Study of antigens specificity and its application for the immunodiagnosis of angiostrongyliasis

Submitted by Caroline Xavier ([email protected]) on 2017-06-30T17:21:25Z No. of bitstreams: 1 TES_BIANCA_BARBIERI_COGNATO_COMPLETO.pdf: 2150300 bytes, checksum: 6c0ebf29cb36a3ae7a825cf9c0060dc4 (MD5)Made available in DSpace on 2017-06-30T17:21:25Z (GMT). No. of bitstreams: 1 TES_BIANCA_BARBIERI_COGNATO_COMPLETO.pdf: 2150300 bytes, checksum: 6c0ebf29cb36a3ae7a825cf9c0060dc4 (MD5) Previous issue date: 2017-03-21Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPESConselho Nacional de Pesquisa e Desenvolvimento Cient?fico e Tecnol?gico - CNPqTwo nematode species belonging to the Metastrongyloidea superfamily are capable to produce disease in humans: Angiostrongylus cantonensis and Angiostrongylus costaricensis. Both are rodent parasites and human infection is considered accidental. Eosinophilic meningoencephalitis, caused by A. cantonensis, raises concern due to the expanding number of cases and geographical area of occurrence. Molecular and immunological methods for the diagnosis are crucial, however, after many studies with different antigenic molecules, has a specific and sensitive test to discriminate the angiostrongyliasis to others parasitoses is lacking. Cross-reactivity with other helminthes, which may cause similar symptoms, and eosinophilic meningitis, has been a problem for the satisfactory performance of specificity in serological tests. In order to improve the diagnosis of angiostrongyliasis, a comparative analysis of protein recognition from different extracts from A. cantonensis, Toxocara canis, Schistosoma mansoni and Strongyloides stercoralis against positive serum for Angiostrongylus.spp was performed. Through extraction kit, one-dimensional and two-dimensional electrophoresis, western blot and mass spectrometry, 149 proteins were identified. Among these, 34 were exclusive to A. cantonensis, COI being present only in the A. cantonensis extract, having no similarities with any other parasite compared in NCBI (nr database) and WormBase Database. Additionally, nine proteins were recognized by more than one parasite and extract, being important cross reactivity markers in parasitic infections. With the data obtained in this study, we suggest the use of the follow proteins as cross-reactivity markers: Galectin 1, HSPA-5 and Ifa1. In addition, immunogenic proteins of A. cantonensis ES-7, Lec-5 and 14-3-3, were recombinant expressed in two cell types, CHO and HEK. Their potential diagnostic values were verified by uni and bidimensional electrophoresis, western and dot blot, and N-glycosidase F (PNGase F) treatment using serum from patients infected with A. cantonensis, negative serum for parasites, and positive for other parasites. ES7 protein expressed in HEK and CHO cells and Lec-5 expressed in CHO were recognized only by Angiostrongylus-positive serum and not by negative control and specificity control in 2D and 1D tests, respectively. In the 2D analyzes Lec-5 showed a weak recognition with negative serum. However, the 14-3-3 protein didn?t show any specificity against A. cantonensis serum, since it was recognized by all tested sera. Antigen-antibody recognition was found to be dependent on the glycogenic portions, since when treated with N-glycosidase F (PNGase F), recognition between proteins and serum disappeared. The heterologous expression, using mammalian cells, as well as the identification of shared and/or specific molecules, may represent a promising source of antigens for the diagnosis of eosinophilic meningoencephalitis, and molecular diagnostic tests become necessary.Duas esp?cies de nemat?deos pertencentes a superfam?lia Metastrongyloidea, s?o capazes de produzir doen?a em humanos: Angiostrongylus cantonensis e Angiostrongylus costaricensis. Ambos s?o parasitas pr?prios de roedores e a infec??o humana ? considerada acidental. A meningite eosinof?lica, causada por A. cantonensis, tem gerado preocupa??o na comunidade cient?fica pela expans?o da ?rea geogr?fica de ocorr?ncia. M?todos moleculares e imunol?gicos para o diagn?stico desta infec??o s?o cruciais para o diagn?stico, entretanto, ap?s muitos estudos com diferentes mol?culas antig?nicas, at? hoje n?o foi desenvolvido um teste de diagn?stico que seja espec?fico e sens?vel o suficiente para discriminar as angiostrongil?ases de outras parasitoses. A reatividade cruzada tem sido o principal problema encontrado nos estudos j? desenvolvidos para este prop?sito. Com o objetivo de aprimorar o diagn?stico das angiostrongil?ases, foi realizado neste estudo an?lise comparativa do reconhecimento de prote?nas de diferentes extratos teciduais de A. cantonensis, Toxocara canis, Schistosoma mansoni e Strongyloides stercoralis contra soro positivo para Angiostrongylus spp Atrav?s de kit de extra??o, eletroforese unidimensional e bidimensional, western blot e espectrometria de massas foram identificadas 149 prote?nas. Dentre estas, 34 foram exclusivas para A. cantonensis, sendo que COI estava presente apenas no extrato de A. cantonensis n?o possuindo similaridades com nenhum outro parasito comparado no NCBI (nr database) e WormBase Database. Estas prote?nas podem ser consideradas promissoras como marcadores de reatividade humoral espec?fica para o parasito. Todavia, outras nove prote?nas foram reconhecidas por mais de um parasito em mais de um extrato testado, sendo importantes marcadores de reatividade cruzada em infec??es parasit?rias. Com os dados obtidos neste estudo, sugerimos como marcadores de reatividade cruzada o uso das prote?nas: Galectin 1, HSPA-5 e Ifa1. Al?m disso, prote?nas imunog?nicas de A. cantonensis ES-7, Lec-5 e 14-3-3, foram expressas de forma recombinante em dois tipos celulares, CHO e HEK, e o potencial uso diagn?stico destas prote?nas foi verificado atrav?s de eletroforese uni e bidimensional, western e dot blot, e tratamento por N-glycosidase F (PNGase F), utilizando soro positivo para Angiostrongylus spp., soro negativo e positivo para outras parasitoses. Prote?na ES-7 expressa em c?lulas HEK e CHO, e Lec-5 expressa em CHO foram reconhecidas apenas pelo soro positivo para Angiostrongylus spp. e n?o pelo soro negativo e controles de especificidade em testes 2D e 1D, respectivamente. J? nas an?lises em 2D, Lec-5 apresentou fraco reconhecimento com soro negativo. J? a prote?na 14-3-3 n?o apresentou nenhuma especificidade pelo soro de A. cantonensis, j? que foi reconhecida por todos os soros testados. O reconhecimento ant?geno-anticorpo se mostrou dependente das por??es glic?dicas, j? que quando tratadas com N-glycosidase F (PNGase F), o reconhecimento das prote?nas pelo soro desapareceu. A express?o heter?loga, utilizando c?lulas de mam?feros, assim como a identifica??o de mol?culas compartilhadas e/ou espec?ficas, podem representar uma promissora fonte de ant?genos para o diagn?stico da meningite eosinof?lica, requerendo aprimorados testes moleculares para seu diagn?stico

Estudo do compartilhamento de ant?genos entre Angiostrongylus spp. e outros helmintos

Made available in DSpace on 2015-04-14T13:09:38Z (GMT). No. of bitstreams: 1 448197.pdf: 1838361 bytes, checksum: 7924b0b15d7d6da94d6a3c9d188d1048 (MD5) Previous issue date: 2013-02-28Two species of parasites nematode of Angiostrongylidae family, genus Angiostrongylus intra-arterial localization are capable of human desease production: Angiostrongylus costaricensis and Angiostrongylus cantonensis. Both species are rodent s parasites and the human infection is considered incidental. On humans, A. cantonensis cause eosinophilic meningitis and A. costaricensis cause abdominal angiostrongiliase. Since there is no elimination of parasitic forms in human infection, diagnosis becomes difficult and molecular methods are necessary. After years of discovery of angiostrongil?ases, there are still many efforts in the study and development of a specific and sensitive diagnostic test capable of discriminating Angiostrongyliases of the other parasites. In this context, cross-reactivity becomes a problem in specificity of serologic tests. The main objective of this study was to analyze the sharing of antigens between Angiostrongylus spp. and parasites Strongyloides spp., Fasciola hepatica, Ascaris lumbricoides, Hymenolepis diminuta and Toxocara canis, and identify these molecules shared. To obtain antigens, some rats were captures and their feces analyzed by the method of Baerman addition to being seeded Agar Plates. Were obtained larvae of Strongyloides spp. and Angiostrongylus spp. In pulmonary arteries were found 11 female worms and 2 male worms of A. cantonensis, which led to the first report of the occurrence of this parasite in Rio Grande do Sul. In the small intestine was obtained a Strongyloides spp worm and analysis of small intestine were obtained Hymenolepis diminuta worms. From the adult worms of parasites were obtained antigens used in this study. Furthermore, we used antigens of allergens such as peanut, tomato, strawberry and pollen. To identify shared antigens, proteins were separated by one and two dimensional electrophoresis technique assayed by Western blot using sera of patients with angiostrongyliases. All antigens were recognized by sera from patients infected with angiostrongyliases, being a total of fourteen bands identified as being immunogenic. The fourteen bands were cut out of polyacrylamide gels and analyzed by mass spectrometry. The proteins identified were: Phosphoenolpyruvate carboxykinase, heat shock protein 70 (HSP70), DAPPUDRAFT hypothetical protein, 60 kDa Chaperonin 5, Glyceraldehyde-3-phosphate dehydrogenase, sigma class GST Chain A, Glucose-6-phosphate isomerase, pyruvate dehydrogenase subunit E1, Glutamate dehydrogenase 2, arachin Ahy-1, Full = Allergen Ara h 1, clone P41B, Gly1. The data generated in this study show that there is sharing of antigens between organisms of different taxonomic groups, but also with allergens tested. Moreover, the description and analysis of shared molecular components can help in understanding the evolutionary history and phylogeny of these organisms.Duas esp?cies de parasitos nemat?deos da fam?lia Angiostrongylidae, do g?nero Angiostrongylus de localiza??o intra-arterial s?o capazes de produzir doen?a em humanos: Angiostrongylus costaricensis e Angiostrongylus cantonensis. Ambas as esp?cies s?o parasitos pr?prios de roedores e a infec??o humana ? considerada acidental. No homem, A. cantonensis ? o causador da meningite eosinof?lica e A. costaricensis causador da angiostrongil?ase abdominal. Como n?o h? elimina??o de formas parasit?rias na infec??o humana, o diagn?stico se torna dif?cil e m?todos moleculares se tornam necess?rios. Depois de anos da descoberta das angiostrongil?ases, ainda h? muitos esfor?os no estudo e desenvolvimento de um teste de diagn?stico espec?fico e sens?vel capaz de discriminar as Angiostrongil?ases de outras parasitoses. Neste contexto, a reatividade cruzada se torna um problema na especificidade de testes sorol?gicos. O objetivo principal deste trabalho foi analisar o compartilhamento de ant?genos entre Angiostrongylus spp. e os parasitos Strongyloides spp., Fasciola hepatica, Ascaris lumbricoides, Hymenolepis diminuta e Toxocara canis, assim como identificar essas mol?culas compartilhadas. Para obten??o de ant?genos, algumas ratazanas foram capturas e suas fezes analisadas atrav?s do m?todo de Baerman al?m de terem sido semeadas em Placas de ?gar. Foram obtidas larvas de Strongyloides spp. e Angiostrongylus spp. Nas art?rias pulmonares de uma ratazana foram encontrados 11 vermes f?meas e 2 vermes machos de A. cantonensis, que deu origem ao primeiro relato da ocorr?ncia deste parasito no Rio Grande do Sul. No intestino delgado foi obtido um verme de Strongyloides spp e da an?lise do intestino delgado foram obtidos vermes de Hymenolepis diminuta. A partir dos vermes adultos dos parasitos, foram obtidos os ant?genos utilizados neste trabalho. Al?m disso, foram utilizados ant?genos de alergenos como o amendoim, tomate, p?len e morango. Para identifica??o dos ant?genos compartilhados, as prote?nas foram separadas por eletroforese uni e bidimensional ensaiadas pela t?cnica de Western-Blot utilizando-se soro de pacientes com angiostrongil?ases. Todos os ant?genos foram reconhecidos pelo soro de pacientes infectados com angiostrongil?ases, sendo um total de quatorze bandas identificadas como imunog?nicas. As quartorze bandas foram recortadas dos g?is de poliacrilamida e analisadas por espectrometria de massas. As prote?nas identificadas foram: Fosfoenolpiruvato carboxiquinase, Prote?na de choque t?rmico 70 (HSP70), Prote?na hipot?tica DAPPUDRAFT, 60 kDa Chaperonina 5, Gliceralde?do-3-fosfato-desidrogenase, Cadeia A sigma classe GST, Glucose-6-fosfato isomerase, Piruvato desidrogenase subunidade E1, Glutamato desidrogenase 2 , arachin Ahy-1, Full=Allergen Ara h 1, clone P41B , Gly1. Os dados gerados no presente trabalho demonstram que h? compartilhamento de ant?genos entre organismos de diferentes grupos taxon?micos, como tamb?m com os alergenos testados. Al?m disso, a descri??o e a an?lise de componentes moleculares compartilhados podem ajudar na compreens?o da hist?ria evolutiva e da filogenia destes organismos