Submitted by Hiolanda Rêgo (hiolandarego@gmail.com) on 2014-07-23T17:41:42Z No. of bitstreams: 1 Dissertação_ICS_ Gabriela de Sales Guerreiro Britto.pdf: 1677695 bytes, checksum: 4fb745911831e57d13c8725d6f4e2689 (MD5) / Made available in DSpace on 2014-07-23T17:41:42Z (GMT). No. of bitstreams: 1 Dissertação_ICS_ Gabriela de Sales Guerreiro Britto.pdf: 1677695 bytes, checksum: 4fb745911831e57d13c8725d6f4e2689 (MD5) / A asma é uma enfermidade crônica, que afeta cerca de 235 milhões de pessoas no mundo. Em uma pesquisa realizada através do ISAAC fase I (International Study of Asthma and Allergies in Childhood), o Brasil está entre os oito países do mundo que demonstraram prevalência mais alta de asma. Helmintos, principalmente aqueles que habitam a intimidade do corpo do hospedeiro, têm sido relatados como fortes moduladores do sistema imune em humanos e animais experimentais, e associados com a redução de doenças alérgicas e autoimunes. Crianças que vivem em áreas sem "higiene" são mais propensas a ter, não só uma, mas várias infecções por helmintos, ao mesmo tempo, mas a maneira como eles afetam atopia e asma ainda é controversa. O objetivo do presente trabalho foi investigar como coinfecções por parasitos intestinais (A. lumbricoides e T. trichiura) e Toxocara spp. afetam os seguintes desfechos: eosinofilia, IgE total e específica, reatividade cutânea a aeroalérgenos, sibilância atópica e não atópica e produção de citocinas por células sanguíneas das crianças estudadas, estimuladas ou não estimuladas por A. lumbricoides. O estudo foi realizado em 1.271 crianças de um grande centro urbano do Nordeste, Brasil. Os dados sobre sintomas de sibilância foram coletados por um questionário adaptado do ISAAC fase II. O sangue foi coletado para detectar IgE total e alérgeno específica, eosinofilia, anticorpos anti-IgG de Toxocara e produção de citocinas (IFN-γ, IL-5, IL-13 e IL-10). Outras avaliações foram realizadas, incluindo testes de puntura cutâneo (SPT) para aeroalérgenos, além disso, as infecções por helmintos intestinais foram diagnosticadas através do exame microscópico das amostras fecais. Considerando a prevalência de infecções helmínticas, 36,4% das crianças tinham apenas uma infecção, 12,7% tinham duas e 5,2% tinham três infecções. Eosinofilia >4% e >10% foi encontrada em 74,3% e 25,5% das crianças, respectivamente. IgE total >0,2 ug/mL ocorreu em 59,7%. IgE específica (sIgE) ≥0,70 kU/L e positividade para SPT, para pelo menos um alérgeno, foram encontrados em 37,1% e 30% das crianças, respectivamente. Um total de 22,7% das crianças apresentava sibilo em geral, 12% apresentaram sibilância não atópica, 10,7% tinham sibilo atópico e 26% eram não-sibilantes atópicas. A infecção por uma espécie de helminto foi associada com aumento de eosinofilia e IgE total e menor SPT e coinfecções mostraram uma associação dose- dependente com esses resultados. Helmintos, especialmente em coinfecções, foram associados com uma típica resposta imune Th2 modificada (evidenciada pela produção de IL-5; IL- 13 e IL-10), mas não foi encontrada associação com sibilo (atópico e não atópico). Coinfecções por helmintos induziram uma resposta imune Th2 melhorada e dose-dependente, evidenciada pela produção de IL-5, IL-13, o que explica a associação com o aumento de IgE total e de eosinofilia. No entanto, eles também ativaram uma rede regulatória conduzindo a uma resposta Th2-modificada, com a produção de IL-10 que, juntamente com a ativação de IgE policlonal, pode ter suprimido a reatividade na pele. No entanto, eles não foram capazes de afetar nem sibilo atópico, nem não atópico. Estudos adicionais devem ser realizados para elucidar os mecanismos moleculares da presente imunomodulação exercida pelos helmintos sob a atopia.

One of the purposes of allergen-specific immunotherapy (SIT) is to modulate the humoral immune response against allergens with significant increases in allergen-specific IgG1 and IgG4 levels. These antibodies are associated with blocking activity by preventing IgE binding to allergen and leading to reduced inflammatory responses. This study aimed to investigate in vitro blocking activity of allergen-specific IgG antibodies on IgE reactivity to D. pteronyssinus (Dpt) in sera from atopic patients. Dpt-specific IgG antibodies were obtained from atopic sera and irrelevant IgG from non-atopic sera. IgG antibodies were purified by ammonium sulfate precipitation followed by Protein-G affinity chromatography and evaluated with regards to purity by SDS-PAGE and immunoreactivity by slot-blot and immunoblot assays. The blocking activity was evaluated by inhibition ELISA. The electrophoretical profile after salting-out precipitation showed an enrichment of high molecular weight proteins in the precipitated fraction and strongly stained bands in the ligand fraction after chromatography, compatible with molecular weight of human IgG. It was detected strong immunoreactivity to IgG, negligible to IgA, and no reactivity to IgE and IgM. Dpt-specific IgG fraction was capable to significantly reduce levels of IgE anti-Dpt, resulting in 35-51% inhibition of IgE reactivity to Dpt in atopic patient sera. Allergen-specific IgG antibodies purified using available and standardized methodology are able to inhibit IgE reactivity to Dpt allergen extract. In addition to the clinical symptoms improvement (subjective parameter), this approach reinforces that the intermittent measurement of serum allergen-specific IgG antibodies will be an important objective laboratorial parameter that will help specialists to follow their patients under SIT. / Uma das propostas da imunoterapia alérgeno específica é a de modular a resposta imune humoral contra alérgenos, com aumento significativo nos níveis de IgG1 e IgG4 específicos. Esses anticorpos estão associados com uma atividade bloqueadora, impedindo a ligação de anticorpos IgE ao alérgeno e levando a uma redução nas respostas inflamatórias. Esse estudo objetivou investigar a atividade bloqueadora, in vitro, de anticorpos IgG específicos sobre a reatividade de IgE a D. pteronyssinus (Dpt) em soros de pacientes atópicos. Anticorpos IgG específicos foram obtidos de soros de pacientes atópicos, e IgG irrelevante a partir de soros de não atópicos, e depois purificados por precipitação com sulfato de amônio, seguido de cromatografia de afinidade em Proteina G-agarose. A pureza desses anticorpos foi avaliada por SDS-PAGE, a imunoreatividade por ensaios de slot-blot e immunoblot, e a atividade bloqueadora por ELISA inibição. O perfil eletroforético, após precipitação com sulfato de amônio, mostrou um enriquecimento de proteínas de alto peso molecular na fração precipitada,e bandas fortemente coradas na fração ligante após a cromatografia, compatíveis com o peso molecular de IgG humana. Foi detectada uma forte imunoreatividade para IgG, leve para IgA, e nenhuma reatividade para IgE e IgM. A Fração IgG específica foi capaz de reduzir significantemente os níveis de IgE anti-Dpt, resultando em 35-51% de inibição da reatividade de IgE a Dpt em pools de soros de pacientes atópicos. Anticorpos IgG específicos purificados, através de uma metodologia disponível e padronizada, são capazes de inibir a reatividade de IgE ao extrato alergênico Dpt. Além da melhoria da sintomatologia clínica, considerada um parâmetro subjetivo, essa abordagem reforça que a avaliação intermitente de anticorpos IgG alérgeno-específicos pode ser uma ferramenta importante, auxiliando especialistas a acompanharem seus pacientes em processo de imunoterapia específica. / Mestre em Ciências da Saúde

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

Signal Transducers and Activators of Transcription (STATs) are latent transcription factors that mediate several cellular responses. This protein family consists of seven members, STAT1 – 6 including two closely related molecules, STAT5a and STAT5b, that show 96% amino acid sequence homology and are critical for lymphoid, myeloid and erythroid cell development and function. Activated STAT proteins dimerize and translocate to the nucleus, where they bind to high-affinity DNA motifs to modulate gene expression. We recently identified STAT5b as the critical regulator of IgE-mediated cytokine production in mast cells. STAT5b knockout (KO) cells show decreased sensitivity to IgE-mediated passive systemic anaphylaxis accompanied with decreased production of IL-6 and IL-13 compared to wild type counterparts. Interestingly, STAT5b KO mice demonstrated elevated levels of serum IgE but a normal response to histamine-mediated passive systemic anaphylaxis. The current work demonstrates that STAT5b regulates mast cell function both in vivo and in vitro. Additionally, activated STAT proteins can also form tetramers through an N-terminal domain-mediated oligomerization process when bound to low-affinity tandem motifs. Dr. Warren Leonard’s laboratory generated STAT5a-STAT5b double knock-in (DKI) mice in which STAT5 proteins are phosphorylated and can form dimers but not tetramers. We have now found that bone marrow-derived mast cells from STAT5 DKI mice are defective in IgE-induced cytokine and chemokine production and exhibit defective stem cell factor (SCF)-induced migration and survival responses in vitro. Similarly, IgE-mediated passive systemic anaphylaxis is decreased in STAT5 DKI mice. These data indicate that Stat5 tetramers are critical for some aspects of mast cell function in allergic and inflammatory disease.

Chronic spontaneous urticaria (CSU) is defined as itchy hives with or without angioedema with a burning sensation that last for six weeks or longer and have no apparent external trigger. The disease occurs in about 1 % of the population. The itching and burning symptoms affect the patient’s quality of life in a negative way which makes a treatment highly needed. The first-line medication for the treatment of CSU is non-sedating H1-antihistamines in recommended doses and the second line of treatment is an increased dose of H1-antihistamines, up to four-fold the approved doses. However, many patients do not response to these therapies whereas a third-line treatment, an add-on therapy with omalizumab, is necessary. Omalizumab is a monoclonal anti-IgE-antibody that binds to free IgE and prevents them to attach to FcεRI-receptors on inflammatory cells like mast cells. This leads to a lower activation of mast cells and less histamine gets released. The definite mechanism of action of CSU-symptoms relief is still unclear but it is thought to be due to decreased levels of IgE and FcεRI-receptors. The symptoms of CSU can be scored using weekly itch severity score (ISS7) which scores the pruritus, weekly hive severity score (HSS7) which scores the number of hives and weekly urticaria activity score (UAS7) that scores both pruritus and number of hives together. The aim of this literature review was to evaluate the efficacy and safety of omalizumab in patients diagnosed with CSU. A search on PubMed was conducted with the search terms "omalizumab" AND "chronic spontaneous urticaria" and "omalizumab" AND "chronic idiopathic urticaria". The articles were limited to randomized, controlled and double-blinded trials that were published 2010 or later. A total of five studies were included for further analysis; MYSTIQUE, ASTERIA II, ASTERIA I, POLARIS and GLACIAL. The studies showed that omalizumab 300 mg and 150 mg reduced the symptoms of CSU significantly compared with placebo, whereas 300 mg had the best efficacy in decreasing the values of ISS7, HSS7 and UAS7. The patients went from having severe urticaria to mild urticaria after treatment with omalizumab. The degree of itching improved from severe itching to mild itching. The side effects were mild to moderate, the most common were nasopharyngitis, headache, arthralgia and upper respiratory tract infection. Omalizumab showed to be effective and safe as an add-on therapy for the treatment of patients with chronic spontaneous urticaria who did not respond adequately to treatment with H1-antihistamines.

Mast cells are tissue resident members of the immune system. They have a wide range of functions and receptors including the FcεRI receptor, which gets activated by binding to IgE bound to an antigen. When the cells are activated in this manner, a process termed the LysRS- Ap4A-MITF signalling pathway occurs, resulting in the translocation of the Lys tRNA synthetase into the nucleus and an activation of its moonlighting activity - the production of diadenosine tetraphosphate (Ap4A). Ap4A is a dinucleoside polyphosphate, a type of ubiquitous molecule present in all domains of life. They are made up of two nucleosides joined together by a 5' to 5' phosphodiester bridge of variable lengths. Recently, these molecules have been shown to serve as non-canonical initiating nucleotides during bacterial transcription, where they function as 5' RNA caps, similar to the well-known 7- methylguanosine eukaryotic mRNA cap. In this thesis, I present proof of existence of Ap 4A capped RNA in mast cells, a previously unknown 5' RNA structure in eukaryotic cells, and I attempt to pinpoint its role in the activation of these cells and in the wider context of mast cell mediated immune response. Keywords: mast cells, RNA caps, Dinucleoside polyphosphates, Ap 4A, RNA modification, IgE, FcεRI receptor, Lysine tRNA synthetase

The purpose of this thesis was to demonstrate device functionality of 3-D paper-based, multiplex platforms, µPADs, without the use of coupling agents between layers. Previously, these platforms were fabricated with double-sided tape and cellulose powder to try to augment proper fluid routing, but difficulties with this method occurred. An acrylic housing unit with strategically placed pressure tabs was designed to aid horizontal and vertical fluid routing through the platform, thus eliminating the inconsistencies associated with coupling agents. Channel characterization studies, a COMSOLTM simulation, and development time studies were performed to aid device design and demonstrate device functionality. The implementation of this µPAD platform as a diagnostic instrument was validated via lateral flow immunoassays utilizing both biotinylated antibodies and biotinylated aptamers as capture reagents. Successful detection of the target analyte, IgE, as well as successful fluid routing through multiple layers of membrane was demonstrated by immunoassays performed on 3-D, multiplex platforms. Another important result determined the aptamers’ ability to detect IgE to be statistically the same as the antibodies’ ability; thus confirming aptamers as viable capture reagent alternatives to antibodies in lateral flow assays. Overall, this research project was performed to develop and validate via experiment a prototype paper-based microfluidic diagnostic device, µPAD, with the capability to detect multiple biomarkers on one platform.

No description available.

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.

Allergic asthma is an immunological disease, mediated by CD4+ Th2 cells, and its prevalence has increased over recent decades. Features of allergic asthma include airway hyperresponsiveness (AHR), airway eosinophilia, excessive airway mucus production, and increased IgE and Th2 cytokine levels. Airway remodeling with pulmonary fibrosis is noted in the progress of asthma. In this thesis, a murine model of allergic asthma was used to investigate the effect of cyclophosphamide (CY) on asthma and the involvement of regulatory T cells (Treg), and the role of Granulocyte-macrophage colony stimulating-factor (GM-CSF) in allergic asthma by using GM-CSF knockout mice. CY is a cytotoxic agent, which paradoxically augments several immune responses. The first part of this thesis was aimed to study the effects of CY in a murine model of allergic airway inflammation. BALB/c mice were immunized with ovalbumin (OVA) on days 0 and 14, and challenged with aerosolized OVA from days 21 to 27. Some mice additionally received CY on days -2 and 12. In the CY-treated animals, pronounced worsening of inflammatory features was noted, including increases in eosinophil infiltration, epithelial thickness, mucus occlusion and eosinophil numbers in bronchoalveolar lavage fluid (BALF). Increased total and OVA-specific serum IgE were also noted in the CY-treated animals. In cell cultures from peritracheal lymph nodes, the Th2 cytokines IL-4 and IL-5 were elevated in animals treated with CY. It was hypothesized that the effects of CY could be caused by reduced immunosuppression mediated by Treg. mRNA expression of the immunosuppressive cytokines IL-10 and TGF-beta was reduced in the lungs of CY-treated mice. The expression of FoxP3, a marker of naturally occurring Treg, was significantly reduced in spleens, thymuses and peritracheal lymph nodes after the second injection of CY, and in the lung tissue after allergen challenge in CY-treated mice. Furthermore, lung IL-10-producing CD4+ T cells and CTLA-4+-bearing CD4+ T cells were reduced after allergen aerosol challenge in CY-treated mice. Thus CY worsened the features of allergic pulmonary inflammation in this model, in association with increased production of IgE and Th2 cytokines. The reduction in expression of FoxP3 and immunosuppressive cytokines by CY suggests that toxicity to Treg may contribute to the increased inflammation. GM-CSF plays a role in the growth, development, and maturation of bone marrow hemopoietic cells into mature blood cells, and has been proposed to be involved in potentiating the function of inflammatory cells in allergic inflammation. In the second part of this thesis, GM-CSF knockout (KO) mice were used to investigate the role of GM-CSF. In allergic KO mice, airway eosinophils were only shown in the perivascular, but not peribronchial areas in the lung, compared to the allergic wild-type (WT) mice in which eosinophil infiltration appeared in both areas. Eosinophil numbers were drastically reduced in the bronchoalveolar lavage fluid (BALF) of KO mice. IL-5 production in the lung tissue and BALF in allergic KO mice was reduced; similar results were also found in peritracheal draining lymph nodes after in vitro stimulation assays. However, IL-4 and IL-13 production, airway hyperresponsiveness (AHR), and serum IgE production were not affected in allergic KO mice. Surprisingly, lung IFN-gamma mRNA and BALF levels were increased in allergic KO mice. Lung mRNA levels of CCR3, a key chemokine receptor on eosinophils, were significantly reduced in allergic KO mice, whereas expression of the chemokines eotaxin and RANTES were at similar levels in allergic KO and WT mice. Lung mRNA levels of the IFN-gamma-inducible chemokines Mig (CXCL9) and IP-10 (CXCL10), which are antagonists of CCR3, and their receptor CXCR3 were increased in allergic KO mice, compared with allergic WT mice. Data obtained from flow cytometry showed more eosinophils survived in the lung of WT mice than KO mice. Another allergy model, a peritoneal allergy model was performed to investigate inflammation in a different model. Leukocyte subpopulations such as neutrophils, eosinophils, macrophages, and lymphocytes were reduced in the peritoneal lavage fluid of allergic KO mice. The findings revealed that GM-CSF is essential for IL-5 production, pulmonary airway eosinophilia and eosinophil survival. In the absence of GM-CSF, over-production of IFN-???? may induce chemokines, including Mig and IP-10, which are antagonists for CCR3 and may reduce airway eosinophil infiltration. In this thesis, a murine model of allergic asthma has been used to obtain novel findings on the regulation of allergic inflammation. The results with CY are relevant to the treatment of asthma patients with CY and other cytotoxic agents. The findings in the GM-CSF KO mice suggest that GM-CSF is a potential therapeutic target in asthma, and that in assessment of new therapeutic agents for asthma, effects on GM-CSF should be considered.