ABSTRACT
OBJECTIVE: Intestinal inflammation resulting from manipulation-induced mast cell activation is a crucial mechanism in the pathophysiology of postoperative ileus (POI). Recently it has been shown that spleen tyrosine kinase (Syk) is involved in mast cell degranulation. Therefore, we have evaluated the effect of the Syk-inhibitor GSK compound 143 (GSK143) as potential treatment to shorten POI. DESIGN: In vivo: in a mouse model of POI, the effect of the Syk inhibitor (GSK143) was evaluated on gastrointestinal transit, muscular inflammation and cytokine production. In vitro: the effect of GSK143 and doxantrazole were evaluated on cultured peritoneal mast cells (PMCs) and bone marrow derived macrophages. RESULTS: In vivo: intestinal manipulation resulted in a delay in gastrointestinal transit at t=24 h (Geometric Center (GC): 4.4 ± 0.3). Doxantrazole and GSK143 significantly increased gastrointestinal transit (GC doxantrazole (10 mg/kg): 7.2 ± 0.7; GSK143 (1 mg/kg): 7.6 ± 0.6), reduced inflammation and prevented recruitment of immune cells in the intestinal muscularis. In vitro: in PMCs, substance P (0-90 µM) and trinitrophenyl (0-4 µg/ml) induced a concentration-dependent release of ß-hexosaminidase. Pretreatment with doxantrazole and GSK143 (0.03-10 µM) concentration dependently blocked substance P and trinitrophenyl induced ß-hexosaminidase release. In addition, GSK143 was able to reduce cytokine expression in endotoxin-treated bone marrow derived macrophages in a concentration-dependent manner. CONCLUSIONS: The Syk inhibitor GSK143 reduces macrophage activation and mast cell degranulation in vitro. In addition, it inhibits manipulation-induced intestinal muscular inflammation and restores intestinal transit in mice. These findings suggest that Syk inhibition may be a new tool to shorten POI.
Subject(s)
Aniline Compounds/therapeutic use , Ileus/prevention & control , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Postoperative Complications/prevention & control , Protein Kinase Inhibitors/therapeutic use , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrimidines/therapeutic use , Aniline Compounds/administration & dosage , Aniline Compounds/pharmacology , Animals , Cell Degranulation/drug effects , Cells, Cultured , Cytokines/biosynthesis , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical/methods , Gastrointestinal Transit/drug effects , Ileus/physiopathology , Macrophage Activation/drug effects , Mast Cells/drug effects , Mast Cells/physiology , Mice , Mice, Inbred C57BL , Ovalbumin/antagonists & inhibitors , Ovalbumin/pharmacology , Phosphodiesterase Inhibitors/therapeutic use , Postoperative Complications/physiopathology , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/pharmacology , Pyrimidines/administration & dosage , Pyrimidines/pharmacology , Substance P/antagonists & inhibitors , Substance P/pharmacology , Syk Kinase , Thioxanthenes/therapeutic use , Xanthones/therapeutic useABSTRACT
AIMS: Several gastrointestinal motility diseases are associated with altered numbers of interstitial cells of Cajal (ICC), and testing for alterations in numbers of ICC has been proposed as one way to improve routine diagnosis in motility diseases. However, the protocols currently used to visualize ICC in formalin-fixed paraffin-embedded (FFPE) tissue using antibodies to CD117 have not been optimized for studying motility disorders. The aims of this study were therefore to determine the optimal protocol using FFPE tissue, determine normal values for ICC in non-neoplastic human colon, and compare results with those obtained using immunofluorescence (IF). METHODS AND RESULTS: Non-neoplastic tissue was collected from patients undergoing resection for colonic cancer and fixed for both light (FFPE) and IF testing. Sections were processed for standard immunohistochemistry using different primary antibodies in conjunction with variations in antigen retrieval [ethylenediamine tetraacetricacid (EDTA), citrate], antibody dilution, blocking and detection (Mach2, Mach3, Envision+). Best results were obtained with EDTA retrieval, the DAKO CD117 antibody and Mach3 detection. CONCLUSIONS: The optimized protocol presented improved CD117 detection in FFPE tissues and showed good concordance with overall localization of CD117-immunoreactive ICC as detected by IF. As such, this protocol may be more useful than current diagnostic procedures in motility disorders.
