Nicolas A. Pardon

Impaired duodenal mucosal integrity and low-grade inflammation in functional dyspepsia

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.

A weakly acidic solution containing deoxycholic acid induces esophageal epithelial apoptosis and impairs integrity in an in vivo perfusion rabbit model

Impaired esophageal mucosal integrity may be an important contributor in the pathophysiology of gastroesophageal reflux disease (GERD). Nevertheless, the effect of potentially harmful agents on epithelial integrity is mainly evaluated in vitro for a short period of time and the possible induction of epithelial apoptosis has been neglected. Our objective was to assess the effect of an acidic and weakly acidic solution containing deoxycholic acid (DCA) on the esophageal epithelium in an in vivo rabbit model of esophageal perfusion and to evaluate the role of the epithelial apoptosis. The esophagus of 55 anesthetized rabbits was perfused for 30 min with different solutions at pH 7.2, pH 5.0, pH 1.0, and pH 5.0 containing 200 and 500 μM DCA. Thereafter, animals were euthanized immediately or at 24 or 48 h after the perfusion. Transepithelial electrical resistance, epithelial dilated intercellular spaces, and apoptosis were assessed in Ussing chambers, by transmission electron microscopy, and by TUNEL staining, respectively. No macroscopic or major microscopic alterations were observed after the esophageal perfusions. The acidic and weakly acidic solution containing DCA induced similar long-lasting functional impairment of the epithelial integrity but different ultrastructural morphological changes. Only the solution containing DCA induced epithelial apoptosis in vivo and in vitro in rabbit and human tissue. In contrast to acid, a weakly acidic solution containing DCA induces epithelial apoptosis and a long-lasting impaired mucosal integrity. The presence of apoptotic cells in the esophageal epithelium may be used as a marker of impaired integrity and/or bile reflux exposure.

Partial Restraint Stress Induces Increased Permeability and Low Grade Inflammation in the Rat Ileum, but Not in the Jejunum

Mixed-lineage kinase-3 (MLK3) activates multiple MAPK pathways and has been shown to initiate apoptosis, proliferation, migration or differentiation in several cell types. MLK3 signaling is also required for breast cancer cell invasion. However, the role of MLK3 signaling in regulation of intestinal epithelial cell sheet migration In Vivo and In Vitro is not known. Given its role in cancer cell migration, we sought to investigate whether MLK3 signaling is important in intestinal mucosal healing and epithelial cell motility. We compared the healing of jejunal mucosal ulcers induced by topical serosal acetic acid in different age groups of MLK3 null mice with that in age-matched wild type mice and assessed the role of MLK3 signaling in human Caco-2 intestinal epithelial migration across type I collagen substrates. In older mice (30-42 weeks of age), ulcer healing at 5 days after the initial insult was reduced by 47% (p<0.05) in MLK3 null and by 29% in MLK3 heterozygous mice when compared with wild-type littermate controls. In younger animals (8-12 weeks), ulcer healing in MLK3 null mice was 25% less at day 3 (n=7, p<0.05) and 23% less at day 5 (n=7, p<0.05) than in wild-type mice. In Vitro, an MLK3 inhibitor significantly decreased closure of circular wounds in Caco-2 cell monolayers after 24 hours compared with to vehicle-treated control by 16.3% and 20.1 % respectively (p<0.05). Proliferative blockade using hydroxyurea (30mM) did not abolish the effect of MLK3 inhibitor on Caco-2 wound closure, suggesting a true effect on cell motility rather than an effect on cell proliferation. The reduction in cell motility by MLK3 inhibitor reduced level of pERK by 79% (p<0.05) and pJNK by 12% (p<0.05) revealing suppression of ERK and JNK signaling. Total MLK3 was also compared in confluent Caco-2 cells to cell that have been migrating for 96 hours. MLK3 was 53% (p<0.05) higher in migrating vs confluent cells further indicating the importance of MLK3 signaling in migrating intestinal epithelial cells. These studies reveal a novel role for MLK3 signaling in the regulation of intestinal epithelial migration and suggest that MLK3 may be an important target to influence intestinal mucosal healing.