Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 5 uppsatser.

Made available in DSpace on 2015-07-22T20:49:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-27 / Uma cisteína proteinase recombinante, rLdccys1, obtida pela expressão do gene Ldccys1 de Leishmania (Leishmania) chagasi em sistema bacteriano, foi utilizada em ensaios de imunodiagnóstico por ELISA e DTH em cães de uma região endêmica de leishmaniose visceral (LV), Teresina, Piauí. A rLdccys1 também foi utilizada para a imunização de cães e a avaliação da possível proteção dos animais imunizados contra o desafio com a L. (L.) chagasi. Os ensaios por ELISA dos soros de cães com LV mostraram que a sensibilidade para a detecção de anticorpos anti-L. (L.) chagasi utilizando a rLdccys1 e os extratos de promastigotas e amastigotas de L. (L.) chagasi foi de 98%, 86% e 89%, respectivamente. A rLdccys1 não apresentou reatividade cruzada com os soros de cães com doenças comuns nas regiões endêmicas de LV, tais como erliquiose, babesiose e doença de Chagas e a especificidade dos ensaios de ELISA utilizando-se a rLdccys1 e os extratos de amastigotas e promastigotas de L. (L.) chagasi foi de 96%, 69% e 68%, respectivamente. As respostas de DTH foram avaliadas nos cães após a injeção subcutânea da rLdccys1 ou do extrato dos amastigotas de L. (L.) chagasi. Todos os cães assintomáticos apresentaram resposta de DTH positiva à rLdccys1 com a formação de nódulos em torno de 10 mm 48 horas após a injeção do antígeno, enquanto que os cães sintomáticos não apresentaram reatividade significante à rLdccys1 nos ensaios de DTH. As respostas de DTH foram mais intensas quando se utilizou a rLdccys1 comparadas às observadas com o extrato dos parasitas. A análise histológica mostrou áreas de necrose e hemorragia nos nódulos induzidos pelo extrato dos parasitas e reação granulomatosa típica, com predomínio de células mononucleares, nos cortes dos nódulos induzidos pela rLdccys1. A análise dos dados obtidos nos ensaios de ELISA e DTH realizados com a rLdccys1 e os extratos de amastigotas de L. (L.) chagasi mostrou a correlação inversa entre as respostas humoral e celular no desencadeamento da LV canina. As respostas celulares induzidas nos cães pela rLdccys1 foram avaliadas após a imunização dos animais com o antígeno recombinante juntamente com a Propionibacterium acnes. Os linfócitos de sangue periférico dos cães imunizados com a rLdccys1 + P. acnes apresentaram significantes índices de estimulação quando reestimulados in vitro com a rLdccys1 ou o extrato dos amastigotas de L. (L.) chagasi. A dosagem de citocinas nos sobrenadantes dessas culturas mostrou a secreção de níveis significantes de IFN-γ, enquanto IL-10 não foi detectada. Nos experimentos em que cães foram imunizados com a rLdccys1 + P. acnes e desafiados com a injeção intraperitoneal de 1x104 amastigotas isolados de cão infectado com a L. (L.) chagasi 3 de 4 animais sobrevieram dez semanas após o desafio, enquanto que os animais controles que receberam PBS e a P. acnes morreram em no máximo 4 e 6 semanas, respectivamente, após o desafio. Nos cães imunizados com a rLdccys1 o número de amastigotas de L. (L.) chagasi foi significantemente reduzido no baço, fígado e medula óssea comparado ao observado nos controles. A dosagem sérica de IFN-γ nos animais imunizados com a rLdccys1 mostrou a secreção significante dessa linfocina, enquanto que níveis basais de IL-10 foram detectados. Todos os cães imunizados com a rLdccys1 e desafiados pela picada das fêmeas de Lutzomyia longipalpis sobreviveram até dezesseis semanas após o desafio e os animais controles injetados com PBS e P. acnes morreram sete e nove semanas, respectivamente, após o desafio. Os cães controles apresentaram número significante de amastigotas de L. (L.) chagasi no fígado e baço, porém nos animais imunizados com a rLdccys1 nenhum parasita foi observado. No grupo imunizado com a rLdccys1 houve aumento crescente dos níveis séricos de IFN-γ durante a imunização que atingiu um pico uma semana após o desafio, enquanto que e a concentração de IL-10 foi mantida em níveis basais. Os dados do presente trabalho mostraram o potencial da rLdccys1 de L. (L.) chagasi para o diagnóstico e a imunoprofilaxia da LV canina, abrindo perspectivas para a imunização dos cães em larga escala nas regiões endêmicas de LV e a avaliação do impacto da proteção conferida nos animais imunizados na incidência da doença. / A recombinant protein, rLdccys1, produced by expression of the gene encoding a 30 kDa cysteine proteinase from Leishmania (Leishmania) chagasi, was used to detect specific antibodies in serum by enzyme-linked immunosorbent assays and to test for reactivity in delayed-type hipersensitivity (DTH) responses of dogs from an endemic region of visceral leishmaniasis (VL), Teresina, Piauí State, Brazil. The recombinant protein was also used for immunization of dogs and evaluation of its possible protective role against L. (L.) chagasi infection. The sensitivity for detection of specific antibodies to L. (L.) chagasi using rLdccys1 and lysates from L. (L.) chagasi promastigotes and amastigotes was 96%, 68%, and 69%, respectively. No cross reactivity between rLdccys1 and Chagas disease was observed, and little reactivity was found with sera from dogs with babesiosis and ehrlichiosis. The specificity of ELISA assays using rLdccys1, lysates from L. (L.) chagasi promastigotes and amastigotes was 96%, 69%, and 68%, respectively. DTH responses were determined after subcutaneous injection of rLdccys1 or L. (L.) chagasi amastigote extract and the induration area was measured at 24, 48 and 72 h after injection. All asymptomatic dogs showed a positive intradermal response to rLdccys1 (10 mm) which peaked at 48 h, whereas no significant reactivity to the recombinant antigen was found in the symptomatic group. DTH responses to rLdccys1 were higher than those induced by amastigote extract. Histological analysis of the intradermal induration showed a predominance of necrotic and hemorrhagic areas in sections from asymptomatic dogs injected with L. (L.) chagasi amastigote extract, whereas a typical granulomatous reaction mediated by mononuclear cells was observed in sections from asymptomatic animals injected with rLdccys1. Data analysis from ELISA and DTH assays with rLdccys1 and L. (L.) chagasi amastigote extracts showed that humoral and cellular responses were inversely correlated during the development of canine VL. Cellular immune responses induced by the recombinant antigen were evaluated after immunization of dogs with rLdccys1 plus Propionibacterium acnes. Peripheral blood mononuclear cells isolated from rLdccys1-immunized dogs showed significant stimulation indexes after in vitro incubation with either rLdccy1 or L. (L.) chagasi amastigote extracts. Cytokine dosages in the supernatants from lymphocyte cultures showed significant levels of IFN-γ, whereas IL-10 was not detected. Whereas 3 from 4 dogs immunized with rLdccys1 plus P. acnes and challenged by intraperitoneal injection of 1x104 L. (L.) chagasi amastigotes survived ten weeks after challenge, control dogs which received either PBS or P. acnes died after four and six weeks, respectively. The load of L. (L.) chagasi amastigotes in spleen, liver, and bone marrow from rLdccys1-immunized dogs was significantly reduced in comparison to that of non immunized controls. A significant concentration of IFN-γ and basal levels of IL-10 were detected in sera from dogs immunized with rLdccys1. All dogs immunized with rLdccys1 plus P. acnes and challenged by the bite of L. (L.) chagas infected Lutzomyia longipalpis survived until sixteen weeks after challenge, whereas control dogs injected with PBS or P. acnes died after seven and nine weeks, respectively. Control dogs showed a significant number of L. (L.) chagasi amastigotes in liver and spleen, but no parasites were found in rLdccys1- immunized dogs. During immunization with rLdccys1 there was an increase of serum levels of IFN-γ in the immunized dogs that peaked one week after challenge. In contrast, a very low concentration of IL-10 was detected in these animals. Overall, these findings indicate that L. (L.) chagasi recombinant cysteine proteinase is potentially useful for diagnosis and immunoprophylaxis of canine VL. We believe that results obtained open perspectives for immunization of dogs in the field and evaluation of the impact on the disease incidence. / TEDE / BV UNIFESP: Teses e dissertações

Made available in DSpace on 2015-07-22T20:49:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-01-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Support Produtos Nutricionais Ltda / Objetivo: Avaliar o conhecimento e a prática de profissionais pediatras, gastroenterologistas pediátricos, alergistas e nutricionistas quanto à prevenção da alergia alimentar. Métodos: Trata-se de um estudo descritivo e transversal, do qual participaram pediatras (n=80), gastro-pediatras (n=120), alergistas (n=100) e nutricionistas (n=220), totalizando 520 profissionais. O instrumento de coleta foi um questionário auto-administrado elaborado a partir das recomendações atuais das principais sociedades de especialidades sobre a prevenção da alergia alimentar. Resultados: A média de idade dos profissionais foi 29,1±5,8 anos. A maioria dos profissionais era do sexo feminino, constituindo um total de 90,4% (n = 470) dos entrevistados. A média do tempo de graduação dos profissionais foi 7,2±5,8 anos. Quanto à recomendação de dieta de exclusão alimentar na gestação, 89 (17,1%) profissionais concordaram com tal prática, sendo maior a indicação por parte dos nutricionistas. Os gastro-pediatras diferenciaram-se estatisticamente dos demais profissionais por apresentarem a menor taxa de recomendação (p< 0,001). A recomendação de dieta de exclusão alimentar materna na lactação foi indicada por maior número de profissionais, sendo recomendada por 212 (40,8%) entrevistados, verificando-se novamente maior recomendação dos nutricionistas e também dos pediatras em comparação aos alergistas e gastroenterologistas (p< 0,001). A duração de aleitamento materno exclusivo considerada ideal foi o período de 4 a 5,9 meses indicado por 275 (52,9%) profissionais, sendo o período de escolha da maioria dos nutricionistas, pediatras e gastro-pediatras. No entanto, 65 alergistas (65,0%) indicaram a faixa de 6 a 7,9 meses. Quanto à época de introdução da alimentação complementar, 218 (41,9%) profissionais afirmaram modificar a idade de introdução com o objetivo de prevenir o desenvolvimento da alergia alimentar. O principal período indicado foi a idade entre 6 a 7,9 meses, assinalada por 118 (54,1%) entrevistados. Comparando a indicação da época de introdução da alimentação complementar entre os profissionais, 70 (70,0%) dos alergistas afirmam modificar a época de introdução da alimentação complementar (p< 0,001). As principais fórmulas infantis especiais indicadas nos casos de impossibilidade de aleitamento materno com o objetivo de prevenir o aparecimento da alergia alimentar foram: Alfaré® (70,6%), Neocate® (66,2%), Pregomin® (57,5%), e Nan HA® (42,1%). Conclusões: Esse estudo revelou que existem erros conceituais no que tange a prevenção da alergia alimentar entre todas as categorias de profissionais. Nossos resultados confirmam que a avaliação do conhecimento e da prática dos profissionais contribui na definição de conteúdos que devem fazer parte dos programas de educação continuada na área da alergia alimentar. / Objective: To evaluate the knowledge and practice of pediatricians, gastroenterologists pediatricians, allergists and nutritionists regarding the primary prevention of food allergy. Methods: A descriptive cross-sectional study that enrolled pediatricians (n = 80), gastro-pediatricians (n = 120), allergists (n = 100) and nutritionists (n = 220), an amount of 520 professionals. The instrument collects used was a self-administered questionnaire prepared by the authors based on current recommendations of the major committees and societies related to prevention of food allergy. Results: The average age of professionals was 29.1 ± 5.8 years. The vast majority of the professionals was female, constituting a total 90.4% (n = 470) of respondents. The average time of graduation of professionals was 7.2 ± 5.8 years. Regarding the recommendation of exclusion diet on pregnancy, 89 (17.1%) professionals agreed with such practice, being the most indication by nutritionists. The gastro-pediatricians statistically differ from the others professionals by submitting the lowest rate of recommendation (p < 0.001). The recommendation of maternal exclusion diet on lactation was indicated by more number of professionals, constituting 40.8% (n = 212) of the sample, with the highest recommendation by nutritionists and pediatricians compared to pediatricians and allergists (p < 0.001). The duration of exclusive breastfeeding considered ideal was the period of 4 to 5.9 months indicated by 52.9% (n = 275) of professionals, according with the choices of most nutritionists, pediatricians and pediatricians. However, most allergists (65%; n = 65) indicated the range of 6 to 7.9 months. Regarding the time of introduction of complementary feeding, 218 (41.9%) professionals recommended modify the age of introduction to prevent the development of food allergy. The main period indicated was aged 6 to 7.9 months, indicated by 118 (54.1%) of respondents. Comparing the indication of the time of introduction of complementary feeding among professionals, 70 (70%) of allergists affirmed modify the time of introduction of complementary feeding (p < 0.001). The majority of special infant formulas indicated in case of impossibility of breastfeeding to prevent the development of food allergies were: Alfare® (70.6%), Neocate® (66.2%), Pregomin® (57.5%) and Nan HA® (42.1%). Conclusions: This study revealed there are misconceptions regarding the prevention of food allergy among all the categories of professionals. Our results confirm that the evaluation of knowledge and professional practice contributes to the definition of content that should be part of continuing education programs in the area of food allergy. / TEDE / BV UNIFESP: Teses e dissertações

A identificação de reações adversas a medicamentos (RAM) nos hospitais constitui uma importante medida da morbidade associada a medicamentos e de seu ônus sobre o sistema de saúde. Este estudo observacional não intervencionista teve por objetivo avaliar a incidência de RAM em pacientes hospitalizados, as características clínicas das reações e fatores de risco associados. Foram avaliados 472 pacientes de cinco clínicas do Hospital das Clínicas da FMUSP (Clínica Médica, Cirurgia Geral, Neurologia, Geriatria, Alergia e Imunologia Clínica), com formação de coorte prospectiva, analisando as características demográficas, comorbidades, número de medicações utilizadas antes e durante a hospitalização e tempo de internação. A prevalência das RAM foi de 1,7% e a incidência geral de RAM foi 16,2%, variando conforme a clínica avaliada, sendo maior na Clínica Médica (30%). As reações mais frequentes foram as do tipo A, predominando as manifestações gastrointestinais. A maior parte das reações foi classificada de gravidade moderada. O maior número de medicações utilizadas por paciente, insuficiência renal crônica e tempo de internação foram fatores de risco para RAM, porém não houve associação das reações com idade avançada. Antecedente de RAM anterior à internação foi identificado como fator de proteção. A incidência de reações de hipersensibilidade a medicamentos (RHM) foi de 3,2%, com maior número de medicações utilizadas por paciente como único fator de risco isolado, sem associação com as clínicas avaliadas ou gênero dos pacientes. As medicações mais associadas às RAM e RHM foram os antibióticos, opióides e contrastes iodados. Os medicamentos mais prescritos foram os sintomáticos. O estudo concluiu que as RAM são frequentes e potencialmente evitáveis. O conhecimento da incidência e dos fatores associados pode estimular a prevenção. A prescrição de medicações para pacientes internados deve ser mais criteriosa, especialmente para os mais susceptíveis, evitando a polifarmácia / The detection of adverse drug reactions (ADRs) in hospitalized patients is an important measure of morbidity associated with drugs and its burden on the health system. The objective of this non-interventionist observational study was to assess the incidence of ADRs in hospitalized patients, the clinical characteristics of reactions and associated risk factors. We evaluated 472 patients from five medical specialties of the Hospital das Clínicas-FMUSP (Internal Medicine, General Surgery, Neurology, Geriatrics, Clinical Immunology and Allergy). We performed a prospective cohort, analyzing the demographics features, comorbidities, number of medications used before and during hospitalization and length of stay in the hospital. The prevalence of ADRs was 1.7% and the overall incidence of ADRs was 16.2%, varying according to the specialty assessed, higher in the Internal Medicine (30%). The most frequent reactions were type A, with gastrointestinal manifestations being the most frequent. Most of the reactions were classified as moderate in severity. The greater number of drugs used, chronic renal failure and longer hospital stays were risk factors for ADRs, but there was no association between reactions and age. History of previous ADRs to admission was identified as a protective factor. The incidence of hypersensitivity drug reactions (HDRs) was 3.2%, with the greater number of medications used per patient as the sole isolated risk factor, without association with specialty or patient\'s gender. The main medications associated with ADRs and HDRs were antibiotics, opioids and iodinated contrast media. The most commonly prescribed medications were symptomatic ones. The study concluded that the ADRs are frequent and potentially preventable. Knowledge of the incidence and associated factors can stimulate prevention. The pharmacotherapy of in-patients should be more careful, especially for the more susceptible patients, avoiding polypharmacy

INTRODUÇÃO: O câncer constitui-se na principal causa de mortalidade entre indivíduos de 45 a 84 anos, configurando-se em um dos principais problemas de saúde pública dos países em desenvolvimento. As reações de hipersensibilidade aos quimioterápicos têm aumentado, impedindo muitas vezes a utilização de terapias de primeira linha no tratamento de neoplasias primárias ou recidivantes. O procedimento de dessensibilização é uma abordagem alternativa, por meio do qual o paciente passa a tolerar a medicação que antes desencadeava reações potencialmente letais. Quimioterápicos do grupo das platinas são exemplos de drogas passíveis de readministração por meio do processo de dessensibilização, no entanto faltam biomarcadores preditivos de reações durante o procedimento. OBJETIVOS: O objetivo principal do estudo foi avaliar o papel do teste de ativação de basófilos (BAT) como biomarcador para reações de hipersensibilidade ocorridas durante a dessensibilização em pacientes alérgicas às platinas. Como objetivo secundário, avaliou-se a prevalência e o impacto da mutação dos genes BRCA 1 e 2 em pacientes com hipersensibilidade imediata à carboplatina submetidas à dessensibilização. MÉTODOS: Padronizou-se o BAT, com análise da expressão de CD63 e CD203c na superfície de basófilos de pacientes com hipersensibilidade imediata às platinas submetidas à dessensibilização. Foram realizados BATs em 15 pacientes portadoras de neoplasias malignas submetidas a 27 dessensibilizações devido à anafilaxia a quimioterápico do grupo das platinas e em 12 indivíduos de dois grupos controle (Grupo 1: seis pacientes tolerantes às platinas e Grupo 2: seis voluntários sadios que nunca foram expostos às platinas). Os resultados dos BATs foram comparados entre os três grupos. Correlacionou-se o BAT com a ocorrência ou não de reação durante a dessensibilização e com os níveis de triptase sérica. Para análise da prevalência e impacto da mutação dos genes BRCA 1 e 2 nas dessensibilizações, realizou-se análise retrospectiva de prontuários de 138 portadoras de neoplasias malignas ginecológicas submetidas à dessensibilização à carboplatina. RESULTADOS: O BAT foi positivo em 11 das 15 pacientes alérgicas (n= 11; 73,3%), com aumento de expressão de CD203c e CD63 em 11 (73,3%) e 6 (40%) pacientes, respectivamente. Todos os participantes dos grupos controles apresentaram testes negativos. Maior expressão de CD63 foi observada em pacientes com reações iniciais mais graves. O BAT foi positivo em 92,3% das reações ocorridas durante as dessensibilizações (n=12/13), sendo positivo em todas as reações que apresentaram aumento concomitante de triptase sérica (n=5). Com relação à mutação dos genes BRCA 1 e 2, sua prevalência foi de 34% nas pacientes com hipersensibilidade às platinas (n=47/138), sendo que 51% das portadoras reagiram durante a dessensibilização. CONCLUSÕES: O BAT positivo, com aumento da expressão de CD63 e/ou CD203c na superfície do basófilo, identificou pacientes alérgicos às platinas com especificidade de 100% e sensibilidade de 73,3%. O BAT e a mutação dos genes BRCA 1 e 2 identificaram pacientes mais propensos a reagir durante o procedimento de dessensibilização. A utilização de biomarcadores preditores de reações durante a dessensibilização aos quimioterápicos do grupo das platinas pode aumentar a segurança do procedimento e auxiliar na manutenção do esquema quimioterápico de primeira linha do paciente / INTRODUCTION: Cancer is the leading cause of death in the age group of 45 to 84 years, and one of the main public health issues in developing nations. Hypersensitivity reactions to chemotherapeutic agents have been increasing, sometimes hindering the use of first-line therapies in the treatment of primary or relapsed tumors. Rapid drug desensitization (RDD) is an alternative approach, through which a patient becomes tolerant to the medication that once triggered a potentially lethal hypersensitivity reaction. Platinum-based compounds are examples of drugs that can be readministered through the desensitization procedure, but currently there are no known biomarkers that could help predict reactions during RDD. OBJECTIVES: The main goal of our study was to assess the basophil activation test (BAT) as a biomarker of breakthrough reactions occurred during RDD in patients allergic to platinum-based agents. As a secondary goal, we evaluated the prevalence and impact of the BRCA 1/2 mutation in carboplatin-allergic patients undergoing RDD. METHODS: We standardized the BAT by evaluating CD63 and CD203c expressions on the basophils of patients with immediate hypersensitivity reactions to platinum-based agents undergoing RDD. We analyzed BATs of 15 patients with malignant neoplasms who had undergone 27 RDD procedures due to anaphylaxis to platinum-based agents, and of 12 control subjects (Group 1: six patients tolerant to platinum-based agents, and Group 2: six healthy volunteers who had never been exposed to platinum-based agents). BAT results were compared among the three groups. We correlated BAT results with the occurrence of breakthrough reactions during RDD and with serum tryptase levels. To conduct the analysis of the BRCA 1/2 mutation prevalence and its impact on RDD, a retrospective review of 138 medical records of patients with gynecological malignancies who underwent RDD to carboplatin was performed. RESULTS: BAT was positive in 11/15 allergic patients (73.3%), with increased expression of CD203c and CD63 in 11 (73.3%) and 6 (40%) patients, respectively. All control subjects presented negative BATs. A higher CD63 expression was observed in patients with severe initial reactions. BAT was positive in 92.3% of the breakthrough reactions occurred during RDD (n=12/13), and in all reactions with concomitant increased tryptase levels (n=5). Regarding the BRCA1/2 mutation, its prevalence was 34% in patients allergic to platinum-based agents (n=47/138), and 51% of the mutation carriers had breakthrough reactions during RDD. CONCLUSIONS: A positive BAT, with an increased expression of CD63 and/or CD203c, identified patients allergic to platinum-based agents with a specificity of 100% and a sensitivity of 73.3%. The BAT and the BRCA 1/2 mutation helped identify patients at risk of breakthrough reactions during RDD. The use of predictive biomarkers of breakthrough reactions during RDD to platinum-based agents might enhance RDD safety and help maintain a patient`s first-line treatment

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.

La voie des RLR (RIG-I like Receptors) joue un rôle essentiel dans la détection des virus à ARN, déclenchant une réponse immunitaire antivirale grâce au recrutement de la protéine adaptatrice mitochondriale MAVS (Mitochondrial AntiViral Signaling protein). Nous avons mis en évidence que les souris déficientes pour la protéine MAVS (MAVS KO) présentaient un phénotype proallergénique dans un modèle d'inflammation stérile d'hypersensibilité retardée de contact (HSRC) qui reproduit la dermatite allergique de contact (DAC) chez l'homme. Nous avons caractérisé le système immunitaire des souris MAVS KO en condition d'équilibre et durant la réponse d'HSRC. Nous avons identifié un rôle du microbiote intestinal des souris MAVS KO dans l'exacerbation de réponse d'HSRC et mis en évidence une dysbiose du microbiote bactérien. Nous avons démontré que la dysbiose était responsable du phénotype inflammatoire observé, phénotype transmissible à des souris sauvages par des expériences de cohébergement et de transplantation fécale. Cette dysbiose induit une augmentation de la perméabilité intestinale chez les souris MAVS KO lors de la réponse d'HSRC, aboutissant à une translocation bactérienne dans les organes lymphoïdes et à la modulation des réponses immunitaires à l'origine de l'exacerbation de réponse d'hypersensibilité. La 2ème partie de ma thèse vise à étudier les conséquences de la déficience en MAVS sur le métabolisme glucidique. Nos expériences ont démontré que les souris MAVS KO développaient une surcharge pondérale et une insulino-résistance sous régime riche en lipides et sucrose, dépendants de la dysbiose intestinale. Au niveau cellulaire, une altération des interactions aux points de contact entre la mitochondrie et le réticulum endoplasmique a été observée. Nos résultats permettent d'envisager le développement de nouvelles approches thérapeutiques des pathologies allergiques et métaboliques humaines par la modulation du microbiote intestinal / RIG-I like receptors (RLRs) play a major role in response to cytosolic viral RNAs by initiating an antiviral immune response through the recruitment of the mitochondrial adaptor protein MAVS (Mitochondrial AntiViral Signaling protein). We showed that MAVS-deficient mice developed an exacerbated response in a sterile inflammatory model of Contact Hypersensitivity (CHS), that reproduces the pathophysiology of allergic contact dermatitis (ACD) in human. We characterized the immune system of MAVS KO mice at steady state and during CHS response. We found that MAVS deficiency leads to changes in the gut bacterial composition suggesting an unexpected role of the RLR pathway in the regulation of intestinal homeostasis. We demonstrated that intestinal dysbiosis is responsible for the increased CHS response, and showed that the inflammatory phenotype of MAVS KO mice can be transferred to WT mice by cohousing and fecal transplantation. We demonstrated that the dysbiotic gut microbiota exerts its effect due to an increased intestinal permeability during DTH sensitization. The ensuing bacterial translocation within lymphoid organs enhances characteristic cytokines production that increases CHS response. The 2nd part of my thesis aimed to study the consequences of MAVS deficiency on glucose metabolism. Our experiments showed that MAVS KO mice exhibit disorders of glucose homeostasis during high fat diet (HFD) associated with the development of overweight and insulin resistance. We also observed alterations of MAM (Mitochondria-associated endoplasmic reticulum membranes), contact poins between mitochondria and endoplasmic reticulum. Recent preliminary data suggested that the metabolic disorders associated with MAVS deficiency are due to intestinal dysbiosis. Our results highlight a new role for the RLR pathway and allow to consider the development of new therapeutic approaches to human allergic and metabolic diseases by modulation of the intestinal microbiota

La tolérance orale permet la modulation de la réponse immunitaire à l’égard des antigènes exogènes présents dans la lumière intestinale. Essentiels à l’établissement d’une relation symbiotique entre le système immunitaire et la flore intestinale, l’induction et le maintien de la tolérance orale reposent sur différents mécanismes immunologiques. Parmi eux, l’induction de cellules T régulatrices par les cellules dendritiques et de mécanismes apoptotiques. Or, la glycoprotéine membranaire CD47 est impliquée, en périphérie, dans ces mécanismes. Cependant, le rôle de CD47 dans la tolérance orale n’est pas connu. À l’aide d’un modèle murin déficient en CD47, nous avons démontré principalement, que l’absence de CD47 est associée à une diminution de 50 % de la proportion de cellules dendrites myéloïdes CD11b+CD103- retrouvées dans les ganglions mésentériques. Suite au transfert adoptif de cellules T antigènes spécifiques dans nos différents modèles expérimentaux, on a, aussi, observé une diminution de 45 % de leur niveau d’activation dans les ganglions mésentériques. Malgré les effets observés, le CD47 n’est pas impliqué dans l’induction d’une réaction de tolérance orale secondaire à l’administration intragastrique de fortes doses d’ovalbumine. Cependant, nous avons démontré que CD47 est impliquée au niveau de la migration des cellules dendritiques de la peau et de certaines sous-populations retrouvées dans les ganglions mésentériques. / Oral tolerance allows the modulation of the immune response against exogenous antigens present in the intestinal lumen. Essential to establish a symbiotic relationship between the immune system and intestinal flora, the induction and maintenance of oral tolerance rests on different immunological mechanisms. Among them, induction of regulatory T cells by dendritic cells and apoptotic mechanisms. However, the membrane glycoprotein CD47 is involved in the periphery of these mechanisms. However, the role of CD47 in oral tolerance is unknown. With a mouse model deficient in CD47, we showed mainly that the absence of CD47 is associated with a decrease of 50% in the proportion of myeloid dendritic cells CD11b+ CD103- found in the mesenteric lymph nodes. Following the adoptive transfer of antigen specific T cells in our experimental models, we also observed a decrease of 45% of their level of activation in mesenteric lymph nodes. Despite the observed effects, CD47 is not involved in the induction of oral tolerance response secondary to intragastric administration of high doses of ovalbumin. However, we have shown that CD47 is involved in the migration of dendritic cells of the skin and some sub-populations found in mesenteric lymph nodes.

In einem komplexen tierexperimentellen Ansatz wurde das Potenzial der anti huCD4-Antikörper MAX16H5 und MAX12F6 zur Modulierung zellvermittelter Immun-reaktionen in vivo untersucht. Dafür kam ein mehrfach transgenes Mausmodell zur Anwendung, in dem das humane Zielmolekül und dessen physiologischer Ligand als Transgene exprimiert waren. Als T-Zell vermittelte Immunreaktion wurde eine Kon-taktreaktion (delayed type hypersensitivity, DTH) gegen DNFB etabliert und validiert. An der DTH wurde untersucht, ob und wie die verschiedenen Antikörper die Sen-sibilisierungs- und die Auslösungsphase beeinflussen. Die experimentellen Ergeb-nisse zeigen, dass die Antikörper epitop- und isotypabhängig die beiden Phasen der DTH unterschiedlich beeinflussen. Die Applikation der Antikörper während der Sensi-bilisierung führte zu einer unterschiedlich ausgeprägten Suppression der DTH. Dage-gen hatten sie gegensätzliche Effekte auf die Auslösung. Während nach MAX12F6-Behandlung eine stärkere und prolongierte DTH gemessen wurde, verlief die DTH-Reaktion nach MAX16H5-Applikation deutlich abgeschwächt. Mittels flowzytometri-scher Analysen konnte gezeigt werden, dass die Antikörper unterschiedliche Subpo-pulationen der T-Helferzellen depletieren. Darüber hinaus führte MAX16H5 offen-sichtlich zur Induktion regulatorischer T-Zellen. Die Daten erklären unterschiedliche Erfolge aus ersten klinischen Studien mit verschiedenen anti huCD4 Antikörpern. Auch eignet sich CD4 auch als diagnostisches Target zur in vivo Diagnostik T-Zell vermittelter Entzündungsreaktionen. Mit Antikörperfragmenten von MAX16H5 wurde ein immunszintigraphisches Verfahren entwickelt, das die spezifische Darstellung der mit der DTH einhergehenden Entzündungsreaktion ermöglicht.

Les douleurs abdominales, reflet d’une hypersensibilité viscérale (HSV), sont l’un des premiers motifs de consultation en médecine générale et en gastro-entérologie. Ces douleurs abdominales, généralement passagères, peuvent également être causées par des pathologies sous-jacentes plus graves. Parmi ces maladies sont retrouvés notamment le Syndrome de l’Intestin Irritable (SII), et les Maladies Inflammatoires Chroniques de l’Intestin (MICI) qui regroupent la maladie de Crohn (MC) et la RectoColite Hémorragique (RCH).Bien que le SII et les MICI diffèrent sur de nombreux points, des facteurs communs à ces deux pathologies peuvent contribuer au symptôme majeur de ces maladies, à savoir la douleur abdominale. En effet, les facteurs environnementaux (alimentation, stress, etc.) prennent une part importante dans l’étiologie du SII, mais il s’avère aujourd’hui qu’ils participent également à l’aggravation des symptômes associés aux MICI. De même, si de nombreux polymorphismes génétiques et l’atteinte inflammatoire sont les bases de la physiopathologie des MICI, des études récentes montrent que de telles modifications peuvent également être retrouvées au cours du développement du SII (identification de gène de susceptibilité et présence de micro-inflammation). Enfin, les récentes études menées sur le microbiote intestinal mettent en évidence des perturbations de celui-ci aussi bien chez des patients atteints du SII que de MICI.Les MICI se caractérisent par des phases d’inflammation sévère au niveau du tube digestif, pour lesquelles la pharmacopée actuelle, qui consiste à réduire cette inflammation, est plutôt satisfaisante. Ces phases sont entrecoupées par des phases dites « de rémission », durant lesquels l’HSV reste un problème chez un grand nombre de patients (environ 38%). Ces douleurs abdominales, diffuses et irradiantes, peuvent être assimilées à celles retrouvées chez les patients atteints du SII (symptômes SII-like). La prise en charge thérapeutique de ces douleurs est limitée, puisque basée sur d’anciens médicaments qui présentent un ratio bénéfice/ risque faible.Les antiépileptiques, sont des molécules qui ciblent l’excitabilité neuronale en modulant l’activité des canaux ioniques, de récepteurs ou encore de voies de signalisation intracellulaires. Parmi ces molécules, la gabapentine (GBP) et la prégabaline (PGB) (regroupées sous le terme de gabapentinoïdes) sont des ligands des sous-unités α2δ des canaux calciques voltage-dépendants (CCVD). Ces ligands α2δ sont actuellement prescrits dans le cadre de douleurs neuropathiques, au même titre que les antidépresseurs, qui sont également utilisés pour le traitement de l’HSV. / Abdominal pain, which can reflect visceral hypersensitivity (VHS), is one of the primary reasons for consultations in general medicine and gastroenterology. These abdominal pain, usually transient, can also be caused by more serious underlying pathologic conditions, such as irritable bowel syndrome (IBS) and chronic inflammatory bowel disease (IBD), which include Crohn's disease (CD) and ulcerative colitis (UC).Although IBS and IBD differ on many points, common factors to these two conditions may contribute to the major symptoms of these intestinal disorders, the abdominal pain. Indeed, environmental factors (diet, stress) play an important part in the etiology of IBS, but it turns out today that they are also involved in the worsening of symptoms associated with IBD. Similarly, if many genetic polymorphisms and inflammatory disorders are the basis of the IBD pathogenesis, recent studies show that such changes can also be found in the development of IBS (identification of susceptibility gene and the presence of micro-inflammation). Finally, recent studies on intestinal microbiota reveal some disturbances, called dysbiosis, as well in IBS or IBD patients.Inflammatory bowel disease is characterized by short phases of severe intestinal inflammation, for which the current pharmacopoeia consisting of reducing this inflammation is rather satisfactory. These phases are interspersed with so-called "remission" phase, during which the VHS remains a problem for many patients (about 38%). These abdominal, diffuse and radiating pain can be similar to the one observed in IBS patients. The therapeutic management of this pain is limited, as it is based on old drugs that are of low benefit / risk ratio.Antiepileptics are molecules that target neuronal excitability by modulating the activity of ion channels, receptors or intracellular signaling pathways. Among these molecules, gabapentin (GBP) and pregabalin (PGB) (grouped under the term gabapentinoids) are ligands of the α2δ subunit for voltage gated calcium channel (VGCC). These α2δ ligands are currently prescribed for neuropathic pain, as well as antidepressants, which are also used for the treatment of VHS.About visceral pain, current data only relate to the treatment of VHS associated with IBS. Two clinical studies show a profit of GBP or PGB on symptoms associated with IBS. These data have also been found in preclinical animal models of various non-inflammatory VHS. In addition, the analgesic effects of these molecules is been highlighted on somatic inflammatory pain models, but no study is looking into the potential beneficial effect of these ligands on models of inflammatory visceral pain or in patients with IBD.