Christian Tomuschat

Altered expression of a two-pore domain (K2P) mechano-gated potassium channel TREK-1 in Hirschsprung's disease.

Background:The pathophysiology of Hirschsprung’s disease (HSCR) is not fully understood. A significant proportion of patients have persisting bowel symptoms such as constipation, soiling, and enterocolitis despite correctly performed operations. Animal data suggest that stretch-activated 2-pore domain K+ channels play a critical role in the maintenance of intestinal barrier integrity.Methods:We investigated TREK-1 protein expression in ganglionic and aganglionic regions of HSCR patients (n = 10) vs. normal control colon (n = 10). Protein distribution was assessed by using immunofluorescence and confocal microscopy. Gene and protein expression were quantified using quantitative real-time polymerase chain reaction, western blot analysis, and densitometry.Results:Confocal microscopy of the normal colon revealed strong TREK-1 channel expression in the epithelium. TREK-1-positive cells were decreased in aganglionic and ganglionic bowel compared to controls. TREK-1 gene expression levels were significantly decreased in aganglionic and ganglionic bowel compared to controls (P < 0.05). Western blotting revealed decreased TREK-1 protein expression in aganglionic and ganglionic bowel compared to controls.Conclusion:We demonstrate, for the first time, the expression and distribution of TREK-1 channels in the human colon. The decreased TREK-1 expression in the aganglionic and ganglionic bowel observed in HSCR may alter intestinal epithelial barrier function leading to the development of enterocolitis.

Altered expression of ATP-sensitive K(+) channels in Hirschsprung's disease.

PURPOSE Hirschsprungs disease-associated enterocolitis (HAEC) is the most common cause of morbidity and mortality in Hirschsprungs disease (HSCR). Altered intestinal epithelial barrier function has been suggested to play a role in the causation of HAEC. In rodent experimental models of colitis, a decreased expression of K(ATP) channels (Subunits: Kir6.1/6.2 and SUR1/2) is reported. We designed this study to determine if K(ATP) channels exist within the human colon and to investigate the expression of different subunits in Hirschsprungs disease. METHODS We investigated Kir6.1, Kir6.2, SUR1, and SUR2 expression in ganglionic and aganglionic bowel of HD patients (n=5) and controls (n=5). Western blotting and confocal immunofluorescence were performed. MAIN RESULTS Western blot analysis revealed that Kir6.1, Kir6.2, SUR1, and SUR2 are strongly expressed in the normal human colon. Kir6.1, Kir6.2, SUR1, and SUR2 expression was significantly decreased in the aganglionic bowel compared to ganglionic bowel and controls. Kir6.1 and SUR1 expression were also significantly decreased in the ganglionic bowel of HSCR patients compared to controls. CONCLUSION We demonstrate for the first time the existence of K(ATP) channels in the human colon. The decreased K(ATP) channel expression in HSCR specimens suggests that an altered K(ATP) expression may interfere with intestinal epithelium barrier function and predispose to HAEC.

Altered goblet cell function in Hirschsprung’s disease

Aims and objectivesHirschsprung’s disease-associated enterocolitis (HAEC) is the most serious complication of Hirschsprung’s disease (HSCR). HAEC occurs in 17–50% of patients with HSCR and may occur before or after a properly performed pull-through operation. The pathogenesis of HAEC is poorly understood. It is well recognized that a complex mucosal barrier protects, as the first line of defense, the surface of healthy intestinal tract from adhesion and invasion by luminal micro-organisms. Within the intestinal epithelium, goblet cells secrete gel-forming mucins, the major component of mucus, which block the direct attachment of commensal bacteria to the epithelial layer. Mucin 2 (MUC2) is the predominant mucin expressed in humans. Trefoil factor 3 (TFF3) synergizes with mucin and enhances the protective barrier properties of the mucus layer. SAM pointed domain-containing ETS transcription factor (SPDEF) drives terminal differentiation and maturation of secretory progenitors into goblet cells. Krueppel-like factor 4 (KLF4) is a goblet cell-specific differentiation factor in the colon and controls goblet cell differentiation and activates mucin synthesis. We hypothesized that the goblet cell function in the ganglionic pulled-through bowel in HSCR is abnormal and, therefore, we investigated the changes in goblet cell differentiation and functional expression of mucin in the bowel specimens from patients with HSCR.Material and methodsWe investigated MUC2, TFF3, SPDEF and KLF4 expression, and the goblet cell population in the ganglionic and aganglionic bowel of HSCR patients (n = 10) and controls (n = 10) by qPCR, Western blotting, confocal immunofluorescence, and alcian blue staining.ResultsThe qPCR and Western blotting analysis revealed that TFF3, SPDEF and KLF4 expressions were significantly downregulated in the aganglionic and ganglionic colon of patients with HSCR as compared to controls (p < 0.05). Alcian blue staining revealed that the goblet cell population was significantly decreased in aganglionic and ganglionic colon as compared to controls (p < 0.05). Confocal microscopy revealed a markedly decreased expression of TFF3, SPDEF and KLF4 in colonic epithelium of patients with HSCR as compared to controls.ConclusionThis is, to our knowledge, the first report of decreased expression of TFF3, SPDEF, KLF4, and goblet cell population in the colon of patients with HSCR. Altered goblet cell function may result in intestinal barrier dysfunction contributing to the development of HAEC.

NOS-interacting protein (NOSIP) is increased in the colon of patients with Hirschsprungs's disease

PURPOSE Hirschsprungs associated enterocolitis (HAEC) is the most common cause of morbidity and mortality in Hirschsprungs disease (HSCR). Nitric oxide (NO) mediates intestinal homoeostasis and is inhibited by NOSIP, a modulator of NO production. We designed this study to investigate the expression of NOSIP in the colon of patients with HSCR. METHODS We investigated NOSIP, endothelial NO synthase, and neuronal NO synthase expression in both the aganglionic and ganglionic regions of HSCR patients (n=10) versus normal control colon (n=10). Protein distribution was assessed by using immunofluorescence and confocal microscopy. Gene and protein expression were quantified using quantitative real-time polymerase chain reaction (qPCR), Western blot analysis, and densitometry. MAIN RESULTS qPCR and Western blot analysis demonstrate that NOSIP was significantly increased in the aganglionic and ganglionic colon compared to controls (p<0.05). Confocal microscopy revealed a markedly increased expression of NOSIP in the colon epithelium of patients with HSCR compared to controls. CONCLUSION To our knowledge, we demonstrate for the first time the expression of NOSIP in the human colon. While NOSIP expression was increased in HSCR vs. non-HSCR patients, no significant difference was observed in patients with HAEC. The increased expression of NOSIP in the aganglionic and ganglionic bowel of HSCR may contribute to the development of enterocolitis by inhibiting local NO production in patients with Hirschsprungs disease. LEVEL OF EVIDENCE II.

Reduced expression of the NLRP6 inflammasome in the colon of patients with Hirschsprungs's disease

PURPOSE Hirschsprungs associated enterocolitis (HAEC) is the most common cause of morbidity and mortality in Hirschsprungs Disease (HSCR). The pathogenesis of HAEC remains unsatisfactorily understood. Mounting evidence of an altered microbiome in patients with HSCR adds a new angle to the pathogenesis of HAEC. NLRP6 is a member of the nucleotide-binding domain, leucine-rich-repeat-containing (NLR) innate immune receptor family, a multiprotein complex that functions as a sensor of damage-associated molecular patterns. Known as inflammasomes they have been implicated in the regulation of colonic microbial ecology and by alteration, regulators of inflammation. We designed this study to test the hypothesis that NLRP6 expression is altered in the colon of patients with HSCR. METHODS We investigated NLPR6 protein expression in both the aganglionic and ganglionic regions of HSCR patients (n = 10) versus healthy control colon (n = 10). Protein distribution was assessed by using immunofluorescence and confocal microscopy. Gene and protein expressions were quantified using quantitative real-time polymerase chain reaction (qPCR), Western blot analysis, and densitometry. MAIN RESULTS qPCR and Western blot analysis revealed that NLRP6 is expressed in the colon of patients with HSCR. NLRP6 expression was significantly decreased (p < 0.003) in the ganglionic and aganglionic bowel in HSCR compared to controls. Confocal microscopy revealed that NLRP6 expression in colonic epithelium was markedly decreased in HSCR specimens compared to controls. CONCLUSION We demonstrate for the first time the expression and distribution of NLRP6 in patients with HSCR. The observed decreased expression of NLRP6 may contribute to an altered colonic microbiome in patients with HSCR and increases the susceptibility to develop HAEC.

Reduction of hydrogen sulfide synthesis enzymes cystathionine-β-synthase and cystathionine-γ-lyase in the colon of patients with Hirschsprungs's Disease

PURPOSE Hirschsprung associated enterocolitis (HAEC) is the most common cause of morbidity and mortality in Hirschsprung Disease (HSCR). The pathogenesis of HAEC is poorly understood. In recent years, there is increasing evidence that a compromised intestinal barrier function plays a major role in the pathogenesis of HAEC. Hydrogen sulfide, synthesized from L-cysteine by two key enzymes, cystathionine-β-synthase (CBS) and cystathionine-γ-lysase (CSE) is reported to play a key role in regulating gastrointestinal motility and promoting resolution of inflammation. We designed this study to test the hypothesis that CBS and CSE expression is altered in the colon of patients with HSCR. METHODS We investigated CBS and CSE protein expression in both the aganglionic and ganglionic regions of HSCR patients (n=10) versus healthy control colon (n=10). Protein distribution was assessed by using immunofluorescence and confocal microscopy. Gene and protein expression was quantified using quantitative real-time polymerase chain reaction (qPCR), Western blot analysis, and densitometry. MAIN RESULTS qPCR and Western blot analysis revealed that CBS and CSE are expressed in the normal human colon. CBS and CSE expression was significantly decreased (p<0.003) in the ganglionic and aganglionic bowel in HSCR compared to controls. Confocal microscopy revealed that CBS and CSE expression in smooth muscles, interstitial cells of Cajal, platelet-derived growth factor-alpha receptor-positive cells, enteric neurons and colonic epithelium was markedly decreased in HSCR specimens compared to controls. CONCLUSION We demonstrate for the first time the expression and distribution of CBS/CSE in patients with HSCR. The observed decreased expression of CBS and CSE may affect mucosal integrity and colonic contractility and thus render HSCR patients more susceptible to develop HAEC.