<p>Among food allergies, peanut has attracted the most research attention because the allergy is typically lifelong, often severe and potentially fatal. Furthermore, other than epinephrine, there are no treatments available to date. A decade of research has provided a great deal of insight into the factors that promote and regulate the <em>development </em>of allergic responses. However, less in known about the factors involved in the <em>elicitation</em> of the most common and severe manifestation of peanut allergy, namely anaphylaxis. The research in this thesis centers on the investigation of cellular and molecular pathways leading to peanut-induced anaphylaxis (PIA) as well as potential therapeutic targets. Specifically presented are: i) the development and characterization of a mouse model of PIA (Chapter 2), ii) the role of molecules including histamine, leukotrienes (LT) and platelet-activating factor (PAF) (Chapter 3) and, iii) the relative contribution of mast cells, basophils and macrophages as well as IgE and IgG₁(Chapter 4). Our data show that oral sensitization to peanut in C57BL/6 mice generated local and systemic markers of type-2 immunity that was associated with robust and consistent clinical anaphylaxis following antigen challenge. In this context, concurrent blockade of PAF and histamine receptors markedly decreases the severity of these reactions. Moreover, they demonstrate that distinctive immune effector pathways involving activation of mast cells (via IgE and IgG₁) and macrophages (via IgG₁) cooperate to elicit a broad range of systemic reactions to peanut. These findings highlight that concomitant and progressive recruitment of immune-effector pathways leads to a full range of anaphylactic reactions and therefore, therapeutic strategies for PIA may need to target several pathways or, alternatively shared components within these pathways. Combination therapy blocking both PAF and histamine may represent such as a therapeutic approach.</p> / Doctor of Philosophy (Medical Science)

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)

Introdução: Dermatite atópica (DA) é uma doença cutânea crônica, predominante na infância, cujo sintoma principal é o prurido de intensidade variável, e os sinais são classicamente as lesões de padrão eczematoso. Há anormalidades na formação e função da barreira cutânea, que estão presentes não somente nas lesões cutâneas como na pele clinicamente não afetada. Objetivo: Analisar a correlação entre as medidas biofísicas da função de barreira cutânea e os critérios clínicos e intensidade da dermatite, de acordo com os critérios de Rajka e Langeland. Métodos: 231 doentes do Departamento de Dermatologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, com diagnóstico clínico de dermatite atópica segundo os critérios diagnósticos de Rajka e Langeland foram avaliados por exame físico, anamese, medidas biofísicas de grau de hidratação de camada córnea pelo método de capacitância (corneometria) e pelo método de perda de água transepidérmica (TEWL); a medida sérica de IgE também foi solicitada no ato do exame. Resultados: Houve uma relação significativa entre as medidas de corneometria, TEWL e gravidade clínica da dermatite atópica. Os dados demonstraram uma correlação inversamente proporcional entre a corneometria e o TEWL, e houve uma diferença estatisticamente significativa (p<0,001) entre as médias de corneometria e TEWL e grau de DA (leve, moderada ou intensa). Com relação aos níveis séricos de IgE, as medidas de corneometria apresentaram uma correlação negativa significativa; para TEWL, a correlação positiva foi estatisticamente significativa (p<0,001). Conclusão: As medidas biofísicas de barreira cutânea na DA, mesmo em pele aparentemente não lesada, podem funcionar como fator de avaliação do grau clínico da DA e da intensidade do prurido. / Background: Atopic dermatitis (AD) is a chronic dermatosis, predominant in childhood, characterized by pruritus and eczematous type lesions with xerosis as the proeminent clinical sign. Objectives: To analyze the correlation between biophysical measurements of skin barrier function and other assessment criteria of clinical severity according to Rajka and Langelands criteria. Methods: Biophysical measurements (Transepidermal water loss and corneometry) were obtained from 231 patients from the department of dermatology, Hospital das Clinicas FMUSP with the diagnsosis of atopical dermatitis. Serum levels of IgE were also evaluated. Results: A significant correlation between corneometry, TEWL and clinical severity of atopic dermatitis were found. Data showed an inverse correlation between corneometry, TEWL, and AD severity, and a significant difference (p<0,001) between means of corneometry and TEWL and AD severity (mild, moderate and severe). As for IgE levels, corneometry had significant negative correlation, in contrast with TEWL, wich showed a significant positive correlation (p<0,001). Conclusion: Biophysical measurements of skin barrier in non lesional skin of atopic dermatitis may work as an evaluation factor for AD severity and pruritus.

INTRODUÇÃO: A Urticária Crônica é caracterizada pelo aparecimento de pápulas eritematosas, pruriginosas recorrentes e transitórias que duram por mais de seis semanas. Na maioria dos pacientes a causa é indeterminada, definida como idiopática (UCI), entretanto, um sub-grupo apresentam autoanticorpos contra a cadeia alfa do receptor de alta afinidade para IgE (FceRI), que são expressos na superfície de mastócitos e basófilos, tornando-os células alvo nesta doença. OBJETIVOS: Avaliar em pacientes com UCI, submetidos ao teste intradérmico de soro autólogo (ASST), o perfil de ativação dos basófilos, pela intensidade de expressão de marcadores de ativação/desgranulação e pela capacidade dos basófilos em responder aos estímulo com a IL-3 e anticorpo anti-IgE. Além disto, a presença de fator liberador de histamina foi avaliado nos soros dos pacientes. METODOLOGIA: Pacientes com UCI (n= 37) foram selecionados no Ambulatório de Urticária do Departamento de Dermatologia do Hospital das Clínicas da Faculdade de Medicina da USP e submetidos ao ASST. O grupo controle foi constituído por indivíduos saudáveis (n=38). A análise da expressão de FceRI, CD63, CD123 e CD203c em basófilos de sangue periférico foi realizada por citometria de fluxo. No ensaio in vitro de estimulação dos basófilos com anti-IgE, as células foram previamente incubadas com IL-3. O ensaio de liberação de histamina mediada por soros de pacientes com UCI foi realizado com três diferentes doadores de leucócitos e a histamina liberada dosada por ELISA de competição. RESULTADOS: Há um baixo número de basófilos no sangue periférico nos pacientes com UCI, coincidente com o baixo nível sérico de histamina. Os escassos basófilos no sangue periférico mostram elevada expressão de FceRI e uma regulação positiva da expressão de CD203c e CD63, independentemente do ASST. A análise funcional dos basófilos, mostra que somente a incubação com IL-3 recombinante já induz aumento significante da expressão de CD203c e da liberação de histamina dos basófilos de pacientes com UCI, que são intensificados com o estímulo por anticorpos anti-IgE após 15 e 40 minutos em relação ao grupo controle. Já a expressão de CD63 em basófilos após estímulo com anti-IgE, aumentou somente nos basófilos de indivíduos sadios, uma vez que os níveis de expressão basal estavam previamente aumentados. A presença de autoanticorpos liberadores de histamina foi analisada nos soros de pacientes com UCI. Três experimentos independentes foram realizados, evidenciando a ocorrência de 17% de positividade. CONCLUSÕES: Os resultados mostram que na UCI, os basófilos são sensibilizados in vivo, por fatores além dos autoanticorpos anti-FceRI e que funcionalmente, estão hiper-reativos a estímulos imunológicos. A presença de fatores séricos liberadores de histamina, independente do ASST, enfatiza que o teste intradérmico é sugestivo de autorreatividade e não de autoimunidade na UC / INTRODUCTION: Chronic Urticaria is characterized by recurrent, transitory, pruritic and erythematous wheals present for at least six weeks. In most patients the cause is unknown, defined as idiopathic (CIU), however, a sub-group has autoantibodies against the alfa chain of the high affinity IgE receptor (FceRIa) expressed on mast cells and basophils surface making it the target cells in this disease. OBJECTIVES: To evaluate in CIU patients, undergone autologous serum skin test (ASST), the activation profile of the basophils assessed by the expression of activation/degranulation markers and by the ability to release histamine in response to IL-3 priming and cross-linking with anti-IgE antibodies. Furthermore, the presence of histamine releasing factor in sera of patients was evaluated. METHODS: CIU patients (n = 37) were selected from the Dermatological Outpatient Clinic of the Hospital das Clínicas de São Paulo (HC-FMUSP) and submitted to the ASST. The control group consisted of healthy subjects (n=38). The analysis of the expression of FceRI, CD63, CD123 and CD203c on basophils from peripheral blood was assessed by flow cytometry. For the in vitro stimulation with anti-IgE antibodies, the cells were previously primed with human recombinant IL-3. The histamine release assay mediated by sera from patients with CIU was performed with three different donors of leukocytes and released histamine measured by competition ELISA. RESULTS: There is a low number of basophils in peripheral blood of patients with CIU, reflecting a low serum levels of histamine. The scarce basophils in peripheral blood show high expression of FceRI and an up-regulation of CD203c and CD63 marker expression, independently of the ASST. The functional analysis of basophils, revealed that recombinant IL-3 per se induces a significant increase in CD203c expression and the histamine release from basophils of patients with CIU, which are enhanced followed for 15 and 40 minutes of anti-IgE estimulation. The expression of CD63 in basophils upon anti-IgE stimulation, increased only in basophils of healthy individuals, since the baseline levels of CD63 expression on basophils of patients was already up-regulated. The presence of histamine release factors was examined in sera from CIU patients. Three independent experiments were performed, showing the occurrence of 17% of positivity. CONCLUSION: The results show that in CIU, the basophils are primed in vivo due to factor other than autoantibodies anti-FceRI and they have an hyperactive status to the immunological stimuli. The presence of serum histamine releasing factors, independent of the ASST, emphasizes that the intradermal test is suggestive of autorreactivity and not for autoimmunity in UC

INTRODUÇÃO: A Urticária Crônica é caracterizada pelo aparecimento de pápulas eritematosas, pruriginosas recorrentes e transitórias que duram por mais de seis semanas. Na maioria dos pacientes a causa é indeterminada, definida como idiopática (UCI), entretanto, um sub-grupo apresentam autoanticorpos contra a cadeia alfa do receptor de alta afinidade para IgE (FceRI), que são expressos na superfície de mastócitos e basófilos, tornando-os células alvo nesta doença. OBJETIVOS: Avaliar em pacientes com UCI, submetidos ao teste intradérmico de soro autólogo (ASST), o perfil de ativação dos basófilos, pela intensidade de expressão de marcadores de ativação/desgranulação e pela capacidade dos basófilos em responder aos estímulo com a IL-3 e anticorpo anti-IgE. Além disto, a presença de fator liberador de histamina foi avaliado nos soros dos pacientes. METODOLOGIA: Pacientes com UCI (n= 37) foram selecionados no Ambulatório de Urticária do Departamento de Dermatologia do Hospital das Clínicas da Faculdade de Medicina da USP e submetidos ao ASST. O grupo controle foi constituído por indivíduos saudáveis (n=38). A análise da expressão de FceRI, CD63, CD123 e CD203c em basófilos de sangue periférico foi realizada por citometria de fluxo. No ensaio in vitro de estimulação dos basófilos com anti-IgE, as células foram previamente incubadas com IL-3. O ensaio de liberação de histamina mediada por soros de pacientes com UCI foi realizado com três diferentes doadores de leucócitos e a histamina liberada dosada por ELISA de competição. RESULTADOS: Há um baixo número de basófilos no sangue periférico nos pacientes com UCI, coincidente com o baixo nível sérico de histamina. Os escassos basófilos no sangue periférico mostram elevada expressão de FceRI e uma regulação positiva da expressão de CD203c e CD63, independentemente do ASST. A análise funcional dos basófilos, mostra que somente a incubação com IL-3 recombinante já induz aumento significante da expressão de CD203c e da liberação de histamina dos basófilos de pacientes com UCI, que são intensificados com o estímulo por anticorpos anti-IgE após 15 e 40 minutos em relação ao grupo controle. Já a expressão de CD63 em basófilos após estímulo com anti-IgE, aumentou somente nos basófilos de indivíduos sadios, uma vez que os níveis de expressão basal estavam previamente aumentados. A presença de autoanticorpos liberadores de histamina foi analisada nos soros de pacientes com UCI. Três experimentos independentes foram realizados, evidenciando a ocorrência de 17% de positividade. CONCLUSÕES: Os resultados mostram que na UCI, os basófilos são sensibilizados in vivo, por fatores além dos autoanticorpos anti-FceRI e que funcionalmente, estão hiper-reativos a estímulos imunológicos. A presença de fatores séricos liberadores de histamina, independente do ASST, enfatiza que o teste intradérmico é sugestivo de autorreatividade e não de autoimunidade na UC / INTRODUCTION: Chronic Urticaria is characterized by recurrent, transitory, pruritic and erythematous wheals present for at least six weeks. In most patients the cause is unknown, defined as idiopathic (CIU), however, a sub-group has autoantibodies against the alfa chain of the high affinity IgE receptor (FceRIa) expressed on mast cells and basophils surface making it the target cells in this disease. OBJECTIVES: To evaluate in CIU patients, undergone autologous serum skin test (ASST), the activation profile of the basophils assessed by the expression of activation/degranulation markers and by the ability to release histamine in response to IL-3 priming and cross-linking with anti-IgE antibodies. Furthermore, the presence of histamine releasing factor in sera of patients was evaluated. METHODS: CIU patients (n = 37) were selected from the Dermatological Outpatient Clinic of the Hospital das Clínicas de São Paulo (HC-FMUSP) and submitted to the ASST. The control group consisted of healthy subjects (n=38). The analysis of the expression of FceRI, CD63, CD123 and CD203c on basophils from peripheral blood was assessed by flow cytometry. For the in vitro stimulation with anti-IgE antibodies, the cells were previously primed with human recombinant IL-3. The histamine release assay mediated by sera from patients with CIU was performed with three different donors of leukocytes and released histamine measured by competition ELISA. RESULTS: There is a low number of basophils in peripheral blood of patients with CIU, reflecting a low serum levels of histamine. The scarce basophils in peripheral blood show high expression of FceRI and an up-regulation of CD203c and CD63 marker expression, independently of the ASST. The functional analysis of basophils, revealed that recombinant IL-3 per se induces a significant increase in CD203c expression and the histamine release from basophils of patients with CIU, which are enhanced followed for 15 and 40 minutes of anti-IgE estimulation. The expression of CD63 in basophils upon anti-IgE stimulation, increased only in basophils of healthy individuals, since the baseline levels of CD63 expression on basophils of patients was already up-regulated. The presence of histamine release factors was examined in sera from CIU patients. Three independent experiments were performed, showing the occurrence of 17% of positivity. CONCLUSION: The results show that in CIU, the basophils are primed in vivo due to factor other than autoantibodies anti-FceRI and they have an hyperactive status to the immunological stimuli. The presence of serum histamine releasing factors, independent of the ASST, emphasizes that the intradermal test is suggestive of autorreactivity and not for autoimmunity in UC

No description available.

Introdução: Dermatite atópica (DA) é uma doença cutânea crônica, predominante na infância, cujo sintoma principal é o prurido de intensidade variável, e os sinais são classicamente as lesões de padrão eczematoso. Há anormalidades na formação e função da barreira cutânea, que estão presentes não somente nas lesões cutâneas como na pele clinicamente não afetada. Objetivo: Analisar a correlação entre as medidas biofísicas da função de barreira cutânea e os critérios clínicos e intensidade da dermatite, de acordo com os critérios de Rajka e Langeland. Métodos: 231 doentes do Departamento de Dermatologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, com diagnóstico clínico de dermatite atópica segundo os critérios diagnósticos de Rajka e Langeland foram avaliados por exame físico, anamese, medidas biofísicas de grau de hidratação de camada córnea pelo método de capacitância (corneometria) e pelo método de perda de água transepidérmica (TEWL); a medida sérica de IgE também foi solicitada no ato do exame. Resultados: Houve uma relação significativa entre as medidas de corneometria, TEWL e gravidade clínica da dermatite atópica. Os dados demonstraram uma correlação inversamente proporcional entre a corneometria e o TEWL, e houve uma diferença estatisticamente significativa (p<0,001) entre as médias de corneometria e TEWL e grau de DA (leve, moderada ou intensa). Com relação aos níveis séricos de IgE, as medidas de corneometria apresentaram uma correlação negativa significativa; para TEWL, a correlação positiva foi estatisticamente significativa (p<0,001). Conclusão: As medidas biofísicas de barreira cutânea na DA, mesmo em pele aparentemente não lesada, podem funcionar como fator de avaliação do grau clínico da DA e da intensidade do prurido. / Background: Atopic dermatitis (AD) is a chronic dermatosis, predominant in childhood, characterized by pruritus and eczematous type lesions with xerosis as the proeminent clinical sign. Objectives: To analyze the correlation between biophysical measurements of skin barrier function and other assessment criteria of clinical severity according to Rajka and Langelands criteria. Methods: Biophysical measurements (Transepidermal water loss and corneometry) were obtained from 231 patients from the department of dermatology, Hospital das Clinicas FMUSP with the diagnsosis of atopical dermatitis. Serum levels of IgE were also evaluated. Results: A significant correlation between corneometry, TEWL and clinical severity of atopic dermatitis were found. Data showed an inverse correlation between corneometry, TEWL, and AD severity, and a significant difference (p<0,001) between means of corneometry and TEWL and AD severity (mild, moderate and severe). As for IgE levels, corneometry had significant negative correlation, in contrast with TEWL, wich showed a significant positive correlation (p<0,001). Conclusion: Biophysical measurements of skin barrier in non lesional skin of atopic dermatitis may work as an evaluation factor for AD severity and pruritus.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-05-17T14:40:56Z No. of bitstreams: 1 anaclaudiacarvalhogouveia.pdf: 2060044 bytes, checksum: a2757483182ff953fdffbf86f346380b (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-06-28T14:35:33Z (GMT) No. of bitstreams: 1 anaclaudiacarvalhogouveia.pdf: 2060044 bytes, checksum: a2757483182ff953fdffbf86f346380b (MD5) / Made available in DSpace on 2016-06-28T14:35:33Z (GMT). No. of bitstreams: 1 anaclaudiacarvalhogouveia.pdf: 2060044 bytes, checksum: a2757483182ff953fdffbf86f346380b (MD5) Previous issue date: 2012-08-30 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A asma alérgica é uma doença inflamatória crônica das vias aéreas, caracterizada por uma resposta de hipersensibilidade imediata, obstrução brônquica, inflamação pulmonar e níveis elevados de IgE. A doença é mediada principalmente por uma resposta imunológica alérgeno-específica tipo Th2. Nas últimas décadas, a prevalência da asma alérgica tem aumentado significativamente, sobretudo nos países desenvolvidos. A Hipótese da Higiene atribui este aumento a uma menor exposição a determinados microrganismos durante a infância, quando o amadurecimento adequado do sistema imunológico requer estímulos que induzam respostas imunológicas de perfil Th1, fundamentais para o equilíbrio de respostas Th2 exacerbadas. Diversos trabalhos epidemiológicos parecem comprovar esta hipótese, evidenciando a existência de uma relação inversa entre o contato com microrganismos indutores de uma resposta Th1 e o desenvolvimento de asma alérgica. Paralelamente, estudos em modelos murinos constataram que o tratamento com Mycobacterium bovis BCG (BCG) reduz respostas Th2 alérgenoespecíficas. No entanto, os mecanismos pelos quais a micobactéria inibe o desenvolvimento da resposta alérgica são ainda pouco conhecidos. Este estudo avaliou o efeito da administração do BCG sobre a resposta imunológica ocorrida na alergia pulmonar em camundongos BALB/c previamente sensibilizados e desafiados com OVA. Vinte e quatro horas após o último desafio, o sangue e o lavado broncoalveolar foram coletados para análises de imunoglobulinas e contagem de células, respectivamente. Adicionalmente, os pulmões foram submetidos à análise histológica, avaliação da atividade de EPO e dosagens de citocinas e quimiocinas, assim como avaliação da expressão de CTLA-4, Foxp3 e IL-10 por citometria de fluxo. Os resultados obtidos indicam que o tratamento com BCG melhorou o processo alérgico através da redução dos principais parâmetros relacionados à resposta Th2, como o infiltrado eosinofílico pulmonar, a atividade de EPO, IL-4, IL-13, CCL11, além de IgE e IgG1 específicas anti-OVA. Por outro lado, a administração da micobactéria aumentou os níveis de IFN-γ, IL-10 e TGF-β, além das expressões de Foxp3 e CTLA-4 pelos linfócitos T CD4+. Paralelamente, houve um aumento na produção de IL-10 pelos linfócitos T CD8+. Esses dados sugerem que, além da indução de uma resposta imune Th1, a ação imunomoduladora do BCG está relacionada também à indução de mecanismos reguladores. / Atopic asthma is a chronic respiratory disease characterized by airway hyperresponsiveness, reversible airway obstruction, lung inflammation, and high levels of allergen-specific IgE, driven by allergen-specific Th2 cells. The increasing prevalence of allergic diseases, particularly in industrialized countries, has led to the hygiene hypothesis, which states that the newborn infant’s immune system is skewed toward Th2 responses and needs timely and appropriate environmental stimulus to create a balanced immune response. Supporting this hypothesis, epidemiological and experimental evidence has shown an inverse correlation between Th1-induced microbial infections and atopic asthma. Similarly, some animal studies have demonstrated that exposure to Mycobacterium tuberculosis or to environmental mycobacteria is able to protect against the development of allergic responses. However the exact mechanism underlying this inhibition still remains poorly understood. This study aimed to evaluate the ability of BCG to suppress an established allergic response in a mouse model of OVA-induced airway inflammation. To achieve this, OVA sensitized and challenged BALB/c mice were twice treated with BCG via nasal and 21 days after the first treatment, mice were rechallenged with OVA. Twenty-four hours after the last challenge, blood samples were collected to detect anti-OVA immunoglobulin isotypes, and bronchoalveolar lavage (BAL) was harvested for cell count. Additionally, lungs were collected for histological analysis, detection of EPO activity and measurement of cytokines and chemokines. The expression of CTLA-4, Foxp3 and IL-10 was also determined in lung tissue by flow cytometry. The data indicated that BCG treatment was able to inhibit an established allergic Th2-response by decreasing the allergen-induced eosinophilic inflammation, EPO activity, levels of IL-4, IL-13, CCL11 and serum levels of IgE and IgG1. Mycobacteria treatment increased lung levels of IFN-γ, IL-10 and TGF-β, and expressions of Foxp3 and CTLA-4 in CD4+T cells. Additionally, an increased production of IL-10 by CD8+ T cells was observed, even though no detectable changes in CD4+IL-10+ was noticed. Altogether, these results suggest that the mechanism underlying the down-regulatory effects of BCG on OVA-induced airway inflammation appear to be associated with the induction of both Th1 and T regulatory immune responses.

Les hypersensibilités allergiques médicamenteuses sont des pathologies mettant en jeu le système immunitaire et induites par la prise de médicaments. Notre travail s’est décomposé en quatre étapes successives : 1- Nous avons classé les β-lactamines en fonction de leurs champs moléculaires, et obtenons un dendrogramme de 4 familles, validé par les données cliniques. Nous avons également réalisé une étude de 3D-QSAR visant à connaître les parties du médicament impliquées dans la pathologie, et à prédire l’allergénicité des β-lactamines. 2- Partant de l’hypothèse que les β-lactamines sont des haptènes, nous avons étudié leur réactivité vis-à-vis d’acides aminés de type lysine et sérine. Nous avons ensuite réalisé des expériences de « docking » afin de définir les interactions entre le médicament et l’albumine sérique humaine. Nous concluons que les sites des lysines 190 et 212 sont les plus adaptés pour la fixation covalente de la drogue et avons validé cette analyse par des méthodes mixtes QM/MM. Enfin, grâce à notre logiciel SuMo, nous avons déterminé d’autres protéines candidates pour l’hapténisation. 3- S’agissant des HyperSensibilités Allergiques Immédiates, nous avons modélisé les différents partenaires que sont les IgE, la β-lactamine portée ou non par une protéine. Nous avons envisagé plusieurs modes de reconnaissance. D’autre part, nous avons analysé les modifications de la protéine, induites par la fixation de la drogue. 4- Concernant les HSA retardées, nous avons émis plusieurs scénarios de reconnaissance de la β-lactamine par le TCR. Nous avons modélisé différents complexes impliquant le TCR, le peptide hapténisé par le médicament, un ion éventuel, ainsi que le CMH, et les soumettons à des dynamiques moléculaires afin d’en étudier la pertinence. D’autre part, nous avons déterminé plusieurs peptides, issus des protéines d’hapténisation et susceptibles de présenter le médicament au TCR, via le CMH. L’ensemble des résultats obtenus est ou sera validé par des expériences in vitro et in vivo. / Drug hypersensitivity is an immune-mediated reaction to a drug. Our work was divided into four stages: 1 - We have classified β-lactam antibiotics based on their molecular fields, and obtained a dendrogram of 4 families, validated by clinical data. We also conducted a 3D-QSAR study to determine what parts of the drug are involved in the pathology and to predict the allergenicity of β- lactams. 2 - Under the assumption that β-lactam antibiotics are haptens, we studied their reactivity in comparison with lysine and serine. We then conducted "docking" experiments to define the interactions between the drug and human serum albumin. We conclude that lysine 190 and 212 are the most suitable sites for the covalent binding of the drugs. We validate this analysis by mixed QM / MM methods. Finally, thanks to our software SuMo, we have found other candidate proteins for haptenization. 3 - Regarding immediate hypersensitivity reactions, we modeled the IgE, the β-lactam and the haptenized protein. We considered several modes of recognition. Secondly, we analyzed the structural changes of the protein induced by the binding of the drug. 4 - Concerning delayed reactions, we considered different scenarios for the recognition of β-lactam by the TCR. We modeled complexes involving the TCR, the peptide haptenized by the β-lactam, a possible ion, and the MHC. We investigated them with molecular dynamics to study their relevance. On the other hand, we have identified many peptides derived from haptenization proteins and able to present the drug to the TCR through the MHC. The validity of the obtained results is or will be confirmed using experiments in vitro and in vivo.

Contexte. Les premières années de vie apparaissent particulièrement propices au développement de la sensibilisation allergénique. Objectifs. Cette thèse vise à : i) décrire les profils de sensibilisation allergénique chez le nourrisson et l’enfant, ii) étudier l’association entre ces profils et la morbidité allergique et iii) identifier les facteurs de risque de cette sensibilisation. Méthodes. Dans le cadre du suivi de la cohorte prospective de naissances en population générale Pollution and Asthma Risk: an Infant Study (PARIS), la sensibilisation allergénique a été évaluée chez 1 860 nourrissons à 18 mois et 1 007 enfants à 8/9 ans par dosage des IgE spécifiques dirigées contre 16 et 19 allergènes, respectivement. Les informations concernant la santé et le cadre de vie des enfants ont été recueillies par questionnaires standardisés répétés. Des profils de sensibilisation et des profils de morbidité ont été identifiés par classification non supervisée et mis en relation par régression logistique multinomiale. Enfin, les facteurs associés à la sensibilisation allergénique chez le nourrisson ont été étudiés par régression logistique multivariée. Résultats. Dès 18 mois, 13,8% des enfants étaient sensibilisés et 6,2%, multi-sensibilisés. À 8/9 ans, ces prévalences étaient de 34,5% et 19,8%, respectivement. Les profils de sensibilisation identifiés chez le nourrisson (3) et dans l’enfance (5) différaient au regard de la morbidité allergique. L’analyse étiologique a permis de préciser le rôle des expositions précoces aux allergènes et aux microorganismes sur la sensibilisation allergénique. Conclusion. Cette thèse contribue à une meilleure compréhension de l’histoire naturelle de la sensibilisation allergénique, et ce, dès les premières années de vie. Cette connaissance est essentielle à la prévention des maladies allergiques qui en découlent. / Background. The first years of life appear to be critical for the development of allergic sensitization. Objectives. This thesis aims: i) to describe allergic sensitization profiles in infants and children, ii) to assess the link between these sensitization profiles and allergic morbidity, and iii) to identify risk factors for allergic sensitization. Methods. This work concerns children involved in the Pollution and Asthma Risk: an Infant Study (PARIS) population-based prospective birth cohort. Allergic sensitization was assessed in 1,860 18-month-old infants and 1,007 8/9-year-old children by specific IgE measurements towards 16 and 19 allergens, respectively. Lifelong health and living condition data were collected by repeated standardized questionnaires. Sensitization profiles and morbidity profiles were identified using unsupervised classification, and related to each other by multinomial logistic regression. Finally, risk factors for early allergic sensitization were assessed by multivariate logistic regression. Results. As soon as 18 months of age, 13.8% of children were sensitized and 6.2%, multi-sensitized. When 8/9 years old, corresponding prevalence was 34.5% and 19.8%, respectively. Sensitization profiles identified in infancy (3) and in childhood (5) differed in terms of allergic morbidity. Risk factor analysis allowed to clarify the role of early exposure to allergens and microorganisms on allergic sensitization. Conclusion. This thesis improves the natural history of allergic sensitization understanding, as soon as the first years of life. This knowledge is essential for subsequent disease preventing.