Epithelial cell extrusion leads to breaches in the intestinal epithelium.

BACKGROUND: Two distinct forms of intestinal epithelial cell (IEC) extrusion are described: 1 with preserved epithelial integrity and 1 that introduced breaches in the epithelial lining. In this study, we sought to determine the mechanism underlying the IEC extrusion that alters the permeability of the gut epithelium. METHODS: IEC extrusions in polarized T84 monolayer were induced with nigericin. Epithelial permeability was assessed with transepithelial electrical resistance and movements of latex microspheres and green fluorescent protein-transfected Escherichia coli across the monolayer. In vivo IEC extrusion was modulated in wild-type and a colitic (interleukin-10 knock-out) mouse model with caspase-1 activation and inhibition. Luminal aspirates and mucosal biopsies from control patients and patients with inflammatory bowel disease were analyzed for caspase-1 and caspase-3&7 activation. RESULTS: Caspase-1-induced IEC extrusion in T84 monolayers resulted in dose-dependent and time-dependent barrier dysfunction, reversible with caspase-1 inhibition. Moreover, the movements of microspheres and microbes across the treated epithelial monolayers were observed. Increased caspase-1-mediated IEC extrusion in interleukin-10 knock-out mice corresponded to enhanced permeation of dextran, microspheres, and translocation of E. coli compared with wild type. Caspase-1 inhibition in interleukin-10 knock-out mice resulted in a time-dependent reduction in cell extrusion and normalization of permeability to microspheres. Increased IEC extrusion in wild-type mice was induced with caspase-1 activation. In human luminal aspirates, the ratio of positively stained caspase-1 to caspase-3&7 cells were 1:1 and 2:1 in control patients and patients with inflammatory bowel disease, respectively; these observations were confirmed by cytochemical analysis of mucosal biopsies. CONCLUSIONS: IEC extrusion mediated by caspase-1 activation contributes to altered intestinal permeability in vitro and in vivo.

Sirolimus drug-eluting, hydrogel-impregnated polypropylene mesh reduces intra-abdominal adhesion formation in a mouse model.

BACKGROUND: Prosthetic mesh is used frequently in abdominal wall hernia reconstruction but is prone to postoperative adhesion formation. Complications resulting from intra-abdominal adhesions represent a considerable clinical and cost burden. We, herein, investigate the antiproliferative and antiadhesiogenic properties of sirolimus and hydrogel-impregnated, drug-eluting mesh to decrease such complications in a mouse model of abdominal wall hernia repair. METHODS: A 1 × 1cm(2) polypropylene mesh from 1 of 3 groups (group 1, plain control; group 2, hydrogel [2% agarose]; and group 3, hydrogel + 10 mcg sirolimus) was implanted operatively into the peritoneal cavity of BALB/c mice and followed for up to 4 weeks. Adhesions were scored by percent surface area of mesh (range, 0-100%), severity (range, 0-3), and tenacity (range, 0-4). Representative samples were assessed by scanning electron microscopy. RESULTS: Mesh impregnated with the combination of hydrogel and sirolimus led to a significant decrease in adhesion formation. The percent surface area of adhesional attachment to mesh was decreased from 100.0 ± 0% in the plain mesh control group versus 18 ± 8% (P < .001) in the combined impregnated mesh group. Similarly, adhesion severity scores were decreased from a score of 2.9 ± 0.1 (plain mesh) versus 1.4 ± 0.1 (sirolimus/hydrogel-impregnated mesh) (P < .001). Scores for tenacity were also decreased markedly from 3.5 ± 0.2 (plain mesh) versus 1.5 ± 0.1 (sirolimus/hydrogel-impregnated mesh (P < .001). CONCLUSION: Creation of a sirolimus drug-eluting and hydrogel-impregnated polypropylene mesh resulted in marked decrease of adhesion formation in this mouse model, was well tolerated without side effects, and has potential for clinical application.

Increased epithelial gaps in the small intestines of patients with inflammatory bowel disease: density matters.

BACKGROUND: Epithelial gaps created by shedding of epithelial cells in the small intestine can be visualized by using confocal laser endomicroscopy (CLE). The density of epithelial gaps in the small bowels of patients with inflammatory bowel disease (IBD) and controls without IBD is unknown. OBJECTIVE: To determine whether the epithelial gap density in patients with IBD is different from that in controls. DESIGN: Prospective, controlled, cohort study. SETTING: A tertiary-care referral center. PATIENTS: This study involved patients with IBD and control patients without IBD undergoing colonoscopy. INTERVENTION: Probe-based CLE (pCLE) was used to image the terminal ileum. MAIN OUTCOME MEASUREMENTS: The primary outcome of the study was gap density, defined as the total number of gaps per 1000 cells counted in adequately imaged villi by using pCLE. The pCLE images were blindly reviewed, and the number of epithelial gaps and cells were manually counted. The secondary outcomes were correlation of gap density with disease activity, location, and severity of clinical disease. RESULTS: There were 30 controls and 28 patients with IBD. Of the patients with IBD, 16 had Crohn's disease, and 12 had ulcerative colitis. The median epithelial gap densities for controls and patients with IBD were 18 and 61 gaps/1000 cells, respectively (P < .001). Gap density did not correlate with disease activity. Patients with ulcerative pan-colitis tended toward gap densities lower than those of patients with limited colitis (32 versus 97 gaps/1000 cells, P = .06). Patients with IBD with severe clinical disease also had lower median gap densities (37 vs 90 gaps/1000 cells, P = .04). LIMITATIONS: A single-center study. CONCLUSION: The epithelial gap density was significantly increased in patients with IBD compared with controls. ( CLINICAL TRIAL REGISTRATION NUMBER: NCT00988273.).

Epithelial gaps in a rodent model of inflammatory bowel disease: a quantitative validation study.

OBJECTIVES: Confocal laser endomicroscopy (CLE) is a non-invasive imaging modality of the gastrointestinal tract. Epithelial gaps in the small intestine of patients and rodents have been demonstrated using CLE. The goal of this study was to quantitatively validate the findings of epithelial gap density observed with CLE against confocal microscopy (CM) and light microscopy. METHODS: Two strains of mice (control 129 Sv/Ev and interleukin 10 knockout (IL-10(-/-))) underwent CLE of the terminal ileum. Adjacent ileal tissues were examined using CM and light microscopy. The total number of gaps and cells in the villi were manually counted from the three-dimensional reconstruction of cross-sectional CLE and CM images. The histology specimens were reviewed for epithelial gap and cell counts by a pathologist blinded to the study groups. The inter- and intra-observer variability for cell and gap counts were determined. RESULTS: For CLE, the gap densities (mean±s.d.) in the ileum for control and IL-10(-/-) mice were: 9.5±1.3 gaps per 1,000 cells and 20.6±2.1 gaps per 1,000 cells counted (P<0.001), respectively. For CM, the ileal gap densities were 7.3±1.3 gaps per 1,000 cells and 22.8±6.2 gaps per 1,000 cells (P=0.03), respectively. For light microscopy, the ileal gap densities were 29.2±5.9 gaps per 1,000 cells and 51.5±6.4 gaps per 1,000 cells for the two strains. CONCLUSION: CLE can be used to quantitatively assess epithelial cells and gaps with accuracy comparable to CM and light microscopy. In a mouse model of inflammatory bowel disease, the epithelial gap density in the terminal ileum is significantly increased when examined using all three modalities.

Dietary lipids alter the effect of steroids on transport of glucose after intestinal resection: Part II. Signalling of the response.

BACKGROUND/PURPOSE: Glucocorticosteroids alter the function of the intestine. Budesonide (Bud) increases the jejunal D-glucose uptake, and this effect is prevented through a polyunsaturated fatty acid (PUFA) diet. This study was undertaken to assess the possible signalling effect of budesonide, prednisone (Pred), or dexamethasone (Dex) in animals with a 50% intestinal resection and fed chow or a diet enriched with saturated (SFA) or polyunsaturated fatty acids. METHODS: Northern blots were performed. RESULTS: Steroids reduced the jejunal but not the ileal expression of proglucagon. Ornithine decarboxylase (ODC) expression was reduced in the jejunum. CONCLUSIONS: c-jun, ODC, and proglucagon may be involved in the adaptive response that occurs with steroids and variations in dietary lipids after intestinal resection.

Dietary lipids alter the effect of steroids on the transport of glucose after intestinal resection: Part I. Phenotypic changes and expression of transporters.

BACKGROUND/PURPOSE: Glucocorticosteroids alter the function of the intestine. This study was undertaken to assess the effect on D-glucose uptake of budesonide (Bud), prednisone (Pred), or dexamethasone (Dex) in animals with a 50% intestinal resection and fed chow or a diet enriched with saturated (SFA) or polyunsaturated fatty acids (PUFA). METHODS: In vitro ring uptake technique, Western blots, and Northern blots were performed. RESULTS: Bud increased the jejunal D-glucose uptake, and this effect was prevented by feeding PUFA. SGLT1 and Na+/K+ ATPase protein and mRNA abundance did not correlate with the change in the rate of uptake of glucose. CONCLUSIONS: (1) Bud increased the jejunal glucose uptake, (2) the activity of the sugar transporter does not correlate with the abundance of protein or their respective mRNAs, (3) th Bud effect on glucose uptake is prevented by feeding PUFA. Thus, the desired intestinal adaptive response after intestinal resection may be enhanced further by the administration of the locally acting steroid budesonide and by feeding a saturated compared with a polyunsaturated fatty acid diet.