A Rosztóczy

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.

Pathways involved in gut mucosal barrier dysfunction induced in adult rats by maternal deprivation: corticotrophin-releasing factor and nerve growth factor interplay

Neonatal maternal deprivation (NMD) increases gut paracellular permeability (GPP) through mast cells and nerve growth factor (NGF), and modifies corticotrophin‐releasing factor (CRF) and corticosterone levels. CRF, corticosterone and mast cells are involved in stress‐induced mucosal barrier impairment. Consequently, this study aimed to specify whether corticosteronaemia and colonic expression of both preproCRF and CRF are modified by NMD, and to determine if altered expression may participate in the elevated GPP in connection with NGF and mast cells. Male Wistar rat pups were either separated from postnatal days 2–14, or left undisturbed with their dam. At 12 weeks of age, adult rats were treated with mifepristone (an antagonist of corticoid receptors), α‐helical CRF(9‐41) (a non‐specific CRF receptor antagonist), or SSR‐125543 (CRF‐R1 receptor antagonist). We also determined corticosteronaemia and both colonic preproCRF and CRF expression. Then, control rats were treated by CRF, doxantrazole (mast cell stabilizer), BRX‐537A (a mast cell activator) and anti‐NGF antibody. NMD did not modify colonic CRF level but increased colonic preproCRF expression and corticosteronaemia. Peripheral CRF, via CRF‐R1 receptor, but not corticosterone, was involved in the elevated GPP observed in these rats, through a mast‐cell‐mediated mechanism, since the increase of GPP induced by exogenous CRF was abolished by doxantrazole. Anti‐NGF antibody treatment also reduced the elevated GPP induced by CRF or BRX‐537A. CRF acts through CRF‐R1 receptors to stimulate NGF release from mast cells, which participates in the elevated GPP observed in NMD adult rats. This suggests that early traumatic experience induced neuro‐endocrine dysfunction, involved in alterations of gut mucosal barrier.

Fecal proteases from diarrheic-IBS and ulcerative colitis patients exert opposite effect on visceral sensitivity in mice

ABSTRACT Elevated colonic luminal serine‐protease (Ser‐P) activity of diarrhea‐predominant IBS (IBS‐D) patients evokes a proteinase‐activated receptor (PAR)‐2‐mediated colonic hypersensitivity in mice. Despite similarly elevated Ser‐P levels in feces, patients with IBD exhibit visceral hypo‐ or normosensitivity to rectal distension, as opposed to IBS‐D. To explain these discrepancies we studied the effect of colonic infusion of fecal supernatants from ulcerative colitis (UC) patients to colorectal mechanical sensitivity of mice and explored the involvement of PAR‐4 and its activator Cathepsin‐G (Cat‐G). Fecal protease activities were assayed in healthy subjects, IBS‐D and UC patients in presence or not of antiproteases or Cat‐G inhibitor. Following intracolonic infusion of fecal supernatants from healthy subjects, IBS‐D and UC patients or PAR‐4 activating peptide (PAR‐4‐AP) or Cat‐G, EMG response to colorectal balloon distension was recorded in mice. This nociceptive response was also determined after treatment with pepducin (PAR‐4 antagonist) on UC supernatant or after a preincubation with antiproteases or Cat‐G inhibitor. In contrast to IBS‐D supernatant, UC supernatant promoted colonic hyposensitivity to distension, an effect mimicked by PAR‐4‐AP or Cat‐G. UC supernatant‐induced hypoalgesia was inhibited by a cocktail of antiproteases. However, blockade of PAR‐4 or Cat‐G inhibition resulted in colonic hypersensitivity similar to that observed after IBS‐D supernatant infusion. Despite similarly elevated Ser‐P activities, IBS‐D and UC fecal supernatant display visceral pro‐ and antinociceptive effects in mice, respectively. Visceral hyposensitivity induced by fecal supernatant from UC patients results from PAR‐4 activation by cathepsin‐G, counterbalancing the pronociceptive effect of simultaneous PAR‐2 activation.

Fecal MMP-9: A new noninvasive differential diagnostic and activity marker in ulcerative colitis

Background:Ulcerative colitis (UC) is characterized by frequent relapses, with the presence of colorectal inflammation and mucosal lesions. Matrix-metalloprotease 9 (MMP-9) is elevated in colonic biopsies, urine, and blood plasma of UC patients. MMP-9 has been suggested as a predictor of UC in the urine of children; however, 20% of the controls tested positive. So far, fecal MMP-9 levels have never been measured. Our aims were: 1) to compare fecal MMP-9 levels in UC patients to control subjects and a functional gastrointestinal disorder characterized by diarrhea (IBS-D); 2) to test the correlation between UC disease activity and fecal levels of MMP-9; and 3) to correlate fecal MMP-9 levels with a known fecal marker of UC activity, calprotectin. Methods:UC (n = 47), IBS-D (n = 23) patients, and control subjects (n = 24) provided fecal samples for MMP-9 analysis. In UC patients, disease severity was evaluated by the Mayo score. Fecal MMP-9 and calprotectin levels were measured by enzyme-linked immunosorbent assay and lateral flow assay, respectively. Results:MMP-9 was undetectable or ⩽0.22 ng/mL in the feces of all controls and IBS-D patients. In UC patients, fecal MMP-9 levels significantly correlated with the overall Mayo score (P < 0.001), the endoscopic score (P < 0.001), and the serum C-reactive protein levels (P = 0.002). Additionally, in UC patients fecal MMP-9 levels showed a significant correlation with a known disease activity marker, fecal calprotectin (P = 0.014). Conclusions:These results highlight fecal MMP-9 as a useful tool in the differential diagnosis of diarrheic disorders and in the noninvasive evaluation of disease activity and mucosal healing in UC.

Leaky Gut in Patients with Diarrhea-Predominant Irritable Bowel Syndrome and Inactive Ulcerative Colitis

Background/Aims: Defective epithelial barrier has been implicated in the pathogenesis of irritable bowel syndrome (IBS) and inflammatory bowel diseases. The aim of this study was to investigate gut permeability in patients with inactive ulcerative colitis (UC) and in patients with IBS. Methods: IBS patients of the diarrhea-predominant (IBS-D) and of the constipation-predominant subgroup (IBS-C), patients with inactive UC and healthy subjects were enrolled. Gut permeability was evaluated by measuring 24-hour urine excretion of orally administered 51Cr-EDTA. Clinical symptoms were evaluated in IBS-D patients and correlated to colonic permeability. Results: There was a significant decrease in the proximal small intestinal permeability in IBS-C patients compared to controls (0.26 ± 0.05 vs. 0.63 ± 0.1%; p < 0.05). Distal small intestinal permeability showed no significant difference in the studied group of patients compared to controls. Colonic permeability of IBS-D and inactive UC patients was significantly increased compared to controls (2.68 ± 0.35 and 3.74 ± 0.49 vs. 1.04 ± 0.18%; p < 0.05, p < 0.001). Colonic permeability of IBS-D patients correlated with stool frequency. Conclusions: Elevated gut permeability is localized to the colon both in IBS-D and in inactive UC patients.

Luminal cysteine-proteases degrade colonic tight junction structure and are responsible for abdominal pain in constipation-predominant IBS.

OBJECTIVES:Luminal serine-proteases lead to increased colonic paracellular permeability and visceral hypersensitivity in patients with diarrhea-predominant irritable bowel syndrome (IBS-D). Other proteases, namely cysteine-proteases (CPs), increase airway permeability by digesting epithelial tight junction proteins. In this study, we focused on constipation-predominant IBS (IBS-C) and we aimed to (i) evaluate CP levels in two cohorts of IBS patients, (ii) test if IBS-C fecal supernatant (FSN) affects permeability, and visceral sensitivity after repeated administrations in mice, and (iii) evaluate occludin expression in IBS-C colonic biopsies.METHODS:Fecal CP activity was determined using selective substrate and inhibitor (E64). The effect of papain, as positive control, and IBS-C FSN administrations were evaluated on colonic paracellular permeability and mucosal occludin levels in mice and T84 monolayers. Occludin protein levels were evaluated in IBS-C colonic biopsies. Sensitivity to colorectal distension (CRD) was measured after repeated administrations of IBS-C FSN.RESULTS:We found in a subset of IBS-C patients an enhanced fecal CP activity, in comparison with healthy controls and IBS-D patients. CP activity levels positively correlated with disease severity and abdominal pain scoring. This association was confirmed by receiver operating characteristic curve analysis. In mice, repeated application of IBS-C FSN into colon triggered increased permeability, linked to the enzymatic degradation of occludin, and was associated with enhanced visceral sensitivity to CRD. Finally, occludin levels were found decreased in colonic biopsies from IBS-C patients, and IBS-C FSNs were able to degrade recombinant human occludin in vitro. All these effects were abolished by preincubation of IBS-C FSN with a CP inhibitor, E64.CONCLUSIONS:These data suggest that luminal CPs may represent a new factor contributing to the genesis of symptoms in IBS.

Immediate insulin treatment prevents gut motility alterations and loss of nitrergic neurons in the ileum and colon of rats with streptozotocin-induced diabetes

The streptozotocin-induced diabetic rat model was used to investigate the relation between the deranged gut motility and the segment-specific quantitative changes in the nitrergic myenteric neurons. Additionally, we studied the effectiveness of early insulin replacement to prevent the diabetes-induced changes. Rats were divided into three groups: controls, diabetics and insulin-treated diabetics. Ten weeks after the onset of diabetes, animals were chosen from each group for intestinal transit measurements. The remainder were killed and gut segments were processed for NADPH-diaphorase histochemistry and HuC/HuD immunohistochemistry. The diabetic rats displayed faster transit than that for the controls. In the insulin-treated group, the transit time was the same as that in the controls. In the duodenum of the diabetic rats, the number of nitrergic neurons was decreased, while the total neuronal number was not altered. In the jejunum, ileum and colon, both the total and the nitrergic neuronal cell number decreased significantly. Insulin treatment did not prevent the nitrergic cell loss significantly in the duodenum and jejunum, but it did prevent it significantly in the ileum and colon. These findings comprise the first evidence that the nitrergic neurons located in different intestinal segments exhibit different susceptibilities to a diabetic state and to insulin treatment.

Role of antispasmodics in the treatment of irritable bowel syndrome.

Irritable bowel syndrome (IBS) is a long-lasting, relapsing disorder characterized by abdominal pain/discomfort and altered bowel habits. Intestinal motility impairment and visceral hypersensitivity are the key factors among its multifactorial pathogenesis, both of which require effective treatment. Voltage-gated calcium channels mediate smooth muscle contraction and endocrine secretion and play important roles in neuronal transmission. Antispasmodics are a group of drugs that have been used in the treatment of IBS for decades. Alverine citrate, a spasmolytic, decreases the sensitivity of smooth muscle contractile proteins to calcium, and it is a selective 5-HT1A receptor antagonist. Alverine, in combination with simethicone, has been demonstrated to effectively reduce abdominal pain and discomfort in a large placebo-controlled trial. Mebeverine is a musculotropic agent that potently blocks intestinal peristalsis. Non-placebo-controlled trials have shown positive effects of mebeverine in IBS regarding symptom control; nevertheless, in recent placebo-controlled studies, mebeverine did not exhibit superiority over placebo. Otilonium bromide is poorly absorbed from the GI tract, where it acts locally as an L-type calcium channel blocker, an antimuscarinic and a tachykinin NK2 receptor antagonist. Otilonium has effectively reduced pain and improved defecation alterations in placebo-controlled trials in IBS patients. Pinaverium bromide is also an L-type calcium channel blocker that acts locally in the GI tract. Pinaverium improves motility disorders and consequently reduces stool problems in IBS patients. Phloroglucinol and trimethylphloroglucinol are non-specific antispasmodics that reduced pain in IBS patients in a placebo-controlled trial. Antispasmodics have excellent safety profiles. T-type calcium channel blockers can abolish visceral hypersensitivity in animal models, which makes them potential candidates for the development of novel therapeutic agents in the treatment of IBS.

Prevalence of Respiratory Symptoms and Diseases Associated with Gastroesophageal Reflux Disease

Aim: Investigation of the prevalence of respiratory symptoms and diseases associated with gastroesophageal reflux disease (GERD). Patients and Methods: 299 subjects with GERD were submitted to upper gastrointestinal endoscopy and 24-hour esophageal pH monitoring and a symptom analysis. Results: Chronic respiratory symptoms or diseases were present in 18% (56/299). Chronic cough was observed in 42/56 patients, while typical reflux symptoms such as heartburn and acid regurgitation were observed in 30/56 and 24/56 cases, respectively. The prevalence of airway diseases was chronic bronchitis 12/56, asthma 10/56, recurrent pneumonia 10/56, chronic sinusitis 7/56 and chronic laryngitis 1/56. In patients with respiratory complications pathologic acid reflux was established in 29/51 cases on the basis of the DeMeester score, while 17/51 had pathologic postprandial, nocturnal or diurnal reflux events. Upper gastrointestinal endoscopy revealed a normal esophageal mucosa in 6/56, Savary-Miller stage I esophagitis in 23/56, stage II in 15/56, stage III in 5/56 and stage IV in 6/56 patients. Conclusions: These investigations have demonstrated an abnormal 24-hour pH score in about half of the patients with GERD-associated respiratory complications, and indicated that short reflux events are characteristic of the reflux activity in one third of this population.

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.