Evaluación in vitro de la integridad de la barrera hematoencefálica y su alteración causada por el virus dengue

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dc.contributor.advisorVelandia-Romero, Myriam Lucía
dc.contributor.advisorCastellanos, Jaime
dc.contributor.authorCalderón Peláez, María Angélica
dc.contributor.orcidCalderón Peláez, María Angélica [0000-0003-0788-0795]
dc.contributor.orcidCastellanos, Jaime [0000-0003-1596-8383]
dc.contributor.orcidVelandia-Romero, Myriam Lucía [0000-0002-3340-7304]
dc.date.accessioned2021-02-23T17:02:24Z
dc.date.available2021-02-23T17:02:24Z
dc.date.issued2014
dc.description.abstractEl sistema nervioso (SN), está protegido por una barrera de difusión celular conocida como Barrera Hematoencefálica (BHE), compuesta por diferentes tipos de células que limitan el paso de moléculas desde los capilares hacia el parénquima cerebral, lo cual garantiza la homeostasis del tejido. A pesar de esto, algunas moléculas y agentes infecciosos logran atravesar la BHE e ingresar al tejido nervioso para inducir graves daños en la fisiología de este. Dentro de los patógenos que logran alterar la BHE e infectar el tejido nervioso, se encuentran algunos miembros de la familia flaviviridae típicamente neurotrópicos, sin embargo el virus del dengue (DENV), un virus considerado no neurotrópico, puede infectar y alterar la fisiología del tejido nervioso ocasionando signos como encefalitis, parálisis y alteraciones motoras y cognitivas que pueden ser permanentes. Para comprender los cambios de tropismo y los factores neurológicos e inmunológicos asociados a la neuroinfección por DENV, en nuestro laboratorio se desarrolló un modelo de neuroinfección en ratones lactantes utilizando una cepa de virus neuroadaptado (D4MB-6), que infecta neuronas y otras células del tejido nervioso e induce la alteración de la BHE en ratones lactantes. Por lo tanto, el presente proyecto buscó evaluar en un modelo de BHE in vitro, si la alteración de la permeabilidad de la barrera endotelial cerebrovascular asociada a la infección con el DENV-4 neuroadaptado o no, favorece el paso de virus libre o asociado a células del sistema inmune lo cual, permitiría la infección y dispersión viral en el tejido nervioso. Para esto, se estableció un modelo de BHE in vitro en monocapa o co-cultivo utilizando cultivos primarios de endotelio cerebrovascular con o sin astrocitos obtenidos a partir de cerebros de ratones lactantes, siendo únicamente las células endoteliales susceptibles a la infección con el virus parental o D4MB-6. Los resultados obtenidos mostraron en ambos modelos de BHE que a las 10 horas post-infección (hpi) con cada virus, hubo una disminución en los valores de resistencia transendotelial (TEER), asociada a un aumento en la permeabilidad. Adicionalmente en este mismo tiempo post infección se detectaron partículas virales infecciosas en la cámara inferior de los insertos, lo que sugiere que la infección ocasionó una alteración en la integridad de las células endoteliales, lo que permitió el paso paracelular de las partículas virales. Se encontró que la infección con el D4MB-6 indujo la relocalización -sin afectar la expresión-, de la proteína ZO-1 y un aumento en la expresión de los transcritos para las proteínas VCAM, PECAM, TNFα y MCP-1 comparado con lo observado durante la infección con el virus parental. Esto último está relacionado con un proceso de activación endotelial que al parecer favoreció el proceso de transmigración de monocitos/macrófagos J774 en ambos modelos de BHE estandarizados. Lo anterior convierte el proceso de alteración de la BHE en uno de los posibles mecanismos utilizados por el DENV para ingresar y dispersarse dentro del tejido nervioso.spa
dc.description.abstractenglishThe nervous system (NS) is protected by a cellular diffusion barrier named Blood Brain Barrier (BBB). This barrier is formed by different cell types that limit the passage of molecules from the capillaries into the brain parenchyma, nevertheless some molecules and infectious agents are able to cross the BBB and infect the nervous tissue. Among the viruses that manage to alter the BBB and infect nervous tissue, there are some flaviviridae family members known as neurotropic flavivirus, however Dengue virus (DENV), a no-neurotropic flavivirus, can alter the physiology of the nervous tissue, causing clinical signs such as encephalitis, paralysis and motor and cognitive alterations that might be permanent. To understand some of the changes in the virus tropism, and in the neurological and immunological factors associated with DENV infection in the NS, we developed a neuroinfection model in suckling mice using a strain of neuroadapted virus (D4MB-6) than infects neurons and some others cells of the nervous tissue and can alter the BBB permeability in suckling mice. Therefore, the present project evaluated the permeability alteration of the cerebrovascular endothelial barrier, associated with DENV-4 or D4MB-6 infection in an in vitro BBB model. We tried to answer if this alteration can promote the passage of virus into the nervous tissue allowing infection and viral spread. We establish a BBB in vitro model using primary cultures (isolated from suckling mice) of endothelial cells (BBB model in monolayer) or endothelial cells and astrocytes (co-culture BBB model). The results showed that only the endothelial cells were susceptible to the infection with each evaluated virus; also in both BBB models at 10 hours post-infection, there was a decreased in the transendothelial resistance values (TEER), associated with an increase in the permeability of the model. Additionally at this time, we detected infectious viral particles in the supernatants of the lower chamber of the inserts, suggesting that infection results in an alteration in the integrity of the endothelial cells and permits the paracellular passage of viral particles. We also found that the infection with D4MB-6 induced the relocation of the ZO-1 protein, but it did not affect the expression pattern of this protein. Infection also induced the over expression of primary transcripts of VCAM, PECAM, TNFα y MCP-1 proteins, compared with the observations made when the cells were infected with DENV-4. This is related with an endothelial activation process that favored the transmigration of immune cells (J774) in both BBB models. Our results suggest that the alteration process of the BBB is one of the possible mechanisms used by the DENV to enter and spread in to the nervous tissue.eng
dc.description.degreelevelMaestríaspa
dc.description.degreenameMagíster en Ciencias Básicas Biomédicasspa
dc.format.mimetypeapplication/pdf
dc.identifier.instnameinstname:Universidad El Bosquespa
dc.identifier.reponamereponame:Repositorio Institucional Universidad El Bosquespa
dc.identifier.repourlrepourl:https://repositorio.unbosque.edu.co
dc.identifier.urihttps://hdl.handle.net/20.500.12495/5422
dc.language.isospa
dc.publisher.facultyFacultad de Medicinaspa
dc.publisher.grantorUniversidad El Bosquespa
dc.publisher.programMaestría en Ciencias Básicas Biomédicasspa
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dc.rightsAttribution-NonCommercial-ShareAlike 4.0 International*
dc.rights.accessrightsinfo:eu-repo/semantics/openAccess
dc.rights.accessrightshttps://purl.org/coar/access_right/c_abf2
dc.rights.creativecommons2013
dc.rights.localAcceso abiertospa
dc.rights.urihttps://creativecommons.org/licenses/by-nc-sa/4.0/*
dc.subjectAlteración endotelialspa
dc.subjectBHEspa
dc.subjectInfecciónspa
dc.subjectDENVspa
dc.subjectTransmigración celularspa
dc.subjectTransporte paracelularspa
dc.subject.decsTécnicas in vitrospa
dc.subject.decsVirus del denguespa
dc.subject.decsBarrera hematoencefálicaspa
dc.subject.keywordsEndothelial alterationspa
dc.subject.keywordsBBBspa
dc.subject.keywordsInfectionspa
dc.subject.keywordsDENVspa
dc.subject.keywordsCellular transmigrationspa
dc.subject.keywordsParacellular transportspa
dc.subject.nlmW 50
dc.titleEvaluación in vitro de la integridad de la barrera hematoencefálica y su alteración causada por el virus denguespa
dc.title.translatedIn vitro integrity evaluation of the Blood Brain Barrier and its disturbance caused by dengue virus infectionspa
dc.type.coarhttps://purl.org/coar/resource_type/c_bdcc
dc.type.coarversionhttps://purl.org/coar/version/c_970fb48d4fbd8a85
dc.type.driverinfo:eu-repo/semantics/masterThesis
dc.type.hasversioninfo:eu-repo/semantics/acceptedVersion
dc.type.localTesis/Trabajo de grado - Monografía - Maestríaspa

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Maestría en Ciencias Básicas Biomédicas