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Examining Attitudes and Behaviors in Adolescents with Food Allergy: A Telephone Survey
S240 Abstracts
939
Examining Attitudes and Behaviors in Adolescents with Food Allergy: A Telephone Survey A. S. Murthy1, R. Seshadri1,2, J. A. Pongracic1; 1Children’s Memorial Hospital, Chicago, IL, 2The Mary Ann and J. Milburn Smith Child Health Research Program, Chicago, IL. RATIONALE: This study examines adolescent attitudes and behaviors regarding food allergy. METHODS: We conducted telephone interviews with adolescents, aged 11-18, with physician-diagnosed food allergies. We created a 36-question survey using 4-point Likert scales, focusing on 3 domains regarding food allergies: school, home and social environments. Using Chi-square and Fisher’s Exact analyses, we examined 3 primary outcomes: (1) recent accidental food allergen exposure (1 year), (2) self-reported anxiety and (3) self-perceived control. RESULTS: Of 155 adolescents contacted, 78 (50.3%) met inclusion criteria and completed the interview. Fifty (64%) were male. The mean age was 14.1 61.8 years. Half reported peanut allergy; 33 (42%) reported 3 food allergens. Forty-two (53.8%) reported always carrying EpiPen when with friends, while 15 (19%) never carry EpiPen. There was no association between any answer and recent accidental exposure. Self-reported anxiety was associated with: (1) peanut allergy (p50.039), (2) 3 food allergens (p50.019), (3) coughing or wheezing with prior exposure (p50.015) (4) sitting at allergen-free lunch tables (p50.028), and (5) perceiving life as more complicated than their peers (p<0.001). Perceived control of food allergy was associated with: peers’ awareness of the subject’s food allergy (p50.05), reporting a normal lifestyle despite having food allergies (p50.045), and comprehension that food allergy is potentially fatal (p50.011). CONCLUSIONS: Both objective and subjective measures were associated with adolescents’ perceived anxiety and control regarding food allergies, suggesting that a psychological component contributes to adolescent attitudes toward food allergies. Future studies should include assessment of anxiety when developing strategies for managing food allergies in this vulnerable population. Funding: Children’s Memorial Hospital
940
TUESDAY
Il-13 Is Overexpressed In Eosinophilic Esophagitis And Induces Eotaxin-3 Expression In Esophageal Epithelial Cells C. Blanchard1, M. K. Mingler1, P. Putnam1, M. E. Rothenberg1,2; 1 Cincinnati Children’s Hospital, Cincinnati, OH, 2University of Cincinnati, College of Medicine, Cincinnati, OH. RATIONALE: Eosinophilic esophagitis (EE) is an emerging worldwide disease that mimics gastroesophageal reflux disease. We recently uncovered that eotaxin-3 was the single most dysregulated gene in the esophagus of EE patients. In this study, we aim to uncover the molecular mechanisms involved in eotaxin-3 overexpression in EE patients. METHODS: Real Time PCR analysis was performed on RNA extracted from normal (NL) and EE esophageal biopsies. Esophageal epithelial cells (TE-7 and TE-13) and primary esophageal epithelial cells were stimulated with IL-13 (0 to 100 ng/mL) and or fluticasone propionate (10-6M). Transient transfections were performed with plasmids containing luciferase gene driven by eotaxin-3 promoter fragments and a dominant negative form or a conditionally active form of STAT6. RESULTS: IL-13 was increased by 16 fold in EE patient biopsies compared to NL biopsies. Notably, IL-13 was able to induce eotaxin-3 mRNA expression by 130667-fold in TE-13 and 7106168-fold in TE-7 esophageal cell lines and over 5.362.3-fold in primary epithelial cells. A mutation in the consensus sequence of STAT6, abrogated IL-13-induced eotaxin-3 promoter activity. A conditionally active form of STAT6 induced promoter activity by 3.161.2-fold. Using a dominant negative form of STAT6, IL-13-induced eotaxin-3 expression was shown to be inhibited by 92%. Finally, fluticasone propionate was able to decrease IL-13induced eotaxin-3 expression by 61% and 82% in TE-7 and TE-13 cell lines respectively. CONCLUSIONS: This study demonstrates that IL-13 is overexpressed in Eosinophilic Esophagitis and induces eotaxin-3 expression by a STAT6
J ALLERGY CLIN IMMUNOL JANUARY 2007
dependent and fluticasone propionate-inhibited pathway in esophageal epithelial cells and may have potential to provide targeted therapy for EE. Funding: NIH, FAAN
941
Gastrointestinal Priming and Recruitment of Th2 Lymphocytes in Food Allergy A. K. Knight1, A. B. Blazquez2, S. Zhang2, L. Mayer2, H. A. Sampson2, M. C. Berin2; 1Louisiana State University Health Sciences Center, Shreveport, LA, 2Mount Sinai School of Medicine, New York, NY. RATIONALE: Although a localized Th2 milieu has been described in the gut of humans with food allergy and mice with experimental food allergy, the sequence of events from allergen ingestion to Th2 recruitment remains poorly understood. Our aim was to define the sites of T cell activation and recruitment in response to food challenge. METHODS: Balb/c mice were sensitized to OVA (ip OVA/alum), and repeatedly fed with 50 mg of OVA to induce diarrhea. Cytokine secretion was measured from cultured mesenteric lymph node (MLN) or spleen cells. DO11.10 OVA-transgenic T cells were transferred to Balb/c mice, and activation after OVA feeding was determined by flow cytometry. Cytokine and chemokine gene expression was determined in mouse jejunum by qPCR. RESULTS: Mice sensitized and fed OVA developed an acute severe allergen-induced diarrhea associated with jejunal upregulation of IL-4 (390fold) and IL-13 (1009-fold). T cell priming occurred primarily in the MLN, with a predominant increase in IL-13 (1611 pg/ml), followed by IFN-g (480 pg/ml), IL-10 (291 pg/ml), IL-5 (261 pg/ml), and IL-4 (107 pg/ml); all were undetectable prior to feeding OVA. A single OVA feed induced MLN T cell proliferation, a4b7 upregulation (from 1% to >60%), and CD62L downregulation (from >85% to < 20%) in transferred DO11.10 T cells. Allergic diarrhea was also associated with a significant small intestinal upregulation of the Th2 T cell chemoattractants CCL17 (10-fold), CCL22 (3.5-fold), and CCL1 (495-fold). CONCLUSIONS: T cell priming in the MLN occurs prior to the switch from systemic sensitization to localized gastrointestinal allergic disease, and with intestinal Th2 chemoattractant expression, may be a critical step in the development of local Th2 inflammation in food allergy. Funding: NIH
942
Overexpression of ClC-3 Channels Prevent TGF-b-induced Apoptosis in Human Airway Epithelial Cells G. Cheng1, M. Kim1, B. Chaudhari1, D. K. Agrawal1,2; 1Dept. of Biomedical Science, Creighton University School of Medicine, Omaha, NE, 2 Dept. of Internal Medicine, Creighton University School of Medicine, Omaha, NE. RATIONALE: Widespread damage of airway epithelium and defective epithelial repair are hallmarks of chronic asthma. Excessive apoptosis could be a likely mechanism. TGF-b plays an important role in the pathophysiology of chronic asthma, and voltage-gated chloride channels are critical to cell shape and cell apoptosis. In this study, we investigated the involvement of chloride channels in TGF-b1 induced apoptosis in human airway epithelial cells (AECs). METHODS: Chloride ion currents in AECs were recorded using whole cell patch-clamp technique. Current-voltage relationship of the currents in BEAS-2B cells was examined. Expression of voltage-gated chloride channels was detected by real-time PCR. The effect of TGF-b1 on survival of BEAS-2B cells was determined by Annexin-V and Propidium iodide using flow cytometry. AECs were also transfected with control and ClC3 vector or Bcl-2 vector. RESULTS: Effect of TGF-b1 on the induction of apoptosis in AECs was more pronounced than TGF-b2. Overexpression of ClC-3 in BEAS-2B cells inhibited TGF-b1- induced apoptosis. Both outward and inward chloride currents in BEAS-2B cells were abolished by knocking down ClC-3 using two siRNAs, especially targeted to ClC-3. Treatment of the AECs with TGF-b1 resulted in the down-regulation of ClC-3 mRNA expression. Bcl-2 expression was down-regulated and Bax up-regulated in the AECs after TGF-b1 stimulation. ClC-3 expression was increased in Bcl-2
939
Examining Attitudes and Behaviors in Adolescents with Food Allergy: A Telephone Survey A. S. Murthy1, R. Seshadri1,2, J. A. Pongracic1; 1Children’s Memorial Hospital, Chicago, IL, 2The Mary Ann and J. Milburn Smith Child Health Research Program, Chicago, IL. RATIONALE: This study examines adolescent attitudes and behaviors regarding food allergy. METHODS: We conducted telephone interviews with adolescents, aged 11-18, with physician-diagnosed food allergies. We created a 36-question survey using 4-point Likert scales, focusing on 3 domains regarding food allergies: school, home and social environments. Using Chi-square and Fisher’s Exact analyses, we examined 3 primary outcomes: (1) recent accidental food allergen exposure (1 year), (2) self-reported anxiety and (3) self-perceived control. RESULTS: Of 155 adolescents contacted, 78 (50.3%) met inclusion criteria and completed the interview. Fifty (64%) were male. The mean age was 14.1 61.8 years. Half reported peanut allergy; 33 (42%) reported 3 food allergens. Forty-two (53.8%) reported always carrying EpiPen when with friends, while 15 (19%) never carry EpiPen. There was no association between any answer and recent accidental exposure. Self-reported anxiety was associated with: (1) peanut allergy (p50.039), (2) 3 food allergens (p50.019), (3) coughing or wheezing with prior exposure (p50.015) (4) sitting at allergen-free lunch tables (p50.028), and (5) perceiving life as more complicated than their peers (p<0.001). Perceived control of food allergy was associated with: peers’ awareness of the subject’s food allergy (p50.05), reporting a normal lifestyle despite having food allergies (p50.045), and comprehension that food allergy is potentially fatal (p50.011). CONCLUSIONS: Both objective and subjective measures were associated with adolescents’ perceived anxiety and control regarding food allergies, suggesting that a psychological component contributes to adolescent attitudes toward food allergies. Future studies should include assessment of anxiety when developing strategies for managing food allergies in this vulnerable population. Funding: Children’s Memorial Hospital
940
TUESDAY
Il-13 Is Overexpressed In Eosinophilic Esophagitis And Induces Eotaxin-3 Expression In Esophageal Epithelial Cells C. Blanchard1, M. K. Mingler1, P. Putnam1, M. E. Rothenberg1,2; 1 Cincinnati Children’s Hospital, Cincinnati, OH, 2University of Cincinnati, College of Medicine, Cincinnati, OH. RATIONALE: Eosinophilic esophagitis (EE) is an emerging worldwide disease that mimics gastroesophageal reflux disease. We recently uncovered that eotaxin-3 was the single most dysregulated gene in the esophagus of EE patients. In this study, we aim to uncover the molecular mechanisms involved in eotaxin-3 overexpression in EE patients. METHODS: Real Time PCR analysis was performed on RNA extracted from normal (NL) and EE esophageal biopsies. Esophageal epithelial cells (TE-7 and TE-13) and primary esophageal epithelial cells were stimulated with IL-13 (0 to 100 ng/mL) and or fluticasone propionate (10-6M). Transient transfections were performed with plasmids containing luciferase gene driven by eotaxin-3 promoter fragments and a dominant negative form or a conditionally active form of STAT6. RESULTS: IL-13 was increased by 16 fold in EE patient biopsies compared to NL biopsies. Notably, IL-13 was able to induce eotaxin-3 mRNA expression by 130667-fold in TE-13 and 7106168-fold in TE-7 esophageal cell lines and over 5.362.3-fold in primary epithelial cells. A mutation in the consensus sequence of STAT6, abrogated IL-13-induced eotaxin-3 promoter activity. A conditionally active form of STAT6 induced promoter activity by 3.161.2-fold. Using a dominant negative form of STAT6, IL-13-induced eotaxin-3 expression was shown to be inhibited by 92%. Finally, fluticasone propionate was able to decrease IL-13induced eotaxin-3 expression by 61% and 82% in TE-7 and TE-13 cell lines respectively. CONCLUSIONS: This study demonstrates that IL-13 is overexpressed in Eosinophilic Esophagitis and induces eotaxin-3 expression by a STAT6
J ALLERGY CLIN IMMUNOL JANUARY 2007
dependent and fluticasone propionate-inhibited pathway in esophageal epithelial cells and may have potential to provide targeted therapy for EE. Funding: NIH, FAAN
941
Gastrointestinal Priming and Recruitment of Th2 Lymphocytes in Food Allergy A. K. Knight1, A. B. Blazquez2, S. Zhang2, L. Mayer2, H. A. Sampson2, M. C. Berin2; 1Louisiana State University Health Sciences Center, Shreveport, LA, 2Mount Sinai School of Medicine, New York, NY. RATIONALE: Although a localized Th2 milieu has been described in the gut of humans with food allergy and mice with experimental food allergy, the sequence of events from allergen ingestion to Th2 recruitment remains poorly understood. Our aim was to define the sites of T cell activation and recruitment in response to food challenge. METHODS: Balb/c mice were sensitized to OVA (ip OVA/alum), and repeatedly fed with 50 mg of OVA to induce diarrhea. Cytokine secretion was measured from cultured mesenteric lymph node (MLN) or spleen cells. DO11.10 OVA-transgenic T cells were transferred to Balb/c mice, and activation after OVA feeding was determined by flow cytometry. Cytokine and chemokine gene expression was determined in mouse jejunum by qPCR. RESULTS: Mice sensitized and fed OVA developed an acute severe allergen-induced diarrhea associated with jejunal upregulation of IL-4 (390fold) and IL-13 (1009-fold). T cell priming occurred primarily in the MLN, with a predominant increase in IL-13 (1611 pg/ml), followed by IFN-g (480 pg/ml), IL-10 (291 pg/ml), IL-5 (261 pg/ml), and IL-4 (107 pg/ml); all were undetectable prior to feeding OVA. A single OVA feed induced MLN T cell proliferation, a4b7 upregulation (from 1% to >60%), and CD62L downregulation (from >85% to < 20%) in transferred DO11.10 T cells. Allergic diarrhea was also associated with a significant small intestinal upregulation of the Th2 T cell chemoattractants CCL17 (10-fold), CCL22 (3.5-fold), and CCL1 (495-fold). CONCLUSIONS: T cell priming in the MLN occurs prior to the switch from systemic sensitization to localized gastrointestinal allergic disease, and with intestinal Th2 chemoattractant expression, may be a critical step in the development of local Th2 inflammation in food allergy. Funding: NIH
942
Overexpression of ClC-3 Channels Prevent TGF-b-induced Apoptosis in Human Airway Epithelial Cells G. Cheng1, M. Kim1, B. Chaudhari1, D. K. Agrawal1,2; 1Dept. of Biomedical Science, Creighton University School of Medicine, Omaha, NE, 2 Dept. of Internal Medicine, Creighton University School of Medicine, Omaha, NE. RATIONALE: Widespread damage of airway epithelium and defective epithelial repair are hallmarks of chronic asthma. Excessive apoptosis could be a likely mechanism. TGF-b plays an important role in the pathophysiology of chronic asthma, and voltage-gated chloride channels are critical to cell shape and cell apoptosis. In this study, we investigated the involvement of chloride channels in TGF-b1 induced apoptosis in human airway epithelial cells (AECs). METHODS: Chloride ion currents in AECs were recorded using whole cell patch-clamp technique. Current-voltage relationship of the currents in BEAS-2B cells was examined. Expression of voltage-gated chloride channels was detected by real-time PCR. The effect of TGF-b1 on survival of BEAS-2B cells was determined by Annexin-V and Propidium iodide using flow cytometry. AECs were also transfected with control and ClC3 vector or Bcl-2 vector. RESULTS: Effect of TGF-b1 on the induction of apoptosis in AECs was more pronounced than TGF-b2. Overexpression of ClC-3 in BEAS-2B cells inhibited TGF-b1- induced apoptosis. Both outward and inward chloride currents in BEAS-2B cells were abolished by knocking down ClC-3 using two siRNAs, especially targeted to ClC-3. Treatment of the AECs with TGF-b1 resulted in the down-regulation of ClC-3 mRNA expression. Bcl-2 expression was down-regulated and Bax up-regulated in the AECs after TGF-b1 stimulation. ClC-3 expression was increased in Bcl-2