L’allergie ou l’hypersensibilité de type I est une réponse inappropriée du système immunitaire à une substance étrangère à l’organisme, nommée « allergène ». L’immunothérapie allergénique (ITA) est actuellement le seul traitement sur le marché qui permet de traiter l’étiologie de la maladie allergique par opposition aux traitements symptomatiques qui diminuent temporairement les manifestations allergiques. Son action consiste à réduire la sensibilité de l’organisme vis-à-vis de l’allergène en modulant progressivement la réponse immunitaire dirigée contre ce dernier. L’objectif de cette thèse était de définir des biomarqueurs d’efficacité clinique utilisables dans le cadre des traitements de l’ITA. La stratégie de recherche est basée sur une hypothèse qui consiste à suggérer que les cellules dendritiques (DCs) sont impliquées dans le succès de l’immunothérapie. En particulier, nous supposons que le traitement induit une baisse des DCs de type 2 (DC2), qui induisent des lymphocytes T auxiliaires de type 2 (TH2), et une augmentation des DCs régulatrices (DCreg), qui induisent des lymphocytes T régulateurs. La première partie de cette thèse a consisté à mettre au point des conditions de culture induisant des DC2. Pour cela, un criblage de molécules biologiques et pharmacologiques a été entrepris sur les DCs dérivées des monocytes afin d’induire in vitro des DC2 et a conduit à la mise au point d’un mélange de plusieurs molécules, dont certaines sont impliquées dans les mécanismes de l’allergie. Le phénotype des DC2 obtenu a été étudié ainsi que la polarisation des lymphocytes T induite après co-cultures en comparaison avec des DCs de type 1 (DC1) et des DCreg.La deuxième partie de cette thèse a consisté à analyser, à l’échelle moléculaire, les différents types de DCs induites (DC1, DC2 et DCreg). Pour cela, deux techniques ont été utilisées, une analyse transcriptomique par puces à ADN et une analyse protéomique par spectrométrie de masse sans marquage, pour comparer le transcriptome et le protéome des DCs induites. Le différentiel d’expression des marqueurs les plus pertinents a été validé au niveau transcriptionnel et protéique.Dans la troisième partie de cette thèse, le suivi des marqueurs dans des cellules du sang de patients allergiques traités ou non par ITA lors d’une étude clinique randomisée, contrôlée, en double aveugle, a permis de définir six nouveaux candidats biomarqueurs d’efficacité de l’immunothérapie, dont trois spécifiques des DC2 et trois autres spécifiques des DCreg. Ces marqueurs pourront être suivis lors des traitements d’ITA pour distinguer les patients répondeurs des non-répondeurs. / Allergy or type I hypersensitivity is an inappropriate response of the immune system to a foreign substance in the body, called "allergen". Allergen immunotherapy (AIT) is currently the only treatment on the market that can handle the etiology of allergic disease versus symptomatic treatments that temporarily reduce allergic manifestations. Its action is to reduce the sensitivity of the body against allergens.The aim of this thesis was to define biomarkers of clinical efficacy of AIT. The research strategy is based on the following hypothesis: dendritic cells (DCs) are involved in the success of immunotherapy. In particular, we assume that the treatment induces a decrease in DCs type 2 (DC2), which induce type 2 helper T cells, and an increase of regulatory DCs (DCreg), which induce regulatory T cells.First, we defined optimal culture conditions inducing the polarization of in vitro immature monocyte-derived DCs (MoDCs) toward a DC2 pattern. After screening several biological and pharmaceutical agents, we selected a cocktail of six molecules with some of them are pro-allergenic molecules. The phenotype of those DC2 cells and the CD4+ T cell polarization induced after coculture were characterized extensively in comparison with type 1 DC (DC1) and DCreg.In a second part, we compared the transcriptomes and the proteomes of MoDCs polarized into DC1, DC2 and DCreg by using cDNA microarrays together with label-free mass spectrometry. The differential expression of the most relevant markers was confirmed at the transcriptional and protein level. In the third part, markers were also followed in the peripheral blood from allergic patients enrolled in a randomized, double-blind, placebo-controlled AIT study. The expression of three DC2 markers was down-regulated and of three DCreg markers was up-regulated in patients who responded to the treatment and correlated with clinical efficacy. These markers could be used as follow-up read-outs of AIT efficacy in order of to discriminate responders from nonresponders.

A atopia caracteriza-se pela tendência de um indivíduo a produzir IgE em quantidade elevada, em resposta a um alérgeno específico, levando ao desenvolvimento de asma, rinite ou eczema. Todavia, a manifestação do fenótipo da alergia depende da interação de fatores genéticos e exposição a alérgenos ambientais. Desta forma, o alérgeno é processado e apresentado aos linfócitos T, que desenvolvem uma resposta imune Th2, exacerbada característica da atopia. A principal célula que está envolvida na comunicação entre a imunidade inata e adaptativa é a célula dendrítica (DC) cuja função é capturar, processar e apresentar o antígeno aos linfócitos. As DCs imaturas capturam o antígeno e migram do tecido para o órgão linfóide periférico, onde elas se diferenciam em DCs maduras e apresentam o antígeno aos linfócitos T naive. Assim, os linfócitos T naive podem se diferenciar em subtipos de linfócitos efetores como: linfócitos Th1 e Th2. Esses linfócitos auxiliam na produção de anticorpos pelos linfócitos B na resposta imune humoral contra patógenos específicos. O sistema imune humoral compreende cinco classes de imunoglobulinas: IgG, IgM, IgD, IgE e IgA e sua produção sofre influência da imunidade celular. Desta forma, alérgenos provenientes de ácaros como, Dermatophagoides pteronyssinus, podem levar à inflamação alérgica, alterando a produção de anticorpos. Tendo em vista evidências que demonstram a interação das DCs com anticorpos, propomos investigar sua influência na apresentação dos principais alérgenos da poeira domiciliar e sua modulação sobre a resposta imune em indivíduos alérgicos e não alérgicos. / Atopy is characterized by the trend of an individual to produce high amounts of IgE in response to a specific allergen, leading to the development of asthma, rhinitis or eczema. However, the manifestation of the allergy phenotype depends on the interaction of genetic factors and exposure to environmental allergens. In this way, the allergen is processed and presented to the T lymphocytes, which develop a Th2 immune response, exacerbated characteristic of atopy. The main cell that is involved in the communication between innate and adaptive immunity is the dendritic cell (DC) whose function is to capture, process and present the antigen to lymphocytes. Immature DCs capture the antigen and migrate from the tissue to the peripheral lymphoid organ, where they differentiate into mature DCs and present the antigen to naive T lymphocytes. Thus, naive T lymphocytes can differentiate into subtypes of effector lymphocytes such as Th1 and Th2 lymphocytes. These lymphocytes assist in the production of antibodies by B lymphocytes in the humoral immune response against specific pathogens. The humoral immune system comprises five classes of immunoglobulins: IgG, IgM, IgD, IgE and IgA and their production is influenced by cellular immunity. In this way, allergens from mites such as Dermatophagoides pteronyssinus, can lead to allergic inflammation, altering the production of antibodies. In view of evidence demonstrating the interaction of DCs with antibodies, we propose to investigate their influence on the presentation of the main house dust allergens and their modulation on the immune response in allergic and non-allergic individuals.

Resumo: Os venenos dos insetos da ordem Hymenoptera contêm uma variedade de proteínas alergênicas. As ferroadas desses insetos podem induzir reações alérgicas e ocasionalmente, anafilaxias fatais. Há um crescente interesse nos componentes químicos dos venenos desses insetos, sobretudo no campo da alergia e imunologia clínica. As principais reações desses venenos são as reações inflamatórias e/ou imunológicas em suas vítimas, podendo ocorrer alguns efeitos sistêmicos. Dentre os Hymenoptera sociais, os venenos de abelhas e vespas têm sido extensivamente estudados e muitos de seus componentes moleculares já foram isolados e identificados. Fosfolipase, hialuronidase, antígeno-5 e serinoprotease são proteínas antigênicas de elevada massa molecular que, quando injetadas durante o ato de ferroar, iniciam uma resposta imune peculiar, sensibilizando alguns indivíduos. Porém, poucos estudos têm sido realizados para a identificação e o mapeamento dos epítopos desses alérgenos. O conhecimento sobre a interação molecular dos principais alérgenos destes venenos certamente deverá contribuir para o aperfeiçoamento dos diagnósticos de alergia, bem como para o desenvolvimento de tratamentos mais seletivos de reações alérgicas mediadas por IgE. No presente estudo identificamos e mapeamos os epítopos lineares de células-B do antígeno-5 do veneno da vespa social Polybia paulista. O antígeno-5 é conhecido como um dos principais alérgenos do veneno de Hymenoptera com massa molecular em torno de 23 kDa e função biológica desconhecida, porém muitos estudos demonstram a sua alergenicidade. Tendo conhecimento da sequência primária do antígeno-5, a identificação e o mapeamento dos epítopos foram realizados através da síntese múltipla de peptídeos, utilizando as técnicas do SPOT-Synthesis, imunodetecção e método indireto do ELISA com soro de pacientes sensíveis... (resumo completo, clicar acesso eletrônico abaixo) / Abstract: The venom insects of Hymenoptera order contain a variety of allergenic proteins. The stings of these insects can induce allergic reactions and occasionally fatal anaphylaxis. There is a growing interest in the knowledge about the chemical components of the venom of these insects, especially in the field of allergy and clinical immunology. The main reactions of these venoms are inflammation and / or the induction of immune process in the victims; sometimes systemic effects also may be observed. Among the social Hymenoptera, the venoms of bees and wasps have been extensively studied and many of their molecular components have been isolated and identified. Phospholipase, hyaluronidase, antigen-5 and serine protease are antigenic proteins of high molecular weight, which may initiate a peculiar immune response to sensitize some individuals. However, few studies have been performed for the identification and mapping of the epitopes these allergens. The knowledge about the molecular interaction of the major allergens of these venoms with IgG and/or IgE will certainly contribute for improving the diagnosis of allergy, as well as to develop more selective treatments of reactions IgE-mediated allergy. In this study we identified and mapped the linear epitopes of B-cells of the antigen-5 from the venom of the social wasp Polybia paulista. The antigen-5 is known as one of the major allergens from Hymenoptera venoms with molecular weight around 23 kDa and unknown biological function, but many studies show its allergenicity. The aim of this study was to identify and to map the linear epitopes of B-cells of this allergen. Taking into account the knowledge the primary sequence of antigen-5, the identification and mapping of epitopes was performed by using multiple synthesis of peptides with the combination of different protocols: SPOT-Synthesis, immunoblotting and indirect method of ELISA with the sera... (Complete abstract click electronic access below) / Orientador: Mario Sergio Palma / Coorientador: Lucilene Delazari dos Santos / Banca: Salvatore Giovanni de Simone / Banca: Luisa Karla de Paula Arruda / Mestre

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.

<p>A new European labeling directive (2003/89/EC) states that certain foods and products derived thereof must always be declared. Among the tree nuts specified is Macadamia nut (Macadamia spp.). During the last few years, cases of IgE-allergic reactions, even severe anaphylaxes, have been reported. Reliable methods for the detection of this nut are needed.</p><p>Protein from Macadamia nuts was isolated. Polyacrylamide gel electrophoresis in SDS revealed two main protein bands of about 20 and 50kDa. These protein bands were cut and extracted from the gel and rabbits were immunized with each protein.</p><p>Immunoblotting showed dominant reactivity with the respective antigens. The antisera were further tested for specificity in immunodiffusion and in rocket immunoelectrophoresis.</p><p>In addition, a specific DNA-method was developed, based on Real-Time PCR using Macadamia vicilin as target sequence. Two different primer pairs were tested. Specificity was tested against potentially related nuts. Optimisation of primer and probe concentrations was performed. The limit of detection was 2-4 pg DNA, corresponding to a macadamia nut concentration of 50 to 100 μg per g. In a background of soybean DNA, down to 0,01 % macadamia DNA could be detected.</p>

<p>Reliable methods to analyze food for the presence of almond are important – not only for those allergic to almond, but also for monitoring the compliance with labelling regulations (EG directive 2003/89). Until now the Swedish National Food Administration has used methods like rocket immunoelectrophoresis and real-time PCR to detect almond in food. These methods are, however, not sensitive enough for protecting the most sensitive individuals. Therefore, the performance of a commercial ELISA kit was tested with regard to specificity/cross reactivity and limit of detection for almond both in solution and in different matrixes.</p><p>The limit of quantitation was at least 3,1 ppm (mg/kg) in solution and similar concentrations were measured in bisquits and chocolate. The ELISA method was about 100-fold more sensitive than rocket immunoelectrophoresis and PCR.</p><p>The specificity of the test kit was evaluated against a number of different nuts and seeds. No important cross reactivity was found. The antibodies against almond used in the kit can not differentiate between almond and apricot kernel. For such purposes the PCR method could be used.</p>

<p>Directive EC 2003/89/EC of the European Parliament and of the Council states that certain</p><p>ingredients and products derived there of known to cause allergen reactions must always be</p><p>declared. Furthermore labelling is mandatory irrespective of the amount included. The National</p><p>Food Administration therefore needs methods for monitoring the presence of allergens in food.</p><p>Methods already exist for most of the allergens on the EU-list, but an operational method for</p><p>celery (Apium graveolens) is missing.</p><p>A specific DNA-method was developed, based on TaqMan Real-Time PCR with the celery</p><p>mannitol dehydrogenase gene as target sequence. The analysis was started with homogenisation</p><p>of the sample followed by extraction of DNA. The Real-Time PCR method was shown to be</p><p>specific for celery, producing a 113 bp fragment with two celery varieties and negative results</p><p>with other closely selected species commonly present together with celery in food products (12</p><p>samples). The detection limit was 2-20 pg DNA, which corresponds to 1-7 haploid genome</p><p>copies. When evaluated with model samples of celery in meat, a detection limit of less than</p><p>0,01 % was determined. When used to analyse food products from the market, six out of seven</p><p>products declared to contain celery were correctly identified as positive.</p>

<p>The aim was to study the prevalence of asthma, eczema, allergy and respiratory symptoms among pupils in Shanxi province, China, in relation to home and school environment and outdoor air pollution. In one study there was a low prevalence of self-reported asthma, eczema and pollen or pet allergy among pupils (9-20y). Rural childhood and consumption of fruit and fish were negatively associated with asthma or allergy, while current urban residency and consumption of hamburgers tended to be risk factors. In another study in junior high school pupils, similar low prevalence of asthma and allergy was found. Compared with pupils at the same age in Uppsala, Sweden, asthma and allergy were less common while daytime attacks of breathlessness were more common in Chinese pupils. Parental asthma or allergy was a predictor of asthma symptoms. Factors in the home environment such as new floor, new furniture and ETS exposure were risk factors for asthma symptoms. Crowdedness, dust amount, CO₂, temperature and air humidity were negatively associated with respiratory symptoms. Microbial chemical components like muramic acid and ergosterol, markers for bacteria and fungi, were negatively associated with wheeze or daytime attacks of breathlessness. The associations with endotoxin varied depending on the length of 3-hydroxy fatty acids of the lippopolysaccharides (LPS). Among outdoor air pollutants, SO₂ and formaldehyde were positively associated with asthma symptoms or respiratory infections. In addition, indoor SO₂, NO₂ and formaldehyde were positively associated with asthma symptoms and respiratory infections. In conclusion, rural childhood and dietary factors can be protective for asthma and allergy. ETS and chemical emissions from new material at home can be risk factors for asthmatic symptoms. In the school environment, factors of indoor origin seemed to be generally protective for respirator symptoms while factors of outdoor origin seemed to be risk factors.</p>

The aim was to study the prevalence of asthma, eczema, allergy and respiratory symptoms among pupils in Shanxi province, China, in relation to home and school environment and outdoor air pollution. In one study there was a low prevalence of self-reported asthma, eczema and pollen or pet allergy among pupils (9-20y). Rural childhood and consumption of fruit and fish were negatively associated with asthma or allergy, while current urban residency and consumption of hamburgers tended to be risk factors. In another study in junior high school pupils, similar low prevalence of asthma and allergy was found. Compared with pupils at the same age in Uppsala, Sweden, asthma and allergy were less common while daytime attacks of breathlessness were more common in Chinese pupils. Parental asthma or allergy was a predictor of asthma symptoms. Factors in the home environment such as new floor, new furniture and ETS exposure were risk factors for asthma symptoms. Crowdedness, dust amount, CO2, temperature and air humidity were negatively associated with respiratory symptoms. Microbial chemical components like muramic acid and ergosterol, markers for bacteria and fungi, were negatively associated with wheeze or daytime attacks of breathlessness. The associations with endotoxin varied depending on the length of 3-hydroxy fatty acids of the lippopolysaccharides (LPS). Among outdoor air pollutants, SO2 and formaldehyde were positively associated with asthma symptoms or respiratory infections. In addition, indoor SO2, NO2 and formaldehyde were positively associated with asthma symptoms and respiratory infections. In conclusion, rural childhood and dietary factors can be protective for asthma and allergy. ETS and chemical emissions from new material at home can be risk factors for asthmatic symptoms. In the school environment, factors of indoor origin seemed to be generally protective for respirator symptoms while factors of outdoor origin seemed to be risk factors.

Reliable methods to analyze food for the presence of almond are important – not only for those allergic to almond, but also for monitoring the compliance with labelling regulations (EG directive 2003/89). Until now the Swedish National Food Administration has used methods like rocket immunoelectrophoresis and real-time PCR to detect almond in food. These methods are, however, not sensitive enough for protecting the most sensitive individuals. Therefore, the performance of a commercial ELISA kit was tested with regard to specificity/cross reactivity and limit of detection for almond both in solution and in different matrixes. The limit of quantitation was at least 3,1 ppm (mg/kg) in solution and similar concentrations were measured in bisquits and chocolate. The ELISA method was about 100-fold more sensitive than rocket immunoelectrophoresis and PCR. The specificity of the test kit was evaluated against a number of different nuts and seeds. No important cross reactivity was found. The antibodies against almond used in the kit can not differentiate between almond and apricot kernel. For such purposes the PCR method could be used.