Aspectos fisiológicos e imunológicos de Pythium insidiosum e atividade de antimicrobianos frente a Conidiobolus lamprauges
Autor(a) principal: | |
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Data de Publicação: | 2016 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | Manancial - Repositório Digital da UFSM |
Texto Completo: | http://repositorio.ufsm.br/handle/1/18079 |
Resumo: | Conidiobolomycosis and pythiosis are important infections that affect animals and humans, which presents difficulties in diagnosis and treatment. The conidiobolomycosis is caused by the fungi Conidiobolus coronatus, C. incongruus and C. lamprauges, affecting sheep, horses, dogs and humans; pythiosis is caused by the oomycete Pythium insidiosum and affects wild and domestic mammals, particularly horses and humans. There is no standard pharmacological approach to the treatment of infections and the surgical rescission is a method often needed in both cases. In this context, this thesis aimed to (a) determine the in vitro susceptibility of C. lamprauges against different antimicrobial drugs, as well as identify possible synergistic associations; (b) the use of standardized disk diffusion technique with minocycline as a screening method for the presumptive identification of P. insidiosum; (c) quantify, extract and evaluate the immunomodulatory potential of β-glucans from P. insidiosum in vitro in cell cultures in vitro and in mice; (d) develop an experimental pythiosis model in mice and evaluate the immune response to infection. As results we achieved: i) assessing the in vitro susceptibility of C. lamprauges isolated from sheep infections, there is reduced susceptibility to most antimicrobial drugs tested, with terbinafine the drug with improved activity (MIC <0.06- 0.5 μg/mL). The highest rates of synergism were observed with the combination of sulfamethoxazole and trimethoprim (100%) and between the terbinafine with azole antifungal drugs (71%); ii) the standardization of the use of disk diffusion technique with minocycline disks as a screening method for the presumptive identification of P. insidiosum was effective because there was no mycelial growth of P. insidiosum around the minocycline disk during the seven days of incubation, thus differentiating it from C. lamprauges and other true fungi; iii) β-glucan content from P. insidiosum enzymatically assessed was 23.09 ± 3.71% of total glucan, divided into α-glucans (4.10 ± 0.83%) and β-glucans (18.99 ± 3.59); iv) β-glucan extract from P. insidiosum yielded an extract containing 82% of β-glucans and 18% of residual amino acids and peptides, and the structural analysis revealed that it was a (1,3)(1,6)-β-glucan; This (1,3)(1,6)-β-glucan demonstrates a potential to stimulate the immune system in vitro, as observed by the increase in equine, human and mouse lymphocyte proliferation and ability to induce Th17 type response when administered to mice; vi) the use of BALB/c mice immunosuppressed with cyclophosphamide and hydrocortisone association was shown to be an effective experimental model for the study of pythiosis, with 60% of mortality; vii) The cytokine production observed during the development of the experimental pythiosis in mice was characterized by an expression of IL6, IL-10 and TNF-α, indicating an inflammatory response and possible suppression of the host immune response as responsible for the infection property. |
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Aspectos fisiológicos e imunológicos de Pythium insidiosum e atividade de antimicrobianos frente a Conidiobolus lampraugesPhysiological and immunological aspects of Pythium insidiosum and antimicrobial activity of antimicrobial agents against Conidiobolus lampraugesConidiobolus lampraugesSuscetibilidadeβ-glucanasImunoterapiaSusceptibilityβ-glucansImmunotherapyPythium insidiosumCNPQ::CIENCIAS DA SAUDE::FARMACIAConidiobolomycosis and pythiosis are important infections that affect animals and humans, which presents difficulties in diagnosis and treatment. The conidiobolomycosis is caused by the fungi Conidiobolus coronatus, C. incongruus and C. lamprauges, affecting sheep, horses, dogs and humans; pythiosis is caused by the oomycete Pythium insidiosum and affects wild and domestic mammals, particularly horses and humans. There is no standard pharmacological approach to the treatment of infections and the surgical rescission is a method often needed in both cases. In this context, this thesis aimed to (a) determine the in vitro susceptibility of C. lamprauges against different antimicrobial drugs, as well as identify possible synergistic associations; (b) the use of standardized disk diffusion technique with minocycline as a screening method for the presumptive identification of P. insidiosum; (c) quantify, extract and evaluate the immunomodulatory potential of β-glucans from P. insidiosum in vitro in cell cultures in vitro and in mice; (d) develop an experimental pythiosis model in mice and evaluate the immune response to infection. As results we achieved: i) assessing the in vitro susceptibility of C. lamprauges isolated from sheep infections, there is reduced susceptibility to most antimicrobial drugs tested, with terbinafine the drug with improved activity (MIC <0.06- 0.5 μg/mL). The highest rates of synergism were observed with the combination of sulfamethoxazole and trimethoprim (100%) and between the terbinafine with azole antifungal drugs (71%); ii) the standardization of the use of disk diffusion technique with minocycline disks as a screening method for the presumptive identification of P. insidiosum was effective because there was no mycelial growth of P. insidiosum around the minocycline disk during the seven days of incubation, thus differentiating it from C. lamprauges and other true fungi; iii) β-glucan content from P. insidiosum enzymatically assessed was 23.09 ± 3.71% of total glucan, divided into α-glucans (4.10 ± 0.83%) and β-glucans (18.99 ± 3.59); iv) β-glucan extract from P. insidiosum yielded an extract containing 82% of β-glucans and 18% of residual amino acids and peptides, and the structural analysis revealed that it was a (1,3)(1,6)-β-glucan; This (1,3)(1,6)-β-glucan demonstrates a potential to stimulate the immune system in vitro, as observed by the increase in equine, human and mouse lymphocyte proliferation and ability to induce Th17 type response when administered to mice; vi) the use of BALB/c mice immunosuppressed with cyclophosphamide and hydrocortisone association was shown to be an effective experimental model for the study of pythiosis, with 60% of mortality; vii) The cytokine production observed during the development of the experimental pythiosis in mice was characterized by an expression of IL6, IL-10 and TNF-α, indicating an inflammatory response and possible suppression of the host immune response as responsible for the infection property.A conidiobolomicose e a pitiose são infecções importantes que acometem animais e humanos e que apresentam dificuldades tanto no diagnóstico quanto no tratamento. A conidiobolomicose é causada pelos fungos Conidiobolus coronatus, C. incongruus e C. lamprauges, afetando ovinos, equinos, caninos e humanos, já a pitiose é causada pelo oomiceto Pythium insidiosum e atinge mamíferos selvagens e domésticos, principalmente equinos, e humanos. Não existe uma abordagem farmacológica padrão para o tratamento dessas infecções e em ambos os casos a rescisão cirúrgica é uma abordagem muitas vezes necessária. Neste contexto, esta tese objetivou (a) determinar a suscetibilidade in vitro de C. lamprauges frente a diferentes fármacos antimicrobianos, bem como identificar possíveis associações sinérgicas; (b) padronizar o uso da técnica de disco difusão utilizando a minociclina como um método de screening para a identificação presuntiva de P. insidiosum; (c) quantificar, extrair e avaliar o potencial imunomodulador das β-glucanas de P. insidiosum in vitro em cultivo de linfócitos e in vivo em camundongos; (d) desenvolver um modelo experimental de pitiose em camundongos e avaliar a resposta imunológica à infecção. Como resultados obtivemos: i) na avaliação da suscetibilidade in vitro de C. lamprauges isolados de infecções em ovinos, observou-se uma suscetibilidade reduzida a maioria dos fármacos testados, sendo a terbinafina o fármaco com melhor atividade (CIM < 0,06-0,5 μg/mL). As maiores taxas de sinergismo foram observadas com a associação de sulfametoxazol com trimetoprima (100%) e entre terbinafina com fármacos antifúngicos azólicos (71%); ii) a padronização do uso da técnica de disco difusão com discos de minociclina como um método de screening para a identificação presuntiva de P. insidiosum mostrou-se eficaz, pois não houve crescimento miceliano de P. insidiosum em torno do disco de minociclina durante os sete dias de incubação, diferenciando-o assim de C. lamprauges e outros fungos verdadeiros; iii) o conteúdo de β-glucanas de P. insidiosum avaliado enzimaticamente foi de 23,09% ± 3,71 de glucanas totais, divididas em α-glucanas (4,10% ± 0,83) e β-glucanas (18,99% ± 3,59); iv) a extração de β-glucanas de P. insidiosum produziu um extrato contendo 82% de β-glucanas e 18% de aminoácidos e peptídeos residuais e a análise estrutural mostrou tratar-se de uma (1,3)(1,6)- β-glucana; v) esta (1,3)(1,6)- β-glucana demonstrou potencial para estimular o sistema imune in vitro observado pelo aumento na proliferação de linfócitos de equinos, humanos e camundongos e de induzir resposta do tipo Th17 quando administrada aos camundongos; vi) o uso de camundongos BALB/c imunossuprimidos com a associação de ciclofosfamida e hidrocortisona mostrou ser um modelo experimental eficaz para o estudo da pitiose, com mortalidade de 60% dos animais infectados; vii) a produção de citocina observada durante o desenvolvimento da pitiose experimental em camundongos caracterizou-se pela expressão de IL6, IL-10 e TNF- α, indicando uma resposta inflamatória e uma possível supressão da resposta imune do hospedeiro como responsável pelo estabelecimento da infecção.Universidade Federal de Santa MariaBrasilFarmacologiaUFSMPrograma de Pós-Graduação em FarmacologiaCentro de Ciências da SaúdeSanturio, Janio Moraishttp://lattes.cnpq.br/6316012260769979Leal, Daniela Bitencourt Rosahttp://lattes.cnpq.br/3639683273462361Mario, Débora Alves Nuneshttp://lattes.cnpq.br/0731595522786356Zanette, Régis Adrielhttp://lattes.cnpq.br/3166371422900794Alves, Sydney Hartzhttp://lattes.cnpq.br/0330782478769631Tondolo, Juliana Simoni Moraes2019-08-29T17:52:15Z2019-08-29T17:52:15Z2016-03-28info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisapplication/pdfhttp://repositorio.ufsm.br/handle/1/18079porAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccessreponame:Manancial - Repositório Digital da UFSMinstname:Universidade Federal de Santa Maria (UFSM)instacron:UFSM2019-08-30T06:02:36Zoai:repositorio.ufsm.br:1/18079Biblioteca Digital de Teses e Dissertaçõeshttps://repositorio.ufsm.br/ONGhttps://repositorio.ufsm.br/oai/requestatendimento.sib@ufsm.br||tedebc@gmail.comopendoar:2019-08-30T06:02:36Manancial - Repositório Digital da UFSM - Universidade Federal de Santa Maria (UFSM)false |
dc.title.none.fl_str_mv |
Aspectos fisiológicos e imunológicos de Pythium insidiosum e atividade de antimicrobianos frente a Conidiobolus lamprauges Physiological and immunological aspects of Pythium insidiosum and antimicrobial activity of antimicrobial agents against Conidiobolus lamprauges |
title |
Aspectos fisiológicos e imunológicos de Pythium insidiosum e atividade de antimicrobianos frente a Conidiobolus lamprauges |
spellingShingle |
Aspectos fisiológicos e imunológicos de Pythium insidiosum e atividade de antimicrobianos frente a Conidiobolus lamprauges Tondolo, Juliana Simoni Moraes Conidiobolus lamprauges Suscetibilidade β-glucanas Imunoterapia Susceptibility β-glucans Immunotherapy Pythium insidiosum CNPQ::CIENCIAS DA SAUDE::FARMACIA |
title_short |
Aspectos fisiológicos e imunológicos de Pythium insidiosum e atividade de antimicrobianos frente a Conidiobolus lamprauges |
title_full |
Aspectos fisiológicos e imunológicos de Pythium insidiosum e atividade de antimicrobianos frente a Conidiobolus lamprauges |
title_fullStr |
Aspectos fisiológicos e imunológicos de Pythium insidiosum e atividade de antimicrobianos frente a Conidiobolus lamprauges |
title_full_unstemmed |
Aspectos fisiológicos e imunológicos de Pythium insidiosum e atividade de antimicrobianos frente a Conidiobolus lamprauges |
title_sort |
Aspectos fisiológicos e imunológicos de Pythium insidiosum e atividade de antimicrobianos frente a Conidiobolus lamprauges |
author |
Tondolo, Juliana Simoni Moraes |
author_facet |
Tondolo, Juliana Simoni Moraes |
author_role |
author |
dc.contributor.none.fl_str_mv |
Santurio, Janio Morais http://lattes.cnpq.br/6316012260769979 Leal, Daniela Bitencourt Rosa http://lattes.cnpq.br/3639683273462361 Mario, Débora Alves Nunes http://lattes.cnpq.br/0731595522786356 Zanette, Régis Adriel http://lattes.cnpq.br/3166371422900794 Alves, Sydney Hartz http://lattes.cnpq.br/0330782478769631 |
dc.contributor.author.fl_str_mv |
Tondolo, Juliana Simoni Moraes |
dc.subject.por.fl_str_mv |
Conidiobolus lamprauges Suscetibilidade β-glucanas Imunoterapia Susceptibility β-glucans Immunotherapy Pythium insidiosum CNPQ::CIENCIAS DA SAUDE::FARMACIA |
topic |
Conidiobolus lamprauges Suscetibilidade β-glucanas Imunoterapia Susceptibility β-glucans Immunotherapy Pythium insidiosum CNPQ::CIENCIAS DA SAUDE::FARMACIA |
description |
Conidiobolomycosis and pythiosis are important infections that affect animals and humans, which presents difficulties in diagnosis and treatment. The conidiobolomycosis is caused by the fungi Conidiobolus coronatus, C. incongruus and C. lamprauges, affecting sheep, horses, dogs and humans; pythiosis is caused by the oomycete Pythium insidiosum and affects wild and domestic mammals, particularly horses and humans. There is no standard pharmacological approach to the treatment of infections and the surgical rescission is a method often needed in both cases. In this context, this thesis aimed to (a) determine the in vitro susceptibility of C. lamprauges against different antimicrobial drugs, as well as identify possible synergistic associations; (b) the use of standardized disk diffusion technique with minocycline as a screening method for the presumptive identification of P. insidiosum; (c) quantify, extract and evaluate the immunomodulatory potential of β-glucans from P. insidiosum in vitro in cell cultures in vitro and in mice; (d) develop an experimental pythiosis model in mice and evaluate the immune response to infection. As results we achieved: i) assessing the in vitro susceptibility of C. lamprauges isolated from sheep infections, there is reduced susceptibility to most antimicrobial drugs tested, with terbinafine the drug with improved activity (MIC <0.06- 0.5 μg/mL). The highest rates of synergism were observed with the combination of sulfamethoxazole and trimethoprim (100%) and between the terbinafine with azole antifungal drugs (71%); ii) the standardization of the use of disk diffusion technique with minocycline disks as a screening method for the presumptive identification of P. insidiosum was effective because there was no mycelial growth of P. insidiosum around the minocycline disk during the seven days of incubation, thus differentiating it from C. lamprauges and other true fungi; iii) β-glucan content from P. insidiosum enzymatically assessed was 23.09 ± 3.71% of total glucan, divided into α-glucans (4.10 ± 0.83%) and β-glucans (18.99 ± 3.59); iv) β-glucan extract from P. insidiosum yielded an extract containing 82% of β-glucans and 18% of residual amino acids and peptides, and the structural analysis revealed that it was a (1,3)(1,6)-β-glucan; This (1,3)(1,6)-β-glucan demonstrates a potential to stimulate the immune system in vitro, as observed by the increase in equine, human and mouse lymphocyte proliferation and ability to induce Th17 type response when administered to mice; vi) the use of BALB/c mice immunosuppressed with cyclophosphamide and hydrocortisone association was shown to be an effective experimental model for the study of pythiosis, with 60% of mortality; vii) The cytokine production observed during the development of the experimental pythiosis in mice was characterized by an expression of IL6, IL-10 and TNF-α, indicating an inflammatory response and possible suppression of the host immune response as responsible for the infection property. |
publishDate |
2016 |
dc.date.none.fl_str_mv |
2016-03-28 2019-08-29T17:52:15Z 2019-08-29T17:52:15Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
format |
doctoralThesis |
status_str |
publishedVersion |
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http://repositorio.ufsm.br/handle/1/18079 |
url |
http://repositorio.ufsm.br/handle/1/18079 |
dc.language.iso.fl_str_mv |
por |
language |
por |
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Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/ info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/ |
eu_rights_str_mv |
openAccess |
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application/pdf |
dc.publisher.none.fl_str_mv |
Universidade Federal de Santa Maria Brasil Farmacologia UFSM Programa de Pós-Graduação em Farmacologia Centro de Ciências da Saúde |
publisher.none.fl_str_mv |
Universidade Federal de Santa Maria Brasil Farmacologia UFSM Programa de Pós-Graduação em Farmacologia Centro de Ciências da Saúde |
dc.source.none.fl_str_mv |
reponame:Manancial - Repositório Digital da UFSM instname:Universidade Federal de Santa Maria (UFSM) instacron:UFSM |
instname_str |
Universidade Federal de Santa Maria (UFSM) |
instacron_str |
UFSM |
institution |
UFSM |
reponame_str |
Manancial - Repositório Digital da UFSM |
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Manancial - Repositório Digital da UFSM |
repository.name.fl_str_mv |
Manancial - Repositório Digital da UFSM - Universidade Federal de Santa Maria (UFSM) |
repository.mail.fl_str_mv |
atendimento.sib@ufsm.br||tedebc@gmail.com |
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1805922075788967936 |