Jose G. P. Ferraz

Role for protease activity in visceral pain in irritable bowel syndrome

Mediators involved in the generation of symptoms in patients with irritable bowel syndrome (IBS) are poorly understood. Here we show that colonic biopsy samples from IBS patients release increased levels of proteolytic activity (arginine cleavage) compared to asymptomatic controls. This was dependent on the activation of NF-kappaB. In addition, increased proteolytic activity was measured in vivo, in colonic washes from IBS compared with control patients. Trypsin and tryptase expression and release were increased in colonic biopsies from IBS patients compared with control subjects. Biopsies from IBS patients (but not controls) released mediators that sensitized murine sensory neurons in culture. Sensitization was prevented by a serine protease inhibitor and was absent in neurons lacking functional protease-activated receptor-2 (PAR2). Supernatants from colonic biopsies of IBS patients, but not controls, also caused somatic and visceral hyperalgesia and allodynia in mice, when administered into the colon. These pronociceptive effects were inhibited by serine protease inhibitors and a PAR2 antagonist and were absent in PAR2-deficient mice. Our study establishes that proteases are released in IBS and that they can directly stimulate sensory neurons and generate hypersensitivity symptoms through the activation of PAR2.

Hydrogen sulphide synthesis in the rat and mouse gastrointestinal tract

AIMSnHydrogen sulphide (H2S) exerts several anti-inflammatory effects, accelerates the healing of experimental gastric ulcers, and can stimulate intestinal secretion. Little is known about H2S synthesis in the gastrointestinal tract. The aim of this study was to characterize H2S synthesis throughout the gastrointestinal tract.nnnMETHODSnH2S synthesis in various gastrointestinal tissues of rats and mice was determined. The effects and selectivity of inhibitors of two key enzymes for H2S synthesis, cystathionine-gamma-lyase and cystathionine-beta-synthase, were examined. Cystathionine-gamma-lyase and cystathionine-beta-synthase expression was evaluated by Western blotting and immunohistochemistry. Cystathionine-gamma-lyase and cystathionine-beta-synthase expression in biopsies of human colon was also examined.nnnRESULTSnH2S synthesis was variable throughout the gastrointestinal tract in parallel with variations in cystathionine-gamma-lyase and cystathionine-beta-synthase expression. The efficacy of cystathionine-beta-synthase and cystathionine-gamma-lyase inhibitors to reduce H2S synthesis in these tissues was also variable. Cystathionine-beta-synthase is the predominant source of H2S synthesis in the colon of rodents. Cystathionine-gamma-lyase and cystathionine-beta-synthase were also expressed in healthy human colon biopsies.nnnCONCLUSIONSnThe capacity for H2S synthesis varies throughout the rodent gastrointestinal tract, as does the distribution and contribution of the two key enzymes. Investigation of additional enzymatic sources of H2S and the development of more selective inhibitors are suggested.

Impaired hydrogen sulfide synthesis and IL-10 signaling underlie hyperhomocysteinemia-associated exacerbation of colitis.

Significance Inflammatory bowel diseases (IBDs) are debilitating conditions with no known cure. Recent evidence suggests that elevated intestinal hydrogen sulfide (H2S) synthesis promotes healing and reduces inflammation. H2S is synthesized from cysteine largely via vitamin B6-dependent enzymes. People with IBD are also at increased risk of hyperhomocysteinemia, a condition that is often caused by vitamin B deficiency. Dietary induction of vitamin B deficiency markedly increased serum homocysteine levels and worsened colitis in rodents. The latter was due to the absence of the typical injury-induced elevation of H2S synthesis. Interleukin-10 plays a key role in increasing H2S synthesis, attenuating the severity of colitis, and reducing serum homocysteine levels. The H2S–interleukin 10 axis may be a viable target for therapy of IBD. Vitamin B deficiencies, which can lead to hyperhomocysteinemia (Hhcy), are commonly reported in patients with inflammatory bowel disease (IBD) and may be a causative underlying factor. However, the mechanism for this effect is not known. Hydrogen sulfide (H2S) is a gaseous mediator that promotes tissue repair and resolution of inflammation. In experimental colitis, a marked increase in colonic H2S synthesis drives ulcer healing and resolution of inflammation. Because H2S synthesis is in part dependent upon enzymes that require vitamin B6 as a cofactor, we tested the hypothesis that Hhcy in rodent models would increase the susceptibility to colitis. In all three models tested, diet-induced Hhcy significantly exacerbated colitis. The usual elevation of colonic H2S synthesis after induction of colitis was absent in all three models of colitis. Administration of an H2S donor to Hhcy rats significantly decreased the severity of colitis. Compared with wild-type mice, interleukin (IL) 10-deficient mice on a normal diet had decreased levels of colonic H2S synthesis, a 40% increase in serum homocysteine, and a phenotype similar to wild-type mice with Hhcy. IL-10–deficient mice fed the vitamin B-deficient diet exhibited more severe colonic inflammation, but the normal elevation of colonic H2S synthesis was absent. Administration of IL-10 to the IL-10–deficient mice restored colonic H2S synthesis and significantly decreased serum homocysteine levels. These results suggest that the exacerbation of colitis in Hhcy is due in part to impaired colonic H2S synthesis. Moreover, IL-10 plays a novel role in promoting H2S production and homocysteine metabolism, which may have therapeutic value in conditions characterized by Hhcy.

Giardia duodenalis Cathepsin B Proteases Degrade Intestinal Epithelial Interleukin-8 and Attenuate Interleukin-8-Induced Neutrophil Chemotaxis

ABSTRACT Giardia duodenalis (syn. G. intestinalis, G. lamblia) infections are a leading cause of waterborne diarrheal disease that can also result in the development of postinfectious functional gastrointestinal disorders via mechanisms that remain unclear. Parasite numbers exceed 106 trophozoites per centimeter of gut at the height of an infection. Yet the intestinal mucosa of G. duodenalis-infected individuals is devoid of signs of overt inflammation. G. duodenalis infections can also occur concurrently with infections with other proinflammatory gastrointestinal pathogens. Little is known of whether and how this parasite can attenuate host inflammatory responses induced by other proinflammatory stimuli, such as a gastrointestinal pathogen. Identifying hitherto-unrecognized parasitic immunomodulatory pathways, the present studies demonstrated that G. duodenalis trophozoites attenuate secretion of the potent neutrophil chemoattractant interleukin-8 (CXCL8); these effects were observed in human small intestinal mucosal tissues and from intestinal epithelial monolayers, activated through administration of proinflammatory interleukin-1β or Salmonella enterica serovar Typhimurium. This attenuation is caused by the secretion of G. duodenalis cathepsin B cysteine proteases that degrade CXCL8 posttranscriptionally. Furthermore, the degradation of CXCL8 via G. duodenalis cathepsin B cysteine proteases attenuates CXCL8-induced chemotaxis of human neutrophils. Taken together, these data demonstrate for the first time that G. duodenalis trophozoite cathepsins are capable of attenuating a component of their hosts proinflammatory response induced by a separate proinflammatory stimulus.

Up-regulation of Annexin-A1 and lipoxin A(4) in individuals with ulcerative colitis may promote mucosal homeostasis.

Background One of the characteristics of an active episode of ulcerative colitis (UC) is the intense mucosal infiltration of leukocytes. The pro-resolution mediators Annexin-A1 (AnxA1) and lipoxin A4 (LXA4) exert counter-regulatory effects on leukocyte recruitment, however to date, the dual/cumulative effects of these formyl peptide receptor-2 (FPR2/ALX) agonists in the context of human intestinal diseases are unclear. To define the contribution of these mediators, we measured their expression in biopsies from individuals with UC. Methods Colonic mucosal biopsies were collected from two broad patient groups: healthy volunteers without (‘Ctrl’ nu200a=u200a20) or with a prior history of UC (‘hx of UC’ nu200a=u200a5); individuals with UC experiencing active disease (‘active’ nu200a=u200a8), or in medically-induced remission (‘remission’ nu200a=u200a16). We assessed the mucosal expression of LXA4, AnxA1, and the FPR2/ALX receptor in each patient group using a combination of fluorescence microscopy, biochemical and molecular analyses. Results Mucosal expression of LXA4 was elevated exclusively in biopsies from individuals in remission (3-fold, P<0.05 vs. Ctrl). Moreover, in this same group we observed an upregulation of AnxA1 protein expression (2.5-fold increase vs. Ctrl, P<.01), concurrent with an increased level of macrophage infiltration, and an elevation in FPR2/ALX mRNA (7-fold increase vs. Ctrl, P<.05). Importantly, AnxA1 expression was not limited to cells infiltrating the lamina propria but was also detected in epithelial cells lining the intestinal crypts. Conclusions Our results demonstrate a specific up-regulation of this pro-resolution circuit in individuals in remission from UC, and suggest a significant role for LXA4 and AnxA1 in promoting mucosal homeostasis.

Enhanced Synthesis and Diminished Degradation of Hydrogen Sulfide in Experimental Colitis: A Site-Specific, Pro-Resolution Mechanism

Hydrogen sulfide (H2S) is produced throughout the gastrointestinal tract, and it contributes to maintenance of mucosal integrity, resolution of inflammation, and repair of damaged tissue. H2S synthesis is elevated in inflamed and damaged colonic tissue, but the enzymatic sources of that synthesis are not completely understood. In the present study, the contributions of three enzymatic pathways to colonic H2S synthesis were determined, with tissues taken from healthy rats and rats with colitis. The ability of the colonic tissue to inactivate H2S was also determined. Colonic tissue from rats with hapten-induced colitis produced significantly more H2S than tissue from healthy controls. The largest source of the H2S synthesis was the pathway involving cysteine amino transferase and 3-mercaptopyruvate sulfurtransferase (an α-ketoglutarate-dependent pathway). Elevated H2S synthesis occurred specifically at sites of mucosal ulceration, and was not related to the extent of granulocyte infiltration into the tissue. Inactivation of H2S by colonic tissue occurred rapidly, and was significantly reduced at sites of mucosal ulceration. This correlated with a marked decrease in the expression of sulfide quinone reductase in these regions. Together, the increased production and decreased inactivation of H2S at sites of mucosal ulceration would result in higher H2S levels at these sites, which promotes of resolution of inflammation and repair of damaged tissue.

Underlying mechanisms of portal hypertensive gastropathy.

Gastric mucosal lesions are frequently observed in patients with liver cirrhosis and portal hypertension. Similar lesions can be observed in experimental portal hypertension. This review summarizes our current knowledge of the pathophysiology of portal hypertensive gastropathy, with a particular focus on the microcirculatory disturbances that characterize this condition. The stomach of cirrhotic patients exhibits an increased susceptibility to injury induced by several irritants. Similarly, the stomach of portal hypertensive animals is less resistant to injury. One of the most important factors contributing to the increased susceptibility to damage is an impaired hyperemic response when the epithelium is exposed to irritants. This appears to be related to a reduction in mucosal prostaglandin production and to altered microcirculatory responsiveness to nitric oxide. Nitric oxide overproduction in portal hypertension may have direct effects on gastric blood flow regulation. Elevated production of tumor necrosis factor-alpha by gastric mucosa in portal hypertensive rats has also been shown to contribute to mucosal injury. A better understanding of the pathogenesis of portal hypertensive gastropathy may lead to development of specific therapeutic interventions for this condition.

Pathogenesis of portal hypertensive gastropathy: translating basic research into clinical practice

Portal hypertensive gastropathy (PHG) is often seen in patients with portal hypertension, and can lead to transfusion-dependent anemia as well as acute, life-threatening bleeding episodes. This Review focuses on the mechanisms that underlie the pathogenesis of PHG that provide reasonable grounds for the treatment of this condition, and ultimately enable translation of basic research into clinical practice. Increased portal pressure associated with cirrhosis and liver dysfunction is critical for the development of clinically significant PHG, and leads to impaired gastric mucosal defense mechanisms that render the stomach susceptible to mucosal injury. The use of pharmacological agents such as β-blockers reduces the frequency of bleeding episodes in PHG. As a last resort, surgical decompression of the portal system, transjugular intrahepatic stent placement and liver transplantation can resolve this condition. Elimination of known risk factors for gastric injury such as alcohol, aspirin and traditional NSAIDs is critical. The role of Helicobacter pylori colonization of the gastric mucosa in PHG is not clear. Careful and critical interpretation of human and experimental data can be helpful to establish a rationale for the medical management of this important condition.

Targeted Biopsies Identify Larger Proportions of Patients With Colonic Neoplasia Undergoing High-Definition Colonoscopy, Dye Chromoendoscopy, or Electronic Virtual Chromoendoscopy.

BACKGROUND & AIMSnIt is unclear what are the best and most appropriate endoscopic procedures for detecting colonic neoplasia in patients with long-term colonic inflammatory bowel disease (IBD). Dye chromoendoscopy (DCE) is the standard used in IBD surveillance colonoscopies. However, studies are needed to determine the optimal endoscopic technique for detecting dysplastic lesions. We investigated current practices used in surveillance colonoscopies by IBD gastroenterologists at a single tertiary center. We also determined the rate of neoplasia detection among different surveillance endoscopic techniques in an analysis of random or targeted biopsies.nnnMETHODSnWe collected data on 454 patients with IBD (54.5% male; mean age, 50 y; mean disease duration, 14.5 y; 55.9% with ulcerative colitis, 42.7% with Crohns disease, and 1.3% with indeterminate colitis) who underwent surveillance colonoscopy from April 2011 through March 2014 at the University of Calgary in Canada. Subjects were examined using white-light standard-definition endoscopy (WLE), high-definition (HD) colonoscopy, virtual electronic chromoendoscopy (VCE), or DCE; random or targeted biopsy specimens were collected. Endoscopic and histologic descriptions with suspected neoplasia were recorded. Rates of neoplasia detection by the different endoscopic procedures were compared using chi-square analysis.nnnRESULTSnOf the patients analyzed, 27.7% had WLE endoscopy with random collection of biopsy specimens, 27.3% had HD colonoscopy with random collection of biopsy specimens, 14.1% had VCE with random collection of biopsy specimens, 0.9% had DCE with random collection of biopsy specimens, 12.8% had HD colonoscopy with collection of targeted biopsy specimens, 11.9% had VCE with collection of targeted biopsy specimens, and 5.3% had DCE with collection of targeted biopsy specimens. Neoplastic lesions were detected in 8.2% of the procedures performed in the random biopsy group (95% confidence interval, 5.6-11.7) and 19.1% of procedures in the targeted biopsy group (95% confidence interval, 13.4-26.5) (P < .001). Neoplasias were detected in similar proportions of patients by HD colonoscopy, VCE, or DCE, with targeted biopsy collection.nnnCONCLUSIONSnIn a large cohort of IBD patients undergoing surveillance colonoscopy, targeted biopsies identified greater proportions of subjects with neoplasia than random biopsies. Targeted collection of biopsy specimens appears to be sufficient for detecting colonic neoplasia in patients undergoing HD colonoscopy, DCE, or VCE, but not WLE.

New Pharmacologic Therapies in Gastrointestinal Disease

Many gastrointestinal diseases remain poorly responsive to therapies, and even in the cases of conditions for which there are many effective drugs, there is still considerable room for improvement. This article is focused on drugs for digestive disorders that have entered the marketplace recently, or are expected to reach the marketplace within the next 1 to 2 years. Although advances have been made in understanding gastrointestinal motility, visceral pain, mucosal inflammation, and tissue repair, the major gastrointestinal diseases remain as significant therapeutic challenges.