David E. Reed

Mast cell tryptase and proteinase‐activated receptor 2 induce hyperexcitability of guinea‐pig submucosal neurons

Mast cells that are in close proximity to autonomic and enteric nerves release several mediators that cause neuronal hyperexcitability. This study examined whether mast cell tryptase evokes acute and long‐term hyperexcitability in submucosal neurons from the guinea‐pig ileum by activating proteinase‐activated receptor 2 (PAR2) on these neurons. We detected the expression of PAR2 in the submucosal plexus using RT‐PCR. Most submucosal neurons displayed PAR2 immunoreactivity, including those colocalizing VIP. Brief (minutes) application of selective PAR2 agonists, including trypsin, the activating peptide SL‐NH2 and mast cell tryptase, evoked depolarizations of the submucosal neurons, as measured with intracellular recording techniques. The membrane potential returned to resting values following washout of agonists, but most neurons were hyperexcitable for the duration of recordings (> 30 min–hours) and exhibited an increased input resistance and amplitude of fast EPSPs. Trypsin, in the presence of soybean trypsin inhibitor, and the reverse sequence of the activating peptide (LR‐NH2) had no effect on neuronal membrane potential or long‐term excitability. Degranulation of mast cells in the presence of antagonists of established excitatory mast cell mediators (histamine, 5‐HT, prostaglandins) also caused depolarization, and following washout of antigen, long‐term excitation was observed. Mast cell degranulation resulted in the release of proteases, which desensitized neurons to other agonists of PAR2. Our results suggest that proteases from degranulated mast cells cleave PAR2 on submucosal neurons to cause acute and long‐term hyperexcitability. This signalling pathway between immune cells and neurons is a previously unrecognized mechanism that could contribute to chronic alterations in visceral function.

FODMAPs alter symptoms and the metabolome of patients with IBS: a randomised controlled trial

Objective To gain mechanistic insights, we compared effects of low fermentable oligosaccharides, disaccharides and monosaccharides and polyols (FODMAP) and high FODMAP diets on symptoms, the metabolome and the microbiome of patients with IBS. Design We performed a controlled, single blind study of patients with IBS (Rome III criteria) randomised to a low (n=20) or high (n=20) FODMAP diet for 3 weeks. Symptoms were assessed using the IBS symptom severity scoring (IBS-SSS). The metabolome was evaluated using the lactulose breath test (LBT) and metabolic profiling in urine using mass spectrometry. Stool microbiota composition was analysed by 16S rRNA gene profiling. Results Thirty-seven patients (19 low FODMAP; 18 high FODMAP) completed the 3-week diet. The IBS-SSS was reduced in the low FODMAP diet group (p<0.001) but not the high FODMAP group. LBTs showed a minor decrease in H2 production in the low FODMAP compared with the high FODMAP group. Metabolic profiling of urine showed groups of patients with IBS differed significantly after the diet (p<0.01), with three metabolites (histamine, p-hydroxybenzoic acid, azelaic acid) being primarily responsible for discrimination between the two groups. Histamine, a measure of immune activation, was reduced eightfold in the low FODMAP group (p<0.05). Low FODMAP diet increased Actinobacteria richness and diversity, and high FODMAP diet decreased the relative abundance of bacteria involved in gas consumption. Conclusions IBS symptoms are linked to FODMAP content and associated with alterations in the metabolome. In subsets of patients, FODMAPs modulate histamine levels and the microbiota, both of which could alter symptoms. Trial registration number NCT01829932.

Stress increases descending inhibition in mouse and human colon

A relationship between stress and the symptoms of irritable bowel syndrome (IBS) has been well established but the cellular mechanisms are poorly understood. Therefore, we investigated effects of stress and stress hormones on colonic descending inhibition and transit in mouse models and human tissues.

Co-expression of μ and ∂ opioid receptors by mouse colonic nociceptors

To better understand opioid signalling in visceral nociceptors, we examined the expression and selective activation of μ and δ opioid receptors by dorsal root ganglia (DRG) neurons innervating the mouse colon.

Protease-mediated suppression of DRG neuron excitability by commensal bacteria.

Peripheral pain signaling reflects a balance of pronociceptive and antinociceptive influences; the contribution by the gastrointestinal microbiota to this balance has received little attention. Disorders, such as inflammatory bowel disease and irritable bowel syndrome, are associated with exaggerated visceral nociceptive actions that may involve altered microbial signaling, particularly given the evidence for bacterial dysbiosis. Thus, we tested whether a community of commensal gastrointestinal bacteria derived from a healthy human donor (microbial ecosystem therapeutics; MET-1) can affect the excitability of male mouse DRG neurons. MET-1 reduced the excitability of DRG neurons by significantly increasing rheobase, decreasing responses to capsaicin (2 μm) and reducing action potential discharge from colonic afferent nerves. The increase in rheobase was accompanied by an increase in the amplitude of voltage-gated K+ currents. A mixture of bacterial protease inhibitors abrogated the effect of MET-1 effects on DRG neuron rheobase. A serine protease inhibitor but not inhibitors of cysteine proteases, acid proteases, metalloproteases, or aminopeptidases abolished the effects of MET-1. The serine protease cathepsin G recapitulated the effects of MET-1 on DRG neurons. Inhibition of protease-activated receptor-4 (PAR-4), but not PAR-2, blocked the effects of MET-1. Furthermore, Faecalibacterium prausnitzii recapitulated the effects of MET-1 on excitability of DRG neurons. We conclude that serine proteases derived from commensal bacteria can directly impact the excitability of DRG neurons, through PAR-4 activation. The ability of microbiota-neuronal interactions to modulate afferent signaling suggests that therapies that induce or correct microbial dysbiosis may impact visceral pain. SIGNIFICANCE STATEMENT Commercially available probiotics have the potential to modify visceral pain. Here we show that secretory products from gastrointestinal microbiota derived from a human donor signal to DRG neurons. Their secretory products contain serine proteases that suppress excitability via activation of protease-activated receptor-4. Moreover, from this community of commensal microbes, Faecalibacterium prausnitzii strain 16-6-I 40 fastidious anaerobe agar had the greatest effect. Our study suggests that therapies that induce or correct microbial dysbiosis may affect the excitability of primary afferent neurons, many of which are nociceptive. Furthermore, identification of the bacterial strains capable of suppressing sensory neuron excitability, and their mechanisms of action, may allow therapeutic relief for patients with gastrointestinal diseases associated with pain.

Emerging Studies of Human Visceral Nociceptors

Animal studies have led to significant advances in our understanding of pain mechanisms in the intestine that could lead to altered signaling in disorders such as irritable bowel syndrome. However, how these translate to the human afferent nervous system is unclear. Recent studies have demonstrated that it is possible to use a variety of techniques, including electrophysiological recordings, to begin to examine these concepts in humans. This mini-review examines these studies to explore how well animal studies translate to humans suffering from irritable bowel syndrome, highlights some of the advantages and technical limitations of these approaches, and identifies some priorities for future studies using human tissues.

Sa2059 Afferent Activation in the Proximal Colon by Enteroendocrine Mediators and Nutrients

Background: Despite the importance of gastric inhibitory polypeptide (GIP) as a physiological incretin, postprandial circulating glucose levels in GIP receptor null (GIPR-/-) and wild-type (wt) mice are similar. However, unlike wt mice, GIPR-/mice do not develop diet-induced obesity, suggesting a potential role for GIPR antagonists in the treatment of obesity. While specific GIPR antagonists, such as N-terminal truncated peptides and peptides with a Glu to Pro substitution at position 3 (Pro3-GIP), have been identified, their short circulating T1/2 has hampered their development. We hypothesize that the T1/2 of these antagonists can be increased significantly by fusing them to the Fc-fragment of IgG, a strategy that has been employed previously for this purpose. Aim: To develop long-acting antagonists to GIPR by fusion to the IgG Fc-fragment. Methods: PCR and molecular cloning were used to produce chimeric genes encoding GIP(6-42)-Fc-IgG and Pro3-GIP-Fc-IgG. After expression in HEK 293 cells, protein A agarose chromatography was used to purify protein from conditioned media. Fc-fusions were identified by Western analysis using a specific GIP antibody, and biological activity was assessed in the presence and absence of 1 nM synthetic GIP(1-42) using a GIP-specific reporter cell line. Results: Transgenic HEK 293 cells produced protein with the predicted molecular size for the Fc fusion and that possessed GIP-specific immunoreactivity. Purified Pro3-GIP-Fc-IgG induced reporter expression with an EC50 of 0.5 μM, whereas GIP(6-42)-Fc-IgG did not. Both GIP(6-42)-Fc-IgG and Pro3-GIP-Fc-IgG antagonized report gene expression induced by 1 nM GIP, with IC50 values of 4 μM and 20 μM, respectively. Summary and Conclusion: Pro3-GIP-Fc-IgG, but not GIP(6-42)-FcIgG, induced reporter gene expression, while both fusion products inhibited reporter gene expression induced by 1 nM GIP, indicating that the former is a partial agonist, while only the latter functions as an antagonist. The IC50 values for GIP(6-42)-Fc-IgG and Pro3-GIPFc-IgG were higher than the corresponding values reported for synthetic GIP(6-42) and Pro3-(GIP), indicating the possibility of partial interference of ligand binding by the Fc fragment. Nevertheless, the increase in T1/2 imparted by the fusion of N-terminal truncated GIP peptides the Fc fragment may thereby provide viable candidates for treating obesity and related disorders.

Tu1997 A Novel In Vivo Method to Measure Gastrointestinal Transit in Mice

INTRODUCTION The intestine is a significant site of gluconeogenesis, with high expression of glucose 6 phosphatase. Abnormal forms of glycogen, including polyglucosan bodies, occur in the muscularis propria in a subset of patients with gastrointestinal motility disorders. Little is known, however, of the distribution of glycogen metabolic enzymes and related proteins in normal gastrointestinal tract, or effects of deficiencies of these proteins on the bowel. METHODS Key enzymes in glycogen metabolism, including glycogen synthase (GS), glycogen branching enzyme (GBE), malin and laforin were examined in stomach, ileum and colon of two mouse strains and in normal human bowel using immunohistochemistry with semiquantitiative analysis of epithelium, muscle, submucosal and myenteric plexus, validated by western blotting and positive and negative controls. The pathological effects of GBE, laforin and malin gene knockout on mouse gastrointestinal tract were also studied. RESULTS GS was present at high levels in the smooth muscle of muscularis propria, GBE in the majority of large ganglion cells of the submucosal and myenteric plexuses, and laforin and malin in submucosal ganglia. Analysis of human bowel showed no significant regional differences in the distribution of GBE, laforin and malin, GS was more prominent in the muscularis propria of the small intestine, compared with stomach and colon. GBE knockout produced a progressive severe myopathy with numerous polyglucosan bodies and vacuolation. Polyglucosan bodies were also present in both laforin and malin knockout models, but with a less severe phenotype than that seen in the GBE knockout, and were not seen in controls. CONCLUSION The distribution of glycogen metabolic proteins in the bowel wall of mouse and man shows a specific neuromuscular distribution for each pathway component. Defects in glycogen metabolism in mouse knockout models reproduce histological features of polyglucosan body myopathy in human digestive motility disorders.

Comparing the Quality of Reporting in Randomized Controlled Trial Abstracts at Two International Gastroenterology Meetings in 2010

BACKGROUND: Osmotic and stimulant laxatives are the mainstay of treatment for children suffering with functional constipation. We systemically evaluated studies which had investigated osmotic and stimulant laxatives for functional constipation in children. METHODS: Randomised controlled trials (RCTs), published between 1966 and October 2010, which compared osmotic and stimulant laxatives with either placebo or other interventions were included. Data sources were MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials, Cochrane Inflammatory Bowel Disease and functional Bowel Disorders Specialised Register and reference lists of retrieved articles. Data extraction and assessment of methodological quality were performed independently by two reviewers. RESULTS: 16 RCTs met the inclusion criteria. Meta-analysis of 4 studies with 338 participants comparing polyethylene glycol (PEG) with Lactulose showed significantly greater stools per week (stools/wk) with PEG (Mean difference 0.98, 95% confidence interval (CI) 0.49 to 1.46). Those on PEG were less likely to require additional laxative therapies (Odds Ratio 0.49, 95% CI 0.27 to 0.89). No serious adverse events were reported with either agent. Incidence of gastrointestinal symptoms, such as abdominal pain, nausea and vomiting, did not differ between groups. Heterogeneity amongst studies was noted, probably attributable to differences in concomitant medications and follow up times between studies. Meta-analysis of 2 studies with 173 participants comparing PEG with Milk of magnesia showed that the stools/wk was significantly greater with PEG (Mean difference 0.69, 95% CI 0.48 to 0.89). One child was noted to be allergic to PEG, but there were no other serious adverse events reported. 2 studies compared PEG with placebo, but there was insufficient data to allow meta-analysis. Both studies reported a significantly increased number of stools/wk with PEG. Meta-analysis of 2 studies with 287 patients comparing Paraffin with Lactulose showed that the stools/wk following treatment was significantly greater with Paraffin (Mean difference 4.94, 95% CI 4.28 to 5.61). Again, no serious adverse events were reported. CONCLUSIONS: The available evidence suggests that PEG is more effective than placebo and that both liquid paraffin and PEG are more effective than Lactulose for treating children with functional constipation.