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sMo1759

Neutrophils From Healthy Individuals but Not Celiac Disease Patients ShowChemotactic Activity to PT-GliadinSunaina Khandelwal, Mirkka Janka-Junttila, Karen M. Lammers, Marcello Chieppa, ElaineL. Leonard Puppa, Carole Parent, Vincenzo Casolaro, Alessio Fasano

Background: Celiac disease (CD) is triggered by the ingestion of gliadin, the immunogeniccomponent of gluten-containing grains. We have observed that exposure to gliadin inducesrapid and massive influx of neutrophils to the murine gut mucosa, strongly implying thatgliadin itself is a chemoattractant factor for neutrophils. Aim: To study whether gliadin haschemo-attractant properties for human neutrophils and, if so, whether neutrophils fromhealthy individuals (HC) and CD patients respond differently to gliadin. Methods: Neutro-phils were isolated from venous blood of HC and CD patients on a gluten-free diet (CD-GFD) for application in different chemotaxis assays. Neutrophils from HC (n=2) were appliedto the Taxi-scan assay, an In Vitro model that allows real-time monitoring of chemotaxis topepsin- trypsin-digested gliadin (PTG) or N-formyl-methionyl-leucyl-phenylalanine (fMLP)as a positive control. Subsequently, neutrophils isolated from HC (n=20 for PTG, n=14 forfMLP) and CD-GFD (n=16 PTG, n=12 fMLP) were labeled with calcein, applied to an under-agar migration assay for 2 h at 37°C with PTG or fMLP as stimulus, and immediately afterincubation analyzed using fluorescent microscopy. Results: With the Taxi-scan assay weshow that PT-gliadin was also a chemoattractant factor for human neutrophils. We thencompared the chemotactic response of neutrophils from HC and CD-GFD patients to PTGand fMLP in the under-agar assay. Unexpectedly, CD-GFD neutrophil migration to PTGwas markedly reduced compared to HC (0.403 ± 0.259 vs. 5.772 ± 1.338 net neutrophilmigration, respectively, P=0.0005). A similar, albeit non-significant difference was alsoobserved in the migration of CD-GFD vs. HC neutrophils to fMLP (4.790 ± 1.080 vs. 11.93± 2.886 net neutrophil migration, respectively, P=0.067). Conclusion: We here show thatPT-gliadin is a chemoattractant factor also for human neutrophils. We also show that,surprisingly, the chemotactic response of CD-GFD neutrophils to PT-gliadin is strongly andsignificantly impaired when compared to that of HC neutrophils. Compared to HC neutro-phils, CD-GFD neutrophils also show a trend toward reduced responsiveness to fMLP. Thesefindings support a new paradigm, by which a suboptimal, rather than exaggerated, innateimmune response, resulting in delayed arrival of neutrophils at the site of gluten-promotedinflammation, may critically contribute to the autoimmune pathogenesis of CD.

Mo1760

IL-15 Maintains Gliadin Peptide Influx During Long Term IFN-γ and TNF-αChallenge of CACO-2 MonolayersAnn Chua, Guy R. Sander

Background and Aims: The cytokines interferon-γ (IFN-γ), tumor necrosis factor (TNF-α)and interleukin-15 (IL-15) play different roles in the pathogenesis of coeliac disease (CD)but little is known of their combined role in regulating gliadin peptide influx across theintestinal epithelium. Our aim was to determine the combined effect of all three cytokineson gliadin peptide influx across an In Vitro model of the small intestinal epithelium duringperiods of short and long term cytokine challenge. Methods: Influx of a fluorescentlylabelled gliadin peptide p57-89 (FAM-p57-89) and ionic permeability across Caco-2 intestinalepithelial monolayers were determined during a 2 week cytokine challenge time-course.Caco-2 cells were grown on semi-permeable Transwells and challenged individually or incombination with IFNγ (10ng/ml and 1ng/ml), TNF-α (0.3ng/ml) and IL-15 (1ng/mL).Results: IFNγ (10ng/ml) significantly increased ionic permeability (6 fold +/-1) and the influxof a gliadin peptide (2.2 fold +/-0.2) across Caco-2 monolayers following 7 days of challenge.No change was detected following a shorter challenge period of 2 days. Neither IL-15 orTNF-α alone increased the rate of gliadin peptide influx during the 14 day challenge period.Addition of a low concentration of TNF-α (0.3ng/ml) in combination with IFNγ (10ng/ml)increased the rate of gliadin peptide influx (5 fold) following 7 days of challenge. In thepresence of IL-15, the increased rate of influx (5 fold) was maintained following 14 daysof IFNγ (10ng/ml) and TNF-α challenge but was reduced to 2 fold in the absence of IL-15. Conclusions: IFNγ, but not IL-15 or TNF-α is the cytokine that increases the influx ofgliadin peptides across the intestinal epithelium. IL-15 maintains an increased rate of gliadinpeptide influx during long term IFNγ and TNF-α challenge possibly by protecting the epithe-lium.

Mo1761

Investigation of the Cell Adhesion Molecules and the Components of theExtracellular Matrix in Celiac DiseaseNatalya Vorobyova, Sergey G. Khomeriki

Introduction: It is known, that the disturbance of histogenesis is the base of changes in thesmall intestine mucosa in celiac disease (CD). This process depends on the relationshipbetween the epithelium and the extracellular matrix. It is performed with the intercells andcells-matrix interactions. At the present time the state of these components in CD is studiedpoorly. Aims & methods: The aim of this study is to evaluate the expression of the celladhesion molecules and the components of the extracellular matrix in CD. They wereassessed immunohistohemically in the duodenal mucosa of the patients with CD (n= 25,from 18 to 45 years) and healthy controls (n=10) by means of antibody to E-cadherin(1:100), β-catenin (1:800), CD44 (RTU), collagen IY (RTU) LabVision, MMP-9 (1:250,Epitomics). The tissue expression was detected and visualized by “UltraVision LP Value HRPPolymer”, DAB+ (LabVision). Results: Total villous atrophy was found in all biopsy specimensof patients with CD (Marsh III). The strong regular expression of collagen IY was markedon the epithelium basement membranes of the crypts and villous in the normal duodenalmucosa. In cases CD there was a weak irregular staining of the basement membranes of theepithelium. The expression of E-cadherin and β-catenin was reduced on the cell membraneof the superficial and cryptal epithelium in celiac mucosa as compared with normal one.Therefore, in 30% of biopsy specimens with CD the expression of E-cadherin was observedin the cell cytoplasm and revealed the uneven distribution on the cell membrane. CD44

S-644AGA Abstracts

expression was enhanced on the membrane of the stromal cells and intraepithelial lympho-cytes in CD as compared with normal controls. The rate of MMP-9 in the stromal cellcytoplasm in the celiac duodenal mucosa was higher than one in the normal mucosa. Therised expression of MMP-9 was determined in the extracellular matrix in CD as well.Conclusion: The alterations of the intercells and cells-matrix interactions were revealed inthe duodenal mucosa from the patients with CD. These alterations result in the enhancedpermeability of the epithelium and the typical atrophic and proliferative changes of themucosa. The further studies of the molecular aspects of CD pathogenesis will discover newpossibilities for the diagnosis and prognosis of this disease.

Mo1762

Difference in HLA DQ2/DQ8 Haplotype and FOX3 Gene Expression BetweenCeliac and Non-Celiac Down Syndrome PatientsGloria Serena, Davide Libreri, Craig Sturgeon, Debby Kryszak, Karen M. Lammers,Alessio Fasano

Background: Down syndrome (DS), trisomy 21, is associated with an increased prevalenceof autoimmune disorders including celiac disease (CD). While the prevalence of celiac diseaseis 0.5-1% worldwide in normal population, its prevalence among DS patients rises up to5-15%. CD is an immune mediated enteropathy triggered by gliadin, a protein included ingluten-containing cereals among genetically susceptible individuals (95% has HLA DQ2haplotype, 5% HLA DQ8). CD4+CD25+FoxP3+ regulatory T cells (Tregs) constitute asubpopulation of CD4+ T cells that plays an important role in maintaining immune homeost-asis and self-tolerance. Dysfunction of Tregs is associated with autoimmunity and is hypothes-ized to result from a lack of exons within the FOXP3 gene that encode important functionalsites. Aim: To study putative genetic correlations among DS patients that account for thehigher prevalence of CD in this population. Methods: Venous blood was drawn from 89DS patients and 16 DS patients with CD (DS-CD) in Department of Pediatrics in Palermoand HLA DQ2/DQ8 haplotype was evaluated using DQ-CD Typing Plus Kit of BioDiagene.RNA was extracted from a subset of these samples (19 DS, 5 DS-CD) with the Midi RneasyLipid Tissue Kit (Qiagen). Real-time RT-PCR (SYBRgreen) was run to detect gene expressionof total FoxP3, its full length and Δ2 isoforms. Results: Thirty-two out of 89 DS patients(35.9%) and 9 out of 16 DS-CD patients (56.3%) had the HLA DQ2/DQ8 haplotype. Thereal-time RT-PCR analysis showed that expression of total FoxP3 and isoforms was higherexpressed in DS patients vs. DS-CD. Conclusions: Frequency of HLA DQ2/DQ8 haplotypein DS patients was similar to that of the normal population (30%), however, HLA DQ2/DQ8 haplotype was less frequent in DS-CD patients compared to non-Down CD population,suggesting the implication of an alternative HLA haplotype in DS-CD. Our preliminary datashowing higher expression on FoxP3 (isoforms) gene expression in DS vs. DS-CD patientssuggest a lack of functionality of autoimmune regulation in DS-CD.

Mo1763

Are TG2 Inhibitors Able to Decrease Gliadin-Induced Toxicity Related toCeliac Disease - A Proof-of-Concept StudyKatri Lindfors, Tiina Rauhavirta, Rami Kivistö, Pekka T. Männistö, Arturo GraciaHorsman, Martin Griffin, Markku Mäki, Katri Kaukinen

INTRODUCTION: Celiac disease is an autoimmune-mediated enteropathy characterized byan immune response to dietary gluten in wheat, rye and barley in genetically susceptibleindividuals. Gluten-derived gliadin peptides are deamidated by transglutaminase 2 (TG2),which leads to immune response in the small-intestinal mucosa. Therefore, it has beensuggested that TG2 inhibitors could substitute the gluten-free diet as a treatment for celiacdisease in the future. AIM: The aim of this study is to investigate whether TG2 inhibitorscan prevent the toxic effects of gliadin In Vitro and ex vivo. MATERIALS AND METHODS:Caco-2 cells used in In Vitro studies were pre-treated with two TG2 inhibitors, R281(extracellular) and R283 (intracellular), and thereafter treated with peptic-tryptic digestedgliadin (PT-gliadin). The change of permeability in response to different compounds wasanalyzed by measuring transepithelial resistance. Effects on cytoskeleton were defined withfluorescence stainings. Small-intestinal biopsies used in ex vivo studies were derived fromceliac disease patients who were either untreated or on gluten-free diet. Biopsies were pre-treated with R281 or R283 and cultured for 24 h or 48 h with or without PT-gliadin.Culture media were collected for measuring the secreted autoantibodies and snap-frozenbiopsies were stained with CD25 antibody to quantitate activated lymphocytes and Ki-67antibody to assess epithelial cell proliferation. RESULTS: Pretreatment with TG2 inhibitorsabolished the PT-gliadin-induced decrease of Caco-2 cell transepithelial resistance and actincytoskeletal rearrangement measured as membrane ruffling. Ex vivo celiac patient smallbowel mucosal biopsy organ culture experiments showed that TG2 inhibitors inhibitedthe gluten-induced increase of CD25-positive lymphocytes and augmented epithelial cellproliferation. However, the TG2 inhibitors were not able to prevent PT-gliadin inducedsecretion of celiac-specific autoantibodies into the culture medium. CONCLUSIONS: TG2inhibitors alleviate the toxic innate effects of gliadin on intestinal Caco-2 epithelial cells.Moreover, the inhibitors are able to prevent the gliadin-induced increase of CD25-positivelymphocytes and Ki-67 positive proliferative epithelial cells in celiac patient mucosal biopsiesbut they do not affect the secretion of celiac disease-specific autoantibodies. Taken together,our results would suggest that TG2 inhibitors decrease some, but not all, of the toxic effectsof gliadin. Therefore, further studies are needed to test the suitability of TG2 inhibitors asan alternative future treatment form for celiac disease.