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Effects Of rs3744262 On DNA Methylation and Symptoms In Participants With Allergic Rhinitis During Grass Pollen Exposure In The Environmental Exposure Unit (EEU)
Abstracts AB89
J ALLERGY CLIN IMMUNOL VOLUME 133, NUMBER 2
IL17RB+ Granulocytes In Asthma Patients Dr. Lin Li, MD1, Dr. Matthew A. Schaller, PhD2, Dr. Alan P. Baptist, MD, MPH, FAAAAI1, Dr. Nicholas W. Lukacs, PhD3; 1University of Michigan, Division of Allergy and Clinical Immunology, Ann Arbor, MI, 2University of Michigan, Department of Pathology, Ann Arbor, MI, 3University of Michigan, Ann Arbor, MI. RATIONALE: IL-25 and its receptor IL-17RB have been shown to promote Th2 type allergic responses. Numerous IL-17RB+ cells that produce Th2 cytokines have been identified. A granulocytic cell population that expresses IL-17RB as well as IL-4 and IL-13 was identified in patients with asthma and termed Type 2 Myeloid (T2M) cells. This study aims to confirm T2M elevation in asthmatic patients and its potential correlation with severity of asthma. METHODS: 1) Flow cytometry. Peripheral blood from subjects with physician diagnosis of asthma (N517) and healthy controls (N58) were collected. Isolated granulocytes were incubated with cell-specific fluorescent antibodies and the percentage of T2M (CD11B+CD16+CD177+IL-17RB+) and IL17RB+ granulocytes were determined. 2) Clinical assessment. Through spirometry testing and patient interview, severity of asthma was assessed (number of exacerbations, FEV1, FEV1/FVC, and asthma control test). RESULTS: Compared to healthy controls, asthma subjects have significantly higher percentage of both IL17RB+ granulocytes (P50.013) and T2M (P50.037). Results identified that the T2M cells were a distinct population of granulocytes separate from eosinophils. Among asthmatics, the percentage of IL17RB+ granulocytes or T2M did not show significant correlation with number of exacerbations, FEV1, FEV1/FVC or ACT scores. CONCLUSIONS: A higher percentage of granulocytes in asthmatic patients expressed the IL-25 receptor, IL17RB. Asthmatics also have significantly more T2M than controls. The presence of either the IL-17RB+ or T2M cell population did not correlate with severity of asthma in patients during their clinic visit. Future studies will examine patients during active asthma exacerbations to determine correlations of T2M cells with disease severity.
311
Pollen From Genetically Modified Bt Maize Does Not Promote Allergic Responses In Mice Mrs. Monica Andreassen1,2, Dr. Elena Rocca3, Dr. Thomas Bøhn3,4, Dr. Odd-Gunnar Wikmark3, Prof. Johnnie Van den Berg5, Prof. Martinus Lovik2, Prof. Terje Traavik3,4, Dr. Unni C. Nygaard2; 1GenØk - Centre for Biosafety, Tromso, Norway, 2Norwegian Institute of Public Health, Oslo, Norway, 3GenØk - Centre for Biosafety, Norway, 4University of Tromsø, Norway, 5North-West University, Potchefstroom, South Africa. RATIONALE: The genome of the genetically modified (GM) maize event MON810 contains a truncated version of a transgene, cry1ab, from the soil bacterium Bacillus thuringiensis (Bt). Hence, the GM plants express a modified Cry1Ab protein, which is toxic for some yield-reducing maize pests. The Cry1Ab protein shares 87% sequence homology with the recognized mucosal adjuvant Cry1Ac, and accordingly possesses potential adjuvant properties. We investigated whether pollen from the Cry1Ab-expressing maize affects airway responses or acts as an adjuvant on antibody production against ovalbumin (OVA) in an airway allergy mouse model. METHODS: On days 0, 1, 2, 21, 22 and 23 mice were intranasally exposed to OVAwith either 0.5 mg of recently shed (<24 hrs) pollen from MON810 or the near isogenic non-GM maize, or the purified Bt-derived protoxin Cry1Ab. OVAwith cholera toxin (CT) was used as positive control for adjuvant effect. Blood and bronchoalveolar lavage fluid (BALf) were collected on day 26, and anti-OVA specific IgE, IgG1, IgG2, anti-Cry1Ab specific IgG1, and Th1/Th2/ Th17/Treg cytokines, respectively, were determined. RESULTS: While this airway allergy model detected adjuvant effects of a known potent adjuvant (CT), anti-OVA specific IgE, IgG1 and IgG2a were not elevated in mice receiving MON810 pollen or the Cry1Ab protoxin. Exposure to protoxin Cry1Ab, but not MON810 pollen, induced an anti-Cry1Ab specific IgG1 response. Cytokine levels in BALf were low in all groups. CONCLUSIONS: In the mouse model employed, exposure to pollen from the genetically modified Bt maize event MON810 does not trigger enhanced allergic sensitization, general immunogenicity or airway responses.
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Effects Of rs3744262 On DNA Methylation and Symptoms In Participants With Allergic Rhinitis During Grass Pollen Exposure In The Environmental Exposure Unit (EEU) Dr. Michelle North, PhD1, Ms. Sarah Mah, BSc2, Mr. Andrew G. Day, MSc3, Dr. Michael Kobor, PhD2,4, Dr. Anne K. Ellis, MD, MSc, FAAAAI1,5; 1Departments of Medicine and Biomedical & Molecular Science, Queen’s University, Kingston, ON, Canada, 2Centre for Molecular Medicine & Therapeutics, Child & Family Research Institute, Vancouver, BC, Canada, 3Clinical Research Centre, Kingston General Hospital, Kingston, ON, Canada, 4University of British Columbia, Vancouver, BC, Canada, 5Allergy Research Unit, Kingston General Hospital, Kingston, ON, Canada. RATIONALE: Genotype can affect epigenetics, as SNPs can alter CpG sites. Surrounding CpG sites may also be affected, but genetic-epigenetic interactions are not well understood. rs3744262 is a CpG-SNP in ephrin-B3. Ephrin-B3 is a transmembrane ligand for receptor tyrosine kinases, involved in bidirectional signaling. Ephrin-B3 is crucial in epithelia and enhances the T cell response. However, the effects of this CpG-SNP on nearby methylation sites and effects on symptoms during allergen challenge in an Environmental Exposure Unit (EEU) have not been examined. METHODS: 38 participants with allergic rhinitis were exposed to grass pollen for 3 hours on two consecutive days. DNA was isolated from blood drawn at baseline, 3H and 27H post-exposure. All participants recorded their total symptom scores (TSS) and peak nasal inspiratory flow (PNIF) at baseline and 30 minute intervals during exposure. All participants were genotyped at rs3744262 and pyrosequencing was performed on 16 participants at the site of interest plus three surrounding CpG sites. Kruskal-Wallis tests and Spearman correlations were used. RESULTS: rs3744262 was associated with DNA methylation at that site (p<0.0001). Genotype was not associated with TSS, PNIF or methylation of surrounding sites. However, one of the CpG sites surrounding rs3744262 was negatively correlated with baseline TSS (p50.03). Another nearby CpG showed a trend towards positive correlation with baseline PNIF (p50.08). CONCLUSIONS: Epigenetic modifications in the region of the EFNB3 gene are associated with allergic rhinitis symptoms. Further studies are needed to examine the effects of genetics and epigenetics on allergic rhinitis.
313
Chronic Spontaneous Urticaria – An Evaluation Of An Indirect Immunofluorescence Method For Detecting Anti-Mast Cell IgG Antibodies Bahar Bahrani, Natasha Gattey, Peter Hull; University of Saskatchewan, Canada. RATIONALE: An autoimmune basis is believed to be responsible for about half of all cases of chronic spontaneous urticaria (CSU) with specific IgG auto-antibodies directed at mast cells. The autologous serum skin test (ASST), basophil histamine release assay and basophil activation test have been used to diagnose autoimmune form of CSU. These tests are limited because they are indirect form of mast cell/basophil degranulation. METHODS: Sections were cut from paraffin embedded blocks from skin biopsied infant with bullous mastocytosis. Serum from 69 patients with CSU was used, and fluorescein conjugated human IgG was used to label fixed antibody. An ASST was performed on 66 of the patients with severe urticaria and was found to be positive in 45.45%. 27 of these patients were receiving intravenous immunoglobulin (IVIG) or received it in the past. RESULTS: A positive indirect immunofluorescence was found in half the patients. It was positive in 22.73% of the patients with a positive ASST, but was also positive in 25.76% with a negative ASST. The sensitivity and specificity of ASST were calculated to be 46.88% and 52.94%, respectively. When IVIG treated patients were omitted from the calculations the sensitivity and specificity was 34.62% and 77.27%, respectively CONCLUSIONS: Positive indirect immunofluorescence was found in half the patients with CSU. When IVIG treated patient were excluded the ASST was associated with is a high specificity but with low sensitivity. Indirect immunofluorescence should be considered as better indicator of the autoimmune form of urticaria.
SUNDAY
310
J ALLERGY CLIN IMMUNOL VOLUME 133, NUMBER 2
IL17RB+ Granulocytes In Asthma Patients Dr. Lin Li, MD1, Dr. Matthew A. Schaller, PhD2, Dr. Alan P. Baptist, MD, MPH, FAAAAI1, Dr. Nicholas W. Lukacs, PhD3; 1University of Michigan, Division of Allergy and Clinical Immunology, Ann Arbor, MI, 2University of Michigan, Department of Pathology, Ann Arbor, MI, 3University of Michigan, Ann Arbor, MI. RATIONALE: IL-25 and its receptor IL-17RB have been shown to promote Th2 type allergic responses. Numerous IL-17RB+ cells that produce Th2 cytokines have been identified. A granulocytic cell population that expresses IL-17RB as well as IL-4 and IL-13 was identified in patients with asthma and termed Type 2 Myeloid (T2M) cells. This study aims to confirm T2M elevation in asthmatic patients and its potential correlation with severity of asthma. METHODS: 1) Flow cytometry. Peripheral blood from subjects with physician diagnosis of asthma (N517) and healthy controls (N58) were collected. Isolated granulocytes were incubated with cell-specific fluorescent antibodies and the percentage of T2M (CD11B+CD16+CD177+IL-17RB+) and IL17RB+ granulocytes were determined. 2) Clinical assessment. Through spirometry testing and patient interview, severity of asthma was assessed (number of exacerbations, FEV1, FEV1/FVC, and asthma control test). RESULTS: Compared to healthy controls, asthma subjects have significantly higher percentage of both IL17RB+ granulocytes (P50.013) and T2M (P50.037). Results identified that the T2M cells were a distinct population of granulocytes separate from eosinophils. Among asthmatics, the percentage of IL17RB+ granulocytes or T2M did not show significant correlation with number of exacerbations, FEV1, FEV1/FVC or ACT scores. CONCLUSIONS: A higher percentage of granulocytes in asthmatic patients expressed the IL-25 receptor, IL17RB. Asthmatics also have significantly more T2M than controls. The presence of either the IL-17RB+ or T2M cell population did not correlate with severity of asthma in patients during their clinic visit. Future studies will examine patients during active asthma exacerbations to determine correlations of T2M cells with disease severity.
311
Pollen From Genetically Modified Bt Maize Does Not Promote Allergic Responses In Mice Mrs. Monica Andreassen1,2, Dr. Elena Rocca3, Dr. Thomas Bøhn3,4, Dr. Odd-Gunnar Wikmark3, Prof. Johnnie Van den Berg5, Prof. Martinus Lovik2, Prof. Terje Traavik3,4, Dr. Unni C. Nygaard2; 1GenØk - Centre for Biosafety, Tromso, Norway, 2Norwegian Institute of Public Health, Oslo, Norway, 3GenØk - Centre for Biosafety, Norway, 4University of Tromsø, Norway, 5North-West University, Potchefstroom, South Africa. RATIONALE: The genome of the genetically modified (GM) maize event MON810 contains a truncated version of a transgene, cry1ab, from the soil bacterium Bacillus thuringiensis (Bt). Hence, the GM plants express a modified Cry1Ab protein, which is toxic for some yield-reducing maize pests. The Cry1Ab protein shares 87% sequence homology with the recognized mucosal adjuvant Cry1Ac, and accordingly possesses potential adjuvant properties. We investigated whether pollen from the Cry1Ab-expressing maize affects airway responses or acts as an adjuvant on antibody production against ovalbumin (OVA) in an airway allergy mouse model. METHODS: On days 0, 1, 2, 21, 22 and 23 mice were intranasally exposed to OVAwith either 0.5 mg of recently shed (<24 hrs) pollen from MON810 or the near isogenic non-GM maize, or the purified Bt-derived protoxin Cry1Ab. OVAwith cholera toxin (CT) was used as positive control for adjuvant effect. Blood and bronchoalveolar lavage fluid (BALf) were collected on day 26, and anti-OVA specific IgE, IgG1, IgG2, anti-Cry1Ab specific IgG1, and Th1/Th2/ Th17/Treg cytokines, respectively, were determined. RESULTS: While this airway allergy model detected adjuvant effects of a known potent adjuvant (CT), anti-OVA specific IgE, IgG1 and IgG2a were not elevated in mice receiving MON810 pollen or the Cry1Ab protoxin. Exposure to protoxin Cry1Ab, but not MON810 pollen, induced an anti-Cry1Ab specific IgG1 response. Cytokine levels in BALf were low in all groups. CONCLUSIONS: In the mouse model employed, exposure to pollen from the genetically modified Bt maize event MON810 does not trigger enhanced allergic sensitization, general immunogenicity or airway responses.
312
Effects Of rs3744262 On DNA Methylation and Symptoms In Participants With Allergic Rhinitis During Grass Pollen Exposure In The Environmental Exposure Unit (EEU) Dr. Michelle North, PhD1, Ms. Sarah Mah, BSc2, Mr. Andrew G. Day, MSc3, Dr. Michael Kobor, PhD2,4, Dr. Anne K. Ellis, MD, MSc, FAAAAI1,5; 1Departments of Medicine and Biomedical & Molecular Science, Queen’s University, Kingston, ON, Canada, 2Centre for Molecular Medicine & Therapeutics, Child & Family Research Institute, Vancouver, BC, Canada, 3Clinical Research Centre, Kingston General Hospital, Kingston, ON, Canada, 4University of British Columbia, Vancouver, BC, Canada, 5Allergy Research Unit, Kingston General Hospital, Kingston, ON, Canada. RATIONALE: Genotype can affect epigenetics, as SNPs can alter CpG sites. Surrounding CpG sites may also be affected, but genetic-epigenetic interactions are not well understood. rs3744262 is a CpG-SNP in ephrin-B3. Ephrin-B3 is a transmembrane ligand for receptor tyrosine kinases, involved in bidirectional signaling. Ephrin-B3 is crucial in epithelia and enhances the T cell response. However, the effects of this CpG-SNP on nearby methylation sites and effects on symptoms during allergen challenge in an Environmental Exposure Unit (EEU) have not been examined. METHODS: 38 participants with allergic rhinitis were exposed to grass pollen for 3 hours on two consecutive days. DNA was isolated from blood drawn at baseline, 3H and 27H post-exposure. All participants recorded their total symptom scores (TSS) and peak nasal inspiratory flow (PNIF) at baseline and 30 minute intervals during exposure. All participants were genotyped at rs3744262 and pyrosequencing was performed on 16 participants at the site of interest plus three surrounding CpG sites. Kruskal-Wallis tests and Spearman correlations were used. RESULTS: rs3744262 was associated with DNA methylation at that site (p<0.0001). Genotype was not associated with TSS, PNIF or methylation of surrounding sites. However, one of the CpG sites surrounding rs3744262 was negatively correlated with baseline TSS (p50.03). Another nearby CpG showed a trend towards positive correlation with baseline PNIF (p50.08). CONCLUSIONS: Epigenetic modifications in the region of the EFNB3 gene are associated with allergic rhinitis symptoms. Further studies are needed to examine the effects of genetics and epigenetics on allergic rhinitis.
313
Chronic Spontaneous Urticaria – An Evaluation Of An Indirect Immunofluorescence Method For Detecting Anti-Mast Cell IgG Antibodies Bahar Bahrani, Natasha Gattey, Peter Hull; University of Saskatchewan, Canada. RATIONALE: An autoimmune basis is believed to be responsible for about half of all cases of chronic spontaneous urticaria (CSU) with specific IgG auto-antibodies directed at mast cells. The autologous serum skin test (ASST), basophil histamine release assay and basophil activation test have been used to diagnose autoimmune form of CSU. These tests are limited because they are indirect form of mast cell/basophil degranulation. METHODS: Sections were cut from paraffin embedded blocks from skin biopsied infant with bullous mastocytosis. Serum from 69 patients with CSU was used, and fluorescein conjugated human IgG was used to label fixed antibody. An ASST was performed on 66 of the patients with severe urticaria and was found to be positive in 45.45%. 27 of these patients were receiving intravenous immunoglobulin (IVIG) or received it in the past. RESULTS: A positive indirect immunofluorescence was found in half the patients. It was positive in 22.73% of the patients with a positive ASST, but was also positive in 25.76% with a negative ASST. The sensitivity and specificity of ASST were calculated to be 46.88% and 52.94%, respectively. When IVIG treated patients were omitted from the calculations the sensitivity and specificity was 34.62% and 77.27%, respectively CONCLUSIONS: Positive indirect immunofluorescence was found in half the patients with CSU. When IVIG treated patient were excluded the ASST was associated with is a high specificity but with low sensitivity. Indirect immunofluorescence should be considered as better indicator of the autoimmune form of urticaria.
SUNDAY
310