Effect of proinfiammtory stimuli on cellular activation and nitric oxide production in human primary enteroglial cells.

Abstracts / Digestive and Liver Disease 41S (2009), S1–S167 S23 to treated CD and normal biopsies. Flow cytometry analysis of T-LPL confirmed that IL-17A is over-produced in CD mucosa, and that both CD4+ and CD8+ cells were major sources of IL-17A (Table). Of the IL-17-producing cells, more than 50% co-expressed IFN-gamma. Blockade of IL-21 activity reduced IL-17A expression in cultures of both untreated CD and PT-stimulated treated CD biopsies. Conclusions: Data indicate that IL-17A expression is up-regulated in CD mucosa, and that IL-21 contributes to sustain IL-17 production. # C. Small bowel diseases 1. Celiac disease OC.05.6 EFFECT OF PROINFLAMMATORY STIMULI ON CELLULAR ACTIVATION AND NITRIC OXIDE PRODUCTION IN HUMAN PRIMARY ENTEROGLIAL CELLS C. Cirillo ∗ , G. Sarnelli, A. Mango, I. Esposito, S. Masone, G. Aprea, R. Cuomo Università degli Studi di Napoli Federico II, Napoli Background and aim: Enteric glial cells (EGC) are involved in intesti- nal inflammation. In humans, there are no in vitro models to study the relationships between EGC and inflammation. Aim: To set up a new method to isolate and purify a primary cul- ture of human EGC and to study EGC activation and responses’ to proinflammatory stimuli. Material and methods: Myenteric plexus preparations were isolated from human ileum of 15 patients undergoing surgery for colon car- cinoma and enzimatically dissociated. Ganglia were plated and cell cultures were grown to subconfluence. After 21 days, EGC were purified of contaminating cells by incubation with the anti-Thy-1.1 ab- coated magnetic beads and separation using a Dynal Magnet ® .Mixed EGC cultures and purified EGC cell line were immunocytochemi- cally characterized with anti-S100B, anti-glial fibrillary acidic protein (GFAP) and anti-alpha-smooth muscle actin antibodies to identify EGC and fibroblasts, respectively. To study the effects of inflammatory stimuli on EGC activation, cells were incubated with lipopolysaccha- ride (LPS, 1 μ g/mL), Tumor Necrosis Factor-alpha (TNF- α , 5ng/ml), interferon-gamma (IFN- γ , 100U/mL) for 32 h alone or in combination. A double labeling immunofluorescence using antibodies to S100B or GFAP was performed to identify EGC expressing c-fos in the nucleus, as a marker of cells activation. S100B mRNA, protein expression and secretion, iNOS expression and NO production were assessed. Results: In mixed EGC cultures, immunocytochemically analysis dis- played that 40% of cultured cells were EGC (S100B and GFAP positives), 50% were fibroblasts and neurons were virtually absent. In contrast, in purified EGC cell line, the presence of contaminating cells was significantly reduced by treatments with Dynabeads ® and the ma- jority of the cells were anti-GFAP and anti-S100B positives. Incubation with LPS and IFN- γ alone, or in combination with TNF- α , resulted in a consistent EGC’s activation (c-fos positive nuclei in S100B/GFAP positive cells), and significantly increased S100B expression, which was able to stimulate iNOS protein expression and NO production. Conclusions: We described for the first time a new method to isolate and purify EGC from human intestine. Proinflammatory cytokines di- rectly activate this cell population and stimulate NO production through the EGC-derived S100B protein