The Ussing chamber is increasingly being used for in vitro studies of human intestinal mucosa, but little attention has been paid to the viability of specimens over time. Ninety-one mucosal specimens from the ileum in 19 patients operated on for colonic cancer were studied in regard to intestinal barrier function, metabolism, electrophysiology and histology during 360 min of incubation in Ussing chambers. Steady-state permeability to 51Cr-EDTA was maintained for 120 min. Mucosal ATP and lactate levels were stable for 180 min and transmucosal glucose flux for 240 min. Lactate dehydrogenase leakage was limited within 120 min. Transepithelial potential difference was 9.0 +/- 3.0 mV at the start, and declined slowly throughout 360 min. Light microscopy revealed epithelial lifting from the basal lamina at 90 min. Transmission electron microscopy demonstrated preserved ultrastructure for 120 min. Specimens with a transepithelial potential difference below 6 mV at the start were associated with increased 51Cr-EDTA permeability and lactate dehydrogenase leakage and more pronounced light microscopy changes. All studied parameters pointed to preserved viability if experiments were kept within a period of 90 min after equilibration. The few specimens with early viability derangement were identified by a transepithelial potential difference below 6 mV at the start. The Ussing chamber provides a tool for in vitro studies of human intestinal epithelium, including permeability. To minimize viability problems, experiments should be limited in time and monitored by measurements of transepithelial potential difference.

No abstract

Starvation was the main cause of increased mucosal permeability in both intestinal segments of the rat, but surgical trauma had an additive effect, which was most pronounced in the jejunum.

Introduction Dietary fi bres may have prebiotic effects mediated by promotion of probiotic bacteria. This study explores the possibility that they may also improve health by inhibiting epithelial adhesion and translocation by pathogenic bacteria. We have focussed on soluble non-starch polysaccharide (NSP) from plantain bananas (Musa spp) since previous studies had shown that this is particularly effective at blocking Escherichia coli epithelial adherence. Methods The ability of plantain NSP to inhibit epithelial cell adhesion and invasion of various bacterial pathogens, their translocation through microfold (M)-cell monolayers (generated by co-culture of Caco2-cl1 cells and Raji B cells) and human Peyer's patches mounted in Ussing chambers has been assessed. Results Plantain NSP showed dose-related inhibition of epithelial adhesion and M-cell translocation by a range of gut pathogens. Plantain NSP at 5 mg/ml, a concentration readily achievable in the human gut lumen, inhibited adhesion to Caco2 cells of Clostridium diffi cile (67.6±12.3%, p<0.001 ANOVA), Salmonella typhimurium (85.0±8.2%, p<0.01), Shigella sonnei (46.6±29.3%, p<0.01) and ETEC (56.1±23.67%, p<0.05) but did not inhibit EPEC adhesion. Plantain NSP (5 mg/ml) also inhibited invasion of Caco2 cells by Salmonella (80.2±9.7%) and Shigella (46.7±13.4%; both p<0.01). Blockade of adherence and invasion to Caco 2 cells by plantain NSP was also confi rmed by Giemsa stain and light microscopy. Plantain NSP, at 5 mg/ml, also inhibited translocation of Salmonella and Shigella across M-cells by 46% and 73% respectively (both P<0.01). Similarly, blockade of Salmonella translocation across follicle-associated epithelium (FAE) of human ileal Peyer's patches was observed with 5 mg/ml plantain NSP (p<0.01; N=3). Conclusion Soluble plant fi bre from plantain bananas inhibits invasion and adhesion of pathogenic bacteria to Caco2 cells, and their translocation across M-cells of the FAE. This represents a novel mechanism by which soluble dietary fi bres could promote intestinal health and prevent infective diarrhoea.

Background The human gut mucosal immune system is compartmentalised in distinct and specialised immune niches. The epithelium and the lamina propria have been proposed as effector sites, while gut-associated lymphoid tissues (GALTs) constitute inductive immune niches. The major mucosal GALTs are the Peyer’s patches in the ileum and the colonic isolated lymphoid follicles (ILFs), scattered in the submucosa of the colon. The majority of studies of human gut immune function in health and disease have analysed unfractionated mucosal tissue samples. Hence, in contrast to mice, little is known about compartmentalised immune cell specialisation in the human gut. The aim of this study was to use novel dissection methods to analyse separate human gut immune niches. Methods Macroscopically healthy margins from colorectal cancer colectomies were obtained at a minimum distance of 10 cm from the tumour border. After faeces, mucus, fat and muscle removal, Peyer’s patches were identified and dissected using a stereomicroscope (based on Keita et al., Lab Invest, 2006). Colonic mucosa and submucosa (containing ILFs) fractions were mechanically separated by forceps (based on the method developed by Fenton et al., Immunity, under revision). Isolation of epithelial and lamina propria fractions from the mucosal compartment was performed by calcium chelation (DTT and EDTA) and enzymatic digestion (Collagenase II and DNAse), respectively. Cell suspensions from each fraction were analysed by flow cytometry (BD LSR-Fortessa and BD FACSymphony). Results As expected, mucosal GALTs were characterised by an enrichment of germinal centre B cells (CD19+CD20+CD38+), lymphoid tissue-like innate lymphoid cells (Lin−CD127+CD117+Nrp1+) and a higher CD4+/CD8+ T-cell ratio vs. mucosa, whereas the mucosal fraction was enriched for plasma cells (CD19+CD20−CD38high) and distinguished by a decreased CD4+/CD8+ T-cell ratio as compared with the GALT in both ileum and colon. CD19+/CD3+ ratios were only higher in Peyer’s patches but not in colonic submucosa enriched with ILFs, possibly due to the smaller size of the B-cell follicles in the latter. The intraepithelial compartment lacked B cells and contained more γδ-T cells as compared with the GALT and lamina propria. Conclusion We have used novel dissection methods in human intestinal tissues that reveal a compartmentalised immune cell specialisation that is in line with what has previously been described in mice. The method will allow for future deeper analysis of the human gut immune niches in health and disease, such as in inflammatory bowel disease.