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Kung, Wing Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 92-115). / Abstracts in English and Chinese. / Abstract --- p.ii / Acknowledgements --- p.vii / Table of contents --- p.ix / List of Tables --- p.xii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter Chapter 1. --- General introduction --- p.1 / Chapter Chapter 2. --- Literature review --- p.4 / Chapter 2.1 --- Food allergy and its prevalence --- p.4 / Chapter 2.2 --- Mechanism and clinical symptoms of food allergy --- p.6 / Chapter 2.3 --- Tropomyosin as the major allergen in shellfish --- p.15 / Chapter 2.4 --- Cross reactivity and epitope mapping of tropomyosin --- p.21 / Chapter 2.5 --- Novel approaches for the treatment of food allergy --- p.29 / Chapter Chapter 3. --- Expression of shrimp recombinant tropomyosin and sensitization of mice --- p.36 / Chapter 3.1 --- Introduction --- p.36 / Chapter 3.2 --- Materials and Methods --- p.40 / Chapter 3.2.1 --- "Recovery of E, coli with tropomyosin-carrying plasmid" --- p.40 / Chapter 3.2.2 --- Preparation of tropomyosin-carrying plasmid --- p.41 / Chapter 3.2.3 --- Confirmation of DNA sequence of the tropomyosin --- p.41 / Chapter 3.2.4 --- Identification of the recombinant protein --- p.43 / Chapter 3.2.5 --- Purification of the recombinant protein --- p.43 / Chapter 3.2.6 --- Sodium dedecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.44 / Chapter 3.2.7 --- Concentration measurement of the recombinant tropomyosin --- p.45 / Chapter 3.2.8 --- Mice --- p.46 / Chapter 3.2.9 --- Mice sensitization and challenging --- p.46 / Chapter 3.2.10 --- Tropomyosin-specific IgE level in blood --- p.47 / Chapter 3.2.11 --- Statistical analysis --- p.49 / Chapter 3.3 --- Results --- p.52 / Chapter 3.3.1 --- DNA sequence of the cloned tropomyosin --- p.52 / Chapter 3.3.2 --- Expression and purification of tropomyosin --- p.52 / Chapter 3.3.3 --- Hypersensitivity symptoms after challenge --- p.53 / Chapter 3.3.4 --- Blood tropomyosin-specific IgE level --- p.53 / Chapter 3.4 --- Discussion --- p.62 / Chapter Chapter 4. --- Identification of T cell epitopes --- p.67 / Chapter 4.1 --- Introduction --- p.67 / Chapter 4.2 --- Materials and methods --- p.67 / Chapter 4.2.1 --- Soluble epitope peptide synthesis --- p.68 / Chapter 4.2.2 --- Isolation of spleen cells from mice --- p.69 / Chapter 4.2.3 --- T cell proliferation assay --- p.70 / Chapter 4.3 --- Results --- p.71 / Chapter 4.3.1 --- Splenocyte proliferation to synthetic peptide --- p.72 / Chapter 4.3.2 --- Splenocyte proliferation to synthetic peptides pool --- p.72 / Chapter 4.4 --- Discussion --- p.77 / Chapter Chapter5 --- General conclusion --- p.89 / References --- p.92

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Background: According to recent studies in children, IgE sensitization not only against perennial allergens, but also against food allergens, is related to asthma risk and increased airway inflammation. During the last decade, a new technique for IgE determination based on allergen components has become available, but its use in epidemiological studies has been limited. Aims: To investigate the relationship between the pattern of IgE sensitization to allergen components and the prevalence of asthma, airway inflammation and hyperresponsiveness in a population-based setting. To examine the relationship of IgE sensitization to allergen extract, and airway inflammation, airway hyperresponsiveness and blood eosinophilia in asthmatics. To examine the natural history of IgE sensitization to food allergens in adults. To compare extract-based and component-based IgE measurements in relation with new-onset respiratory disease and airway inflammation and hyperresponsiveness. Methods: The present thesis is based on cross-sectional and longitudinal analyses of the adult, the population-based study ECRHS (European Community Health Survey) and a cross-sectional, observational study of young subjects with asthma. IgE sensitization was examined by means of both extract-based and component-based tests. Airway inflammation was assessed by exhaled NO and airway hyperresponsiveness with methacholine test. Results: IgE sensitization to food allergens independently related to increased airway inflammation in both a population-based study and a study of asthmatics. Furthermore, a relation was found with increased blood eosinophils in asthmatics. The decrease in prevalence of IgE sensitization against food allergens during a 9-year follow-up was larger than the decrease of aeroallergens. Subjects with IgE sensitization to both cat extract and components showed more frequent airway inflammation, greater bronchial responsiveness and higher likelihood of developing asthma and rhinitis than subjects with IgE sensitization only to cat extract. Conclusions: The presence of IgE antibodies against food allergens was independently associated with airway and systemic inflammation. Both aeroallergens and food allergens should be examined in order to understand the signaling of local and systemic inflammation in asthma. Prevalence of IgE sensitization to food decreased in adults to a larger extent than IgE sensitization against aeroallergens. Measurement of IgE sensitization to cat allergen components appears to have a higher clinical value than extract-based measurement

Purpose: Asthma can have significant adverse effects on the health and quality of life of children, and the prevalence of this condition continues to rise. Breastfeeding may protect against asthma, but some uncertainty remains. The purpose of this study was to further examine the relationship between breastfeeding and the risk of developing asthma in early childhood. Methods: Data were collected from the State and Local Area Integrated Telephone Survey: National Survey of Children's Health, 2003. The study population consisted of 33,315 children ages 0 to 5 years. Prevalence rates of asthma and breastfeeding ,were calculated, as were crude and Mantel-Haenszel summary odds ratios for breastfeeding and other potential confounders including age, race, education, poverty, and tobacco use. Logistic regression models were used to estimate odds ratios and 95% confidence intervals aRer adjustment for these confounders. Results: Breastfeeding (never vs. ever) was significantly associated with an increased odds ratio of asthma among the children surveyed (POR = 1.18, 95% CI = 1.04, 1.34). In addition, children with asthma had a slightly lower mean duration of breastfeeding than did children without asthma. However, a significant trend of increasing odds ratios with increasing duration of breastfeeding was not found. It therefore appears that the act of ever breastfeeding, regardless of duration, exerts some protective effect against the development of asthma in early childhood. Conclusions: Never breastfeeding was found to be significantly associated with the development of asthma in early childhood. Age, race, education, poverty level, and tobacco use were also implicated in this association. While further research is needed to fully determine the effectiveness of breastfeeding in the primary prevention of asthma, public health efforts should focus on promoting breastfeeding as it has the potential improve the overall health of children.

Avaliamos os mecanismos regulatórios desencadeados pela imunização materna na resposta alérgica da prole OVA. A imunização materna com OVA promoveu alterações como o aumento da expressão dos receptores FcgRIIb nos linfócitos B esplênicos dos neonatos aos 3 dias de idade (d.i.), o que se manteve até os 20 d.i. Com a imunização das proles no período neonatal a imunização materna inibiu a produção de anticorpos IgE anti-OVA. Além disso, foi observado na população de linfócitos B da prole o aumento da expressão dos receptores FcgRIIb e CD44. A transferência passiva de IgG de mães imunes no pós-natal mostrou uma inibição da produção de IgE, e no período pré-natal foi capaz de reduzir a expressão das moléculas CD40 e CD23 nos linfócitos B e a secreção de IL-10 em linfócitos T CD4 na prole no período neonatal. As evidências mostram que a imunização pré-concepcional com OVA induz mecanismos que regulam a resposta IgE da prole imunizada no período neonatal, o que foi parcialmente observado com a transferência passiva de anticorpos IgG durante o período pré e pós-natal. / To evaluate the regulatory mechanisms triggered by maternal immunization in the immune response of the offspring, the effect of preconceptional immunization with OVA was evaluated. Maternal immunization with OVA led to early alteration with increased expression of FcgRIIb in B lymphocytes from 3 days old pups. Offspring from immune mother showed diminished percentage of CD4 T cells IL-4+. The immunization of offspring during neonatal period showed that maternal immunization inhibits the production of anti-OVA IgE antibodies. The evaluation of CD4 T cell population revealed diminished IL-4+ cells. Passive IgG transfer from immune mother during neonatal period showed inhibition in the IgE synthesis, during pregnancy showed capacity to reduce the expression of CD40 molecules in B cells from neonatal pups. These evidences show that maternal OVA immunization down regulates the IgE response of offspring including phenotypic and functional alteration in B and CD4 T cells. These alterations were partially observed with IgG transfer during pregnancy or after birth.

Durante o repasto sanguíneo, fêmeas do mosquito Aedes aegypti inoculam saliva na pele de seu hospedeiro vertebrado, modulando suas funções imunológicas e facilitando a transmissão de doenças. Alguns componentes presentes na saliva de Ae. aegypti já foram descritos e caracterizados como alérgenos. No presente estudo, desenvolvemos um modelo de inflamação alérgica em camundongos expostos naturalmente às picadas de mosquitos Ae. aegypti. Nossos dados revelam que a sensibilização e posterior desafio com componentes salivares desse mosquito induz inflamação pulmonar com aumento no número de eosinófilos, células T CD4+, CD19+ e citocinas IL-4 e IL5 no lavado broncoalveolar, produção de muco e colágeno no pulmão, anticorpos IgE total e IgG1 e IgG2a específicos no soro, porém, sem alterações no padrão respiratório e reatividade das vias aéreas. Em conjunto, os resultados gerados neste trabalho confirmam o desenvolvimento de uma resposta alérgica diferente dos modelos clássicos dessa área com a presença de elementos que sugerem tanto um perfil Th1 quanto Th2 de resposta. / During blood feeding, Aedes aegypti female mosquitoes inoculate saliva into the skin of their vertebrate hosts, modulating their immune functions and facilitating disease transmission. Some components presents in the saliva of Ae. aegypti have been described and characterized as allergens. In this study, we developed a model of allergic inflammation in mice naturally exposed to Ae. aegypti mosquito bites. Our data show that the sensitization and subsequent challenged with this mosquito salivary components induces lung inflammation with increased numbers of eosinophils, T CD4+ and CD19+ cells and IL-4 and IL-5 in bronchoalveolar lavage, mucus and collagen production in lung, total IgE and specific IgG1 and IgG2a antibodies in serum, but no changes in breathing pattern and in the airways reactivity. Together, the results generated in the present study confirm the development of an allergic response that is different from classic models of this area with the presence of components which suggest both Th1 and Th2 responses.

As hipersensibilidades do tipo I são caracterizadas por um grupo heterogêneo de manifestações clínicas que atingem mais de 30% da população mundial. Novas reatividades a alérgenos regionais brasileiros têm sido descritas e muitas fontes ainda não são totalmente conhecidas. Dentre os alérgenos mais prevalentes estão os venenos de insetos. A vespa regional Polybia paulista (Hymenoptera vespidae) é endêmica no sudeste do Brasil e é responsável por acidentes graves, causando reações alérgicas que podem levar ao choque anafilático. Alguns componentes dos venenos de vespas de diferentes espécies apresentam mimetismo molecular ou biológico, podendo gerar reação imunológica cruzada, mas muitas vezes não são os responsáveis pelo desencadeamento da resposta alérgica. Isto ocasiona falha no diagnóstico e consequentemente no tratamento indicado, a imunoterapia alérgeno-específica. Diante desses fatos e do grande número de pacientes que procuram o Serviço de Imunologia Clínica e Alergia do Hospital das Clínicas de São Paulo (HCFMUSP) com manifestações clínicas de alergias a ferroadas de insetos, foi desenvolvida uma sistemática de investigação clínica e laboratorial, com ênfase na abordagem proteômica, para identificar e caracterizar físico-química e imunologicamente novos alérgenos do veneno da vespa Polybia paulista e estudar potenciais reatividades cruzadas com alérgenos já conhecidos. Vinte e um pacientes com história de anafilaxia a venenos de vespa foram selecionados para participar do estudo. Foram realizados testes cutâneos e in vitro com veneno de Polistes spp. disponível comercialmente e com o veneno da Polybia paulista, produzido seguindo o protocolo padronizado anteriormente. Os resultados mostraram que a maioria dos pacientes apresentam IgE específica para os dois venenos com maior reatividade ao veneno de Polybia e que o padrão de proteínas reconhecidas entre os dois venenos é diferente, evidenciando a necessidade de veneno de Polybia paulista na prática clínica nas regiões cuja vespa está presente. Foram identificadas mais de 2000 proteínas no extrato total do veneno de Polybia paulista e algumas proteínas alergênicas ainda não descritas. Dentre elas foi identificada uma nova isoforma ao antígeno 5 da vespa Polybia scutellaris relatada como hipoalergênica. A molécula foi produzida na forma recombinante com conformação adequada, pela primeira vez em E. coli. O alérgeno, registrado na IUIS como Poly p 5, foi reconhecido por IgEs no soro dos pacientes testados e apresenta reatividade cruzada com outros antígenos 5 homólogos. Testes de desgranulação de basófilos em linhagem celular de ratos mostraram que o Poly p 5 induziu pouca desgranulação, indicando seu potencial hipoalergênico / Type I hypersensitivity is characterized by heterogeneous clinical manifestations and specialists estimate that today around 30% of the general population suffers from an allergic disease. New allergens are being reported and some sources are not yet identified. Insect venoms are amongst the most prevalent allergen sources. The social wasp Polybia paulista (Hymenoptera vespidae) is endemic in the southeastern of Brazil and is responsible for serious accidents due to their venomous stings, causing allergic reactions that can lead to anaphylactic shock. Several components presenting molecular or biological mimicry can be found in different species of wasps and lead to a cross-immunological reaction but they are not always responsible for the allergic manifestations. Therefore, diagnostic and consequently immunotherapy is unsuccessful, since specific allergen identification is crucial. Considering the high number of patients attended at the \"Serviço de Imunologia Clínica e Alergia do Hospital das Clínicas de São Paulo\" with clinical manifestations of allergies not yet determined or barely studied, an approach involving a systematic clinical, laboratorial and investigative practice through a proteomic analysis was created to identify and characterize new allergens of Polybia paulista venom. Twenty-one patients with clinical history of anaphylaxis to Hymenoptera venoms were selected for this work. Cutaneous and in vitro tests were performed using Polistes venom commercially available as well as Polybia paulista venom, produced following a published protocol. The results shows that the majority of the patients has specific IgE for both venoms with biggest reactivity to Polybia paulista venom and the protein profile recognized in these venoms is different. More than 2000 proteins were identified in the whole venom extract of Polybia paulista and some of the allergenic proteins are not yet described in this venom. Among them, a new isoform that is similar to antigen 5 from Polybia scutellaris, already known as hypoallergenic. The molecule was produced as a recombinant properly folded for the first time in E. coli. The allergen, registered at IUIS as Poly p 5, was recognized by IgEs in the sera of 50% of the patients tested and has cross-reactivity with other homologs of antigen 5. Basophil degranulation tests in rat lineage cells showed that Poly p 5 induced little degranulation, indicating the hypoallergenic potential of this molecule

Diversos estudos em modelo animal indicam claramente que a transferência materna de IgG alérgeno-específica previne a resposta alérgica na prole. A presença de alérgeno no leite também mostrou proteger a prole por induzir tolerância oral. Neste estudo, analisamos em humanos a transferência materna de alérgenos dos ácaros Der p e Blo t e anticorpos específicos durante a gestação e amamentação em uma coorte de 91 amostras pareadas de soro materno, colostro e cordão umbilical. Demonstramos que tanto o Der p 1 como Blo t 5 podem estar presentes em amostras de colostro e cordão umbilical e identificamos a atopia materna como fator crítico para o aumento de IgG específica nestes compartimentos. No cordão umbilical, a maior diferença entre mães atópicas e não atópicas foi para a IgG4. A concentração de Der p 1 e Blo t 5 não apresentou correlação com o estado atópico materno ou concentração de anticorpos específicos. No colostro, apenas para o alérgeno Der p 1 a concentração foi maior em mães atópicas e apresentou correlação com os níveis de anticorpos IgG específicos. / Experimental data in rodents indicate that maternal allergen-specific IgG prevents allergic sensitization in the progeny. Airborne allergens in maternal milk also protects breastfeed mice by oral tolerance induction. In this study, we assessed in humans whether Der p and Blo t allergens and specific antibodies were transferred during pregnancy and breastfeeding in a cohort of 91 paired samples of maternal blood, colostrums and umbilical cord blood. Our study indicates that Der p 1 and Blo t 5 can be found in both, cord blood and colostrums, and identifies maternal atopy as a critical factor for increased levels of allergen-specific IgG in these compartments. In the cord blood the biggest difference between atopic and non atopic mothers was observed for IgG4. Der p 1 and Blo t 5 concentrations were not correlated with maternal atopic status nor specific-antibodies levels. In colostrums, only the concentration of Der p 1 was higher in atopic mothers and correlated with colostrums specific-IgG levels.