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- Avaliação e comparação de extractos de látex comercialmente disponíveis em Portugal para testes cutâneos por picadaPublication . Gabriel, Marta Sofia da Fonseca; Tomaz, Cândida Ascensão TeixeiraA prevalência da alergia ao látex de borracha natural (LBN) tem-se revelado reduzida na população em geral, contudo está descrita como permanecendo um importante problema de saúde em subpopulações consideradas de risco, como é o caso particular dos trabalhadores da área da saúde (TAS) e doentes com espinha bífida (DEB). A principal meta para a prevenção de reacções alérgicas severas baseia-se na correcta e precoce identificação de indivíduos alérgicos e sensibilizados, com propensão para sofrer sintomas após exposição repetida a produtos de látex. De entre as metodologias de diagnóstico disponíveis, os testes cutâneos por picada (TCP) constituem o método mais fiável para o diagnóstico da sensibilização às proteínas do látex, contudo estes testes são grandemente afectados pela qualidade dos reagentes alergénicos aplicados. Actualmente, existem vários extractos de LBN comercialmente disponíveis em Portugal, produzidos por diferentes fabricantes, cuja variabilidade no conteúdo proteico e alergénico pode constituir um factor de influência nos resultados dos TCP. De facto, apesar de ser do conhecimento geral que tais preparações alergénicas são passíveis de apresentar grande heterogeneidade, até à data poucos são os estudos publicados que visaram a sua avaliação, pelo que tais variações não se encontram, actualmente, bem documentadas. Tendo por base estes conhecimentos, estabeleceu-se como objectivo deste projecto, a avaliação do nível real de variabilidade entre extractos comercialmente disponíveis de entidades fabricantes distintas e a análise do efeito de tais variações de conteúdo, na eficiência de indução de reactividade cutânea por TCP. Para tal, foram utilizados extractos actualmente comercializados em Portugal, fornecidos por sete fabricantes distintos. Todos os extractos foram analisados para o seu conteúdo proteico por Bradford e electroforese em gel de poliacrilamida - dodecil sulfato de sódio (SDS-PAGE). Procedeu-se também à quantificação dos quatro alergénios major Hev b 1, Hev b 3, Hev b 5 e Hev b 6.02 utilizando kits comerciais de ensaio imunoenzimático (EIA). Por fim, os extractos foram analisados quanto à sua actividade alergénica in vivo e in vitro, por TCP e microarrays, respectivamente. Os resultados obtidos demonstraram marcada variabilidade no conteúdo em proteína total dos extractos de diferentes fabricantes, sendo que os respectivos valores variaram de teores proteicos inferiores a 8,0 a 526,5 μg/mL, o que corresponde a um surpreendente nível de heterogeneidade de aproximadamente 65 vezes. Estas diferenças acentuadas de conteúdo proteico entre extractos foram qualitativamente confirmadas pelos resultados de SDS-PAGE, sendo obtidos perfis electroforéticos completamente distintos. No que respeita à quantificação dos quatro alergénios major, esta também se demonstrou bastante variável, sendo que em alguns dos extractos não foram detectadas quantidades mensuráveis de Hev b 3 e Hev b 5. Ambos os resultados de TCP e dos ensaios de microarrays demonstraram diferenças notáveis na capacidade alergénica entre os extractos de diferentes fabricantes. Particularmente, pelos resultados de TCP foi claramente observado que na maior parte dos doentes, a variabilidade de eficiência de diagnóstico foi tão acentuada que houve extractos que induziram respostas de TCP positivas e outros que não foram capazes de induzir qualquer reactividade cutânea, num mesmo doente alérgico. Pelas evidências demonstradas ao longo deste trabalho pode concluir-se que os extractos de látex de diferentes fabricantes, comercialmente disponíveis, apresentam uma heterogeneidade de conteúdo proteico e alergénico bastante marcada, o que certamente influencia as respectivas eficiências de diagnóstico, em consequência de diferentes capacidades alergénicas. Assim, sugere-se a necessidade do estabelecimento de metodologias de uniformização e melhoramento dos extractos alergénicos actualmente disponíveis para o diagnóstico da alergia ao látex.
- Evaluation of Alt a 1 as specific marker of exposure to fungal allergenic sources and clinical relevance of a manganese-dependent superoxide dismutase and a serine protease as new Alternaria alternata allergensPublication . Gabriel, Marta Sofia da Fonseca; Tomaz, Cândida Ascensão Teixeira; Marcos, Jorge MartinezAllergic diseases are considered to be one of the epidemics of the century, affecting approximately one-third of the general population. Classically, among the allergenic sources able to induce IgE-mediated reactions, fungi have been one of the less favored areas of study. It has been demonstrated that sensitization to fungal aeroallergens, particularly from Alternaria alternata, represents an unequivocal risk factor for the development, persistence and severity of asthma. Thus, the better understanding and management of fungal allergy, namely by improvements in the actual assessment and diagnostic approaches are needed. Regarding the assessment of fungal exposure, actual methodologies are generally laborious and time-consuming making difficult to establish or exclude a fungal contamination and potential associations with allergic disease. In terms of diagnosis, the main difficulty arises from the high number of patients that are apparently sensitized to multiple fungi in which the identification of primary cause of sensitization is complex. Because A. alternata is one of the most abundant and potent source of airborne allergens, the panel of allergenic components produced by this fungal specie, seems to be a very relevant target of study. Among the several allergens described in this mold, Alt a 1 has been demonstrated to be the most important, sensitizing approximately 80% of A. alternata allergic patients. For this reason, Alt a 1 has been used as the diagnostic marker of genuine sensitization to A. alternata. However, given the complex A. alternata sensitization data, which shows a significant level of polysensitization, due to co-sensitization and/or cross-reactivity to several other phylogenetically related and non-related molds, other allergenic A. alternata components should be studied to explain the whole broad range of reported clinical observations. In the last years, componentresolved diagnosis (CRD) has been shown to be a valuable tool to elucidate clinical observations and to differentiate between cases of co-sensitization and cross-reactivity. Nevertheless, the actual available panel of A. alternata allergens appears to not be enough for achieving an accurate diagnosis and prognosis of sensitization to this mold. Considering the above mentioned facts, the major aims of this work were, on one hand to develop an approach to specifically detect Alternaria and related species by using the A. alternata major allergen, Alt a 1, as a specie-specific molecular marker. And, on other hand, to characterize two new cross-reactive A. alternata allergens belonging to the manganesedependent superoxide dismutase (MnSOD) and serine protease (SP) protein families and to study their role in A. alternata sensitization. The strategies used to accomplish both aims made use of phylogenetic relationships among fungal species that share A. alternata allergen homologues. First, investigating conservation of the genes encoding for Alt a 1 and its homologues which allowed the design of a set of primers in the conserved immunologically relevant Alt a 1 coding sequence region. This primer set, together with a pair of primers to amplify the complete Alt a 1 encoding gene, was applied in a polymerase chain reaction (PCR)-based system. This approach yielded two rapid, sensitive and specific methodologies with high potential to be applied both for the detection of Alt a 1 and Alt a 1 homologues and for specific identification of the existence of contamination by the very close taxonomically related species A. alternata and A. tenuissima. The investigation of conservation of the genes encoding for A. alternata protein homologues, was also employed to design primers in the invariant region of fungal MnSOD and SP nucleotide sequences available in public databases. The aforementioned primers along with rapid amplification of cDNA ends (RACE) and sequencing assays allowed for the isolation of the full-length cDNA encoding for A. alternata MnSOD and SP. Both proteins were then produced as recombinant proteins in E. coli and evaluated for IgE immunoreactivity using a comprehensive panel of sera from patients clinically labeled as to be sensitized to A. alternata. Immunoblotting analysis showed that IgE antibodies from A. alternata-sensitized patients bound to recombinant A. alternata MnSOD and SP with prevalence of 11.5% (n=61) and 10.2% (n=59), respectively. These results were reported to the World Health Organization and International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub- Committee, and both proteins, respectively named Alt a 14 and Alt a 15, are currently included in the official A. alternata allergen list. By performing immunoblotting inhibition assays it was demonstrated that Alt a 14 and Alt a 15 are able to mediate IgE cross-reactivity with similar homologues allergens from other important allergenic fungal species, such as A. fumigatus and C. lunata, respectively. Furthermore, evidence of reactivity of Alt a 14 to IgE of Allergic Bronchopulmonary Aspergillosis (ABPA)-diagnosed patients was found, thus indicating that sensitization to this A. alternata allergen could play important implications in the development of ABPA. On the other hand, sensitization to Alt a 15 was shown to be restricted to apparently poly-sensitized patients and to justify some cases of sensitization to A. alternata in which there is no evidence of Alt a 1 sensitization. A homologue to Alt a 14, together with a manitol desidrogenase (MtDH), of the edible mushroom A. bisporus were identified to induce an anaphylactic shock reaction in a patient who presented a previous history of respiratory allergic symptoms associated to mold aeroallergens. These results were useful in proving that although minor allergens, A. alternata cross-reacting proteins may be the primary cause of strong allergic reactions to various other allergenic sources, such as mushrooms. Overall, in this work we successfully developed a specific and sensitive PCR method based on the amplification of regions of the gene encoding for the allergenic Alt a 1 and Alt a 1 homologues which it is intended to be applied for environmental monitoring as well as for quality and biosecurity control of food stuffs. Moreover, cloning and characterization of Alt a 14 and Alt a 15 as minor allergens of A. alternata that can trigger cross-reactive IgE response with other important and prevalent allergenic fungal species were also accomplished. The availability of these allergens as recombinant molecules suitable for application in a molecule-based diagnostic approach to fungal allergy can improve the diagnostic process prognosis of clinical manifestations and potential cross-reactivities. Furthermore, this can guide to a more effective specific immunotherapy using a single or a few allergenic molecules. Hence, this work provided valuable findings that can contribute to improving the accuracy of assessment of allergen exposure, diagnosis and management of IgE-mediated fungal diseases.