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Gliadin induces zonulin release, tight junctions disassembly, and increased intestinal permeabillity
determine whether TUNEL+ and Bcl-2÷ cells were T-lymphocytes, seriate sections were incubated with a polyclonal anti-CO3 antibody (DAKO) at 1:2000 dilution. Results: In CO patients with EATLthe percentageof TUNEL÷ LPLs (mean 0.8%) was significantly lower than in biopsied controls (mean 2.4%, p
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701 Refractory Coeliac Disease Type I and II; Pilot-Treatment with Azathioprine and Prednison Cjj Mulder, Pj Wahab, Acitl Tan, Jwr Meijer, Rijnstate Hosp, Arnhem Netherlands Aim: To evaluatethe effect of azathioprine and prednisonetreatment (APT) on clinical and histological parametersin adult patients with refractory coeliac disease(RCD); type 1 without aberrant T-lymphocytesand type 2 with aberrant T-lymphocytes. Methods: Twenty-two COpatients were evaluatedwith intention to treat for RCD. Eighteenpatienswere treated, starting with prednisone 40 mg tapering to a maintenance dose of 10 mg daily after 6 weeks in combination with azathioprine 2 mg/kg daily for 1 year. If possible, prednisone was tapered to 2.5 mg daily. Other causes of malabsorption have been ruled out. RCD was defined as malabsorption due to gluten-related partial, subtotal or total villous atrophy (Marsh III ABC). Before, after 3 months and 12 months after starting treatment, clinical and histological parameterswere monitored. All patients were typed for HLA-DOA1 and DQB1 alleles and had intra-epithelial lymphocyte phenotypingand/or immunehistology from duodenal biopsies. All had abdominal CT-scansand small-bowel X-rays before entry to look for Entempathy-Associated T-cell Lymphomas (EATL). Results: Two patients were excluded from the trial because their compliance to GFO was not sufficient, and 2 patients because of developing EML's during work-up. Six of 8 patientswith RCDtype 1 respondedhistologically (1 yr) and complete normalisation of villi was seen in 5 patients. Ten patients had RCD type 2 and 6 of them developedan EATL3-12 months after starting treatment. However,3 of them had normalised villi at that time. Four others are still alive, 2 with increesing number of aberrantT-celts and 2 have normalised villi with a decreasingnumber of aberrant T-cells. All patients except one (RCDtype 1) carried DO2 or DO8 marker.Conclusion: In our study group of 18 adult refractory coeliacs type 1 and 2, APT induced a histological improvement in 12 patients (66%), in 10 (55%) normalisation of villi was demonstrated.The outcome of treatment in type 1 was very promising, but disappointing in type 2, because6 patients developedEATL during treatment (60%), 2 deterioratedfor their T-cells and only 2 are in "remission". We suggest APT in RCD type 1. In RCD type 2 we suggest to consider chemotherapeutical agents like cladribine or CHOP. 702 Gliadin Induces Zonulin Release, Tight Junctions Disassembly, And Increased Intestinal Permeability. Maria Grazia Clementa, Stefano De Virgilis, Dept of Biota Sci and 6iotech, Un Cagliari, Cagliari Italy; Alessio Fesano, Dept of Pediatric, Univ of Maryland, Baltimore, MD Background:While great progress has beenmade in understandingthe immunological mechanisms of gliadin-induced intestinal damage in celiac disease (CD), the pathways by which gliadin peptidescross the intestinal epithelialbarrier and reachthe submucosato be recognized by antigen presenting cells are still unknown. Objective:To investigatethe potential effect of gliadin on the cytoskeletal-dependentintestinal permeability changes and to analyze the relationship between gliadin and zonulin, a modulator of tight junction permeability that is up-regulated during the acute phase of CD. Methods: The effect of gliadin on the intracellular F-actin was investigatedin cultured rat small bowel enterocytes(IEC-6 cells) by fluorescence microscopic analysis and spectroffuorimetry, while the effects on intestinal permeabilitywere analysed by the Ussing chamber assay. The gliadin experiments were repeated after pretreatment with CGP 41251 (a protein kinase C, PKC, inhibitor), cycloheximide (a protein synthesis inhibitor) and the synthetic peptide FZI/O, which specifically binds and competes with the zonulin receptor. Results: Incubation of cultured cells with gliadin led to a significant reorganization of intracellular actin filaments, with an increment of F-actin polymerization which was detected by both fluorescence microscopy and spectrofluorimetry after only 15 minutes of gliadin incubation. This effect was time- and dose-dependent,was reversible,and was inhibited by a pre-incubation with CGP 41251 but not cycloheximide. The synthetic peptide FZI/O inhibited both the gliadin-induced actin polymerization and the increasead intestinal permeability observed with the Ussing chamber experiments. Conclusions: Gliadin was able to cause an early reorganizationof actin cytoskeleton in normal rat enterocyte ceils and an increase in intestinal permeability.These effects seem mediated by PKC and involve the zonulin pathway, since the synthetic peptide FZI/O inhibited the effects of gliadin on both cell cytoskeleton and intestinal epithelial permeability. 703 increased Rectal Permeability In Celiac Disease Edgardo Smecuol, Emilia Sugai, Ruben Dezi, Sonia I. Niveloni, Ivan Doldan, SiIvia C. Pedreira, Roberto M. Mazure, Horacio Vazquez,Zulema Kogan, Eduardo C. Maurino, Gastroentarology Hosp, Buenos Aires Argentina; Jon B. Meddings, Univ of Calgary, Calgary Canada; Julio C. Bai, GastroenterologyHosp, Buenos Aires Argentina BACKGROUND:The rectal mucosais easilyaccessibleand has beenpreviouslyshown involved in celiac disease, Instillation of gluten-derived peptides into the rectum produces typical immunopathologic changesthat have been used to assessthe presenceof gluten sensitivity. Furthermore, microscopic and collagenous colitis is associated with celiac disease. In the small intestine, celiac diseaseis characteristicallyassociatedwith increasedepithelial permeability. AIM: To determine whether rectal permeability is increased in patients with celiac disease. MATERIAL & METHODS:Sucralose has been demonstrated to be an inert, nondegradablesugarthat is well suited for the determinationof colonic permeability.We examined 5 consecutive patients with celiac disease (age range: 25-51yr.) manifesting as diarrhea and fat malabsorption and 5 healthy subjects (age range: 18-47). After cleaning the rectum with 500 ml of isotonic saline, a solution containing sucralose (5g) in isotonic saline (100 ml) was instilled into the rectum. Patients remained prone for 60 minutes and no evacuationwas permitted for 4 hours after sucralose instillation. All urine passed during the following five hours was collected for urinary sucralose determination using HPLC. Finally, rectal biopsies were taken by an endoscopic forceps (open-cup: 8 mm) at 10 cm from the anal margin. Samples were analyzedblindly by histological and morphometric methods. RESULTS:Mean
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701 Refractory Coeliac Disease Type I and II; Pilot-Treatment with Azathioprine and Prednison Cjj Mulder, Pj Wahab, Acitl Tan, Jwr Meijer, Rijnstate Hosp, Arnhem Netherlands Aim: To evaluatethe effect of azathioprine and prednisonetreatment (APT) on clinical and histological parametersin adult patients with refractory coeliac disease(RCD); type 1 without aberrant T-lymphocytesand type 2 with aberrant T-lymphocytes. Methods: Twenty-two COpatients were evaluatedwith intention to treat for RCD. Eighteenpatienswere treated, starting with prednisone 40 mg tapering to a maintenance dose of 10 mg daily after 6 weeks in combination with azathioprine 2 mg/kg daily for 1 year. If possible, prednisone was tapered to 2.5 mg daily. Other causes of malabsorption have been ruled out. RCD was defined as malabsorption due to gluten-related partial, subtotal or total villous atrophy (Marsh III ABC). Before, after 3 months and 12 months after starting treatment, clinical and histological parameterswere monitored. All patients were typed for HLA-DOA1 and DQB1 alleles and had intra-epithelial lymphocyte phenotypingand/or immunehistology from duodenal biopsies. All had abdominal CT-scansand small-bowel X-rays before entry to look for Entempathy-Associated T-cell Lymphomas (EATL). Results: Two patients were excluded from the trial because their compliance to GFO was not sufficient, and 2 patients because of developing EML's during work-up. Six of 8 patientswith RCDtype 1 respondedhistologically (1 yr) and complete normalisation of villi was seen in 5 patients. Ten patients had RCD type 2 and 6 of them developedan EATL3-12 months after starting treatment. However,3 of them had normalised villi at that time. Four others are still alive, 2 with increesing number of aberrantT-celts and 2 have normalised villi with a decreasingnumber of aberrant T-cells. All patients except one (RCDtype 1) carried DO2 or DO8 marker.Conclusion: In our study group of 18 adult refractory coeliacs type 1 and 2, APT induced a histological improvement in 12 patients (66%), in 10 (55%) normalisation of villi was demonstrated.The outcome of treatment in type 1 was very promising, but disappointing in type 2, because6 patients developedEATL during treatment (60%), 2 deterioratedfor their T-cells and only 2 are in "remission". We suggest APT in RCD type 1. In RCD type 2 we suggest to consider chemotherapeutical agents like cladribine or CHOP. 702 Gliadin Induces Zonulin Release, Tight Junctions Disassembly, And Increased Intestinal Permeability. Maria Grazia Clementa, Stefano De Virgilis, Dept of Biota Sci and 6iotech, Un Cagliari, Cagliari Italy; Alessio Fesano, Dept of Pediatric, Univ of Maryland, Baltimore, MD Background:While great progress has beenmade in understandingthe immunological mechanisms of gliadin-induced intestinal damage in celiac disease (CD), the pathways by which gliadin peptidescross the intestinal epithelialbarrier and reachthe submucosato be recognized by antigen presenting cells are still unknown. Objective:To investigatethe potential effect of gliadin on the cytoskeletal-dependentintestinal permeability changes and to analyze the relationship between gliadin and zonulin, a modulator of tight junction permeability that is up-regulated during the acute phase of CD. Methods: The effect of gliadin on the intracellular F-actin was investigatedin cultured rat small bowel enterocytes(IEC-6 cells) by fluorescence microscopic analysis and spectroffuorimetry, while the effects on intestinal permeabilitywere analysed by the Ussing chamber assay. The gliadin experiments were repeated after pretreatment with CGP 41251 (a protein kinase C, PKC, inhibitor), cycloheximide (a protein synthesis inhibitor) and the synthetic peptide FZI/O, which specifically binds and competes with the zonulin receptor. Results: Incubation of cultured cells with gliadin led to a significant reorganization of intracellular actin filaments, with an increment of F-actin polymerization which was detected by both fluorescence microscopy and spectrofluorimetry after only 15 minutes of gliadin incubation. This effect was time- and dose-dependent,was reversible,and was inhibited by a pre-incubation with CGP 41251 but not cycloheximide. The synthetic peptide FZI/O inhibited both the gliadin-induced actin polymerization and the increasead intestinal permeability observed with the Ussing chamber experiments. Conclusions: Gliadin was able to cause an early reorganizationof actin cytoskeleton in normal rat enterocyte ceils and an increase in intestinal permeability.These effects seem mediated by PKC and involve the zonulin pathway, since the synthetic peptide FZI/O inhibited the effects of gliadin on both cell cytoskeleton and intestinal epithelial permeability. 703 increased Rectal Permeability In Celiac Disease Edgardo Smecuol, Emilia Sugai, Ruben Dezi, Sonia I. Niveloni, Ivan Doldan, SiIvia C. Pedreira, Roberto M. Mazure, Horacio Vazquez,Zulema Kogan, Eduardo C. Maurino, Gastroentarology Hosp, Buenos Aires Argentina; Jon B. Meddings, Univ of Calgary, Calgary Canada; Julio C. Bai, GastroenterologyHosp, Buenos Aires Argentina BACKGROUND:The rectal mucosais easilyaccessibleand has beenpreviouslyshown involved in celiac disease, Instillation of gluten-derived peptides into the rectum produces typical immunopathologic changesthat have been used to assessthe presenceof gluten sensitivity. Furthermore, microscopic and collagenous colitis is associated with celiac disease. In the small intestine, celiac diseaseis characteristicallyassociatedwith increasedepithelial permeability. AIM: To determine whether rectal permeability is increased in patients with celiac disease. MATERIAL & METHODS:Sucralose has been demonstrated to be an inert, nondegradablesugarthat is well suited for the determinationof colonic permeability.We examined 5 consecutive patients with celiac disease (age range: 25-51yr.) manifesting as diarrhea and fat malabsorption and 5 healthy subjects (age range: 18-47). After cleaning the rectum with 500 ml of isotonic saline, a solution containing sucralose (5g) in isotonic saline (100 ml) was instilled into the rectum. Patients remained prone for 60 minutes and no evacuationwas permitted for 4 hours after sucralose instillation. All urine passed during the following five hours was collected for urinary sucralose determination using HPLC. Finally, rectal biopsies were taken by an endoscopic forceps (open-cup: 8 mm) at 10 cm from the anal margin. Samples were analyzedblindly by histological and morphometric methods. RESULTS:Mean