Andre G. Buret

Exacerbation of Inflammation-associated Colonic Injury in Rat through Inhibition of Cyclooxygenase-2

Cyclooxygenase type 1 is constitutively expressed and accounts for synthesis of prostaglandins in the normal gastrointestinal tract. Cyclooxygenase-2 is expressed at sites of inflammation. Selective inhibitors of cyclooxygenase-2 have been suggested to spare gastrointestinal prostaglandin synthesis, and therefore lack the ulcerogenic effects associated with standard nonsteroidal antiinflammatory drugs. However, the effects of cyclooxygenase-2 inhibitors on inflamed gastrointestinal mucosa have not been examined. We examined cyclooxygenase-2 mRNA and protein expression before and after induction of colitis in the rat, the contribution of cyclooxygenase-2 to colonic prostaglandin synthesis during colitis and the effects of selective inhibitors of cyclooxygenase-2 on colonic injury in this model. Cyclooxygenase-2 mRNA expression increased three to sixfold during the period 24 h to 1 wk after induction of colitis, with marked increases in cyclooxygenase-2 protein expression in the lamina propria and muscularis of the colon during colitis. Cyclooxygenase-1 expression (mRNA and protein) was not affected by the induction of colitis. The prostaglandins produced during colitis were largely derived from cyclooxygenase-2. Treatment with selective cyclooxygenase-2 inhibitors resulted in exacerbation of colitis, with perforation occurring when the compounds were administered for a week. These studies demonstrate that suppression of cyclooxygenase-2 can result in exacerbation of inflammation-associated colonic injury.

Early healing events in a porcine model of contaminated wounds: effects of nanocrystalline silver on matrix metalloproteinases, cell apoptosis, and healing.

A porcine model of wound healing was employed to examine the impact of nanocrystalline silver–coated dressings on specific wound healing events. Full‐thickness wounds were created on the backs of pigs, contaminated with an experimental inoculum containing Pseudomonas aeruginosa, Fusobacterium sp., and coagulase‐negative staphylococci, and covered with dressing products either containing silver or not. Nanocrystalline silver‐coated dressings promoted rapid wound healing, particularly during the first several days post‐injury. Healing was characterized by rapid development of well vascularized granulation tissue that supported tissue grafting 4 days post‐injury, unlike control dressed wounds. The proteolytic environment of wounds treated with nanocrystalline silver was characterized by reduced levels of matrix metalloproteinases. Matrix metalloproteinases have been shown to be present in chronic ulcers at abnormally high levels, as compared with acute wounds, and may contribute to the nonhealing nature of these wounds. Cellular apoptosis occurred at a higher frequency in the nanocrystalline silver–treated wounds than in wounds dressed with other products. The results suggest that nanocrystalline silver may play a role in altering or compressing the inflammatory events in wounds and facilitating the early phases of wound healing. These benefits are associated with reduced local matrix metalloproteinase levels and enhanced cellular apoptosis. (WOUND REP REG 2002;10:)

Platelets modulate gastric ulcer healing: Role of endostatin and vascular endothelial growth factor release

Bleeding and delayed healing of ulcers are well recognized clinical problems associated with the use of aspirin and other nonsteroidal antiinflammatory drugs, which have been attributed to their antiaggregatory effects on platelets. We hypothesized that antiplatelet drugs might interfere with gastric ulcer healing by suppressing the release of growth factors, such as vascular endothelial growth factor (VEGF), from platelets. Gastric ulcers were induced in rats by serosal application of acetic acid. Daily oral treatment with vehicle, aspirin, or ticlopidine (an ADP receptor antagonist) was started 3 days later and continued for 1 week. Ulcer induction resulted in a significant increase in serum levels of VEGF and a significant decrease in serum levels of endostatin (an antiangiogenic factor). Although both aspirin and ticlopidine markedly suppressed platelet aggregation, only ticlopidine impaired gastric ulcer healing and angiogenesis as well as reversing the ulcer-associated changes in serum levels of VEGF and endostatin. The effects of ticlopidine on ulcer healing and angiogenesis were mimicked by immunodepletion of circulating platelets, and ticlopidine did not influence ulcer healing when given to thrombocytopenic rats. Incubation of human umbilical vein endothelial cells with serum from ticlopidine-treated rats significantly reduced proliferation and increased apoptosis, effects reversed by an antibody directed against endostatin. Ticlopidine treatment resulted in increased platelet endostatin content and release. These results demonstrate a previously unrecognized contribution of platelets to the regulation of gastric ulcer healing. Such effects likely are mediated through the release from platelets of endostatin and possibly VEGF. As shown with ticlopidine, drugs that influence gastric ulcer healing may do so in part through altering the ability of platelets to release growth factors.

Strain-Dependent Induction of Enterocyte Apoptosis by Giardia lamblia Disrupts Epithelial Barrier Function in a Caspase-3-Dependent Manner

ABSTRACT We recently demonstrated that Giardia lamblia rearranges cytoskeletal proteins and reduces transepithelial electrical resistance. The effect of G. lamblia on enterocyte apoptosis is unknown, and a possible link between microbially induced enterocyte apoptosis and altered epithelial permeability has yet to be established. The aim of this study was to assess whether G. lamblia induces enterocyte apoptosis in duodenal epithelial monolayers and whether this effect increases epithelial permeability. Monolayers of nontransformed human duodenal epithelial cells were incubated with sonicated or live G. lamblia trophozoites (NF, S2, WB, or PB strains) for 8, 24, and 48 h. Cell cultures were assessed for apoptosis by Hoechst fluorescence staining, enzyme-linked immunosorbent assay for apoptotic nucleosomes, and electron microscopy. In separate experiments, monolayers were pretreated with or without 120 μM caspase-3 inhibitor (Z-DEVD-FMK) for 1 h and were assessed for production of apoptotic nucleosomes, tight junctional integrity (with fluorescent ZO-1 staining followed by confocal laser microscopy), and transepithelial permeability for fluorescein isothiocyanate-dextran. G. lamblia strains NF and S2, but not strains WB or PB, induced enterocyte apoptosis within the monolayers, and this effect was inhibited by Z-DEVD-FMK pretreatment. Using the G. lamblia NF isolate, additional experiments investigated the possible link between enterocyte apoptosis and altered epithelial permeability. G. lamblia NF disrupted tight junctional ZO-1 and increased epithelial permeability, but these effects were also prevented by pretreatment with the caspase-3 inhibitor. These findings indicate that strain-dependent induction of enterocyte apoptosis may contribute to the pathogenesis of giardiasis. This effect is responsible for a loss of epithelial barrier function by disrupting tight junctional ZO-1 and increasing permeability in a caspase-3-dependent manner.

Extra-intestinal and long term consequences of Giardia duodenalis infections

Giardiasis is the most common waterborne parasitic infection of the human intestine worldwide. The etiological agent, Giardia duodenalis (syn. G. intestinalis, G. lamblia), is a flagellated, binucleated protozoan parasite which infects a wide array of mammalian hosts. Human giardiasis is a true cosmopolitan pathogen, with highest prevalence in developing countries. Giardiasis can present with a broad range of clinical manifestations from asymptomatic, to acute or chronic diarrheal disease associated with abdominal pain and nausea. Most infections are self-limiting, although re-infection and chronic infection can occur. Recent evidence indicating that Giardia may cause chronic post-infectious gastrointestinal complications have made it a topic of intense research. The causes of the post-infectious clinical manifestations due to Giardia, even after complete elimination of the parasite, remain obscure. This review offers a state-of-the-art discussion on the long-term consequences of Giardia infections, from extra-intestinal manifestations, growth and cognitive deficiencies, to post-infectious irritable bowel syndrome. The discussion also sheds light on some of the novel mechanisms recently implicated in the production of these post-infectious manifestations.

[25] The MBEC assay system: Multiple equivalent biofilms for antibiotic and biocide susceptibility testing

Publisher Summary A number of technologies have been developed to study biofilm growth. Although these technologies produce reproducible biofilms for the study of biofilm growth, structure, and physiology, they have not been amenable for the routine study of biofilm susceptibility to antibiotics and biocides. For this reason, virtually every antibiotic and biocide available has been selected for activity against planktonic organisms. These drugs often have been found to lack activity against microbial biofilms. The MBEC (minimum biofilm eradication concentration) Assay System using the Calgary Biofilm Device provides, for the first time, an assay easily applicable to screening antibiotics and biocides for activity against microbial biofilms. The MBEC Assay System is ideally suited either for screening new putative antibiotics and/or biocides, or for the determination of both the MIC (minimal inhibitory concentration) and MBEC values in clinical situations for the treatment of chronic, recurrent, or device-related infections. The MBEC Assay System produces 96 equivalent biofilms formed under flow conditions, without the need for pumps. Further, as it is based on the standard 96 well platform, it conforms to existing technology available in most laboratories. The MBEC Assay System consists of a two-piece disposable plastic apparatus used for biofilm formation.

PAR2 activation alters colonic paracellular permeability in mice via IFN-γ-dependent and -independent pathways

Activation of colonic proteinase‐activated receptor‐2 (PAR2) caused inflammation and increased mucosal permeability in mouse colon. The present study was aimed at characterizing the possible links between these two phenomena. We evaluated the effects of intracolonic infusion of PAR2‐activating peptide, SLIGRL, on colonic paracellular permeability and inflammation at two different doses, 5 and 100 μg per mouse, in an attempt to discriminate between both PAR2‐mediated effects. We further investigated the possible involvement of interferon γ (IFN‐γ) and calmodulin‐dependent activation of myosin light chain kinase (MLCK), and alterations of zonula occludens‐1 (ZO‐1) localization in PAR2‐induced responses. Thus, at the lower dose, SLIGRL increased colonic permeability without causing inflammation. Western blotting showed phosphorylation of mucosal myosin light chain (MLC) expression after both doses of SLIGRL. Moreover, while the MLCK inhibitor, ML‐7, abolished the permeability effects of the low dose of SLIGRL, it only partially inhibited that of the high dose. In IFN‐γ‐deficient mice (B6 ifng−/−), the increases in permeability were similar for both doses of SLIGRL and prevented by ML‐7. In addition, MLCK immunoprecipitation revealed an increase of calmodulin binding to MLCK in the mucosa of mice treated with either dose of SLIGRL. Finally, we have shown that direct activation of PAR2 on enterocytes is responsible for increased permeability and ZO‐1 disruption. Moreover, SLIGRL at a dose that does not produce inflammation increases permeability via calmodulin activation, which binds and activates MLCK. The resulting tight junction opening does not depend upon IFN‐γ secretion, while the increased permeability in response to the high dose of PAR2 agonist involves IFN‐γ secretion.

Role of CD8+ and CD4+ T Lymphocytes in Jejunal Mucosal Injury during Murine Giardiasis

ABSTRACT T-cell-mediated pathogenesis has been documented in various idiopathic and microbially induced intestinal disorders. Diffuse microvillous shortening seen in giardiasis is responsible for disaccharidase insufficiencies and malabsorption of electrolytes, nutrients, and water. Other mucosal changes include crypt hyperplasia and increased numbers of intraepithelial lymphocytes (IEL). A recent report using an athymic mouse model of infection showed that these epithelial injuries were dependent on T cells. The aim of the present study was to identify which subset of superior mesenteric lymph node (SMLN) T cells were responsible for mucosal alterations in giardiasis. CD4+ and CD8+ T cells, as well as whole lymphocyte populations, were isolated from SMLN of Giardia muris-infected mice for adoptive transfer. Jejunal segments of recipient mice were assessed for brush border ultrastructure, sucrase activity, crypt/villus ratio, and IEL numbers. Mice that received enriched CD8+ and whole SMLN lymphocytes, but not CD4+ T cells, from infected donors showed diffuse shortening of microvilli, loss of brush border surface area, impaired sucrase activity, and increased crypt/villus ratios compared to respective controls. Transfer of whole SMLN lymphocytes, as well as enriched CD4+ or CD8+ T cells, from infected donors led to increased IEL numbers in the recipient jejunum. The findings indicate that loss of intestinal brush border surface area, reduced disaccharidase activities, and increased crypt/villus ratios in giardiasis are mediated by CD8+ T cells, whereas both CD8+ and CD4+ SMLN T cells regulate the influx of IEL.

Proteinase-activated receptor 1 activation induces epithelial apoptosis and increases intestinal permeability.

Proteinase-activated receptor 1 (PAR1)-mediated inflammation remains poorly understood. Here we characterize previously unrecognized effects of PAR1-induced apoptosis signaling, which contributes to epithelial barrier dysfunction. Incubation of epithelial cells with PAR1 agonists induced apoptosis and increased epithelial permeability in a caspase-3-dependent manner. Similarly, studies in vivo demonstrated that intracolonic infusion with PAR1 agonists increased colonic permeability in mice, and that this effect was abolished by pretreatment with a caspase-3 inhibitor. PAR1 agonists induced tight junctional zonula-occludens 1 disruption and apoptotic nuclear condensation. Investigation into signaling pathways showed that these effects were dependent on caspase-3, tyrosine kinase, and myosin light chain kinase. Conversely, the Src kinase inhibitor PP1 augmented zonula-occludens 1 injury and nuclear condensation induced by PAR1 agonists. These results support a role for proteinases and PARs in intestinal disease and provide new directions for possible therapeutic applications of PAR1 antagonists.

SGLT-1-mediated glucose uptake protects intestinal epithelial cells against LPS-induced apoptosis and barrier defects: a novel cellular rescue mechanism?

Excessive apoptosis induced by enteric microbes leads to epithelial barrier defects. This mechanism has been implicated in the pathogenesis of inflammatory bowel diseases (IBD) and bacterial enteritis. The sodium‐dependent glucose cotransporter (SGLT‐1) is responsible for active glucose uptake in enterocytes. The aim was to investigate the effects of SGLT‐1 glucose uptake on enterocyte apoptosis and barrier defects induced by bacterial lipopolysaccharide (LPS). SGLT‐1‐transfected Caco‐2 cells were treated with LPS (50 µg/mL) in low (5 mM) or high (25 mM) glucose media. LPS in low glucose induced caspase‐3 cleavage, DNA fragmentation, and increased paracellular permeability to dextran in epithelial cells. These phenomena were significantly attenuated in high glucose. LPS increased SGLT‐1 activity in high, but not low glucose media. Addition of phloridzin, which competitively binds to SGLT‐1, inhibited the cytoprotection mediated by high glucose. Western blot showed that LPS in high glucose increased the levels of anti‐apoptotic Bcl‐2 and Bcl‐XL, and did not change proapoptotic Bax. Differential extraction of membranous vs. cytosolic cell components demonstrated that high glucose inhibits mitochondrial cytochrome c translocation to cytosol. Collectively, SGLT‐1‐mediated glucose uptake increases anti‐apoptotic proteins, and protects enterocytes from LPS‐induced apoptosis and barrier defects. The understanding of this novel glucose‐mediated rescue mechanism may lead to therapeutic interventions for various enteric diseases. Linda C. H.Yu Andrew N.Flynn Jerrold R.Turner Andre G.Buret SGLT‐1‐mediated glucose uptake protects intestinal epithelial cells against LPS‐induced apoptosis and barrier defects: a novel cellular rescue mechanism?. FASEB J. 19, 1822–1835 (2005)