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Universidade Federal Do Amazonas Instituto De Ciências Biológicas Programa Multi-Institucional De Pós-Graduação Em Biotecnologia UNIVERSIDADE FEDERAL DO AMAZONAS INSTITUTO DE CIÊNCIAS BIOLÓGICAS PROGRAMA MULTI-INSTITUCIONAL DE PÓS-GRADUAÇÃO EM BIOTECNOLOGIA JULIANA LUIZA VARJÃO LAMEIRAS PRODUÇÃO DE SORO HIPERIMUNE PARA Potamotrygon motoro Müller & Henle, 1841 (CHONDRICHTHYES – POTAMOTRYGONINAE): VERIFICAÇÃO DA REAÇÃO-CRUZADA FRENTE ÀS PEÇONHAS DE OUTRAS ESPÉCIES DE ARRAIAS E DA NEUTRALIZAÇÃO DAS ATIVIDADES EDEMATOGÊNICA E MIOTÓXICA Manaus 2018 JULIANA LUIZA VARJÃO LAMEIRAS PRODUÇÃO DE SORO HIPERIMUNE PARA Potamotrygon motoro Müller & Henle, 1841 (CHONDRICHTHYES – POTAMOTRYGONINAE): VERIFICAÇÃO DA REAÇÃO-CRUZADA FRENTE ÀS PEÇONHAS DE OUTRAS ESPÉCIES DE ARRAIAS E DA NEUTRALIZAÇÃO DAS ATIVIDADES EDEMATOGÊNICA E MIOTÓXICA Tese apresentada à Universidade Federal do Amazonas como requisito para obtenção do título de Doutora pelo Programa Multi- institucional de Pós-graduação em Biotecnologia. Área de concentração: Biotecnologia para Saúde. Orientadora: Professora Dra. Maria Cristina dos Santos – UFAM Coorientador: Professor Dr. Oscar Tadeu Ferreira da Costa – UFAM Manaus 2018 ' Poder Executivo Ministerio da Educa�ao Universidade Federal do Amazonas Programa Multi-lnstitucionalde P6s-Graduacao em Biotecnologia 226a. ATADEDEFESADETESE No dia 27 de mar90 (ter9a-feira) de 2018, as 9hs, 11a sala de aula do Bloco ''G'', Setor Sul - UFAM. Juliana Luiza Varjao Lameiras defendeu sua Tese de Doutorado intitulada ''Produ�ao de Soro Hiperimune para Potamotrygon motoro Miiller & Henle, 1841 (Chondrichthyes - potamotrygoninae): verifica�ao da rea'rao-cruzada frente as pe'ronhas de outras especies de arraias e da neutraliza�ao das atividades edematogenica e miotoxica. '' Banca de Examinadores: Membros Parecer AIssina tura I I / ! --. Aprovada (f) Assinatura: ;· c:?M· <.... � � (__ _C, ..._,·, .\ f Prof a. Dra. Maria Cristina dos Santos - (Presidente) Reprovada ( ) CPF: 1.0'61J.f4 ( . t 7 C f ,_ o 2.. I \ .. Aprovada -�- Assinatufa: ���· i Prof. Dr. Wuelton Marcelo Monteiro- FMT/ AM Reprovada ( ) CPF: Cl.\ J . �\c\:Q . 1- �t1 - C E: .,,-- "'· ' Aprovada (. .>") Assinatura: \ e';,_fy(/,C��L� Prof. Dr. Jose Fernando Marques Barcellos- UFAM Reprovada ( ) CPF: ,, \e),0;>.f \ !I,,( b� J-.,,.,...�5 t J Aprovada (><') Assinatur:_ j� · .. Profa. Dra. Tatiana Nayara Liborio Kimura - UFAM Reprovada ( CPF: ) 6��- .... G':!J,r3 _· ::1'3, _ _ O--c:1Q Aprovada (xJ Assinatura: _, j.,� /F 7 l. - ) Ir; / J <_) I ;Lt) Prof. Dr. Luiz Carlos de Lima Ferreira- FMT/AM Reprovada ( ) CPF: 0 3.1. I l 1�' l t '7" � Manaus, 27 de mar90 de 2018. ResultadoFinal: Aprovado( a)( X,) Reprovado( a)( ) • . • � .. .. • * .•. CoordenadorPPGBIOTEC/UFAM ...... ...................................... ....................... ...................................................... ... ..................... ................................... ... ...................................................................... ... ..... ....... ...................... ... ... .................. .............. ........................ .......... ......................... .................................. ................ .... .................. ... ..... ..... .. ... Av.Gal.RodrigoOtavioJordaoRamos,3000,Coroado,CampusUniversitario,BlocoMSetorSul,UFAM CEP:69077-000 - Manaus/AMTelefones:(92)99128-7527e-mail:[email protected] • Ficha Catalográfica Ficha catalográfica elaborada automaticamente de acordo com os dados fornecidos pelo(a) autor(a). Lameiras, Juliana Luiza Varjão L228p Produção de soro hiperimune para Potamotrygon motoro Müller & Henle, 1841 (Chondrichthyes – Potamotrygoninae): verificação da reação-cruzada frente às peçonhas de outras espécies de arraias e da neutralização das atividades edematogênica e miotóxica / Juliana Luiza Varjão Lameiras. 2018 96 f.: il. color; 31 cm. Orientadora: Maria Cristina dos Santos Coorientador: Oscar Tadeu Ferreira da Costa Tese (Doutorado em Biotecnologia) - Universidade Federal do Amazonas. 1. Potamotrygonídeos. 2. ferrão. 3. envenenamentos. 4. antiveneno. 5. soroneutralização. I. Santos, Maria Cristina dos II. Universidade Federal do Amazonas III. Título Aos meus pais, Bernardo e Rosanei, às minhas irmãs, Luciana e Bárbara, às minhas sobrinhas, Bia e Luiza ao meu esposo, Oscar, aos meus avós, Oscarino e Lídia, e às minhas tias, Lídia e Ana Maria, com muito carinho. Amo vocês! AGRADECIMENTOS • À minha orientadora e ‘Mãe Científica’ Professora Doutora Maria Cristina dos Santos, por todo o conhecimento e apoio e por ter um papel fundamental na minha formação profissional e pessoal. Agradeço pela paciência, pelos esclarecimentos nos meus momentos de dúvida e por ter confiado a mim a realização deste projeto. • Ao Coordenador Técnico Leonardo Matos e às técnicas Andressa Encarnação e Tania Sumita do Biotério Central do Instituto Nacional de Pesquisas da Amazônia (INPA) pela apoio técnico recebido no cuidado com os animais dos experimentos. • À valiosa assistência técnica da Sra. Erilene Carmo dos Santos a ao colega Evandro Pessoa nos protocolos histológicos. • À Professora Doutora Michella Lasmar, ao Doutor Allyson Guimarães, à Mestra Cacilda Satomi Yano e ao Professor Doutor Paulo Nogueira pelas valiosas contribuições. • Aos amigos do Laboratório de Imunoquímica, Rafael Sousa e Luana Santana pela ajuda em todos os momentos. • Às amigas Valéria Mourão, Carolina Scheffer, Ilia Gilmara e Thaís Pereira pela carinho e apoio. É muito bom ter a amizade de vocês! • Aos meus pais e melhores amigos, Bernardo Lameiras e Rosanei Varjão, que sempre estiveram ao meu lado. • Às minhas tias, Lídia Lameiras e Ana Maria Lameiras, que sempre me incentivaram a estudar e que nunca me deixaram desistir. • E, finalmente, agradeço ao meu esposo, Professor Doutor Oscar Costa, pela carinho, compreensão, dedicação, paciência, enfim, por tudo. Agradeço a ele, que me acompanhou nas coletas, nos experimentos e me apoiou tantas vezes, discutiu meus resultados comigo, ouviu minhas indagações e me ajudou tanto. Muito obrigada, meu amor! RESUMO Arraias pertencentes à subfamília Potamotrygoninae são peixes cartilaginosos que ocorrem nas bacias hidrográficas da América do Sul. Esses animais são providos de ferrões na base da cauda, próprios para a sua defesa. O ferrão é coberto por uma bainha tegumentar contendo glândulas mucosas e de veneno. As vítimas costumam sofrer envenenamentos por arraias geralmente quando pisam no dorso do animal, escondido sob a areia, na beira da praia. Por consequência, a arraia movimenta a cauda abruptamente e introduz o ferrão no indivíduo, causando uma laceração extremamente dolorida, que geralmente leva à necrose do tecido. Os acidentes costumam ocorrer em lugares distantes, isolados, geralmente sem atendimento médico adequado e, portanto, quase sempre não são notificados. Não há uma terapia definida e eficaz para os envenenamentos por arraias e os profissionais de saúde geralmente não recebem treinamento adequado para cuidar deste tipo de acidente. O tratamento é baseado no uso de analgésicos, anti-inflamatórios e antibióticos, pois ainda não existe antídoto específico. Embora os procedimentos médicos sejam capazes de controlar o quadro clínico dos pacientes, a neutralização das toxinas seria a conduta ideal para impedir a indução dos sinais e sintomas apresentados nos envenenamentos por arraias de água doce. No intuito de obter um imunobiológico capaz de neutralizar as principais atividades apresentadas pelas vítimas, o presente estudo teve por objetivo analisar a neutralização, por soros hiperimunes, das atividades edematogênica e miotóxica induzidas pela peçonha da arraia Potamotrygon motoro. Os soros foram obtidos em camundongos Balb/c por meio de imunização intradérmica usando-se ou o extrato do dorso ou do ferrão de P. motoro adsorvidos em adjuvante hidróxido de alumínio. Por análise de Dot-ELISA e Western Blot, foi possível verificar que o extrato do dorso foi tão imunogênico quanto o extrato do ferrão, induzindo a altos títulos de anticorpos, que reagiram com antígenos homólogos (da mesma espécie) e heterólogos (extrato do dorso ou do ferrão das espécies Paratrygon aiereba, Plesiotrygon iwamae, Potamotrygon orbignyi e Potamotrygon schroederi, todas de água doce), indicando que ambos os tipos de extrato poderiam ser usados na produção de antiveneno para tratar vítimas de envenenamento por arraias de água doce. A partir daí, foi verificado o poder dos soros hiperimunes em neutralizar, in vivo, as atividades edematogênica e miotóxica induzidas pelo extrato do ferrão de P. motoro por meio de dois protocolos: soroneutralização e vacinação. O primeiro consistiu em injetar a peçonha no músculo gastrocnêmio de camundongos Balb/c, e, em seguida, ministrar ou o soro antidorso ou o soro antiferrão via plexo venoso oftálmico. Já, o segundo consistiu em imunizar os camundongos com o extrato do dorso ou do ferrão adsorvidos em hidróxido de alumínio e desafiá-los com a peçonha do ferrão via intramuscular. Os gastrocnêmios foram removidos para análise histopatológica e esterológica e o sangue, coletado via plexo venoso oftálmico para dosagem de citocinas, PCR e CK. Os soros antidorso e antiferrão não neutralizaram a atividade edematogênica, mas os protocolos de soroneutralização e vacinação neutralizaram parcialmente o dano tecidual induzido pela peçonha do ferrão. A rabdomiólise sistêmica só foi neutralizada 100% nos animais vacinados com o extrato do ferrão. A análise das citocinas indicou que o protocolo de soroneutralização induziu a liberação de citocinas dos perfis Th1, Th2, Th17 e Treg, enquanto o protocolo de vacinação induziu à uma resposta Th1. Os resultados indicam que
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