The intestinal barrier is essential in human health and constitutes the interface between the outside and the internal milieu of the body. A functional intestinal barrier allows absorption of nutrients and fluids but simultaneously prevents harmful substances like toxins and bacteria from crossing the intestinal epithelium and reaching the body. An altered intestinal permeability, a sign of a perturbed barrier function, has during the last decade been associated with several chronic conditions, including diseases originating in the gastrointestinal tract but also diseases such as Alzheimer and Parkinson disease. This has led to an intensified interest from researchers with diverse backgrounds to perform functional studies of the intestinal barrier in different conditions. Intestinal permeability is defined as the passage of a solute through a simple membrane and can be measured by recording the passage of permeability markers over the epithelium via the paracellular or the transcellular route. The methodological tools to investigate the gut barrier function are rapidly expanding and new methodological approaches are being developed. Here we outline and discuss, in vivo, in vitro and ex vivo techniques and how these methods can be utilized for thorough investigation of the intestinal barrier.

Objective Functional dyspepsia (FD) is an extremely common functional gastrointestinal disorder, the pathophysiology of which is poorly understood. We hypothesised that impaired intestinal barrier function is involved in the onset and persistence of this disorder by inducing low-grade inflammation. Therefore, our aim was to evaluate duodenal mucosal integrity and low-grade inflammation in patients with FD. Design Duodenal biopsy specimens were obtained from 15 patients with FD fulfilling the Rome III criteria and 15 age- and gender-matched healthy volunteers. Transepithelial electrical resistance (TEER) and paracellular permeability were measured in Ussing chambers. Expression of cell-to-cell adhesion proteins was evaluated by real-time PCR, western blot and/or immunofluorescence. Numbers of mast cells, eosinophils and intraepithelial lymphocytes were assessed by immunohistochemistry. Results Patients with FD displayed lower TEER and increased paracellular passage compared with healthy controls, which is indicative of impaired mucosal integrity. In addition, abnormal expression of cell-to-cell adhesion proteins at the level of tight junctions, adherens junctions and desmosomes was shown. Furthermore, patients were characterised by the presence of low-grade inflammation, as demonstrated by increased infiltration of mucosal mast cells and eosinophils. A significant association between the expression level of several cell-to-cell adhesion proteins, the extent of increased permeability and the severity of low-grade inflammation was found. Conclusions These findings challenge the classical paradigm that patients with FD show no structural changes in the gastrointestinal tract. We suggest that impaired intestinal barrier function is a pathophysiological mechanism in FD. Thus, restoration of intestinal barrier integrity may be a potential therapeutic target for treating patients with FD.

Background: Crohn's disease is common in developed nations where the typical diet is low in fibre and high in processed food. Primary lesions overlie Peyer's patches and colonic lymphoid follicles where bacterial invasion through M-cells occurs. We have assessed the effect of soluble non-starch polysaccharide (NSP) and food emulsifiers on translocation of Escherichia coli across M-cells. Methods: To assess effects of soluble plant fibres and food emulsifiers on translocation of mucosa-associated E coli isolates from Crohn's disease patients and from non-Crohn's controls, we used M-cell monolayers, generated by co-culture of Caco2-cl1 and Raji B cells, and human Peyer's patches mounted in Ussing chambers. Results: E coli translocation increased across M-cells compared to parent Caco2-cl1 monocultures; 15.8-fold (IQR 6.2–32.0) for Crohn's disease E coli (N=8) and 6.7-fold (IQR 3.7–21.0) for control isolates (N=5). Electron microscopy confirmed E coli within M-cells. Plantain and broccoli NSP markedly reduced E coli translocation across M-cells at 5 mg/ml (range 45.3–82.6% inhibition, p<0.01); apple and leek NSP had no significant effect. Polysorbate-80, 0.01% vol/vol, increased E coli translocation through Caco2-cl1 monolayers 59-fold (p<0.05) and, at higher concentrations, increased translocation across M-cells. Similarly, E coli translocation across human Peyer's patches was reduced 45±7% by soluble plantain NSP (5 mg/ml) and increased 2-fold by polysorbate-80 (0.1% vol/vol). Conclusions: Translocation of E coli across M-cells is reduced by soluble plant fibres, particularly plantain and broccoli, but increased by the emulsifier Polysorbate-80. These effects occur at relevant concentrations and may contribute to the impact of dietary factors on Crohn's disease pathogenesis.

Objective: Persistent stress and life events affect the course of ulcerative colitis and irritable bowel syndrome by largely unknown mechanisms. Corticotropin-releasing hormone (CRH) has been implicated as an important mediator of stress-induced abnormalities in intestinal mucosal function in animal models, but to date no studies in human colon have been reported. The aim was to examine the effects of CRH on mucosal barrier function in the human colon and to elucidate the mechanisms involved in CRH-induced hyper-permeability. Design: Biopsies from 39 volunteers were assessed for macromolecular permeability (horseradish peroxidise (HRP), ⁵¹Cr-EDTA), and electrophysiology after CRH challenge in Ussing chambers. The biopsies were examined by electron and confocal microscopy for HRP and CRH receptor localisation, respectively. Moreover, CRH receptor mRNA and protein expression were examined in the human mast cell line, HMC-1. Results: Mucosal permeability to HRP was increased by CRH (2.8±0.5 pmol/cm²/h) compared to vehicle exposure (1.5±0.4 pmol/cm²/h), p = 0.032, whereas permeability to ⁵¹Cr-EDTA and transmucosal electrical resistance were unchanged. The increased permeability to HRP was abolished by α-helical CRH (9-41) (1.3±0.6 pmol/cm²/h) and the mast cell stabiliser, lodoxamide (1.6±0.6 pmol/cm²/h). Electron microscopy showed transcellular passage of HRP through colonocytes. CRH receptor subtypes R1 and R2 were detected in the HMC-1 cell line and in lamina propria mast cells in human colon. Conclusions: Our results suggest that CRH mediates transcellular uptake of HRP in human colonic mucosa via CRH receptor subtypes R1 and R2 on subepithelial mast cells. CRH-induced macromolecular uptake in human colon mucosa may have implications for stress-related intestinal disorders.

Objective. The intestinal microbiota plays a substantial role in the pathogenesis of inflammatory bowel disease (IBD). Faecalibacterium prausnitzii (FP) is underrepresented in IBD patients and have been suggested to have anti-inflammatory effects in mice. Increased intestinal permeability is common in IBD but the relationship between FP and intestinal barrier function has not been investigated. Our aim was to study treatment with FP supernatant on intestinal barrier function in a dextran sodium sulfate (DSS) colitis mice model. Material and methods. C57BL/6 mice received 3% DSS in tap water ad libitum during five days to induce colitis. From day 3 the mice received a daily gavage with FP supernatant or broth during seven days. Ileum and colon were mounted in Ussing chambers for permeability studies with ⁵¹Cr-EDTA and Escherichia coli K-12. Colon was saved for Western blot analyses of tight junction proteins. Results. DSS-treated mice showed significant weight loss and colon shortening. Gavage with FP supernatant resulted in a quicker recovery after DSS treatment and less extensive colonic shortening. Ileal mucosa of DSS mice showed a significant increase in ⁵¹Cr-EDTA-passage compared to controls. ⁵¹Cr-EDTA passage was significantly decreased in mice receiving FP supernatant. No significant differences were observed in passage of E. coli K12. Western blots showed a trend to increased claudin-1 and claudin-2 expressions in DSS mice. Conclusions. Supernatant of FP enhances the intestinal barrier function by affecting paracellular permeability, and may thereby attenuate the severity of DSS-induced colitis in mice. These findings suggest a potential role of FP in the treatment of IBD.