Global prevalence of allergic diseases has been on the rise for the last 30 years. In Canada, this upward trend in allergic diseases has resulted in over 3 million Canadians being affected by allergic asthma. Allergic asthma is triggered by inhalation of environmental allergens resulting in bronchial constriction and inflammation, which leads to clinical symptoms such as wheezing, coughing and difficulty breathing. Asthmatic airway inflammation is initiated by the release of inflammatory mediators (-eg- histamine) released by granulocytic cells (-eg- mast cells and basophils). However, immunoglobulin E (IgE) antibody is also necessary for the initiation of the allergic cascade, and IgE is produced and released exclusively by memory B cells and plasma cells. Allergen crosslinking of IgE:FcεRI complexes on the surface of mast cells and basophils causes degranulation of pro-inflammatory mediators. Acute allergen exposure has also been shown to increase IgE levels in the airways of patients diagnosed with allergic asthma; however, more studies are needed to better understand local airway inflammation. Our group's work, in accordance with the literature, has shown an increase of IgE in the airways of subjects with mild allergic asthma following allergen inhalation challenge. Although regulatory B cells (Bregs) have been shown to modulate IgE-mediated inflammatory processes in allergic asthma pathogenesis, particularly in mouse models of allergic airway disease, the levels and function of these IgE+ B cells and Bregs remain to be elucidated in human models of asthma. The overall objective for this dissertation was to investigate the biology of B cells in allergic asthma pathogenesis, specifically investigating the frequency of IgE+ B cells and Bregs in allergic asthma, and the kinetics of these cells after allergen exposure. First, we characterized IgE+ B cells in the blood and sputum of allergic asthmatics and healthy controls with and without allergies (Chapter 2). We showed that IgE+ B cell levels were higher in sputum, but not blood, of allergic asthmatics compared to controls. We further demonstrated that these findings were consistent across airway IgE+ B cell subsets, which include IgE+ memory B cells and IgE+ plasma cells. Additionally, IgE+ B cells in sputum positively correlated with sputum eosinophils, total IgE and B cell activating factor (BAFF) measured in sputum fluid phase. These findings highlight the association of airway IgE+ B cells with allergic asthma, and suggest that local IgE+ B cell functions contribute to the pathogenesis of asthma. Second, we measured the trafficking of IgE+ B cells in periphery (blood, bone marrow and tonsil) and locally (sputum) in allergic asthmatics following whole lung allergen challenge (Chapter 3). IgE+ B cells only increased in the airways of allergic asthmatics following allergen inhalation challenge; there were no allergen-induced changes in IgE+ B cell levels in blood, bone marrow and tonsil. In addition, we showed allergen-induced increases in BAFF and total IgE, but not allergen-specific IgE in sputum fluid phase. Taken together, chapters 2 and 3 show that allergic asthmatics have elevated levels of IgE+ B cells in the airways, that can be further increased after allergen exposure. Therefore, local B cell production of IgE in the lungs may be an important source of IgE for initiation of acute inflammatory responses in allergic airways. Third, we evaluated the levels of Bregs in allergic asthmatics compared to controls, and examined the kinetics, function and distribution (bone marrow, blood and sputum) of Bregs following allergen inhalation challenge (Chapter 4). We showed that Bregs were 2-fold lower in the blood of allergic asthmatics compared to controls, highlighting a possible dysregulation of this regulatory cell type in allergic asthmatics, which may contribute to disease pathology. Furthermore, after whole lung allergen challenge Bregs decreased in the bone marrow with a co-incident increase in the blood and sputum of allergic asthmatics. This pattern reflects potential trafficking of these cells from bone marrow to the airways after exposure to allergic stimuli. Lastly, we stimulated CD19+ B cells purified from blood of allergic asthmatic with IL-4 in vitro. IL-4 is a type 2 cytokine known to isotype-switch B cells to IgE+ B cells, as well as differentiates naïve T cells into Th2 cells, thus propagating the allergic cascade. We found that IL-4 promoted higher proportions of IL-10+ and FoxP3+ Bregs, which demonstrates that Bregs may have a role in dampening IgE-mediated inflammation in a type 2 environment. However, further functional studies are warranted. Taken together, the findings of this dissertation highlight the local compartmental changes in IgE+ B cells and Bregs following allergen challenge of allergic airways. Better understanding the temporal and compartmental shifts in B cell subpopulations, particularly IgE+ B cells and Bregs, may aid in future development of therapeutics. / Thesis / Doctor of Philosophy (PhD)

Allergic airway diseases such as rhinitis, asthma, and chronic rhinosinusitis are responsible for causing a huge economic burden on patients and society. Patients suffering from asthma often have allergies to pollen, dust mite, and mold. Interestingly, studies have shown that there is a correlation between severe asthma and sensitization to fungi including Aspergillus, Alternaria, Cladosporium, and Penicillium. This project has been focused on studying the innate immunomodulatory activities of the major allergen Alt a 1, from the ubiquitous airborne fungus, Alternaria alternata. In several studies, 90-100% of allergic patients who are sensitized to Alternaria, have Alt a 1 specific IgE antibodies indicating that it is a major and clinically relevant allergen. Although progress has been made over the past few decades regarding elucidating the mechanistic underpinnings of allergic inflammation, more research needs to be done, especially in regards to innate immunity and its role in the sensitization and exacerbation aspects of allergic diseases. Published studies have increasingly made it clear that Toll-like receptors (TLRs) are key players in innate immunity to several allergens. For example, the dust mite allergen, Der p 2, has been shown to mimic the activity of human and mouse MD2 in the presence of LPS to trigger a response through TLR4. Bet v 1, an allergen from Birch tree, has been shown to enter and be transported through lung epithelium in patient cells. It is hypothesized that transcytosis of allergens like Bet v 1 may contribute to sensitization and exacerbation in atopic individuals. This project was focused on two primary aims; (1) Characterize the innate immune response of Alt a 1 in human airway epithelial cells, and (2) Identify if and how Alt a 1 can enter human airway cells. We found that Alt a 1 was able to stimulate innate immune responses in bronchial epithelial cells and this was dependent upon TLR2, TLR4 and the downstream adaptor proteins MyD88 and TIRAP. We also found in our studies that Alt a 1 rapidly enters bronchial epithelial cells. Furthermore, our data suggests that endocytosis of Alt a 1 may be partially dependent upon interaction with phosphatidyl-inositol-3-phosphate (PI-3-P). / Ph. D. / Allergic airway diseases such as rhinitis, asthma, and chronic rhinosinusitis are responsible for causing a huge economic burden on patients and society. Patients suffering from asthma often have allergies to pollen, dust mite, and mold. Interestingly, studies have shown that there is a correlation between severe asthma and allergy to several fungal species including Aspergillus, Alternaria, Cladosporium, and Penicillium. This project has been focused on studying how the allergen, Alt a 1, from the fungus, <i>Alternaria alternata</i>, can cause an allergic response in the human airways. In several studies, 90-100% of allergic patients who have allergy to Alternaria, have proteins in their bloodstream that specifically recognize Alt a 1. This indicates that they are allergic to Alt a 1. Though we know that these patients have allergy to Alt a 1, we do not know how this protein causes the characteristic symptoms of allergy, such as a runny nose, watery eyes, hives, and breathing difficulty. Published studies have increasingly made it clear that molecules on the surface of cells that line the airways are important players in the body’s response to allergens. A dust mite allergen, Der p 2, can interact with one such receptor on human cells. The receptor may not be the only way that allergens can cause a response. Studies have shown that allergens can directly enter human cells. For example, a Birch tree allergen, Bet v 1, has been shown to enter human lung cells. This project was focused on two primary aims; (1) Identify how human airway cells response to Alt a 1, and (2) Identify if and how Alt a 1 can enter human airway cells. We found that Alt a 1 was able to cause human airway cells to produce several molecules that lead to the characteristic symptoms of allergy, and that this response was dependent on a receptor on human airway cells. We also found that Alt a 1 rapidly enters human airway cells.

L’immunothérapie allergénique (ITA) est la seule thérapie capable d’agir sur l’étiologie des allergies. La compréhension des mécanismes d’action de ce traitement et la mise en évidence de biomarqueurs d’efficacité favoriserait l’optimisation de l’ITA. A l’aide des tétramères de classe II, nous avons suivi les lymphocytes T CD4 périphériques spécifiques d’allergènes, acteurs centraux de la réaction allergique, dans des conditions normales, pathologiques ou en cours d’ITA, afin d’établir un lien entre ces trois situations physiologiques. Nous avons mis en évidence des différences entre les réponses lymphocytaires T CD4 spécifiques d’allergènes saisonniers et perannuels, chez les individus sains et allergiques. Puis, lors de 2 études cliniques d’ITA sublinguale, l’une menée chez des adultes allergiques aux pollens de graminées traités pendant 4 mois et l’autre menée chez des enfants allergiques aux acariens traités pendant 1, nous avons respectivement observé une diminution des lymphocytes Th2A et une augmentation de la production d’IFN- γ liées au traitement. Toutefois, ces variations ne corrèlent pas avec l’efficacité clinique de l’ITA observée dans ces deux études. Les limites d’utilisation des tétramères de classe II nous ont amené à rechercher si l’expression de marqueurs d’activation membranaires pouvait remplacer un marquage « tétramère ». Alors qu’une corrélation insuffisante a été observée entre le marquage « tétramère » et l’expression des marqueurs d’activation testés, nous avons mis en évidence 3 populations cellulaires aux propriétés fonctionnelles diverses, soulignant l’hétérogénéité des réponses lymphocytaires spécifiques d’allergènes. De plus, la découverte des lymphocytes Th2A pourrait être une approche prometteuse pour le suivi des réponses lymphocytaires T CD4 spécifiques d’allergènes lors d’ITA à plus long terme. / Allergenic immunotherapy (AIT) is currently the only curative treatment for allergic disease. Whereas efficacy of this treatment is well established, its mechanisms of action are not clearly understood and predictive as well as surrogate biomarkers are needed to further support AIT development. We focused on allergen specific CD4 T cells, highly involved in allergic inflammation, and monitored their responses both in normal and pathologic conditions, or during AIT. Using MHC class II tetramers, we highlighted distinct patterns of polarization between seasonal and perennial allergen-specific CD4 T cells as well as between healthy and allergic individuals. Then, allergen-specific CD4 T cell responses were monitored during 2 double-blind placebo-controlled sublingual AIT clinical trials. After short term AIT (4 months), we observed a decrease of Th2A cells, a newly define subset, thought to contain most allergen-specific CD4+ T cells. IFN-γ production was increased after one year of treatment. However, these variations were not related to AIT clinical efficacy. We further compared the expression of various activation markers and MHC class II tetramer staining following in vitro stimulation in order to circumvent inherent limitation of tetramers. No correlation could be established between tetramer staining and the expression of multiple activation markers in allergen-stimulated CD4 T cells. Combining these methods helps understanding patient heterogeneity regarding CD4 T cell responses. Moreover, Th2A cells detection is likely a promising approach to identify allergen-specific CD4 T cell during long-term AIT.

In rural inland, south-eastern Australia, allergy to the fungus Alternaria is prevalent and an important risk factor for asthma. The aim of the thesis was to investigate the distribution and factors influencing allergens of Alternaria in the air. As airborne allergenic spores were thought to arise from harvesting of nearby crops, two towns with different agricultural practices were studied. Moree has two crop harvesting periods in summer and autumn whilst Wagga Wagga has one harvesting period in summer. Over two years, air was sampled daily in Wagga Wagga and Moree using Burkard traps. The reliability of measurements from a single site to represent the distribution of airborne concentrations of spores across each town was examined using data from three traps simultaneously, sited 2.0 to 4.9 km apart, over four weeks. Substantial intra-class correlation coefficients (ICC) were observed between the three sampling sites across both towns (ICC=0.52, 95% CI 0.30-0.71 to 0.76, 95% CI 0.61-0.87) when counts of Alternaria spores were relatively high. The correlation was poor when counts were low. Of more than 365 trap tapes examined, the two microscopic traverses strongly correlated for counts of Alternaria spores (ICC=0.95, 95% CI 0.94-0.96). Alternaria was detected in both towns throughout the two year period with peaks in spore concentrations reflecting the season of crop harvesting in each region. Individual exposure to spores was examined. Thirty three subjects (adults and children from nine families) wore nasal air samplers and personal air samplers both inside and outside their homes. The effects of activity, location, age on the inhalation of Alternaria spores and variation between individuals in the same environment were determined. Every subject inhaled Alternaria spores. Personal exposure to Alternaria in the home environment varied substantially between subjects. Levels of fungal spores inhaled were higher during periods of activity than during rest, and higher while subjects were outdoors than indoors. During outdoor activity, the number of Alternaria spores inhaled ranged from 4 to 794 (median 11) spores/hr. Sources of airborne spores was investigated by sampling air above wheat and cotton crops near the towns during harvesting and non-harvesting periods, in a grain and cotton seed storage shed, and a cotton gin. Substantially higher concentrations were detected above crops during harvesting periods compared to non-harvesting periods. Peaks were associated with harvesting and other activities where plants were manipulated. By regression analysis spore concentrations in both towns were modelled against those detected above crops and with weather variables. Only one crop sampling period (cotton harvest) independently correlated with concentrations in town. Analysis combining all data showed concentrations of spores above crops correlated with spore concentrations in the town when lagged by one day. Variables of rainfall and maximum temperature influenced concentrations in both towns, and wind direction in Wagga Wagga alone. Parents of asthmatic children were asked by questionnaire in which locations symptoms were provoked. Asthma was reported to be exacerbated at grain farms and with disturbance of local vegetation in town and home gardens. Nasal sampling confirmed that activities that disturbed dust or vegetation increased the inhalation of spores. The factors that release allergen from spores were determined in a modified Halogen immunoassay. Approximately 60% of spores released allergen, and the proportion was influenced by isolate, nutrient availability, viability, and not influenced by sunlight or culture age up to 21 days. Germinating the spores significantly increased the proportion that released total allergen and Alt a 1 (p<0.0001). Alt a 1 appears to be a minor contributor to the total allergen released from spores except when spores have germinated. Conclusions: People living in inland rural regions of Australia are exposed to substantial quantities of allergenic spores of Alternaria. Exposure is a highly personal event and is largely determined by disturbance of local vegetation releasing spores such as from nearby crops by wind, harvesting, slashing, transport and processing of produce, and from within town and home gardens. Most spores inhaled are likely to be allergenic, with potency potentially increasing with viability.

<p>This thesis studied environmental factors in relation to asthma and respiratory symptoms among schoolchildren in two countries. In Sweden, 1014 pupils (5-14 year) in 8 schools participated. Wheeze was reported by 7.8%, current asthma by 5.9%, doctor-diagnosed asthma by 7.7%, cat allergy by 6.8% and dog allergy by 4.8%. Current asthma was less common among those consuming more fresh milk and fish. Doctor-diagnosed asthma was less common among those consuming olive oil. Cat, dog and horse allergens were common in settled dust and related to respiratory symptoms. Pupils consuming butter and fresh milk had less respiratory symptoms in relation to allergen exposure. In schools with increased levels of microbial volatile organic compounds and selected plasticizers (Texanol and TXIB) asthma and respiratory symptoms were more common.</p><p>In Korea, 2365 pupils (9-11 year) in 12 schools participated (96%). In total, wheeze was reported by 8.0%, current asthma by 5.7%, doctor-diagnosed asthma by 5.4%, cat allergy by 2.6% and dog allergy by 4.9%. Contamination of dog and mite (<i>Dermatophagoides farinae</i>) allergen was common while cat allergen was uncommon. Remodelling, changing floor and building dampness at home were positively associated with asthma and respiratory symptoms. The strongest associations were found for floor dampness. Indoor/outdoor concentration of NO₂, formaldehyde and ultrafine particles (UFP) at schools were positively associated with asthma and respiratory symptoms. </p><p>When comparing Sweden and Korea, Korean pupils had more breathlessness and asthma but reported less cat and pollen allergy. Swedish schools had CO₂-levels below 1000 ppm, while most Korean schools exceeded this standard. Since both home and school environment may affect pupil’s asthma and respiratory symptoms, air quality should be an important health issue. Moreover, changes in dietary habits may be beneficial to decrease asthma and allergies. Furthermore, interaction between diet and environment needs to be further investigated.</p>

This thesis studied environmental factors in relation to asthma and respiratory symptoms among schoolchildren in two countries. In Sweden, 1014 pupils (5-14 year) in 8 schools participated. Wheeze was reported by 7.8%, current asthma by 5.9%, doctor-diagnosed asthma by 7.7%, cat allergy by 6.8% and dog allergy by 4.8%. Current asthma was less common among those consuming more fresh milk and fish. Doctor-diagnosed asthma was less common among those consuming olive oil. Cat, dog and horse allergens were common in settled dust and related to respiratory symptoms. Pupils consuming butter and fresh milk had less respiratory symptoms in relation to allergen exposure. In schools with increased levels of microbial volatile organic compounds and selected plasticizers (Texanol and TXIB) asthma and respiratory symptoms were more common. In Korea, 2365 pupils (9-11 year) in 12 schools participated (96%). In total, wheeze was reported by 8.0%, current asthma by 5.7%, doctor-diagnosed asthma by 5.4%, cat allergy by 2.6% and dog allergy by 4.9%. Contamination of dog and mite (Dermatophagoides farinae) allergen was common while cat allergen was uncommon. Remodelling, changing floor and building dampness at home were positively associated with asthma and respiratory symptoms. The strongest associations were found for floor dampness. Indoor/outdoor concentration of NO2, formaldehyde and ultrafine particles (UFP) at schools were positively associated with asthma and respiratory symptoms. When comparing Sweden and Korea, Korean pupils had more breathlessness and asthma but reported less cat and pollen allergy. Swedish schools had CO2-levels below 1000 ppm, while most Korean schools exceeded this standard. Since both home and school environment may affect pupil’s asthma and respiratory symptoms, air quality should be an important health issue. Moreover, changes in dietary habits may be beneficial to decrease asthma and allergies. Furthermore, interaction between diet and environment needs to be further investigated.

Food allergies are an important health problem and affect up to 2% of the adult population and 8% of children worldwide. Under the Food Allergen Labeling and Consumer Protection Act (FALCPA) of 2004, foods that contain or derive from the "Big 8" allergens (milk, egg, finfish, crustacean shellfish, tree nuts, peanuts, wheat, and soybeans) must be declared and the "common or usual name" of the allergen source must be printed on the label of the food product. Currently, the most common used detection methods for food allergens are enzyme-linked immunosorbent assay (ELISA) based. ELISA, a protein-based method, targets specific allergen(s) and detects by colorimetric reaction following binding with a specific-enzyme labeled antibody. However, studies have demonstrated that matrix interference and heat treatment can interfere with the detectability of commercial ELISA kits. An alternative approach to targeting the allergen in soy is to use deoxyribonucleic acid (DNA) as a unique marker that can be used to indicate the presence of soy in food. According to FALCPA the source of an allergen should be declared on the label, therefore identifying an allergen, such as soy, by DNA detection could be a valid means of meeting FALCPA requirements. Real-time polymerase chain reaction (real-time PCR), a DNA-based method, can identify the presence of soy through amplification of specific sequences of DNA through the use of primers. However, the sensitivity of real-time PCR can be influenced by the amplification protocol, primer design and DNA extraction methods. Thus, the main objectives of this study were to 1) verify the specificity of primers designed to detect soy DNA from different soy products, 2) optimize the previously developed real-time PCR protocol to detect soy DNA, 3) investigate the application of two commercially available DNA recovery systems (column and magnetic beads) to recover soy DNA from different forms of soy products using real-time PCR and 4) determine the effect of food matrices and thermal processing on soy detection using DNA and ELISA methods. In this study, Wizard Magnetic DNA Purification system kit (Promega, Madison, WI) was selected as the column DNA recovery system while DNeasy mericon Food Kit (Qiagen, Valencia, CA) was selected as the magnetic beads system. Neogen Veratox for soy allergen was selected as the ELISA system. The evaluations of both DNA recovery systems were conducted on soy protein isolates (SPI), powdered soybean and soymilk. The effect of thermal processing in soy detection was conducted on four different food matrices (protein, fat, carbohydrate and water). Each food matrix was spiked with 10% soy protein isolates and heated at 95ºC for an hour. Both DNA (column and magnetic beads DNA recovery system) and ELISA detection methods were used to detect soy in heated and non-heated food matrices. The limit of detection for column DNA recovery method in soybean, SPI and soymilk can be as low as 20 ppm, while magnetic beads DNA method was matrix dependent. The magnetic beads methods demonstrated a lower detection for soybean sample (1.33 ppm) but higher for soymilk (133.3 ppm). The soy percent recovery for non-heated food matrices was higher in ELISA methods and lower in magnetic beads DNA method. For heated food matrices, percent recovery for both DNA methods was higher than ELISA method. Overall, heat treatment can significantly reduce the ability of the ELISA method to detect soy in all food matrices. However, for DNA methods (column and magnetic beads), water and ranch matrices were the only two that were significantly affected by thermal processing. In terms of food matrices, water matrix (heated and non-heated) has the highest percent recovery of soy for all detection methods. However, percent recovery of soy in flour matrix (non-heated) was the lowest using both DNA methods. / Graduation date: 2013

In rural inland, south-eastern Australia, allergy to the fungus Alternaria is prevalent and an important risk factor for asthma. The aim of the thesis was to investigate the distribution and factors influencing allergens of Alternaria in the air. As airborne allergenic spores were thought to arise from harvesting of nearby crops, two towns with different agricultural practices were studied. Moree has two crop harvesting periods in summer and autumn whilst Wagga Wagga has one harvesting period in summer. Over two years, air was sampled daily in Wagga Wagga and Moree using Burkard traps. The reliability of measurements from a single site to represent the distribution of airborne concentrations of spores across each town was examined using data from three traps simultaneously, sited 2.0 to 4.9 km apart, over four weeks. Substantial intra-class correlation coefficients (ICC) were observed between the three sampling sites across both towns (ICC=0.52, 95% CI 0.30-0.71 to 0.76, 95% CI 0.61-0.87) when counts of Alternaria spores were relatively high. The correlation was poor when counts were low. Of more than 365 trap tapes examined, the two microscopic traverses strongly correlated for counts of Alternaria spores (ICC=0.95, 95% CI 0.94-0.96). Alternaria was detected in both towns throughout the two year period with peaks in spore concentrations reflecting the season of crop harvesting in each region. Individual exposure to spores was examined. Thirty three subjects (adults and children from nine families) wore nasal air samplers and personal air samplers both inside and outside their homes. The effects of activity, location, age on the inhalation of Alternaria spores and variation between individuals in the same environment were determined. Every subject inhaled Alternaria spores. Personal exposure to Alternaria in the home environment varied substantially between subjects. Levels of fungal spores inhaled were higher during periods of activity than during rest, and higher while subjects were outdoors than indoors. During outdoor activity, the number of Alternaria spores inhaled ranged from 4 to 794 (median 11) spores/hr. Sources of airborne spores was investigated by sampling air above wheat and cotton crops near the towns during harvesting and non-harvesting periods, in a grain and cotton seed storage shed, and a cotton gin. Substantially higher concentrations were detected above crops during harvesting periods compared to non-harvesting periods. Peaks were associated with harvesting and other activities where plants were manipulated. By regression analysis spore concentrations in both towns were modelled against those detected above crops and with weather variables. Only one crop sampling period (cotton harvest) independently correlated with concentrations in town. Analysis combining all data showed concentrations of spores above crops correlated with spore concentrations in the town when lagged by one day. Variables of rainfall and maximum temperature influenced concentrations in both towns, and wind direction in Wagga Wagga alone. Parents of asthmatic children were asked by questionnaire in which locations symptoms were provoked. Asthma was reported to be exacerbated at grain farms and with disturbance of local vegetation in town and home gardens. Nasal sampling confirmed that activities that disturbed dust or vegetation increased the inhalation of spores. The factors that release allergen from spores were determined in a modified Halogen immunoassay. Approximately 60% of spores released allergen, and the proportion was influenced by isolate, nutrient availability, viability, and not influenced by sunlight or culture age up to 21 days. Germinating the spores significantly increased the proportion that released total allergen and Alt a 1 (p<0.0001). Alt a 1 appears to be a minor contributor to the total allergen released from spores except when spores have germinated. Conclusions: People living in inland rural regions of Australia are exposed to substantial quantities of allergenic spores of Alternaria. Exposure is a highly personal event and is largely determined by disturbance of local vegetation releasing spores such as from nearby crops by wind, harvesting, slashing, transport and processing of produce, and from within town and home gardens. Most spores inhaled are likely to be allergenic, with potency potentially increasing with viability.

L'allergie de contact est une pathologie en constante augmentation et particulièrement répandue dans les pays industrialisés. Aucune thérapie n'existe actuellement et seule l'éviction totale de l'allergène permet d'éviter toute nouvelle réaction d'allergie. La base de l'allergie de contact est la formation d'une liaison entre l'allergène et les protéines épidermiques. C'est cette étape chimique clé qu' il est important de comprendre afin de développer de nouvelles méthodes dites« alternatives» dans le cadre de l'actuelle législation sur les cosmétiques. Le but de ce travail de thèse a été d'étudier la réactivité de différents allergènes connus face aux acides aminés et protéines d'épidermes reconstruits de type SkinEthic® par la technique RMN HRMAS dérivée de la RMN du solide. Six allergènes ont ainsi été étudiés et leur réactivité a été comparée a celle observée en solution face à une protéine modèle. / Contact dermatitis is one of the most common health problem and highly prevalent in industrialized countries. No therapy currently exists and only the total eviction of the allergen can prevent further allergie reaction.The key molecular event in skin sensitization is the formation of a bond between the allergen and the epidermal proteins. Due to the recent legislation on cosmetic and to help avoid the inappropriate use of new allergens, the understanding of this key step has to be expanded in order to develop new alternative metbods.The aim of this PhD work is to study the reactivity of some allergens towards amino acids and proteins presents in reconstructed human epidermis like SkinEthic® using the HRMAS NMR technique. Six allergens have been studied and their reactivity was compared to that observed ln solution with a model protein.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Background: Seasonal allergic rhinoconjunctivitis or hay fever is caused due to the sensitization to pollen allergens, usually from grasses. Lolium multiflorum (Lm) is one of the most important grass pollen allergens in Southern Brazil. Objectives: To evaluate three different pollen extraction methods and to analyze IgE, IgG1 and IgG4 responses to Lm pollen antigens in pollinosis patients. Methods: Three different Lm pollen extracts were prepared (LmPBS extract, Lme-PBS extract and LmNH4HCO3 extract) and analyzed in 13.5% SDS-PAGE. Serum samples from 62 patients with seasonal allergic rhinoconjunctivitis and/or asthma (Lm+ group), 30 patients with perennial allergy rhinitis (Lm- group) and 30 non-atopic subjects (NA) were tested for IgE reactivity to three Lm extracts by ELISA. Lm-specific IgG1 and IgG4 antibodies were also evaluated by using LmPBS extract only in ELISA. Results: By SDS-PAGE, the three extracts were very similar, showing bands ranging from 20 to 100 kDa. By ELISA-IgE, the results revealed higher IgE levels in pollinosis patients when LmPBS extract was used. LmPBS extract was chosen to evaluate the IgE, IgG1 and IgG4 responses and their levels were higher in the Lm+ patients when compared to Lm and NA groups. In the Lm+ group, IgE (EI = 17.6) levels were higher than IgG1 (EI = 2.6) and IgG4 (EI = 3.6) levels, while in other groups there were not any differences in the antibody levels. Conclusions: LmPBS extract was effective in detecting IgE, IgG1 and IgG4 responses to Lolium multiflorum pollen antigens. These antibody classes are in a higher level in pollinosis patients when compared to non-sensitized subjects. Lm allergen extracts for in vivo and in vitro using should be standardized. / Introdução: A rinite alérgica estacional ou doença polínica se deve à sensibilização aos alérgenos de pólens, geralmente de gramíneas. Lolium multiflorum (Lm) é uma gramínea com pólens de elevado potencial alergênico, sendo a principal gramínea causadora de polinose na região Sul do Brasil. Objetivos: Analisar três diferentes métodos de extração antigênica pela reatividade IgE de cada extrato e as respostas anticórpicas IgE, IgG1 e IgG4 específicas aos antígenos de pólen de Lm. Material e Métodos: Três extratos de pólen de Lm foram preparados (extratos LmPBS, Lme- PBS e LmNH4HCO3) e analisados por SDS-PAGE a 13,5%. Amostras de soro de 62 pacientes com rinite alérgica sazonal e/ou asma brônquica (grupo Lm+), 30 pacientes com rinite alérgica perene (grupo Lm-) e 30 indivíduos não-atópicos (grupo NA) foram testadas para a reatividade IgE frente aos três extratos por ELISA. Anticorpos IgG1 e IgG4 específicos a Lm foram avaliados empregando-se somente o extrato LmPBS, por ELISA. Resultados: O perfil protéico dos extratos foi muito semelhante por SDS-PAGE, apresentando bandas protéicas de 20 a100 kDa. Níveis de IgE foram maiores em pacientes com polinose ao se utilizar o extrato LmPBS. O extrato LmPBS foi utilizado para avaliar os níveis de IgE, IgG1 e IgG4, que foram maiores nos pacientes com polinose que em pacientes Lm- e indivíduos NA. No grupo Lm+, os níveis médios de IgE (IE = 17,6) foram maiores que os níveis de IgG1 (IE = 2,6) e IgG4 (IE = 3,6) (p < 0,001). Conclusões: O extrato LmPBS foi eficiente em detectar as respostas IgE, IgG1 e IgG4 a antígenos de pólen de Lm. Essas classes de anticorpos estão em níveis maiores em pacientes com polinose. Extratos antigênicos de Lm para uso in vivo e in vitro devem ser padronizados. / Mestre em Imunologia e Parasitologia Aplicadas