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Nitrogen Dioxide Exposure in School Classrooms Is Associated with Airflow Obstruction in Students with Asthma
AB174 Abstracts
555
Nitrogen Dioxide Exposure in School Classrooms Is Associated with Airflow Obstruction in Students with Asthma
SUNDAY
Jonathan M. Gaffin, MD, MMSc1,2, Marissa Hauptman, MD3,4, Peggy Lai, MD, MPH1,5, Carter R. Petty, MS3,6, Jack M. Wolfson7, ChoongMin Kang7, William J. Sheehan, MD1,3, Sachin N. Baxi, MD1,8, Lisa M. Bartnikas, MD1,3, Perdita Permaul, MD, FAAAAI1,9, Diane R. Gold, MD10,11, Brent Coull, PHD7, Petros Koutrakis, PHD7, and Wanda Phipatanakul, MD, MS, FAAAAI1,12; 1Harvard Medical School, Boston, MA, 2Division of Respiratory Diseases, Boston Children’s Hospital, Boston, MA, 3Boston Children’s Hospital, Boston, MA, 4Region 1 New England Pediatric Environmental Health Specialty Unit, Boston, MA, 5 Massachusetts General Hospital, Boston, MA, 6Clinical Research Center, Boston Children’s Hospital, Boston, MA, 7Harvard TH Chan School of Public Health, Boston, MA, 8Division of Allergy and Immunology, Boston Children’s Hospital, Boston, MA, 9Division of Pediatric Allergy/ Immunology, Massachusetts General Hospital, Boston, MA, 10Harvard T.H. Chan School of Public Health, Boston, MA, 11Channing Laboratory, Brigham and Women’s Hospital, Boston, MA, 12Division of Pediatric Allergy/Immunology, Boston Children’s Hospital, Harvard University School of Medicine, Boston, MA. RATIONALE: Air pollution has been associated with asthma development and morbidity. We aimed to determine the effect of indoor classroom Nitrogen dioxide (NO2) on the lung function of inner city schoolchildren with asthma. METHODS: Children enrolled in the School Inner City Asthma Study were followed for one academic year. At baseline, subjects underwent phenotypic characterization, allergy testing, fractional exhalation of nitric oxide (FeNO) and spirometry. Spirometry and FeNO were repeated twice during the school year. Classroom NO2 was collected by passive sampling technique for 1 week periods, twice per year. Generalized estimating equation models assessed lung function parameters (FEV1/FVC, FEV1% predicted, and FEF25-75% predicted) with the classroom NO2 level temporally closest to the outcome assessment. The primary outcome was FEV1/FVC ratio as the most sensitive measure of airflow obstruction in children. RESULTS: In total, complete exposure and outcome data was available for 188 subjects from 135 classrooms across 29 schools. After adjusting for race and season (spirometry reference values standardized by age, height and gender), NO2 was highly associated with increased airflow obstruction such that each 10ppb rise in NO2 exposure was associated with a 3% decline in FEV1/FVC (b: -0.03, 95% confidence interval (CI) [-0.05, -0.00], p50.03). FEF25-75% predicted was also lower with higher NO2 exposure (b: -14.1, 95%CI [-25.1, -3.0], p50.01). There was no association of NO2 with FEV1% predicted (b: -1.6, 95%CI [-6.7, 3.5], p50.5) or FeNO (b: -2.9, 95%CI [-14.1, 8.4], p50.6). CONCLUSIONS: In children with asthma, indoor classroom NO2 may be associated with increase airflow obstruction.
556
Multiple Reactions Monitoring (MRM) Mass Spectrometry for Absolute Quantification of Allergens in German Cockroach (GCr) Allergen Extracts
Samuel T. Mindaye, Jelena Spiric, Natalie A. David, Ronald L. Rabin, MD, FAAAAI, and Jay E. Slater, MD; CBER/FDA, Silver Spring, MD. RATIONALE: Mass spectrometry (MS) offers a unique approach for rapid compositional characterization and absolute quantification of individual allergens and allergen isoforms in complex allergen extracts, and thus has the potential to transform the ongoing efforts of allergen standardization. METHODS: We developed an assay to evaluate GCr extracts, based on liquid chromatography (LC) and MRM MS, by systematically optimizing the digestion conditions (buffer, protein denaturation, digestion time, and
J ALLERGY CLIN IMMUNOL FEBRUARY 2017
trypsin quantity), LC gradient, and MS parameters (collision energy, dwell time, precursor-fragment pairs). RESULTS: In silico simulation, followed by recombinant-allergen and extract digestion and high resolution MS analysis allowed us to identify 47 proteotypic and quantotypic reference peptides for use as calibrators and internal standards for quantification of endogenous peptides. The peptide list represents 9 allergens and 8 iso-allergens and variants. Since the initial phase of the project aimed to generate a template-method using selected GCr allergens, twenty-six peptides (13 native and 13 isotopically-labelled) representing Bla g 1, 2, 3, 4 and 5 were chemically synthesized and used in LC-MRM/MS method development. Robustness and suitability of the overall protocol was extensively assessed following the ICH (Q2(R1) and FDA guidelines. The protocol offers excellent precision (CV < 10%), linear response (r > 0.99), dynamic range (>3), detection, and recovery. The applicability of the method was further demonstrated using eight commercial GCr extracts from four different vendors. CONCLUSIONS: The LC-MRM/MS method proved to be a useful tool for absolute quantification of allergens in GCr extracts and has promise for the standardization of complex allergen extracts.
557
Bisphenol A(BPA) alters molecular signaling in Immune cells that promotes the development of childhood asthma
Youko Murakami, MD, Barun K. Choudhury, PhD, Randall M. Goldblum, MD, and Terumi Midoro-Horiuti, MD, PhD FAAAAI; University of Texas Medical Branch, Pediatrics, Galveston, TX. RATIONALE: Early exposure to the environmental estrogen (EE) BPA has been associated with increased prevalence of asthma. However, the mechanism(s) that underlie this relationship are not known. The purpose of this study is to test the hypothesis that fetal exposure to EEs induce epigenetic alterations that promote the development of asthma. METHODS: T cell lines (CCL-119 and TIB-152) and human cord blood (CB) CD4+T cells were exposed to varying concentrations of estradiol (E2) or BPA for 5-120 min. The phosphorylation of PI3K/AKT, Enhancer of zeste homolog 2 (EZH2) and methylation of histone H3K27 (H3K27me3) were analyzed by Western blotting. RESULTS: BPA and E2 exposure initiated phosphorylation of AKT, PI3K and EZH2 on CCL-119 and CB CD4+T cells. BPA stimulation of CCL-119 rapidly stimulated phosphorylation of pAKT, pPI3K and pEZH2 by 23% with 10-10M, 162% with 10-9M and 45% with 10-10 M, respectively. In E2 stimulation for 5min, pAKT and pPI3K 36% with 10-12M and 56% with 10-11 M, respectively. BPA stimulation increased H3K27me3 94% with 10-11M and 120% with 10-7M at 120min. E2 stimulated H3K27me3 95% with 10-11M and 139% with 10-6M. CONCLUSIONS: These data suggest that the effects of early exposure to EEs on the increasing prevalence of childhood asthma will require further investigation of the combinatorial effects of E2 and EE. Understanding the molecular and cellular basis for EE’s asthma promoting effects, through immune cells may allow the design future prevention measures, and potentially new molecular-based treatments for childhood asthma.
555
Nitrogen Dioxide Exposure in School Classrooms Is Associated with Airflow Obstruction in Students with Asthma
SUNDAY
Jonathan M. Gaffin, MD, MMSc1,2, Marissa Hauptman, MD3,4, Peggy Lai, MD, MPH1,5, Carter R. Petty, MS3,6, Jack M. Wolfson7, ChoongMin Kang7, William J. Sheehan, MD1,3, Sachin N. Baxi, MD1,8, Lisa M. Bartnikas, MD1,3, Perdita Permaul, MD, FAAAAI1,9, Diane R. Gold, MD10,11, Brent Coull, PHD7, Petros Koutrakis, PHD7, and Wanda Phipatanakul, MD, MS, FAAAAI1,12; 1Harvard Medical School, Boston, MA, 2Division of Respiratory Diseases, Boston Children’s Hospital, Boston, MA, 3Boston Children’s Hospital, Boston, MA, 4Region 1 New England Pediatric Environmental Health Specialty Unit, Boston, MA, 5 Massachusetts General Hospital, Boston, MA, 6Clinical Research Center, Boston Children’s Hospital, Boston, MA, 7Harvard TH Chan School of Public Health, Boston, MA, 8Division of Allergy and Immunology, Boston Children’s Hospital, Boston, MA, 9Division of Pediatric Allergy/ Immunology, Massachusetts General Hospital, Boston, MA, 10Harvard T.H. Chan School of Public Health, Boston, MA, 11Channing Laboratory, Brigham and Women’s Hospital, Boston, MA, 12Division of Pediatric Allergy/Immunology, Boston Children’s Hospital, Harvard University School of Medicine, Boston, MA. RATIONALE: Air pollution has been associated with asthma development and morbidity. We aimed to determine the effect of indoor classroom Nitrogen dioxide (NO2) on the lung function of inner city schoolchildren with asthma. METHODS: Children enrolled in the School Inner City Asthma Study were followed for one academic year. At baseline, subjects underwent phenotypic characterization, allergy testing, fractional exhalation of nitric oxide (FeNO) and spirometry. Spirometry and FeNO were repeated twice during the school year. Classroom NO2 was collected by passive sampling technique for 1 week periods, twice per year. Generalized estimating equation models assessed lung function parameters (FEV1/FVC, FEV1% predicted, and FEF25-75% predicted) with the classroom NO2 level temporally closest to the outcome assessment. The primary outcome was FEV1/FVC ratio as the most sensitive measure of airflow obstruction in children. RESULTS: In total, complete exposure and outcome data was available for 188 subjects from 135 classrooms across 29 schools. After adjusting for race and season (spirometry reference values standardized by age, height and gender), NO2 was highly associated with increased airflow obstruction such that each 10ppb rise in NO2 exposure was associated with a 3% decline in FEV1/FVC (b: -0.03, 95% confidence interval (CI) [-0.05, -0.00], p50.03). FEF25-75% predicted was also lower with higher NO2 exposure (b: -14.1, 95%CI [-25.1, -3.0], p50.01). There was no association of NO2 with FEV1% predicted (b: -1.6, 95%CI [-6.7, 3.5], p50.5) or FeNO (b: -2.9, 95%CI [-14.1, 8.4], p50.6). CONCLUSIONS: In children with asthma, indoor classroom NO2 may be associated with increase airflow obstruction.
556
Multiple Reactions Monitoring (MRM) Mass Spectrometry for Absolute Quantification of Allergens in German Cockroach (GCr) Allergen Extracts
Samuel T. Mindaye, Jelena Spiric, Natalie A. David, Ronald L. Rabin, MD, FAAAAI, and Jay E. Slater, MD; CBER/FDA, Silver Spring, MD. RATIONALE: Mass spectrometry (MS) offers a unique approach for rapid compositional characterization and absolute quantification of individual allergens and allergen isoforms in complex allergen extracts, and thus has the potential to transform the ongoing efforts of allergen standardization. METHODS: We developed an assay to evaluate GCr extracts, based on liquid chromatography (LC) and MRM MS, by systematically optimizing the digestion conditions (buffer, protein denaturation, digestion time, and
J ALLERGY CLIN IMMUNOL FEBRUARY 2017
trypsin quantity), LC gradient, and MS parameters (collision energy, dwell time, precursor-fragment pairs). RESULTS: In silico simulation, followed by recombinant-allergen and extract digestion and high resolution MS analysis allowed us to identify 47 proteotypic and quantotypic reference peptides for use as calibrators and internal standards for quantification of endogenous peptides. The peptide list represents 9 allergens and 8 iso-allergens and variants. Since the initial phase of the project aimed to generate a template-method using selected GCr allergens, twenty-six peptides (13 native and 13 isotopically-labelled) representing Bla g 1, 2, 3, 4 and 5 were chemically synthesized and used in LC-MRM/MS method development. Robustness and suitability of the overall protocol was extensively assessed following the ICH (Q2(R1) and FDA guidelines. The protocol offers excellent precision (CV < 10%), linear response (r > 0.99), dynamic range (>3), detection, and recovery. The applicability of the method was further demonstrated using eight commercial GCr extracts from four different vendors. CONCLUSIONS: The LC-MRM/MS method proved to be a useful tool for absolute quantification of allergens in GCr extracts and has promise for the standardization of complex allergen extracts.
557
Bisphenol A(BPA) alters molecular signaling in Immune cells that promotes the development of childhood asthma
Youko Murakami, MD, Barun K. Choudhury, PhD, Randall M. Goldblum, MD, and Terumi Midoro-Horiuti, MD, PhD FAAAAI; University of Texas Medical Branch, Pediatrics, Galveston, TX. RATIONALE: Early exposure to the environmental estrogen (EE) BPA has been associated with increased prevalence of asthma. However, the mechanism(s) that underlie this relationship are not known. The purpose of this study is to test the hypothesis that fetal exposure to EEs induce epigenetic alterations that promote the development of asthma. METHODS: T cell lines (CCL-119 and TIB-152) and human cord blood (CB) CD4+T cells were exposed to varying concentrations of estradiol (E2) or BPA for 5-120 min. The phosphorylation of PI3K/AKT, Enhancer of zeste homolog 2 (EZH2) and methylation of histone H3K27 (H3K27me3) were analyzed by Western blotting. RESULTS: BPA and E2 exposure initiated phosphorylation of AKT, PI3K and EZH2 on CCL-119 and CB CD4+T cells. BPA stimulation of CCL-119 rapidly stimulated phosphorylation of pAKT, pPI3K and pEZH2 by 23% with 10-10M, 162% with 10-9M and 45% with 10-10 M, respectively. In E2 stimulation for 5min, pAKT and pPI3K 36% with 10-12M and 56% with 10-11 M, respectively. BPA stimulation increased H3K27me3 94% with 10-11M and 120% with 10-7M at 120min. E2 stimulated H3K27me3 95% with 10-11M and 139% with 10-6M. CONCLUSIONS: These data suggest that the effects of early exposure to EEs on the increasing prevalence of childhood asthma will require further investigation of the combinatorial effects of E2 and EE. Understanding the molecular and cellular basis for EE’s asthma promoting effects, through immune cells may allow the design future prevention measures, and potentially new molecular-based treatments for childhood asthma.