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.

Intracolonic infusion of fecal supernatants from ulcerative colitis patients triggers altered permeability and inflammation in mice: role of cathepsin G and protease-activated receptor-4.

BACKGROUND: Cathepsin G (Cat-G) is a neutrophil serine-protease found in the colonic lumen of ulcerative colitis (UC) patients. Cat-G is able to activate protease-activated receptor-4 (PAR(4) ) located at the apical side of enterocytes, leading to epithelial barrier disruption. However, the mechanisms through which Cat-G triggers inflammation are not fully elucidated. The aims of our study were to evaluate in vivo the effects of UC fecal supernatants and Cat-G on epithelial barrier function and inflammation, and the connection between these two parameters. METHODS: Male balb/c mice were used in this study. We evaluated the effect of a 2-hour intracolonic infusion of 1) fecal supernatants from UC patients pretreated or not with specific Cat-G inhibitor (SCGI); 2) PAR(4) -activating peptide (PAR(4) -AP); and 3) Cat-G on colonic myeloperoxidase (MPO) activity and paracellular permeability (CPP). The involvement of PAR(4) was assessed by pretreating animals with pepducin P4pal-10, which blocks PAR(4) signaling. We investigated the role of myosin light chain (MLC) kinase by using its inhibitor, ML-7, and we determined phosphorylated MLC (pMLC) levels in mice colonic mucosa. RESULTS: UC fecal supernatants, Cat-G, and PAR(4) agonist increased both CPP and MPO activity in comparison with healthy subjects fecal supernatants. ML-7 inhibited the CPP increase triggered by Cat-G by 92.3%, and the enhanced MPO activity by 43.8%. Intracolonic infusion of UC fecal supernatant determined an increased phosphorylation level of MLC. CONCLUSIONS: These observations support that luminal factors such as Cat-G play an important proinflammatory role in the pathogenesis of colitis, mainly depending on CPP increase by MLC phosphorylation.

Cancer-associated adipocytes exhibit an activated phenotype and contribute to breast cancer invasion.

Early local tumor invasion in breast cancer results in a likely encounter between cancer cells and mature adipocytes, but the role of these fat cells in tumor progression remains unclear. We show that murine and human tumor cells cocultivated with mature adipocytes exhibit increased invasive capacities in vitro and in vivo, using an original two-dimensional coculture system. Likewise, adipocytes cultivated with cancer cells also exhibit an altered phenotype in terms of delipidation and decreased adipocyte markers associated with the occurrence of an activated state characterized by overexpression of proteases, including matrix metalloproteinase-11, and proinflammatory cytokines [interleukin (IL)-6, IL-1ß]. In the case of IL-6, we show that it plays a key role in the acquired proinvasive effect by tumor cells. Equally important, we confirm the presence of these modified adipocytes in human breast tumors by immunohistochemistry and quantitative PCR. Interestingly, the tumors of larger size and/or with lymph nodes involvement exhibit the higher levels of IL-6 in tumor surrounding adipocytes. Collectively, all our data provide in vitro and in vivo evidence that (i) invasive cancer cells dramatically impact surrounding adipocytes; (ii) peritumoral adipocytes exhibit a modified phenotype and specific biological features sufficient to be named cancer-associated adipocytes (CAA); and (iii) CAAs modify the cancer cell characteristics/phenotype leading to a more aggressive behavior. Our results strongly support the innovative concept that adipocytes participate in a highly complex vicious cycle orchestrated by cancer cells to promote tumor progression that might be amplified in obese patients.

Luminal cathepsin g and protease-activated receptor 4: a duet involved in alterations of the colonic epithelial barrier in ulcerative colitis.

Impairment of the colonic epithelial barrier and neutrophil infiltration are common features of inflammatory bowel disease. Luminal proteases affect colonic permeability through protease-activated receptors (PARs). We evaluated: (i) whether fecal supernatants from patients with ulcerative colitis (UC) trigger alterations of colonic paracellular permeability and inflammation, and (ii) the roles of cathepsin G (Cat-G), a neutrophil serine protease, and its selective receptor, PAR(4), in these processes. Expression levels of both PAR(4) and Cat-G were determined in colonic biopsies from UC and healthy subjects. The effects of UC fecal supernatants on colonic paracellular permeability were measured in murine colonic strips. Involvement of Cat-G and PAR(4) was evaluated using pepducin P4pal-10 and specific Cat-G inhibitor (SCGI), respectively. In addition, the effect of PAR(4)-activating peptide was assessed. UC fecal supernatants, either untreated or pretreated with SCGI, were infused into mice, and myeloperoxidase activity was determined. PAR(4) was found to be overexpressed in UC colonic biopsies. Increased colonic paracellular permeability that was triggered by UC fecal supernatants was blocked by both SCGI (77%) and P4pal-10 (85%). Intracolonic infusion of UC fecal supernatants into mice increased myeloperoxidase activity. This effect was abolished by SCGI. These observations support that both Cat-G and PAR(4) play key roles in generating and/or amplifying relapses in UC and provide a rationale for the development of new therapeutic agents in the treatment of this disease.

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

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.

Distribution of beta-glucosidase and beta-glucuronidase activity and of beta-glucuronidase gene gus in human colonic bacteria.

beta-Glycosidase activities present in the human colonic microbiota act on glycosidic plant secondary compounds and xenobiotics entering the colon, with potential health implications for the human host. Information on beta-glycosidases is currently limited to relatively few species of bacteria from the human colonic ecosystem. We therefore screened 40 different bacterial strains that are representative of dominant bacterial groups from human faeces for beta-glucosidase and beta-glucuronidase activity. More than half of the low G+C% Gram-positive firmicutes harboured beta-glucosidase activity, while beta-glucuronidase activity was only found in some firmicutes within clostridial clusters XIVa and IV. Most of the Bifidobacterium spp. and Bacteroides thetaiotaomicron carried beta-glucosidase activity. A beta-glucuronidase gene belonging to family 2 glycosyl hydrolases was detected in 10 of the 40 isolates based on degenerate PCR. These included all nine isolates that gave positive assays for beta-glucuronidase activity, suggesting that the degenerate PCR could provide a useful assay for the capacity to produce beta-glucuronidase in the gut community. beta-Glucuronidase activity was induced by growth on d-glucuronic acid, or by addition of 4-nitrophenol-glucuronide, in Roseburia hominis A2-183, while beta-glucosidase activity was induced by 4-nitrophenol-glucopyranoside. Inducibility varied between strains.

Effect of the electrical currents generated by the intestinal smooth muscle layers on pancreatic enzyme activity: an in vitro study.

Gut enzymes in the small intestine are exposed to extremely low electrical currents (ELEC) generated by the smooth muscle. In the present study, the in vitro tests were undertaken to evaluate the effect of these electric currents on the activity of the proteolytic pancreatic digestive enzymes. A simulator generating the typical electrical activity of pig gut was used for these studies. The electric current emitted by the simulator was transmitted to the samples, containing enzyme and its substrate, using platinum plate electrodes. All samples were incubated at 37 degrees C for 1 h. The changes in optical density, corresponding to enzyme activity, in samples stimulated for 1 h with ELEC was compared with that not exposed to ELEC. The obtained results show that the electrical current with the characteristics of the myoelectrical migrating complex (MMC) has an influence on pancreatic enzyme activity. Increased endopeptidase and reduced exopeptidase activity was noticed in samples treated with ELEC. This observation can be of important as analyzed factors which can alter enzymatic activity of the gut, can thus also affect feed/food digestibility.