Os problemas decorrentes de doenças alérgicas é tema em destaque atualmente devido ao aumento de pessoas que vêm apresentando sintomas e sofrendo as consequências de mudanças aceleradas nos costumes da nossa sociedade. Apesar das diversas terapias oferecidas, pacientes alérgicos ainda sofrem com efeitos colaterais decorrentes do uso de medicamentos anti-alérgicos. Assim, a busca por novas alternativas que possam amenizar os sintomas alérgicos torna-se relevante. As substâncias naturais tem sido alvo de intensa investigação devido às propriedades farmacológicas no tratamento de diversas doenças incluindo as alérgicas. Neste contexto, este trabalho foi focado no extrato Vimang e seu componente majoritário, a mangiferina (Mgf), que tem apresentado atividades biológicas diversas. Sabendo-se que a atividade biológica de um extrato pode ser potencializada pelo sinergismo entre seus componentes, foi avaliado o efeito sinérgico de substâncias isoladas do Vimang, tais como mangiferina, quercetina, catequina, ácido gálico e benzóico. Esses componentes foram combinados e as misturas avaliadas quanto à capacidade em inibir a desgranulação mastocitária. O Vimang, a quercetina e mangiferina inibem a desgranulação mastócitária enquanto catequina, ácido gálico e benzóico não são ativos; e porisso, não foram incluídos na determinação do isobolograma de aditividade, adotado no estudo de sinergismo. O isobolograma de aditividade mostra sinergismo para as concentrações de mangiferina iguais a 5, 10, 20 e 40 ?M e quercetina iguais a 0,2, 1 e 1,6 ?M; e mostra antagonismo para mangiferina 80 ?M e quercetina 0,4 e 0,8 ?M. Tais resultados sugerem que altas concentrações de mangiferina atrapalham a interação sinérgica entre estas substâncias e altas concentrações de quercetina exercem um efeito contrário, ou seja, favorecem o sinergismo entre quercetina e mangiferina. A baixa solubilidade de flavonóides em meio fisiológico levou ao estudo da interação da Mgf com lipossomos de Dimiristoil-L-?-Fosfatidilcolina (DMPC) para aplicação nos ensaios biológicos. A Interação da Mgf com lipossomos de DMPC, bem como a estabilidade lipossomal, foram avaliadas quanto aos efeitos de pH, força iônica e presença de colesterol. O pH influencia a eficiência de incorporação (EI) da Mgf que é maior em pHs ácidos. Em soluções de pH ácido ou fisiológico há aumento de tamanho do lipossomo na presença de Mgf, o que reforça a hipótese de interação desta com a bicamada lipídica. O aumento da força iônica, na ausência de Mgf, diminui a estabilidade do lipossomo. Na ausência de sal, a Mgf favorece a formação de agregados e diminui a estabilidade dos lipossomos e na presença de sal, a Mgf previne a agregação e estabiliza os agregados lipossomais. A EI da Mgf nos lipossomos de DMPC preparados pelo método da injeção etanólica, em pH fisiológico, foram ao redor de 13%. A tentativa de otimizar a EI foi realizada pela adição de colesterol em diferentes proporções lipídio:colesterol. Um aumento de 13% para 17% foi observado para a proporção DMPC:colesterol (9:1). A Mgf livre inibe a desgranulação dos mastócitos de forma concentração-dependente; mas a presença de lipossomos, preparados pelo método da injeção etanólica não altera significativamente esse perfil. A EI da mangiferina em lipossomos de DMPC:colesterol (9:1), preparados pelo método do filme lipídico, foi igual a 45%. Os resultados de potencial antialérgico, para este caso, mostraram que os lipossomos de DMPC favorecem o potencial antialérgico da Mgf em concentrações acima de 25 ?M. Esses resultados abrem perspectivas na busca de sistemas lipossomais mais estáveis e que possam ser aplicados em ensaios biológicos in vitro e in vivo. / The problems due to allergic diseases are currently a highlighted topic due to the increase of people who are showing symptoms and suffering the consequences of rapid changes in the habits of our society. Despite various therapies offered, allergic patients still suffer side effects from the use of anti-allergic drugs. Thus, the search for new alternatives that can alleviate the allergic symptoms becomes relevant. Natural substances have been the subject of intense research due to the pharmacological properties in the treatment of several diseases, including allergic ones. In this context, this work was focused on Vimang extract and its major component, the mangiferin (Mgf), which has shown several biological activities. Given that the biological activity of an extract can be enhanced by the synergism between its components, the synergistic effect of compounds isolated from Vimang such as mangiferin, quercetin, catechin, gallic acid and benzoic acid was evaluated. These components were combined and the mixtures tested for their ability to inhibit mast cell degranulation. The Vimang, quercetin and mangiferin inhibit mast cell degranulation while catechin, gallic acid and benzoic acid are not active and for that reason they were not included in the determination of the isobologram adopted in the synergism study. The isobologram of additivity shows synergism for the concentration of mangiferin equal to 5, 10, 20 and 40 ?M and quercetin equal 0.2, 1 and 1.6 ?M, and shows antagonism for mangiferin 80 ?M and quercetin 0.4 and 0.8 ?M. The low solubility of flavonoids in physiological medium led to the study of the interaction of Mgf with liposomes of L-?-dimyristoyl-phosphatidylcholine (DMPC) for application in biological assays. The interaction of Mgf with DMPC liposomes as well as the liposomal stability was evaluated considering the effects of pH, ionic strength and cholesterol presence. The pH influences the incorporation efficiency (IE) of mangiferin, which is higher in acidic pHs. In solutions of acidic or physiological pH, the liposome size increases in the presence of Mgf, which reinforces the hypothesis of interaction of Mgf with the lipid bilayer. The increased ionic strength in the absence of Mgf decreases the stability of the liposome. In the absence of salt, Mgf favors the formation of aggregates and decreases the stability of the liposomes. When in presence of salt, Mgf prevents the aggregation and stabilizes the liposomal aggregates. The IE of mangiferin in DMPC prepared by the ethanol injection method, at physiological pH, were around 13%. The attempt to optimize the IE was performed by the addition of cholesterol in different proportions lipid:cholesterol. An increase from 13% to 17% was observed for the proportion of DMPC:cholesterol (9:1). Free mangiferin inhibits mast cells degranulation in a dose-dependent manner, but the presence of liposomes prepared by the ethanol injection method does not significantly change this profile. The IE of mangiferin in liposomes of DMPC:cholesterol (9:1), prepared by the method of lipidic film was equal to 45%.The results of antiallergic potential in this case show that DMPC liposomes favor the mangiferin antiallergic potential at concentrations above 25 mM. These results open perspectives in the search for more stable liposomal systems and can be applied to biological assays in vitro and in vivo.

O ovo é o alimento naturalmente mais completo, uma vez que possui todos os nutrientes necessários, como vitaminas, aminoácidos e minerais essenciais para manter uma vida. Porém, em contra partida, possui várias proteínas promotoras de alergias em considerável parcela da população mundial. Para determinar as proteínas dos alimentos alergênicos, um dos testes mais utilizados é o imunoensaios tais como ELISA (ensaio imunoenzimático - enzyme linked immunosorbent assay), onde o anticorpo reconhece o antígeno e essa conexão é mostrada por um sistema enzimático, em outras palavras, a densidade óptica. O objetivo deste estudo foi determinar a eficiência do anticorpo policlonal, produzido em laboratório, para identificar a presença do antígeno ovomucóide em ovos tratados por irradiação gama para a sua desativação. Para avaliar os tratamentos, o anticorpo policlonal foi produzido em quatro (04) coelhos da raça Nova Zelândia, do sexo feminino, com 45 dias de vida, imunizadas com ovomucóide bioconjugado. Foi utilizado o adjuvante de Freund completo na primeira imunização e a solução tampão PBS, foram realizadas, posteriormente, quatro imunizações a cada quinze dias, mais um reforço 48 horas antes da retirada do plasma sanguíneo. O soro sangüíneo foi titulado por PTA-ELISA (Plate trapped antigen). Todos os procedimentos foram aprovados pelo Comitê de Ética e Experimentação Animal do Instituto de Ciência Animal e Pastagens (IZ) e precedida de acordo com as normas europeias para o bem-estar e ética animal. Foram utilizados ovos comerciais in natura, fornecidos pelo Departamento de Genética da Universidade de Agricultura \"Luiz de Queiroz\" - ESALQ / USP. As amostras foram submetidas à radiação gama proveniente de uma fonte de Co60, do tipo Multipropósito no Instituto de Pesquisas Energéticas e Nucleares (IPEN), sob uma taxa de dose de 19,4 e 31.8Gy/hora, nas doses: 0 (controle); 10kGy; 20KGy e 30KGy, em todas as taxas. Pelo teste de ELISA, foi encontrado o alérgeno ovomucóide das amostras ovo e, pelo resultado apresentados, constatou-se que o tratamento da radiação não mostrou alterações significativas, quando avaliado por anticorpos policlonais. Assim, podemos concluir que o anticorpo produzido é capaz de identificar a proteína alergênica ovomucóide e, a irradiação gama em tais taxas não apresenta mudanças na estrutura da proteína, por esta forma de avaliação. Porém, apresentou algumas alterações na cor e viscosidade visual das amostras de ovos / The egg is the most complete natural food; it has all the necessary nutrients such as vitamins, aminoacids and essential minerals to maintain a life. However, although, has several proteins that promote allergies in considerable part of the world population. To determine allergenic food proteins, one of the most used tests is the immunoassays such as ELISA (enzyme linked immunosorbent assay), where the antibody recognizes the antigen and this connection is showed by an enzymatic system, in other words, optical density. The aim of this study was to determine the polyclonal antibody efficiency, produced in laboratory, to identify the presence the ovomucoid antigen in treated eggs by gamma irradiation for its inactivation. To evaluate the treatments, polyclonal antibody was produced in four New Zealand female rabbits, at 45 days old, immunized with bioconjugated ovomucoid. Was used Freund Complete Adjuvant at first immunization and PBS Buffer at four subsequently immunizations every fifteen days, plus a booster 48 hours before the blood retreated. The blood serum was tittered by PTAELISA (Plate trapped antigen). All procedures were approved by Institute of Animal Science and Pastures (IZ)´s Committee of Ethical and Animal Experimentation and preceded according to European Norms for ethical and animal welfare. It was used, in nature, commercial laying eggs, from the Genetic Department of Agricultural University Luiz de Queiroz ESALQ/USP. So the samples were submitted to the gamma radiation coming from a source of Co60, type Multipurpose at the Energetically Researches and Nuclear Institute (IPEN), under a dose rate of 19.4 and 31.8Gy/hour, in the doses: 0 (control); 10KGy; 20KGy and 30KGy, in all rates. By the ELISAs test we can find the egg allergen ovomucoid and the radiation treatment do not showed considerable changes. So we can concluded that the antibody produced is capable of identify the ovomucoid allergenic protein and the gamma irradiation in such rates does not shows changes in that protein, therefore showed some changes in the color and visual viscosity of the egg samples

All of the above findings demonstrated that eosinophil activation in allergic inflammation could be sensitively regulated by diverse stimuli. Besides, highly redundant functional effects and underlying signaling mechanisms were observed among different mediators. Elucidation of the mechanisms of eosinophil activation could improve our understanding of its complex and active role in the pathogenesis of allergic diseases, thereby providing biochemical basis for the development of more effective therapeutic strategies for treating the diseases. (Abstract shortened by UMI.) / Apart from the cytokines produced by T lymphoctyes, leptin, a cytokine produced by adipocytes, was also demonstrated to activate eosinophils. It was reported that the plasma levels of leptin were elevated in both obese and allergic patients. We found that leptin could activate eosinophils for survival enhancement, adhesion and migration, and secretion of cytokines and chemokines. Besides, we showed that the MAPKs and NF-kappaB pathways were involved in eosinophil adhesion, migration and mediator release induced by leptin, while Janus kinases (JAK)-signal transducers and activators of transcription (STAT) pathway was responsible for leptin-induced eosinophil survival. Our study indicated a potential correlation between obesity and exacerbation of allergic inflammation. / Eosinophilia is a hallmark pathological feature of allergic diseases and it has been targeted as a novel therapeutic strategy for allergic diseases. / In atopic dermatitis (AD), dermal infiltration of eosinophils is one of the pathological features of this disease. IL-31 is a novel Th2 cytokine reported to induce pruritus and skin dermatitis resembling human AD. Our study on the co-culture system of eosinophils and keratinocyte cell line HaCaT illustrated the production of pro-inflammatory cytokines and chemokines from the co-culture system of eosinophils and HaCaT cells, under the stimulation of IL-31. In co-culture system, surface expression of CD18 and intercellular adhesion molecule (ICAM)-1 on eosinophils and HaCaT cells was also up-regulated respectively, implying a direct interaction between the two cell types through their cell surface adhesion molecules. The interaction of eosinophils and HaCaT cells under IL-31 stimulation was shown to be mediated through MAPKs, NF-kappaB and PI3K pathways. These findings therefore elucidate the immunological roles of IL-31, eosinophils and keratinocytes in AD. / In the present study, we investigated the mechanisms of eosinophil activation induced by various stimuli including novel T helper type 2 (Th2) cytokines, adipokine, microbial products and direct interaction with tissue cells. The activation of eosinophils was studied in terms of survival enhancement, modulation of adhesion and migration, and the release of inflammatory mediators including cytokines, chemokines, granular proteins and superoxide. Using pharmacological and molecular approaches, we further investigated the intracellular signaling mechanisms regulating the eosinophil activation mediated by various stimuli. / Increasing evidence has indicated that bacterial and viral infections could intensify allergic responses. Our findings demonstrated that eosinophil activation could be elicited by microbes through toll-like receptors (TLRs), the recently discovered receptors for the recognition of conserved motifs in pathogens. We found that eosinophils could be activated by the ligands of TLR2, 5 and 7 in enhancing survival, adhesion and migration, release of pro-inflammatory cytokines, chemokines, granular proteins and superoxides. These stimulatory effects, mediated by TLR2, 5 and 7, were differentially regulated by MAPKs, NF-kappaB and phosphatidylinositol 3-kinase (PI3K) pathways. Moreover, an important finding of our study is the common involvement of focal adhesion kinase (FAK)-dependent extracellular-regulated protein kinase (ERK) phosphorylation in the signaling of TLR 2, 5 and 7, implying a special role of FAK in linking TLR signaling with MAPKs cascade in human eosinophils. Our study on microbe-induced eosinophil activation provided a potential explanation for linking infection with exacerbation of allergic diseases. / The interleukin (IL)-17 family is a newly discovered group of cytokines which was reported to be important in allergic inflammation. We studied the roles of two IL-17 family members, IL-175E/IL-25 and IL-17F on eosinophil activation. Both cytokines were found to induce the secretion of inflammatory cytokines and chemokines from eosinophils, in which IL-25 could also enhance eosinophil survival and adhesion. Besides, we found that the stimulatory effects induced by both IL-25 and IL-17F were mediated through mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-kappaB) pathways. The main source of IL-17F has recently been reported to be a novel T lymphocyte population, Th17, which is specifically driven by IL-23. An important finding of our present study was the synergistic effects of IL-17F and its potent inducer, IL-23, on cytokine release from eosinophils. Since IL-23 was produced by macrophages and dendritic cells upon microbial stimulation, the synergistic effect of IL-17F and IL-23 on eosinophil activation might imply a potential role in linking microbial infection and allergic inflammation. Our findings also provide further support to the crucial role of the IL-17 family and Th17 lymphocytes in the amplification of allergic diseases. / Cheung, Fung Yi. / Source: Dissertation Abstracts International, Volume: 69-03, Section: B, page: 1552. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 205-221). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

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.

Asma é uma doença inflamatória pulmonar crônica usualmente associada com imunidade do tipo 2, eosinofilia pulmonar, hiperreatividade brônquica (AHR), hiper-produção de muco e altos níveis de IgE. Indíviduos asmáticos podem responder aos alérgenos por duas distintas fases: uma fase imediata (EPR) e uma fase tardia (LPR), ambas não reproduzidas na maioria dos modelos murinos de asma. No presente trabalho utilizamos camundongos AIRmax e AIRmin. Verificamos que camundongos AIRmin respondem com uma AHR intrínseca a metacolina. Esta resposta intensa correlacionou-se com uma menor expressão de receptores muscarínicos do tipo 2. Camundongos AIRmax sensibilizados e desafiados com OVA, ao contrário dos AIRmin, desenvolveram LPR e AHR a MCh. Os AIRmax apresentam um denso infiltrado inflamatório com predominância de eosinófilos, uma elevada produção de muco, citocinas (IL-5 e IL-13) e anticorpos anafiláticos IgE e IgG1. De forma surpreendente animais AIRmax desenvolvem quadro alérgico pulmonar crônico que cursa com AHR a MCh e uma inflamação pulmonar com infiltrado de eosinófilos com deposição de colágeno no tecido pulmonar além de uma produção elevada de anticorpos anafiláticos. Em conclusão desenvolvemos um novo modelo murino de asma. / Asthma is a chronic inflammatory lung disease usually associated to Type 2 T helper cells, lung eosinophilia, airway hyper-reactivity (AHR), mucus hyper-secretion and increased titers of IgE. Asthmatic individuals may react to allergens by two distinct phases: an immediate phase (EPR) and a late phase (LPR) both phases were not reproduced in classical murine models of asthma. In the present study we use AIRmax AIRmin mice. We found that AIRmin mice exposed to methacholine presented intense intrinsic AHR. This intense reaction was related to a lower expression of muscarinic type 2 receptors. AIRmax mice sensitized and challenged with OVA, unlike AIRmin developed LPR and AHR to MCh. The AIRmax also developed a dense inflammatory infiltrate containing predominantly eosinophils, hyper-secretion of mucus, cytokines (IL-5 and IL-13) and anaphylactic antibodies (IgE and IgG1). Surprisingly AIRmax mice develop chronic pulmonary allergic framework that leads to AHR to MCh and lung inflammation with eosinophilic infiltration with collagen deposition in lung tissue and a high production of anaphylactic antibodies. In conclusion, we developed a new murine model of asthma.

Objetivo: Avaliar a prevalência e fatores de risco associados a reações adversas a alimentos (RAA) e diagnóstico médico de alergia alimentar referidas pelos pais em crianças de um a três anos de vida. Métodos: Estudo de coorte prospectivo de crianças nascidas em Ribeirão Preto, São Paulo e São Luís, Maranhão, no ano de 2010, e avaliadas para esse estudo nos três primeiros anos de vida. Em Ribeirão Preto, foram incluidas 3740 das 7702 crianças nascidas no ano de 2010. Em São Luís, participaram do estudo 3320 das 5166 crianças avaliadas ao nascimento no mesmo ano. Os responsáveis pelas crianças responderam questões referentes à ocorrência de reações adversas após a ingestão de alimentos e diagnóstico médico prévio de alergia alimentar. O estudo das associações entre os desfechos e as variáveis independentes de interesse foi feito por meio de análise univariada e por modelos log-binomiais ajustados, obtendo-se medidas de risco relativo (RR) e seus intervalos de confiança (IC). Resultados: A prevalência de reações adversas a alimentos (RAA) referidas pelos pais e de diagnóstico médico de alergia a pelo menos um alimento foi, respectivamente, de 10,7% (399/3740) e 4,4% (164/3716) em Ribeirão Preto, e de 6,4% (216/3320) e 2,7% (88/3320) em São Luís. Reações e diagnóstico de alergia ao leite de vaca ocorreram, respectivamente em 6,3% (223/3533) e 3,4% (119/3516) das crianças de Ribeirão Preto, e em 3,2% (102/3166) e 1,6% (52/3155) em São Luís. Nas duas cidades, houve concordância entre os desfechos reação a pelo menos um alimento e diagnóstico médico de alergia a pelo menos um alimento (índice kappa de 0,52 em RP e 0,51 em SL). O mesmo ocorreu para o relato de reação ao leite de vaca e diagnóstico médico de alergia ao leite (índice kappa de 0,52 em RP e 0,47 em SL). Crianças cujas mães não frequentaram universidades apresentaram menor risco de reação a pelo menos um alimento, tanto em Ribeirão Preto, 9 quanto em São Luís, e menor risco de diagnóstico médico de alergia a algum alimento em Ribeirão Preto. Os fatores associados a maior risco de diagnóstico médico de alergia a pelo menos um alimento foram a situação conjugal sem companheiro, idade de início à creche menor que 6 meses e histórico de alergia nas crianças, somente em Ribeirão Preto. O aleitamento materno exclusivo até os 6 meses associou-se a menor risco de diagnóstico médico de alergia ao leite de vaca em Ribeirão Preto. O início do leite artificial antes dos 6 meses e o início à creche antes dessa idade foram identificados como fatores de risco para diagnóstico médico de alergia ao leite de vaca nas crianças de Ribeirão Preto. Conclusões: O presente estudo contribuiu para o conhecimento da prevalência de RAA, incluindo a AA, referida pelos pais, em crianças brasileiras de até 3 anos de vida. Diferenças sociais, econômicas e culturais podem explicar as diferenças de prevalência e fatores associados a RAA e AA em RP e SL. / Objective: To evaluate the prevalence and risk factors associated with parent reported adverse reactions to food (ARF) and medical diagnosis of food allergy in 1-3 year old children. Methods: Prospective cohort study of children born in Ribeirão Preto, São Paulo and São Luís, Maranhão, in 2010, and evaluated for this study at 1-3 years of age. In Ribeirão Preto, 3740 of the 7702 children born in 2010 were included. In São Luís, 3320 of the 5166 evaluated at birth were included. Caregivers answered questions regarding the occurrence of adverse reactions after ingestion of food allergy and previous medical diagnosis of food allergy. Associations between end points and independent variables were determined by univariate analysis and log-binomial adjusted models. Relative risks (RR) and confident intervals were calculated (IC). Results: The prevalence of parent reported ARF and medical diagnosis of allergy to at least one food was, respectively, 10.7% (399/3740) and 4.4% (164/3716) in Ribeirão Preto, and 6.4% (216/3320) and 2.7% (88/3320) in São. Luís. Reactions and medical diagnosis of allergy to cow\'s milk were reported, respectively, in 6.3% (223/3533) and 3.4% (119 / 3516) of the children from Ribeirão Preto and in 3.2% (102/3166) and 1.6% (52/3155) in São Luís. In both cities, there was agreement between the reported reactions to at least one food and medical diagnosis of allergy to at least one food (kappa of 0.52 and 0.51 in RP in SL). The same happened for reported reactions to cow\'s milk and medical diagnosis of milk allergy (kappa of 0.52 and 0.47 in RP in SL). Children whose mothers did not attend universities were less likely to report reaction to at least one food, both in Ribeirão Preto and São Luís, and had lower risk of medical diagnosis of allergy to any food in Ribeirão Preto. Factors associated with increased risk of medical diagnosis of allergy to at least one food were the marital status without partners, going to a at 11 daycare center before 6 months of age and a history of allergy in children, only in Ribeirão Preto. Exclusive breastfeeding until 6 months was associated with a lower risk of medical diagnosis of allergy to cow\'s milk in Ribeirão Preto. The beginning of the artificial milk before 6 months and going to daycare centers before this age were identified as risk factors for medical diagnosis of allergy to cow\'s milk in children of Ribeirão Preto. Conclusions: This study contributes to the knowledge of the prevalence parent reported ARF, including food allergy , in Brazilian children up to 3 years. Social, economic and cultural differences can explain the differences in prevalence and associated risk factors for ARF and AA in RP and SL.

Tang Chi-Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 89-112). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.vi / Table of contents --- p.viii / List of Tables --- p.xi / List of Figures --- p.xii / List of Abbreviations --- p.xiv / Chapter Chapter 1. --- General introduction --- p.1 / Chapter Chapter 2. --- Literature review --- p.4 / Chapter 2.1 --- History and prevalence of food allergy --- p.4 / Chapter 2.2 --- Mechanism and clinical symptoms of food allergy --- p.6 / Chapter 2.3 --- Tropomyosin as a major shellfish allergen --- p.13 / Chapter 2.4 --- Use of animal model in the studies of food allergy --- p.22 / Chapter 2.5 --- Future approaches for treatment of food allergy --- p.27 / Chapter Chapter 3. --- Cloning and expression of recombinant tropomyosin --- p.30 / Chapter 3.1 --- Introduction --- p.30 / Chapter 3.2 --- Materials and Methods --- p.31 / Chapter 3.2.1 --- Design of PCR primers for amplification of tropomyosin gene --- p.31 / Chapter 3.2.2 --- Cloning of PCR-amplified cDNA into vector --- p.32 / Chapter 3.2.3 --- Transformation of competent E. coli Ml5 cells --- p.34 / Chapter 3.2.4 --- Confirmation of DNA sequence of the cloned vector --- p.34 / Chapter 3.2.5 --- Induction of the recombinant protein --- p.35 / Chapter 3.2.6 --- Purification and storage of the recombinant protein under native condition --- p.36 / Chapter 3.2.7 --- Concentration measurement and storage of the recombinant protein --- p.37 / Chapter 3.2.8 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.38 / Chapter 3.2.9 --- Regeneration of the Ni-NTA column --- p.40 / Chapter 3.3 --- Results and discussion --- p.42 / Chapter 3.3.1 --- DNA sequence of the cloned vector --- p.42 / Chapter 3.3.2 --- Expression of the recombinant protein --- p.42 / Chapter 3.3.3 --- Sodium dodecyl sulfate polyacrylamide gel eletrophoresis (SDS-PAGE) --- p.43 / Chapter Chapter 4. --- Induction of hypersensitive response to shrimp tropomyosin in mice --- p.47 / Chapter 4.1 --- Introduction --- p.47 / Chapter 4.2 --- Materials and methods --- p.52 / Chapter 4.2.1 --- Mice and reagents --- p.52 / Chapter 4.2.2 --- Animal sensitization and challenge --- p.53 / Chapter 4.2.3 --- Morphological and behavioral changes --- p.54 / Chapter 4.2.4 --- Tropomyosin-specific IgE level --- p.55 / Chapter 4.2.5 --- Passive cutaneous anaphylaxis (PCA) reaction --- p.56 / Chapter 4.2.6 --- Tropomyosin-specific cellular proliferation level of splenocytes --- p.56 / Chapter 4.2.7 --- Cytokine profiles of splenoctyes --- p.58 / Chapter 4.2.8 --- Histological examination of small intestine --- p.59 / Chapter 4.2.9 --- Statistical analysis --- p.59 / Chapter 4.3 --- Results --- p.63 / Chapter 4.3.1 --- Morphological and behavioral changes after challenge --- p.63 / Chapter 4.3.2 --- Tropomyosin-specific IgE level --- p.63 / Chapter 4.3.3 --- Passive cutaneous anaphylaxis (PCA) --- p.64 / Chapter 4.3.4 --- Tropomyosin-specific cellular proliferation level of splenocytes --- p.68 / Chapter 4.3.5 --- Cytokine profiles of splenocytes --- p.70 / Chapter 4.3.6 --- Histology of small intestines --- p.76 / Chapter 4.4 --- Discussion --- p.79 / Chapter Chapter 5. --- General conclusion --- p.88 / References --- p.89

Foodservice is integral to safe, adequate and satisfactory food allergy management in the hospital setting. To date, research focused on objective measures of energy and protein provision, implementation and evaluation of food allergy guidelines and assessment of process failures leading to allergen exposure. There is a lack of focus on taking a person-centred approach to understanding the barriers and enablers to optimal food allergy management. A mixed methods phenomenological approach was espoused at a tertiary acute care hospital in Melbourne, Victoria. Data collection techniques included 24-hr diet recall, foodservice satisfaction questionnaires and semi-structured qualitative interviews, all delivered by an Accredited Dietitian. Statistical and thematic analysis was conducted, followed by convergence of the results from each phase of the study. Mean energy intake indicated 64% of requirements and 81% of protein requirements were met; which included external food intake to supplement the hospital diet for around half of the patients. Most patients rated their overall food service satisfaction as ‘Good’, with food quality being the lowest rated foodservice dimension. Addressing sensory, variety and communications aspects of foodservice were focus areas identified by the study patients. These included improving bland flavours and plain appearance, restricted menu choices and automated allergen interface between systems. With a person-centred care focus, the study identified new findings on the views and attitudes of patients with a food allergy on foodservice management in hospital. Within the Food and Meal Science field, the study may be seen as an initial exploratory enquiry for future research on food allergy.

Made available in DSpace on 2016-04-28T20:15:24Z (GMT). No. of bitstreams: 1 2006- Victor do Espirito Santo Cunha.pdf: 1132781 bytes, checksum: 5edd69d626ede6488b658d61c07e111b (MD5) Previous issue date: 2006-02-22 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The objective of this case-control study was to evaluate whether allergenic extracts from five species of house dust mites standardized for humans may be taken into account in the diagnosis of the canine atopic dermatitis. Extracts of Dermatophagoides pteronyssinus, D. farinae, Blomia tropicalis, Lepidoglyphus destructor and Tyrophagus putrescentiae have been evaluated through intradermal testing on 45 dogs, from which 20 belonged to the control group and 25 suffered from allergic dermatitis. There was a significant difference on the response pattern between the two groups (p<0,05). Only one dog (5%) from the control group has reacted to the intradermal test, whereas from the allergic group, 14 dogs (56%) have presented at least one positive reaction (odds ratio = 24,2). Most of the positive reactions observed in the allergic group were to the extracts of T. putrescentiae or L. destructor, each one inducing reactions on ten dogs (40%). The D. farinae, D. pteronyssinus e B. tropicalis extracts were responsible for positive reactions on 7 (28%), 3 (12%) and 3 (12%) dogs, respectively. The intradermal testing sensitivity and specificity were 56% and 95%, respectively, and the positive predictive value and the negative predictive value were 93% and 63%, respectively. / O presente estudo do tipo caso-controle teve como objetivo avaliar se extratos alerg?nicos de cinco esp?cies de ?caros da poeira domiciliar padronizados para humanos podem ser utilizados no diagn?stico da dermatite at?pica canina. Extratos de Dermatophagoides pteronyssinus, D. farinae, Blomia tropicalis, Lepidoglyphus destructor e Tyrophagus putrescentiae foram avaliados atrav?s de testes intrad?rmicos em 45 c?es, dos quais 20 controles e 25 com dermatite al?rgica. Uma diferen?a significativa foi observada no padr?o de respostas entre os dois grupos (p<0,05). Apenas um animal (5%) do grupo controle reagiu ao teste cut?neo, enquanto que no grupo dos al?rgicos 14 c?es (56%) apresentaram pelo menos uma rea??o positiva (odds ratio = 24,2). As maiores freq??ncias de rea??es positivas observadas no grupo dos al?rgicos foram aos extratos de T. putrescentiae ou L. destructor, cada um induzindo rea??es em 10 (40%) c?es. Os extratos de D. farinae, D. pteronyssinus e B. tropicalis foram respons?veis por rea??es positivas em 7 (28%), 3 (12%) e 3 (12%) c?es, respectivamente. A sensibilidade e a especificidade dos testes intrad?rmicos foram de 56% e 95%, respectivamente e, o valor preditivo positivo e valor preditivo negativo de 93% e 63%, respectivamente. .