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DQ8 Haplotype and FOX3 Gene Expression Between Celiac and Non-Celiac Down Syndrome Patients
Mo1759
expression was enhanced on the membrane of the stromal cells and intraepithelial lymphocytes in CD as compared with normal controls. The rate of MMP-9 in the stromal cell cytoplasm in the celiac duodenal mucosa was higher than one in the normal mucosa. The rised 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 in the duodenal mucosa from the patients with CD. These alterations result in the enhanced permeability of the epithelium and the typical atrophic and proliferative changes of the mucosa. The further studies of the molecular aspects of CD pathogenesis will discover new possibilities for the diagnosis and prognosis of this disease.
AGA Abstracts
Neutrophils From Healthy Individuals but Not Celiac Disease Patients Show Chemotactic Activity to PT-Gliadin Sunaina Khandelwal, Mirkka Janka-Junttila, Karen M. Lammers, Marcello Chieppa, Elaine L. Leonard Puppa, Carole Parent, Vincenzo Casolaro, Alessio Fasano Background: Celiac disease (CD) is triggered by the ingestion of gliadin, the immunogenic component of gluten-containing grains. We have observed that exposure to gliadin induces rapid and massive influx of neutrophils to the murine gut mucosa, strongly implying that gliadin itself is a chemoattractant factor for neutrophils. Aim: To study whether gliadin has chemo-attractant properties for human neutrophils and, if so, whether neutrophils from healthy individuals (HC) and CD patients respond differently to gliadin. Methods: Neutrophils were isolated from venous blood of HC and CD patients on a gluten-free diet (CDGFD) for application in different chemotaxis assays. Neutrophils from HC (n=2) were applied to the Taxi-scan assay, an In Vitro model that allows real-time monitoring of chemotaxis to pepsin- 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 for fMLP) and CD-GFD (n=16 PTG, n=12 fMLP) were labeled with calcein, applied to an underagar migration assay for 2 h at 37°C with PTG or fMLP as stimulus, and immediately after incubation analyzed using fluorescent microscopy. Results: With the Taxi-scan assay we show that PT-gliadin was also a chemoattractant factor for human neutrophils. We then compared the chemotactic response of neutrophils from HC and CD-GFD patients to PTG and fMLP in the under-agar assay. Unexpectedly, CD-GFD neutrophil migration to PTG was markedly reduced compared to HC (0.403 ± 0.259 vs. 5.772 ± 1.338 net neutrophil migration, respectively, P=0.0005). A similar, albeit non-significant difference was also observed 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 that PT-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 and significantly impaired when compared to that of HC neutrophils. Compared to HC neutrophils, CD-GFD neutrophils also show a trend toward reduced responsiveness to fMLP. These findings support a new paradigm, by which a suboptimal, rather than exaggerated, innate immune response, resulting in delayed arrival of neutrophils at the site of gluten-promoted inflammation, may critically contribute to the autoimmune pathogenesis of CD.
Mo1762 Difference in HLA DQ2/DQ8 Haplotype and FOX3 Gene Expression Between Celiac and Non-Celiac Down Syndrome Patients Gloria Serena, Davide Libreri, Craig Sturgeon, Debby Kryszak, Karen M. Lammers, Alessio Fasano Background: Down syndrome (DS), trisomy 21, is associated with an increased prevalence of autoimmune disorders including celiac disease (CD). While the prevalence of celiac disease is 0.5-1% worldwide in normal population, its prevalence among DS patients rises up to 5-15%. CD is an immune mediated enteropathy triggered by gliadin, a protein included in gluten-containing cereals among genetically susceptible individuals (95% has HLA DQ2 haplotype, 5% HLA DQ8). CD4+CD25+FoxP3+ regulatory T cells (Tregs) constitute a subpopulation of CD4+ T cells that plays an important role in maintaining immune homeostasis and self-tolerance. Dysfunction of Tregs is associated with autoimmunity and is hypothesized to result from a lack of exons within the FOXP3 gene that encode important functional sites. Aim: To study putative genetic correlations among DS patients that account for the higher prevalence of CD in this population. Methods: Venous blood was drawn from 89 DS patients and 16 DS patients with CD (DS-CD) in Department of Pediatrics in Palermo and 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 Rneasy Lipid Tissue Kit (Qiagen). Real-time RT-PCR (SYBRgreen) was run to detect gene expression of 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. The real-time RT-PCR analysis showed that expression of total FoxP3 and isoforms was higher expressed in DS patients vs. DS-CD. Conclusions: Frequency of HLA DQ2/DQ8 haplotype in 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 data showing higher expression on FoxP3 (isoforms) gene expression in DS vs. DS-CD patients suggest a lack of functionality of autoimmune regulation in DS-CD.
Mo1760 IL-15 Maintains Gliadin Peptide Influx During Long Term IFN-γ and TNF-α Challenge of CACO-2 Monolayers Ann 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 the intestinal epithelium. Our aim was to determine the combined effect of all three cytokines on gliadin peptide influx across an In Vitro model of the small intestinal epithelium during periods of short and long term cytokine challenge. Methods: Influx of a fluorescently labelled gliadin peptide p57-89 (FAM-p57-89) and ionic permeability across Caco-2 intestinal epithelial monolayers were determined during a 2 week cytokine challenge time-course. Caco-2 cells were grown on semi-permeable Transwells and challenged individually or in combination 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 influx of 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 or TNF-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 the presence of IL-15, the increased rate of influx (5 fold) was maintained following 14 days of IFNγ (10ng/ml) and TNF-α challenge but was reduced to 2 fold in the absence of IL15. Conclusions: IFNγ, but not IL-15 or TNF-α is the cytokine that increases the influx of gliadin peptides across the intestinal epithelium. IL-15 maintains an increased rate of gliadin peptide influx during long term IFNγ and TNF-α challenge possibly by protecting the epithelium.
Mo1763 Are TG2 Inhibitors Able to Decrease Gliadin-Induced Toxicity Related to Celiac Disease - A Proof-of-Concept Study Katri Lindfors, Tiina Rauhavirta, Rami Kivistö, Pekka T. Männistö, Arturo Gracia Horsman, Martin Griffin, Markku Mäki, Katri Kaukinen INTRODUCTION: Celiac disease is an autoimmune-mediated enteropathy characterized by an immune response to dietary gluten in wheat, rye and barley in genetically susceptible individuals. Gluten-derived gliadin peptides are deamidated by transglutaminase 2 (TG2), which leads to immune response in the small-intestinal mucosa. Therefore, it has been suggested that TG2 inhibitors could substitute the gluten-free diet as a treatment for celiac disease in the future. AIM: The aim of this study is to investigate whether TG2 inhibitors can 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 digested gliadin (PT-gliadin). The change of permeability in response to different compounds was analyzed by measuring transepithelial resistance. Effects on cytoskeleton were defined with fluorescence stainings. Small-intestinal biopsies used in ex vivo studies were derived from celiac disease patients who were either untreated or on gluten-free diet. Biopsies were pretreated 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-frozen biopsies were stained with CD25 antibody to quantitate activated lymphocytes and Ki-67 antibody to assess epithelial cell proliferation. RESULTS: Pretreatment with TG2 inhibitors abolished the PT-gliadin-induced decrease of Caco-2 cell transepithelial resistance and actin cytoskeletal rearrangement measured as membrane ruffling. Ex vivo celiac patient small bowel mucosal biopsy organ culture experiments showed that TG2 inhibitors inhibited the gluten-induced increase of CD25-positive lymphocytes and augmented epithelial cell proliferation. However, the TG2 inhibitors were not able to prevent PT-gliadin induced secretion of celiac-specific autoantibodies into the culture medium. CONCLUSIONS: TG2 inhibitors 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-positive lymphocytes and Ki-67 positive proliferative epithelial cells in celiac patient mucosal biopsies but 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 effects of gliadin. Therefore, further studies are needed to test the suitability of TG2 inhibitors as an alternative future treatment form for celiac disease.
Mo1761 Investigation of the Cell Adhesion Molecules and the Components of the Extracellular Matrix in Celiac Disease Natalya Vorobyova, Sergey G. Khomeriki Introduction: It is known, that the disturbance of histogenesis is the base of changes in the small intestine mucosa in celiac disease (CD). This process depends on the relationship between the epithelium and the extracellular matrix. It is performed with the intercells and cells-matrix interactions. At the present time the state of these components in CD is studied poorly. Aims & methods: The aim of this study is to evaluate the expression of the cell adhesion molecules and the components of the extracellular matrix in CD. They were assessed 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 HRP Polymer”, DAB+ (LabVision). Results: Total villous atrophy was found in all biopsy specimens of patients with CD (Marsh III). The strong regular expression of collagen IY was marked on the epithelium basement membranes of the crypts and villous in the normal duodenal mucosa. In cases CD there was a weak irregular staining of the basement membranes of the epithelium. The expression of E-cadherin and β-catenin was reduced on the cell membrane of 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 observed in the cell cytoplasm and revealed the uneven distribution on the cell membrane. CD44
AGA Abstracts
S-644
expression was enhanced on the membrane of the stromal cells and intraepithelial lymphocytes in CD as compared with normal controls. The rate of MMP-9 in the stromal cell cytoplasm in the celiac duodenal mucosa was higher than one in the normal mucosa. The rised 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 in the duodenal mucosa from the patients with CD. These alterations result in the enhanced permeability of the epithelium and the typical atrophic and proliferative changes of the mucosa. The further studies of the molecular aspects of CD pathogenesis will discover new possibilities for the diagnosis and prognosis of this disease.
AGA Abstracts
Neutrophils From Healthy Individuals but Not Celiac Disease Patients Show Chemotactic Activity to PT-Gliadin Sunaina Khandelwal, Mirkka Janka-Junttila, Karen M. Lammers, Marcello Chieppa, Elaine L. Leonard Puppa, Carole Parent, Vincenzo Casolaro, Alessio Fasano Background: Celiac disease (CD) is triggered by the ingestion of gliadin, the immunogenic component of gluten-containing grains. We have observed that exposure to gliadin induces rapid and massive influx of neutrophils to the murine gut mucosa, strongly implying that gliadin itself is a chemoattractant factor for neutrophils. Aim: To study whether gliadin has chemo-attractant properties for human neutrophils and, if so, whether neutrophils from healthy individuals (HC) and CD patients respond differently to gliadin. Methods: Neutrophils were isolated from venous blood of HC and CD patients on a gluten-free diet (CDGFD) for application in different chemotaxis assays. Neutrophils from HC (n=2) were applied to the Taxi-scan assay, an In Vitro model that allows real-time monitoring of chemotaxis to pepsin- 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 for fMLP) and CD-GFD (n=16 PTG, n=12 fMLP) were labeled with calcein, applied to an underagar migration assay for 2 h at 37°C with PTG or fMLP as stimulus, and immediately after incubation analyzed using fluorescent microscopy. Results: With the Taxi-scan assay we show that PT-gliadin was also a chemoattractant factor for human neutrophils. We then compared the chemotactic response of neutrophils from HC and CD-GFD patients to PTG and fMLP in the under-agar assay. Unexpectedly, CD-GFD neutrophil migration to PTG was markedly reduced compared to HC (0.403 ± 0.259 vs. 5.772 ± 1.338 net neutrophil migration, respectively, P=0.0005). A similar, albeit non-significant difference was also observed 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 that PT-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 and significantly impaired when compared to that of HC neutrophils. Compared to HC neutrophils, CD-GFD neutrophils also show a trend toward reduced responsiveness to fMLP. These findings support a new paradigm, by which a suboptimal, rather than exaggerated, innate immune response, resulting in delayed arrival of neutrophils at the site of gluten-promoted inflammation, may critically contribute to the autoimmune pathogenesis of CD.
Mo1762 Difference in HLA DQ2/DQ8 Haplotype and FOX3 Gene Expression Between Celiac and Non-Celiac Down Syndrome Patients Gloria Serena, Davide Libreri, Craig Sturgeon, Debby Kryszak, Karen M. Lammers, Alessio Fasano Background: Down syndrome (DS), trisomy 21, is associated with an increased prevalence of autoimmune disorders including celiac disease (CD). While the prevalence of celiac disease is 0.5-1% worldwide in normal population, its prevalence among DS patients rises up to 5-15%. CD is an immune mediated enteropathy triggered by gliadin, a protein included in gluten-containing cereals among genetically susceptible individuals (95% has HLA DQ2 haplotype, 5% HLA DQ8). CD4+CD25+FoxP3+ regulatory T cells (Tregs) constitute a subpopulation of CD4+ T cells that plays an important role in maintaining immune homeostasis and self-tolerance. Dysfunction of Tregs is associated with autoimmunity and is hypothesized to result from a lack of exons within the FOXP3 gene that encode important functional sites. Aim: To study putative genetic correlations among DS patients that account for the higher prevalence of CD in this population. Methods: Venous blood was drawn from 89 DS patients and 16 DS patients with CD (DS-CD) in Department of Pediatrics in Palermo and 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 Rneasy Lipid Tissue Kit (Qiagen). Real-time RT-PCR (SYBRgreen) was run to detect gene expression of 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. The real-time RT-PCR analysis showed that expression of total FoxP3 and isoforms was higher expressed in DS patients vs. DS-CD. Conclusions: Frequency of HLA DQ2/DQ8 haplotype in 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 data showing higher expression on FoxP3 (isoforms) gene expression in DS vs. DS-CD patients suggest a lack of functionality of autoimmune regulation in DS-CD.
Mo1760 IL-15 Maintains Gliadin Peptide Influx During Long Term IFN-γ and TNF-α Challenge of CACO-2 Monolayers Ann 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 the intestinal epithelium. Our aim was to determine the combined effect of all three cytokines on gliadin peptide influx across an In Vitro model of the small intestinal epithelium during periods of short and long term cytokine challenge. Methods: Influx of a fluorescently labelled gliadin peptide p57-89 (FAM-p57-89) and ionic permeability across Caco-2 intestinal epithelial monolayers were determined during a 2 week cytokine challenge time-course. Caco-2 cells were grown on semi-permeable Transwells and challenged individually or in combination 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 influx of 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 or TNF-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 the presence of IL-15, the increased rate of influx (5 fold) was maintained following 14 days of IFNγ (10ng/ml) and TNF-α challenge but was reduced to 2 fold in the absence of IL15. Conclusions: IFNγ, but not IL-15 or TNF-α is the cytokine that increases the influx of gliadin peptides across the intestinal epithelium. IL-15 maintains an increased rate of gliadin peptide influx during long term IFNγ and TNF-α challenge possibly by protecting the epithelium.
Mo1763 Are TG2 Inhibitors Able to Decrease Gliadin-Induced Toxicity Related to Celiac Disease - A Proof-of-Concept Study Katri Lindfors, Tiina Rauhavirta, Rami Kivistö, Pekka T. Männistö, Arturo Gracia Horsman, Martin Griffin, Markku Mäki, Katri Kaukinen INTRODUCTION: Celiac disease is an autoimmune-mediated enteropathy characterized by an immune response to dietary gluten in wheat, rye and barley in genetically susceptible individuals. Gluten-derived gliadin peptides are deamidated by transglutaminase 2 (TG2), which leads to immune response in the small-intestinal mucosa. Therefore, it has been suggested that TG2 inhibitors could substitute the gluten-free diet as a treatment for celiac disease in the future. AIM: The aim of this study is to investigate whether TG2 inhibitors can 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 digested gliadin (PT-gliadin). The change of permeability in response to different compounds was analyzed by measuring transepithelial resistance. Effects on cytoskeleton were defined with fluorescence stainings. Small-intestinal biopsies used in ex vivo studies were derived from celiac disease patients who were either untreated or on gluten-free diet. Biopsies were pretreated 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-frozen biopsies were stained with CD25 antibody to quantitate activated lymphocytes and Ki-67 antibody to assess epithelial cell proliferation. RESULTS: Pretreatment with TG2 inhibitors abolished the PT-gliadin-induced decrease of Caco-2 cell transepithelial resistance and actin cytoskeletal rearrangement measured as membrane ruffling. Ex vivo celiac patient small bowel mucosal biopsy organ culture experiments showed that TG2 inhibitors inhibited the gluten-induced increase of CD25-positive lymphocytes and augmented epithelial cell proliferation. However, the TG2 inhibitors were not able to prevent PT-gliadin induced secretion of celiac-specific autoantibodies into the culture medium. CONCLUSIONS: TG2 inhibitors 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-positive lymphocytes and Ki-67 positive proliferative epithelial cells in celiac patient mucosal biopsies but 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 effects of gliadin. Therefore, further studies are needed to test the suitability of TG2 inhibitors as an alternative future treatment form for celiac disease.
Mo1761 Investigation of the Cell Adhesion Molecules and the Components of the Extracellular Matrix in Celiac Disease Natalya Vorobyova, Sergey G. Khomeriki Introduction: It is known, that the disturbance of histogenesis is the base of changes in the small intestine mucosa in celiac disease (CD). This process depends on the relationship between the epithelium and the extracellular matrix. It is performed with the intercells and cells-matrix interactions. At the present time the state of these components in CD is studied poorly. Aims & methods: The aim of this study is to evaluate the expression of the cell adhesion molecules and the components of the extracellular matrix in CD. They were assessed 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 HRP Polymer”, DAB+ (LabVision). Results: Total villous atrophy was found in all biopsy specimens of patients with CD (Marsh III). The strong regular expression of collagen IY was marked on the epithelium basement membranes of the crypts and villous in the normal duodenal mucosa. In cases CD there was a weak irregular staining of the basement membranes of the epithelium. The expression of E-cadherin and β-catenin was reduced on the cell membrane of 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 observed in the cell cytoplasm and revealed the uneven distribution on the cell membrane. CD44
AGA Abstracts
S-644