treatment [1]. Its use is restricted following an increased incidence of ischaemic colitis and this agent is not available in Europe. The serine proteases family of proteolytic enzymes have been identified as the source of increased faecal proteolytic activity in patients with IBS. These enzymes may be mechanistically important via their action on the Protease activated receptor (PAR) -2, inducing increases in permeability and hypersensitivity. Aims: To assess the efficacy of the commonly prescribed 5-HT3 antagonist Ondansetron, in patients with IBS-D and to identify biomarkers that might allow us to predict response defining an Ondansetron responsive endophenotype of IBS. To structurally characterise faecal serine proteases and define the impact of treatment with Ondansetron. Methods: 120 patients meeting Rome III criteria for IBS-D entered a randomised, double-blind, placebo controlled, cross-over study of 5 weeks of Ondansetron 4mg versus placebo with dose titration allowed up to two tablets thrice daily in the first 3 weeks. Patients completed daily bowel symptom diaries documenting stool consistency using the Bristol Stool Form Score (BSFS). Gut transit and small bowel water content were measured in the last week of each treatment. The primary endpoint was average stool consistency in the last 2 weeks of treatment. Faecal samples were obtained from 30 healthy volunteers (HV) and 79 IBS-D patients participating in a trial of Ondansetron versus placebo. Colonic transit was measured using radio-opaque markers. Faecal serine proteases (FSP) 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 colourimetric assay based upon the proteolysis of azo-casein. Results: Ondansetron significantly improved stool consistency In the intention to treat analysis n= 101, with a 1.39 (95% CI1.20-1.58)point decrease on the Bristol stool form scale whilst taking Ondansetron compared to a 0.51 (95% CI 0.32-0.72) point reduction whilst taking placebo p=<0.0001. Compared to placebo patients on Ondansetron experienced fewer days with urgency (p=0.01), lower urgency scores (P<0.001), reduced frequency of defecation (p=0.001) and less bloating (p=0. 25) although pain scores did not change significantly. Protein analysis identified the most abundant FSPs as being of human origin and likely pancreatic juice derived. Functional assays showed increased FSP and faecal amylase in IBS-D compared to HV. Those with higher amylase had significantly higher FSP and greater anxiety. FSP activity correlated negatively with whole gut transit in IBS-D (Spearman r=-0.32, p=0.005) and HV (r=-0.55, p=0.014), but was not affected by treatment with Ondansetron. Conclusions: Ondansetron is an effective and well tolerated treatment in patients with IBS-D with a low number of side effects. It slows whole gut transit, but without a demonstrable difference in small bowel water content. Clinical rather than biochemical indicators predicted responsiveness to Ondansetron best. Patients with less severe symptoms are more likely to respond well to Ondansetron which should prove a useful addition to the current rather limited therapies available for this important group of patients. Previous reports that FSP activity is elevated in some patients with IBS-D has been confirmed. We have increased our understanding of this phenomenon by characterising the proteins responsible for the serine protease activity, showing that most of this activity is likely due to human pancreatic enzymes

The Tumor Microenvironment of Clear-Cell Ovarian Cancer

Some patients with advanced clear-cell ovarian cancer (CCOC) respond to immunotherapy; however, little is known about the tumor microenvironment (TME) of this relatively rare disease. Here, we describe a comprehensive quantitative and topographical analysis of biopsies from 45 patients, 9 with Federation Internationale des Gynaecologistes et Obstetristes (FIGO) stage I/II (early CCOC) and 36 with FIGO stage III/IV (advanced CCOC). We investigated 14 immune cell phenotype markers, PD-1 and ligands, and collagen structure and texture. We interrogated a microarray data set from a second cohort of 29 patients and compared the TMEs of ARID1A-wildtype (ARID1A(wt)) versus ARID1A-mutant (ARID1A(mut)) disease. We found significant variations in immune cell frequency and phenotype, checkpoint expression, and collagen matrix between the malignant cell area (MCA), leading edge (LE), and stroma. The MCA had the largest population of CD138(+) plasma cells, the LE had more CD20(+) B cells and T cells, whereas the stroma had more mast cells and αSMA(+) fibroblasts. PD-L2 was expressed predominantly on malignant cells and was the dominant PD-1 ligand. Compared with early CCOC, advanced-stage disease had significantly more fibroblasts and a more complex collagen matrix, with microarray analysis indicating “TGFβ remodeling of the extracellular matrix” as the most significantly enriched pathway. Data showed significant differences in immune cell populations, collagen matrix, and cytokine expression between ARID1A(wt) and ARID1A(mut) CCOC, which may reflect different paths of tumorigenesis and the relationship to endometriosis. Increased infiltration of CD8(+) T cells within the MCA and CD4(+) T cells at the LE and stroma significantly associated with decreased overall survival

2D-ReS2 & diamond nanoparticles-based sensor for the simultaneous determination of sunset yellow and tartrazine in a multiple-pulse amperometry FIA system

We present a flow injection system with a multiple pulse amperometric detection (FIA-MPA)-based methodology for the simultaneous analysis of sunset yellow and tartrazine. As transducer, we have developed a novel electrochemical sensor based on the synergistic effect of ReS2 nanosheets and diamond nanoparticles (DNPs). Among several transition dichalcogenides for the sensor development, we have selected ReS2 nanosheets since it yields a better response towards both colourants. Scanning probe microscopy characterization shows that the surface sensor is composed by scattered and stacked ReS2 flakes and large aggregates of DNPs. With this system, the gap between the oxidation potential values of sunset yellow and tartrazine is wide enough to allow the simultaneous determination of both dyes. Under the optimum potential pulse conditions (0.8 and 1.2 V) during 250 ms, a flow rate of 3 mL/min and a volume injection of 250 μL, detection limits of 3.51 × 10− 7 M and 2.39 × 10− 7 M for sunset yellow and tartrazine, respectively, were obtained. This method exhibits good accuracy and precision with Er minor than 13% and RSD lower than 8% with a sampling frequency of 66 samples per hour. Pineapple jelly samples were analyzed by the standard addition method, obtaining 53.7 mg/kg and 29.0 mg/kg of sunset yellow and tartrazine, respectively. From the analysis of fortified samples, recoveries of 94% and 105% were obtainedThe authors acknowledge financial support from projects PID2020- 113142RB-C21 and PID2020-113142RB-C22 and TED2021-129416 AI00 funded by MCIN/AEI/10.13039/501100011033 and P2018/NMT4349 (TRANSNANOAVANSENS-CM) funded by the Comunidad Autonoma ´ de Madrid. We thank L. Chico for fruitful discussion

Assessment of motion of colonic contents in the human colon using MRI tagging

Background We have previously reported a non-invasive, semi-automated technique to assess motility of the wall of the ascending colon (AC) using Magnetic Resonance Imaging. This study investigated the feasibility of using a tagged MRI technique to visualise and assess the degree of flow within the human ascending colon in healthy subjects and those suffering from constipation. Methods An open-labelled study of 11 subjects with constipation and 11 subjects without bowel disorders was performed. MRI scans were acquired fasted, then 60 and 120 mins after ingestion of a 500ml macrogol preparation. The amount of free fluid in the small and large bowel was assessed using a heavily T2-weighted MRI sequence. The internal movement of the contents of the AC were visualised using a cine tagged MRI sequence and assessed by a novel analysis technique. Comparisons were made between fasting and postprandial scans within individuals, and between the constipation and control groups. Key results. Macrogol significantly increased the mobile, MR visible water content of the ascending colon at 60 mins post ingestion compared to fasted data (controls p=0.001, constipated group p=0.0039). The contents of the AC showed increased motion in healthy subjects but not in the constipated group with significant differences between groups at 60 minutes (p<0.002) and 120 minutes (p<0.003). Conclusions and inferences. This study successfully demonstrated the use of a novel MRI tagging technique to visualise and assess the motion of ascending colon contents following a 500ml macrogol challenge. Significant differences were demonstrated between healthy and constipated subjects