Pollinosis is sub-diagnosed and rarely studied in tropical countries. Cashew tree pollen has been reported as an allergen source although the knowledge of its immunoglobulin E (IgE)-reactive molecules is lacking. Therefore, this work aimed to identify IgE-reactive molecules and provide a proteomic profile of this pollen. From the 830 proteins identified by shotgun analysis, 163 were annotated to gene ontology, and a list of 39 proteins filtered for high confidence was submitted to the Allfam database where nine were assigned to allergenic families. Thus, 12 patients from the northeast of Brazil with persistent allergic rhinitis and aggravation of symptoms during cashew flowering season were selected. Using a 2D-based approach, we identified 20 IgE-reactive proteins, four already recognized as allergens, including a homolog of the birch isoflavone-reductase (Bet v 6). IgE-reactivity against the extract in native form was confirmed for five patients in ELISA, with three being positive for Bet v 6. Herein, we present a group of patients with rhinitis exposed to cashew tree pollen with the first description of IgE-binding proteins and a proteomic profile of the whole pollen. Cashew tree pollen is considered an important trigger of rhinitis symptoms in clinical practice in the northeast of Brazil, and the elucidation of its allergenic molecules can improve the diagnostics and treatment for allergic patients.

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Background: Delayed food anaphylaxis upon consumption of red meat is attributed to specific IgE-antibodies directed to galactose-α-1,3-galactose (α-Gal). Anaphylactic reactions may occur after ingestion of meat from different mammals, mainly beef and pork, but reactions to lamb, rabbit or horse have also been reported. In particular, pork kidney has been shown to trigger symptoms that were more severe and occurred within a shorter delay. The objective of the present study was the identification and characterization of pork kidney proteins carrying α-Gal carbohydrates and mediating delayed allergic reactions through specific IgE to α-Gal. Materials and methods: A cohort of 59 patients with specific IgE to α-Gal was screened by immunoblot for IgE-reactive proteins in pork kidney extract. Proteins were purified by affinity chromatography and identified by Edman sequencing and peptide mass fingerprinting. Isolated proteins were used in immunoassays using patient sera and α-Gal specific antibodies. Allergenicity was assayed in basophil activation and skin prick test. Results: Multiple IgE-binding proteins were detected in protein extracts of pork kidney by immunoblot using patient sera and an anti-α-Gal antibody. Reactive bands were located in the high molecular weight range of 100 to ≥200 kDa. Two major IgE-binding proteins were identified as porcine angiotensin I converting enzyme (ACE I) and aminopeptidase N (AP-N). IgE-binding to both proteins was lost by periodate treatment, resulting in oxidation of carbohydrates. Addition of α-Gal inhibited IgE-reactivity to both peptidases. Allergenicity was confirmed by activation of patient basophils and positive skin prick tests. Conclusions: Two IgE-reactive cell membrane peptidases carrying α-Gal epitopes were identified in pork kidney, a tissue which is known as potent inducer of red meat-induced anaphylaxis. Allergenicity and clinical relevance of these proteins were confirmed in patients with delayed anaphylaxis to red meat by skin prick test and basophil activation.

À minha orientadora Dra. Keity Souza Santos por me acolher como sua aluna , pela confiança, amizade e atenção. Ao meu grupo: Karine, Anne e Susanne, pela ajuda e amizade. Pessoas importantes sempre dispostas a ajudar: Eleni Arruda, Andreia Kuramoto e todos os colegas do LIM 60 e InCor. À Dra. Fabiane Pomiencinsky pela concepção da ideia para este projeto e pelo acompanhamento clínico dos pacientes deste estudo. Ao Dr. Fabio Fernandes Morato Castro pela concepção da ideia para este projeto, pelas discussões, esclarecimentos de dúvidas e contribuição científica. À Dra. Helen Andrade Arcuri pela contribuição na parte de bioinformática. Ao Prof. Dr. Jorge Kalil por me acolher como aluna do seu departamento. Aos pesquisadores da Universidade de Salzburg, Dra. Gabriele Gadermaier, Dr. Peter Briza e Dra. Fatima Ferreira pela colaboração, sem a qual este trabalho não seria possível. À CAPES e à FAPESP pelo apoio financeiro. Por último e mais importante, minha família. Aos meus pais, Meire e José Roberto, minha irmã, Viviane e meu noivo Lukas, pelo amor e apoio em todos os momentos. Normalização adotadaEsta dissertação está de acordo com as seguintes normas, em vigor no momento desta publicação:Referências: adaptado de "International Committee of Medical Journals Editors" (Vancouver).

UTMB. RATIONALE: Significant information exists on the sequences and structural details of the IgE based responses to peanut allergens. Much less work has been done to explain such a high degree of cross reactivity among various nuts. We have developed computational and experimental approaches to identify common epitopes that could contribute to cross-reactivity. METHODS: IgE and IgG4 immunanalysis of allergen proteins and peptides were performed using antibodies and sera from patients with confirmed peanut and tree nut allergy. The protein extracts of these samples were all normalized according to protein content and subjected to SDS-PAGE, Spot blot, microarray and western blot analysis with sera from multiple patients. Antibody or IgE-reactive bands were identified using mass spectroscopy. Potentially cross-reactive peptides were identified computationally and with peptide-microarray analysis. RESULTS: Previously unknown IgE and IgG4 epitope maps for multiple nut allergens were developed. IgE cross-reactivity was seen at both at protein and peptide levels of the different nuts. Several proteins that showed crossreactivity with various antibodies were observed, excised, and shown to be previously identified allergens in the corresponding nut extracts. Previously unidentified, strongly cross-reactive peptides and peptide motifs with various levels of sequence identity were found to be cross-reactive often in proteins that are not in the same protein families (Pfam). CONCLUSIONS: Extensive IgE cross-reactivity was seen between peanuts and tree nuts within the known major allergen protein families. However, previously unidentified cross-reactive proteins seem to also exist. Novel IgE epitopes and peptide motifs were identified that could account for cross reactivity.

Sublingual immunotherapy (SLIT) is used worldwide to treat house dust mites (HDM) allergy. Epitope specific immunotherapy with peptide vaccines is used far less, but it is of great interest in the treatment of allergic reactions, as it precludes the drawbacks of allergen extracts. The ideal peptide candidates would bind to IgG, blocking IgE-binding. To better elucidate IgE and IgG4 epitope profiles during SLIT, sequences of main allergens, Der p 1, 2, 5, 7, 10, 23 and Blo t 5, 6, 12, 13, were included in a 15-mer peptide microarray and tested against pooled sera from 10 patients pre- and post-1-year SLIT. All allergens were recognized to some extent by at least one antibody isotype and peptide diversity was higher post-1-year SLIT for both antibodies. IgE recognition diversity varied among allergens and timepoints without a clear tendency. Der p 10, a minor allergen in temperate regions, was the molecule with more IgE-peptides and might be a major allergen in populations highly exposed to helminths and cockroaches, such as Brazil. SLIT-induced IgG4 epitopes were directed against several, but not all, IgE-binding regions. We selected a set of peptides that recognized only IgG4 or were able to induce increased ratios of IgG4:IgE after one year of treatment and might be potential targets for vaccines.

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