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Global Gene Expression Profile Analysis in Eosinophilic Gastritis Identifies CDH26
AB216 Abstracts
831
Global Gene Expression Profile Analysis in Eosinophilic Gastritis Identifies CDH26 J. M. Caldwell, M. H. Collins, C. Blanchard, E. M. Stucke, P. E. Putnam, J. P. Franciosi, J. P. Abonia, M. E. Rothenberg; Cincinnati Children’s Hospital Medical Center, Cincinnati, OH. RATIONALE: We aimed to determine the molecular basis of the pathogenesis of eosinophilic gastritis (EG). METHODS: Global transcript analysis was employed to identify genes regulated differentially in the gastric tissue of pediatric patients with active EG compared to normal patients. Further characterization of the gene and protein expression patterns of the most highly upregulated gene, CDH26, was undertaken through RT-PCR, immunohistochemistry, and western blot analysis. Cdh26 protein interactions were examined using transient transfection and immunoprecipitation analysis. RESULTS: We identified 104 transcripts that exhibited differential regulation between normal gastric tissue and that of patients with EG. Of these, 28 were upregulated, while 76 were downregulated. In addition, IL-13 transcript levels were increased on average 375-fold (p<0.01) in the gastric tissue of patients with EG. The protein encoded by the most highly upregulated gene, cadherin-like 26 (CDH26), was highly expressed in the gastric epithelium of patients with EG. Furthermore, IL-13 induced CDH26 expression in the gastric cell line NCI-N87 in vitro. Cdh26, a putative type-I cadherin molecule, exhibited homotypic interaction and additionally interacted with beta-catenin, alpha-catenin, and delta-catenin (p120) when expressed ectopically in 293T cells. CONCLUSIONS: We gained insight into the pathogenesis of EG by identifying a transcript signature of the gastric tissue of EG patients. Our data suggest the most highly upregulated transcript, CDH26, may be regulated in part by IL-13 in gastric epithelial cells. Cdh26 interacts with catenin proteins, which link cadherin molecules to the actin cytoskeleton. Furthermore, Cdh26 exhibits homotypic interaction, consistent with a function of Cdh26 in cell adhesion.
832
MONDAY
Follow-up Of A Single-centre Cohort Of Patients With Eosinophilic Esophagitis: Clinical Features And Prognosis I. N. Fung1, M. Dye2, J. M. Spergel1; 1Children’s Hospital of Philadelphia, Philadelphia, PA, 2College of New Jersey, Ewing, NJ. RATIONALE: To determine the natural history of Eosinophilic Esophagitis (EoE). METHODS: Parents of patients from an established EoE database at the Children’s Hospital of Philadelphia were called regarding their child’s current EoE status. Concomitant atopic disease, diet treatment, EoE medications, gastrointestinal symptoms and disease state by esophageal biopsy were accessed via phone interview. RESULTS: We contacted the parents of 617 patients with an average age of 10.25 years (range 1.0 - 24.2 years) at the time of the call. For the 597 (96.6%) patients with known date of first diagnostic biopsy, average time from diagnosis was 47 months (range 1 - 197 months). Analysis of the total group showed concomitant atopic diseases including asthma (48.6%), allergic rhinitis (43.1%), atopic dermatitis (26.7%), and food-induced anaphylaxis (21.6%). 74.6% were on diet modification and 20.7% were on elemental formula. EoE medications being used were anti-reflux medications (63.2%), swallowed inhaled steroids (34.2%), and oral steroids (3.6%). Gastrointestinal symptoms at present included abdominal pain (38.2%), heartburn (27.2%), poor weight gain (24.3%), vomiting (17.5%), difficulty swallowing (13.1%) and food impaction (7.9%). 39.5% had GERD. Other gastrointestinal disorders were Irritable Bowel Syndrome (5.3%), hiatal hernia (4.5%), Crohn’s Disease(2.9%), Ulcerative Colitis (2.3%), and Barrett’s esophagus (0.6%). One patient had resolved EoE; she also had Celiac Disease and her EoE resolved with a gluten-free diet. CONCLUSIONS: EoE is a chronic disease in which cure is rare. With the exception of GERD, other chronic gastrointestinal diseases are unlikely.
J ALLERGY CLIN IMMUNOL FEBRUARY 2011
833
Milk Protein-specific Cytokine Secretion Profiles In Infant Patients With Fpies And Proctocolitis H. Morita1, I. Nomura2, K. Orihara1, A. Matsuda1, H. Saito1, K. Matsumoto1; 1National Research Institute for Child Health and Development, Tokyo, JAPAN, 2National Medical Center for Children and Mothers, Tokyo, JAPAN. RATIONALE: Food protein-induced enterocolitis syndrome (FPIES) and proctocolitis are thought to be cell-mediated, non-IgE-associated gastrointestinal food allergies. However, the precise pathogenesis of these allergies remains uncertain. In the present in vitro study we investigated the antigenspecific cytokine secretion profiles of peripheral blood mononuclear cells (PBMCs) in these patients. METHODS: PBMCs from 13 and 6 infant patients with FPIES and proctocolitis, respectively (diagnosis was confirmed by oral challenge test), PBMCs from 13 normal infants and cord blood mononuclear cells from 10 normal infants were cultured in the presence and absence of lipopolysaccharide (LPS)-depleted milk proteins. Lymphocyte proliferation was assessed by 3H-thymidine-uptake on day 5 of culture, and the cytokine (TNF-a, IFN-g, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, IL17A, eotaxin and fractalkine) secretion profiles were measured using Milliplex technology on day 6. RESULTS: PBMCs from the patients with FPIES but not the patients with proctocolitis or the control children showed significant proliferative responses to LPS-depleted milk proteins. Inflammatory cytokines (TNF-a, IL-1b and IL-17A) and both Th1/Th2 cytokines (IFN-g, IL-5 and IL-13) were significantly secreted antigen-specifically by PBMCs from patients with FPIES. In contrast, the secretion of these cytokines was only marginal in PBMCs from patients with proctocolitis. CONCLUSIONS: These results suggest that antigen-specific acquired immune response may be involved in the pathogenesis of FPIES but not proctocolits, and that the inflammatory response is not restricted to Th2 cytokines.
834
Dysregulation of the Desmosomal Cadherin Desmoglein-1 in Eosinophilic Esophagitis J. D. Sherrill1, E. M. Stucke1, M. S. Costello1, V. L. Fabry1, M. H. Collins1, P. E. Putnam1, J. P. Franciosi1, J. P. Abonia1, L. J. Martin1, P. M. A. Sleiman2, J. M. Spergel2, H. Hakonarson2, M. E. Rothenberg1; 1Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, 2The Children’s Hospital of Philadelphia, Philadelphia, PA. RATIONALE: The esophageal tissue of eosinophilic esophagitis (EE) patients demonstrates suprabasal acantholysis, dilated intercellular spaces, and expansion of the basal epithelial layer indicating a pronounced defect in cellular adherence. We have shown decreased esophageal expression of the desmosomal cadherin desmoglein-1 (Dsg1) in EE and a SNP within Dsg1 is linked to increased EE susceptibility. Our aim is to further examine the role of the Dsg1 and its effects on the esophageal epithelium in EE pathogenesis. METHODS: Quantitative PCR, immunofluorescence microscopy, and western blotting were used to assess Dsg1 expression in patient biopsies. Esophageal epithelial cell migration was measured using wound healing assays. Dsg1 SNP association analysis was performed using PLINK. Luciferase reporter assays were performed to assess Dsg1 promoter activity. RESULTS: Dsg1 protein exhibits suprabasal expression within the esophagus, which is dramatically downregulated in EE, negatively correlates with esophageal eosinophil levels (p56 3 1023, r2 50.99), does not correlate with IL-13 expression and is not reversed during disease remission. Epidermal growth factor (EGF)-stimulated esophageal epithelial cell migration was reduced in the presence of an anti-Dsg1 antibody recognizing the extracellular domain of the protein. Multiple SNPs in the desmoglein locus associated with increased EE susceptibility (p54.70 3 1022-6.47 3 1024; odds ratio51.27-2.62). Lastly, the Dsg1 promoter is responsive to both serum and the activation of protein kinase C while IL-13 had no effect on Dsg1 promoter activity or protein expression. CONCLUSIONS: These data suggest that IL-13-independent dysregulation of Dsg1 in EE contributes to the pathogenic state of the esophageal epithelium involving an EGF mediated pathway.
831
Global Gene Expression Profile Analysis in Eosinophilic Gastritis Identifies CDH26 J. M. Caldwell, M. H. Collins, C. Blanchard, E. M. Stucke, P. E. Putnam, J. P. Franciosi, J. P. Abonia, M. E. Rothenberg; Cincinnati Children’s Hospital Medical Center, Cincinnati, OH. RATIONALE: We aimed to determine the molecular basis of the pathogenesis of eosinophilic gastritis (EG). METHODS: Global transcript analysis was employed to identify genes regulated differentially in the gastric tissue of pediatric patients with active EG compared to normal patients. Further characterization of the gene and protein expression patterns of the most highly upregulated gene, CDH26, was undertaken through RT-PCR, immunohistochemistry, and western blot analysis. Cdh26 protein interactions were examined using transient transfection and immunoprecipitation analysis. RESULTS: We identified 104 transcripts that exhibited differential regulation between normal gastric tissue and that of patients with EG. Of these, 28 were upregulated, while 76 were downregulated. In addition, IL-13 transcript levels were increased on average 375-fold (p<0.01) in the gastric tissue of patients with EG. The protein encoded by the most highly upregulated gene, cadherin-like 26 (CDH26), was highly expressed in the gastric epithelium of patients with EG. Furthermore, IL-13 induced CDH26 expression in the gastric cell line NCI-N87 in vitro. Cdh26, a putative type-I cadherin molecule, exhibited homotypic interaction and additionally interacted with beta-catenin, alpha-catenin, and delta-catenin (p120) when expressed ectopically in 293T cells. CONCLUSIONS: We gained insight into the pathogenesis of EG by identifying a transcript signature of the gastric tissue of EG patients. Our data suggest the most highly upregulated transcript, CDH26, may be regulated in part by IL-13 in gastric epithelial cells. Cdh26 interacts with catenin proteins, which link cadherin molecules to the actin cytoskeleton. Furthermore, Cdh26 exhibits homotypic interaction, consistent with a function of Cdh26 in cell adhesion.
832
MONDAY
Follow-up Of A Single-centre Cohort Of Patients With Eosinophilic Esophagitis: Clinical Features And Prognosis I. N. Fung1, M. Dye2, J. M. Spergel1; 1Children’s Hospital of Philadelphia, Philadelphia, PA, 2College of New Jersey, Ewing, NJ. RATIONALE: To determine the natural history of Eosinophilic Esophagitis (EoE). METHODS: Parents of patients from an established EoE database at the Children’s Hospital of Philadelphia were called regarding their child’s current EoE status. Concomitant atopic disease, diet treatment, EoE medications, gastrointestinal symptoms and disease state by esophageal biopsy were accessed via phone interview. RESULTS: We contacted the parents of 617 patients with an average age of 10.25 years (range 1.0 - 24.2 years) at the time of the call. For the 597 (96.6%) patients with known date of first diagnostic biopsy, average time from diagnosis was 47 months (range 1 - 197 months). Analysis of the total group showed concomitant atopic diseases including asthma (48.6%), allergic rhinitis (43.1%), atopic dermatitis (26.7%), and food-induced anaphylaxis (21.6%). 74.6% were on diet modification and 20.7% were on elemental formula. EoE medications being used were anti-reflux medications (63.2%), swallowed inhaled steroids (34.2%), and oral steroids (3.6%). Gastrointestinal symptoms at present included abdominal pain (38.2%), heartburn (27.2%), poor weight gain (24.3%), vomiting (17.5%), difficulty swallowing (13.1%) and food impaction (7.9%). 39.5% had GERD. Other gastrointestinal disorders were Irritable Bowel Syndrome (5.3%), hiatal hernia (4.5%), Crohn’s Disease(2.9%), Ulcerative Colitis (2.3%), and Barrett’s esophagus (0.6%). One patient had resolved EoE; she also had Celiac Disease and her EoE resolved with a gluten-free diet. CONCLUSIONS: EoE is a chronic disease in which cure is rare. With the exception of GERD, other chronic gastrointestinal diseases are unlikely.
J ALLERGY CLIN IMMUNOL FEBRUARY 2011
833
Milk Protein-specific Cytokine Secretion Profiles In Infant Patients With Fpies And Proctocolitis H. Morita1, I. Nomura2, K. Orihara1, A. Matsuda1, H. Saito1, K. Matsumoto1; 1National Research Institute for Child Health and Development, Tokyo, JAPAN, 2National Medical Center for Children and Mothers, Tokyo, JAPAN. RATIONALE: Food protein-induced enterocolitis syndrome (FPIES) and proctocolitis are thought to be cell-mediated, non-IgE-associated gastrointestinal food allergies. However, the precise pathogenesis of these allergies remains uncertain. In the present in vitro study we investigated the antigenspecific cytokine secretion profiles of peripheral blood mononuclear cells (PBMCs) in these patients. METHODS: PBMCs from 13 and 6 infant patients with FPIES and proctocolitis, respectively (diagnosis was confirmed by oral challenge test), PBMCs from 13 normal infants and cord blood mononuclear cells from 10 normal infants were cultured in the presence and absence of lipopolysaccharide (LPS)-depleted milk proteins. Lymphocyte proliferation was assessed by 3H-thymidine-uptake on day 5 of culture, and the cytokine (TNF-a, IFN-g, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, IL17A, eotaxin and fractalkine) secretion profiles were measured using Milliplex technology on day 6. RESULTS: PBMCs from the patients with FPIES but not the patients with proctocolitis or the control children showed significant proliferative responses to LPS-depleted milk proteins. Inflammatory cytokines (TNF-a, IL-1b and IL-17A) and both Th1/Th2 cytokines (IFN-g, IL-5 and IL-13) were significantly secreted antigen-specifically by PBMCs from patients with FPIES. In contrast, the secretion of these cytokines was only marginal in PBMCs from patients with proctocolitis. CONCLUSIONS: These results suggest that antigen-specific acquired immune response may be involved in the pathogenesis of FPIES but not proctocolits, and that the inflammatory response is not restricted to Th2 cytokines.
834
Dysregulation of the Desmosomal Cadherin Desmoglein-1 in Eosinophilic Esophagitis J. D. Sherrill1, E. M. Stucke1, M. S. Costello1, V. L. Fabry1, M. H. Collins1, P. E. Putnam1, J. P. Franciosi1, J. P. Abonia1, L. J. Martin1, P. M. A. Sleiman2, J. M. Spergel2, H. Hakonarson2, M. E. Rothenberg1; 1Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, 2The Children’s Hospital of Philadelphia, Philadelphia, PA. RATIONALE: The esophageal tissue of eosinophilic esophagitis (EE) patients demonstrates suprabasal acantholysis, dilated intercellular spaces, and expansion of the basal epithelial layer indicating a pronounced defect in cellular adherence. We have shown decreased esophageal expression of the desmosomal cadherin desmoglein-1 (Dsg1) in EE and a SNP within Dsg1 is linked to increased EE susceptibility. Our aim is to further examine the role of the Dsg1 and its effects on the esophageal epithelium in EE pathogenesis. METHODS: Quantitative PCR, immunofluorescence microscopy, and western blotting were used to assess Dsg1 expression in patient biopsies. Esophageal epithelial cell migration was measured using wound healing assays. Dsg1 SNP association analysis was performed using PLINK. Luciferase reporter assays were performed to assess Dsg1 promoter activity. RESULTS: Dsg1 protein exhibits suprabasal expression within the esophagus, which is dramatically downregulated in EE, negatively correlates with esophageal eosinophil levels (p56 3 1023, r2 50.99), does not correlate with IL-13 expression and is not reversed during disease remission. Epidermal growth factor (EGF)-stimulated esophageal epithelial cell migration was reduced in the presence of an anti-Dsg1 antibody recognizing the extracellular domain of the protein. Multiple SNPs in the desmoglein locus associated with increased EE susceptibility (p54.70 3 1022-6.47 3 1024; odds ratio51.27-2.62). Lastly, the Dsg1 promoter is responsive to both serum and the activation of protein kinase C while IL-13 had no effect on Dsg1 promoter activity or protein expression. CONCLUSIONS: These data suggest that IL-13-independent dysregulation of Dsg1 in EE contributes to the pathogenic state of the esophageal epithelium involving an EGF mediated pathway.