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Background and Rationale: Asthma and overweight are public health concerns. Lifestyle, including dietary and activity patterns, is associated with overweight and asthma. Moreover, an association between these two diseases has been described. Yet, few studies have considered these associations longitudinally in youth. Methods: Based on data from the 1995 Manitoba Birth Prospective Cohort (n=723, 404 [55.9%] boys), we designed a series of studies to address the question: “Do obesity and related lifestyle behaviours influence asthma and airway hyperresponsivess (AHR) outcomes in children?” Following protocol for a mixed methods sequential explanatory design study, we first considered this research question using quantitative methods. Exposure variables included weight status (body mass index (BMI); BMI z-scores; normal weight vs. overweight), diet, physical activity and screen time. Outcome variables included asthma and AHR at 8-10 years old and at 12-13 years old. Quantitative findings provided direction for the qualitative investigations. That is, we sought to further explain some of the quantitative findings using qualitative methods. For the qualitative portion of this dissertation, 15-16 year old youth were purposively selected (Winnipeg residency, asthma status, gender) from the 1995 Manitoba Prospective Birth Cohort. Due to recruitment challenges, participation was supplemented with youth from the Canadian Asthma Primary Prevention Study, using the same purposive selection criteria. Quantitative Results: Overweight at 12-13 years old was associated with a two-fold increased odds of persistent asthma in girls. In contrast, boys within the highest BMI quartile at 8-10 years old were nearly twice as likely to have remittent asthma at 12-13 years old. High vegetable intake was protective against allergic asthma and moderate-to-severe AHR by 50% and 42%, respectively. High screen time at 8-10 years old, particularly amongst overweight youth, was associated with an increased odds of asthma, but not AHR at 8-10 years and 12-13 years; there were no associations between physical activity, asthma and AHR. Qualitative Results: Youth spoke of asthma as a condition that neither limits physical activity, nor is an excuse for refraining from physical activity. Conclusions: Modest evidence that some quantitatively-measured weight and related lifestyle behaviours during the pubertal years is associated with asthma. Yet, qualitative data suggest that youth with asthma believe that physical activity is achievable despite their condition, although some describe that asthma interferes with physical activity.

Background and Rationale: Asthma and overweight are public health concerns. Lifestyle, including dietary and activity patterns, is associated with overweight and asthma. Moreover, an association between these two diseases has been described. Yet, few studies have considered these associations longitudinally in youth. Methods: Based on data from the 1995 Manitoba Birth Prospective Cohort (n=723, 404 [55.9%] boys), we designed a series of studies to address the question: “Do obesity and related lifestyle behaviours influence asthma and airway hyperresponsivess (AHR) outcomes in children?” Following protocol for a mixed methods sequential explanatory design study, we first considered this research question using quantitative methods. Exposure variables included weight status (body mass index (BMI); BMI z-scores; normal weight vs. overweight), diet, physical activity and screen time. Outcome variables included asthma and AHR at 8-10 years old and at 12-13 years old. Quantitative findings provided direction for the qualitative investigations. That is, we sought to further explain some of the quantitative findings using qualitative methods. For the qualitative portion of this dissertation, 15-16 year old youth were purposively selected (Winnipeg residency, asthma status, gender) from the 1995 Manitoba Prospective Birth Cohort. Due to recruitment challenges, participation was supplemented with youth from the Canadian Asthma Primary Prevention Study, using the same purposive selection criteria. Quantitative Results: Overweight at 12-13 years old was associated with a two-fold increased odds of persistent asthma in girls. In contrast, boys within the highest BMI quartile at 8-10 years old were nearly twice as likely to have remittent asthma at 12-13 years old. High vegetable intake was protective against allergic asthma and moderate-to-severe AHR by 50% and 42%, respectively. High screen time at 8-10 years old, particularly amongst overweight youth, was associated with an increased odds of asthma, but not AHR at 8-10 years and 12-13 years; there were no associations between physical activity, asthma and AHR. Qualitative Results: Youth spoke of asthma as a condition that neither limits physical activity, nor is an excuse for refraining from physical activity. Conclusions: Modest evidence that some quantitatively-measured weight and related lifestyle behaviours during the pubertal years is associated with asthma. Yet, qualitative data suggest that youth with asthma believe that physical activity is achievable despite their condition, although some describe that asthma interferes with physical activity.

BACKGROUND:Airway hyperresponsiveness (AHR), a primary characteristic of asthma, involves increased airway smooth muscle contractility in response to certain exposures. We sought to determine whether common genetic variants were associated with AHR severity.METHODS:A genome-wide association study (GWAS) of AHR, quantified as the natural log of the dosage of methacholine causing a 20% drop in FEV1, was performed with 994 non-Hispanic white asthmatic subjects from three drug clinical trials: CAMP, CARE, and ACRN. Genotyping was performed on Affymetrix 6.0 arrays, and imputed data based on HapMap Phase 2, was used to measure the association of SNPs with AHR using a linear regression model. Replication of primary findings was attempted in 650 white subjects from DAG, and 3,354 white subjects from LHS. Evidence that the top SNPs were eQTL of their respective genes was sought using expression data available for 419 white CAMP subjects.RESULTS:The top primary GWAS associations were in rs848788 (P-value 7.2E-07) and rs6731443 (P-value 2.5E-06), located within the ITGB5 and AGFG1 genes, respectively. The AGFG1 result replicated at a nominally significant level in one independent population (LHS P-value 0.012), and the SNP had a nominally significant unadjusted P-value (0.0067) for being an eQTL of AGFG1.CONCLUSIONS:Based on current knowledge of ITGB5 and AGFG1, our results suggest that variants within these genes may be involved in modulating AHR. Future functional studies are required to confirm that our associations represent true biologically significant findings.

Perfluorinated compounds, non-degradable xenobiotics in many consumer products, can cause developmental toxicity in animals, and human exposure is associated with asthma symptoms. We tested the hypothesis that sustained chronic exposure to perfluorooctanoic acid (PFOA), fluorotelomer alcohol (FTOH) or perfluorooctanesulfonic acid (PFOS) induces lung dysfunction that exacerbates allergen-induced airway hyperresponsiveness (AHR) and inflammation. Mice were exposed to the chemicals from early gestation day to adulthood. Some pups were sensitized and challenged with ovalbumin. Serum PFOA was analyzed by liquid chromatograph-tandem mass spectrometry. Lung function was measured using a small animal ventilator. We assayed inflammatory cells in the lung, performed PCR for lung cytokines, and examined bronchial goblet cell hyperplasia by histology. Here we show that either PFOA or FTOH exposure can induce AHR, but neither one predisposes for exaggerated allergic lung inflammation or AHR. FTOH or PFOS exposure appears to suppress allergic lung inflammation, but does not affect allergic lung dysfunction.

Airway hyperresponsiveness (AHR) is one of the primary features of allergic airways disease. Despite continuous allergen exposure atopic asthmatics do not develop progressively worsening AHR. The mechanism(s) that limit AHR are unknown. Two valid candidates are regulatory T cells (Treg) and antigen presenting cells (APC). Dendritic cells (DC) are the main APC within the airways. Presentation of allergens to T cells can result in the differentiation and expansion of different subsets of T cells including effector Treg cells. The precise role of Treg and DC in the attenuation of allergen-induced AHR remains unknown. The general aim of this thesis is to investigate mechanisms to limit AHR in a murine model of atopic asthma. Specific aims are to: 1. develop a murine model of allergen-induced attenuation of AHR, 2. determine the potential role of regulatory T cells (Treg) in allergen-induced AHR attenuation, and 3. determine the potential role of airway dendritic cells (DC) in allergen-induced AHR attenuation. Balb/c mice were sensitised with intraperitoneal Ovalbumin (OVA) in aluminium hydroxide and challenged with a single, 3-weeks or 6-weeks of OVA aerosols. Aerosols were 1% OVA in sterile saline delivered for 30 minutes for three days per week. Animals were sacrificed 24 hours after the final aerosol for measurements of lung function and Methacholine (MCh) responsiveness (low-frequency forced oscillation technique), collection of bronchoalveolar lavage fluid (BALF) and serum. '...' In contrast, 6-weeks of OVA challenges decreased Treg numbers back to control levels. Adoptive transfer of 1x106 Treg taken from DLN of 3-week challenged mice attenuated AHR in single-OVA recipients (p<0.05). Furthermore, in vivo depletion of Treg in 3-week OVA challenged mice restored AHR (p<0.05 compared with control). Similar proportions of CD4+ T cells became activated following both aerosol regimes, however total numbers of airway CD4+ T cells were decreased (p<0.05), and OVA-specific CD4+ T cell proliferation in DLN was reduced (p<0.05) after 3-weeks versus one OVA aerosol. Analysis of antigen handling by airway APC populations showed antigen uptake (OVA-647) and processing (DQ-OVA) by macrophages and airway DC subsets to be down-regulated (p<0.05) after 3-weeks of OVA aerosols. In addition, adoptive transfer of Treg into single-OVA recipients did not affect antigen handling by airway APC populations. These data suggest that Treg are responsible for allergen-induced attenuation of AHR in vivo in established airways disease. AHR attenuation was associated with an altered function of airway DC, resulting in reduced antigen capture and processing, leading to limited clonal expansion of antigen-specific CD4+ T cells with limited production of Th2 cytokines. Furthermore, Treg were not directly responsible for the down-regulation of allergen capture in the airways. In conclusion, knowledge of the role of Treg and DC in attenuation of AHR could potentially result in improved and more directed therapies for the attenuation of AHR in atopic asthmatics.

We report herein that IL-10-treated dendritic cells (DC) can be used effectively to reverse established severe asthma-like disease in a mouse model. Our lab had shown previously that allergen-presenting splenic CD8¦Á+ DCs could ¡Ö50% reduce airway hyper responsiveness (AHR), eosinophilia, and Th2 responses in asthma-phenotype mice, but only marginally reduce IgE/IgG1 levels. We now show that bone marrow-derived DCs that have been differentiated in the presence of IL-10 (DCIL-10) are effective in reversing the asthma phenotype. Co-culture of DCIL-10 with T memory (TM) cells from asthma-phenotype mice was associated with lack of Th2 responses, and this was partially reversed by IL-2. Immunostimulatory DC activated these Th2 cells. <i>In vivo</i>, delivery of allergen-pulsed DCIL-10, either into the airway or intraperitoneally abrogated AHR from weeks 3-10 post-treatment, and ameliorated lung eosinophilia and Th2 (IL-4, -5, -9, & -13, IgE) responses, as well as circulating allergen-specific IgE responses for at least 32 weeks following treatment. Repeated OVADCIL-10 treatments kept AHR normalized for 8 weeks as well as Th2 responses significantly low. In vivo, delivery of anti-IL-10R, but not anti-TGF-¦Â from day 12-21 after treatment had moderate effects on DCIL-10-driven tolerance, but 1-methyl tryptophan (inhibitor of indoleamine-2,3-dioxygenase) treatment had significant effects on Th2 responses. The mechanisms mediating tolerance in vivo are likely complex, but we speculate that infectious tolerance sustains this regulatory activity during the 32-week period in which we have observed tolerance to be in place.

We report herein that IL-10-treated dendritic cells (DC) can be used effectively to reverse established severe asthma-like disease in a mouse model. Our lab had shown previously that allergen-presenting splenic CD8¦Á+ DCs could ¡Ö50% reduce airway hyper responsiveness (AHR), eosinophilia, and Th2 responses in asthma-phenotype mice, but only marginally reduce IgE/IgG1 levels. We now show that bone marrow-derived DCs that have been differentiated in the presence of IL-10 (DCIL-10) are effective in reversing the asthma phenotype. Co-culture of DCIL-10 with T memory (TM) cells from asthma-phenotype mice was associated with lack of Th2 responses, and this was partially reversed by IL-2. Immunostimulatory DC activated these Th2 cells. <i>In vivo</i>, delivery of allergen-pulsed DCIL-10, either into the airway or intraperitoneally abrogated AHR from weeks 3-10 post-treatment, and ameliorated lung eosinophilia and Th2 (IL-4, -5, -9, & -13, IgE) responses, as well as circulating allergen-specific IgE responses for at least 32 weeks following treatment. Repeated OVADCIL-10 treatments kept AHR normalized for 8 weeks as well as Th2 responses significantly low. In vivo, delivery of anti-IL-10R, but not anti-TGF-¦Â from day 12-21 after treatment had moderate effects on DCIL-10-driven tolerance, but 1-methyl tryptophan (inhibitor of indoleamine-2,3-dioxygenase) treatment had significant effects on Th2 responses. The mechanisms mediating tolerance in vivo are likely complex, but we speculate that infectious tolerance sustains this regulatory activity during the 32-week period in which we have observed tolerance to be in place.

Spleen tyrosine kinase (Syk) plays a critical role in regulation of immune and inflammatory responses. This thesis investigated the role of Syk in the development of the asthma phenotype in acute and chronic mouse models of allergic airways inflammation. Airway hyperresponsiveness (AHR) to methacholine and inflammation increased significantly in HDM-induced compared with the saline control mice. We demonstrated that in vivo inhibition of Syk by selective Syk inhibitors, and genetic deletion of Syk using conditional Syk knockout mice attenuated AHR despite of inflammatory cell influx in the lung. Histological analysis showed airway remodeling in the chronic model, which was attenuated to some degree by deletion of Syk. This study identified a role of Syk in airway hyperresponsiveness and remodeling without significantly affecting leukocyte recruitment in HDM model of airways disease. My results support the improvement of therapeutic strategies in asthma by targeting the Syk pathway.

Spleen tyrosine kinase (Syk) plays a critical role in regulation of immune and inflammatory responses. This thesis investigated the role of Syk in the development of the asthma phenotype in acute and chronic mouse models of allergic airways inflammation. Airway hyperresponsiveness (AHR) to methacholine and inflammation increased significantly in HDM-induced compared with the saline control mice. We demonstrated that in vivo inhibition of Syk by selective Syk inhibitors, and genetic deletion of Syk using conditional Syk knockout mice attenuated AHR despite of inflammatory cell influx in the lung. Histological analysis showed airway remodeling in the chronic model, which was attenuated to some degree by deletion of Syk. This study identified a role of Syk in airway hyperresponsiveness and remodeling without significantly affecting leukocyte recruitment in HDM model of airways disease. My results support the improvement of therapeutic strategies in asthma by targeting the Syk pathway.