Lionel Bueno

Induction of Intestinal Inflammation in Mouse by Activation of Proteinase-Activated Receptor-2

Proteinase-activated receptor (PAR)-2, a G-protein-coupled receptor for trypsin and mast cell tryptase, is highly expressed in the intestine. Luminal trypsin and tryptase are elevated in the colon of inflammatory bowel disease patients. We hypothesized that luminal proteinases activate PAR-2 and induce colonic inflammation. Mice received intracolonically PAR-2 agonists (trypsin, tryptase, and a selective PAR-2-activating peptide) or control drugs (boiled enzymes, inactive peptide) and inflammatory parameters were followed at various times after this treatment. Colonic administration of PAR-2 agonists up-regulated PAR-2 expression and induced an inflammatory reaction characterized by granulocyte infiltration, increased wall thickness, tissue damage, and elevated T-helper cell type 1 cytokine. The inflammation was maximal between 4 and 6 hours and was resolved 48 hours after the intracolonic administration. PAR-2 activation also increased paracellular permeability of the colon and induced bacterial trans-location into peritoneal organs. These proinflammatory and pathophysiological changes observed in wild-type mice were not detected in PAR-2-deficient mice. Luminal proteinases activate PAR-2 in the mouse colon to induce inflammation and disrupt the integrity of the intestinal barrier. Because trypsin and tryptase are found at high levels in the colon lumen of patients with Crohns disease or ulcerative colitis, our data may bear directly on the pathophysiology of human inflammatory bowel diseases.

Mediators and Pharmacology of Visceral Sensitivity: From Basic to Clinical Investigations

Over the last decade, the role of visceral sensitivity has been largely recognized in the pathophysiology of functional digestive disorders, particularly in the irritable bowel syndrome. These studies have highlighted the role of afferent pathways arising from the gut as a possible target for new treatments intended to relieve pain or modify altered reflexes present in such patients. These pharmacological targets have been identified mainly by studies on animal models of visceral hyperalgesia of various origins including local inflammation. Locally, several mediators are of paramount importance for sensitization of nerve endings: 5-hydroxytryptamine, bradykinin, tachykinins, calcitonin gene-related peptide, and neurotrophins. Selective antagonists to various subtypes of their receptors are currently available and have been shown to be active in these animal models. Other substances, such as somatostatin, opiold peptides, cholecystokinin, oxytocin, and adenosine, modulate the transmission of nociceptive inputs from the gut to the brain and are of clinical interest. This article reviews the current understanding of these mediators. Although these agents seem to be promising tools for the treatment of visceral hyperalgesia and its consequences (abdominal pain and disturbed reflexes), their clinical efficacy remains to be shown. A better understanding of the nature and the location of the defect in the sensory pathways may permit the selection of subgroups of patients for treatment according to the pharmacological properties of these new therapeutic agents.

Stress‐induced visceral hypersensitivity to rectal distension in rats: role of CRF and mast cells

Background: psychological factors have long been implicated in the aetiology of irritable bowel syndrome often associated with abdominal pain. This work was designed to study, in rats, the influence of partial restraint stress on the abdominal cramps induced by rectal distension and to determine the role of corticotropin releasing factor (CRF) and mast cells degranulation in this response. Methods: abdominal contractions were electromyographically recorded. Thirty minutes after stress or intracerebroventricular CRF, rectal distension was performed by inflation of a balloon (0.4–1.2 mL). α‐helical CRF9–41 or doxantrazole were administered centrally (15 min) and intraperitoneally (30 min), respectively, before stress. Histamine release and the number of mast cells were determined in colonic pieces from stressed and control rats. Results: stress and CRF enhanced the number of abdominal cramps evoked by rectal distension without affecting rectal compliance. α‐helical CRF9–41 and doxantrazole antagonized the stress and CRF‐induced enhancement of abdominal cramps. Stress increased the colonic histamine content whereas the number of colonic mast cells was unchanged. Conclusions: stress enhances abdominal contractions in response to rectal distension in rats via pathways involving central CRF and intestinal mast cells.

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.

Neonatal maternal deprivation triggers long term alterations in colonic epithelial barrier and mucosal immunity in rats

Background: Stressful events in the early period of life (for example, maternal deprivation) have been shown to modify adult immune and gastrointestinal tract functions. The present study aimed to establish whether maternal deprivation affects colonic epithelial barrier and the development of an experimental colitis in adult rats. Methods: Male Wistar rat pups were separated during postnatal days 2–14 or left undisturbed with their dam. At 12 weeks of age, we assessed colonic paracellular permeability, bacterial translocation, myeloperoxidase (MPO) activity, mucosal mast cell density, cytokine (interleukin (IL)-1β, IL-2, IL-4, IL-10, and interferon γ (IFN-γ)) mRNA expression, and macroscopic damage. Total gut permeability, MPO activity, and macroscopic damage were also assessed four days after intracolonic administration of 2,4,6-trinitrobenzenesulphonic acid (TNBS). Results: Maternal deprivation triggered a significant increase in colonic permeability associated with bacterial translocation into the mesenteric lymph nodes, liver, and spleen. These alterations were associated with some macroscopic damage and an increase in colonic MPO activity, mucosal mast cell density, and cytokine mRNA expression. Intracolonic infusion of TNBS induced a significantly higher inflammatory reaction in separated animals, as judged by enhanced MPO colonic levels, total gut permeability, and macroscopic lesions. Conclusions: Maternal deprivation promotes long term alterations in the colonic epithelial barrier associated with an exaggerated immune response to an external immune stimulus. This suggests a role for early psychological factors in the regulation of colonic mucosal barrier in later life.

Stress-induced disruption of colonic epithelial barrier: role of interferon-γ and myosin light chain kinase in mice

BACKGROUND & AIMS Stressful life events are supposed to be involved in various diseases such as inflammatory bowel diseases and irritable bowel syndrome. Impairment of the intestinal epithelial barrier function is a suspected consequence of stress, but the underlying mechanisms remain unclear. This study aimed to determine the mechanisms through which stress modulates the colonic epithelial barrier. METHODS Cytokine messenger RNA (mRNA) expression was evaluated in murine colon, liver, and spleen by competitive reverse-transcription polymerase chain reaction after 1-4 days of daily 2-hour stress sessions. Colonic paracellular permeability was measured as the in vivo lumen-to-blood ratio of (51)Cr-ethylenediaminetetraacetic acid. The effect of a myosin light chain (MLC) kinase inhibitor (ML-7) was assessed on stress-induced interferon (IFN)-gamma mRNA expression and colonic epithelial barrier impairment, and MLC phosphorylation was determined by immunoblot. Finally, the incidence of repeated stress sessions on bacterial translocation was determined. RESULTS Repeated stress induced an overexpression of colonic IFN-gamma. In the liver, higher levels of IFN-gamma, interleukin (IL)-4, and IL-10 mRNAs were detected and were associated with bacterial translocation, inflammation, and apoptosis. Stress increased colonic permeability of control mice, but not of SCID and IFN-gamma-deficient mice. ML-7 inhibited the stress-induced increased permeability, bacterial translocation, and cytokine overexpression in the liver and restored a normal histology. Larger amounts of phosphorylated MLC were detected in stressed animals. CONCLUSIONS Repeated stress sessions drive organ-specific cytokine expression patterns and alter colonic mucosal barrier functions associated with bacterial translocation. This effect depends on the presence of CD4(+) T cells and requires IFN-gamma production and MLC phosphorylation.

Rate of flow of digesta and electrical activity of the small intestine in dogs and sheep.

1. Spiking activity of the small intestine in the conscious dog and sheep was recorded continuously from electrodes chronically implanted on the jejunum and summed at intervals of 20 sec. The activity was related to the transit time and flow rate of intestinal contents as estimated by phenol red and by dilution of continuous marker infusions respectively. Also in some sheep the flow of digesta was measured directly from a cannula in the proximal part of the jejunum, and also by use of an electromagnetic flow meter. 2. In the fasted dog and in sheep on a normal diet the intestinal activity was characterized by a migrating myo‐electric complex comprising an irregular phase followed by a regular phase. These migrating myo‐electric complexes occurred regularly after a period of inactivity at a frequency of 15–20/24 hr. In dogs after feeding, a continuous spiking activity appeared and persisted for periods of 7–8 hr. This was associated with much higher rates of flow and shorter transit times than were observed during fasting. In sheep, continuous spiking activity could be induced by intravenous injection of 5‐hydroxytryptophan and this, similarly, was accompanied by a more rapid flow and a shorter transit time than recorded during the control period. 3. In both species the longest transit time occurred when a phenol red bolus was injected during the period of electrical inactivity. Relatively short transit times were observed when the bolus was administered just before the period of regular spiking activity. 4. When relaxation of the bowel was induced by intraperitoneal injection of hypertonic saline there was no spiking activity and the transit time for the infused solution was greatly lengthened, especially in the sheep. A noticeable flow of digestive contents persisted in the dog. 5. In the sheep the intestinal contents flowed intermittently during periods of 10–15 min and at the same frequency as the migrating myo‐electric complex. Two thirds of this flow took place in the 4–6 min immediately preceding the periods of irregular spiking activity. 6. It is concluded that in the fasted dog and in the sheep the migrating myo‐electric complex controls the pressure gradients on which the flow of intestinal contents depends. This is accomplished in the main by the prolonged phase of irregular spiking activity, and it is suggested that the regular spiking activity which follows it, though not in itself propulsive, serves as a barrier to prevent backflow of digesta into the quiescent part of the intestine. When continuous spiking activity is induced, by feeding in the dog and by injection of 5‐hydroxytryptophan in the sheep, no part of the intestine is quiescent and the transit time is shortened by the incessant irregular spiking activity.

Evaluation of colonic sensory thresholds in IBS patients using a barostat. Definition of optimal conditions and comparison with healthy subjects.

To study the role of abnormal visceral perception in the pathophysiology of the irritable bowel syndrome (IBS), we evaluated colonic tone and visceral perception during intracolonic distension using a flaccid balloon connected to a computerized barostat and placed in the descending colon of IBS patients and healthy controls. In the first part of the study, basal colonic tone and response to pharmacological (neostigmine and glucagon) and physiological (1000-kcal meal) stimuli were recorded in nine IBS patients. Colonic tone increased by 72±27% after injection of neostigmine and decreased by 88±62% after glucagon. After the meal, the maximal increase in colonic tone was 76±31% with the total response to the meal lasting 109.7±32.0 min. In the second part of the study, symptomatic responses (discomfort and pain thresholds) and pressure variations were evaluated during two different methods of distension (stepwise and intermittent) in a randomized order in the nine IBS patients and six healthy controls. Each distension method was repeated twice in IBS patients to study reproducibility. In IBS patients, the mean discomfort threshold volume was 172±76 ml when using stepwise and 167±43 ml when using intermittent distension. The mean pain threshold volume was 250±25 ml when using stepwise and 211±22 ml when using intermittent distension, this difference being statistically significant (P<0.02). Discomfort and pain threshold volumes recorded during the first session of the same distension method were not different from those recorded during the second one. When comparing IBS patients to controls, the pain threshold was reached at a volume ≤300 ml in all IBS patients versus only one control when using stepwise distensions (P<0.001) and in all IBS patients versus no control when using intermittent distensions (P<0.001). Intracolonic pressure-volume curves were similar in patients and controls. In conclusion, isovolumic distension of the colon is a reproducible method of evaluating viscerosensitivity, which is significantly increased in IBS patients. This increased viscerosensitivity is not related to abnormal colonic compliance and may, alone or in combination with other colonic abnormalities, explain the symptoms of irritable bowel syndrome.

Evaluation of colonic myoelectrical activity in health and functional disorders.

Electrical activity of the colon was investigated in six healthy volunteers and 22 patients presenting functional colonic disorders associated with pain, constipation, or diarrhoea. Myoelectrical activity during 10 hours, including two daily meals, was recorded from eight groups of nickel-chrome electrodes using a 1.5 m length probe introduced by the rectal route. Electromyograms of volunteers consisted of slow waves at two distinct rhythms, approximately 3 and 10 cycles per minute, during 16 to 28% of the recording time respectively for each site. Action potentials were also recorded as 11 to 80 short spike bursts (SSB) per hour lasting 1.5 to 3.5 s at any site and 20-26 long spike bursts (LSB) per hour, lasting 17 to 21 s, occurring in series of four to eight propagated bursts. Additional high-velocity propagated bursts were recorded during the three hour postprandial period. Three typical changes in spike activity patterns were detected: an increase by 170-420% of the number of SSB associated to a high spiking activity was recorded in 13 patients (group I), the absence of SSB, a low spiking activity level and only 3 to 8 LSB per hour (group II), in six patients; and the postprandial response was absent (group III) in three other investigated patients. Evidence for three groups of motor disturbances and their possible relation to clinical manifestations of functional disorders is presented.

Guanylate cyclase C‐mediated antinociceptive effects of linaclotide in rodent models of visceral pain

Background  Linaclotide is a novel, orally administered investigational drug currently in clinical development for the treatment of constipation‐predominant irritable bowel syndrome (IBS‐C) and chronic idiopathic constipation. Visceral hyperalgesia is a major pathophysiological mechanism in IBS‐C. Therefore, we investigated the anti‐nociceptive properties of linaclotide in rodent models of inflammatory and non‐inflammatory visceral pain and determined whether these pharmacological effects are linked to the activation of guanylate cyclase C (GC‐C).