Conselho Nacional de Desenvolvimento Científico e Tecnológico / A síndrome da imunodeficiência adquirida (AIDS), causada pelo vírus da imunodeficiência humana (HIV), é uma das epidemias mais impactantes do milênio e, desde o início, o número de mulheres jovens infectadas vem aumentando vertiginosamente, principalmente nos países em desenvolvimento onde muitas destas engravidam precocemente. Apesar da grande maioria dos casos de AIDS pediátrico no mundo resultar da transmissão vertical, aproximadamente dois terços dos neonatos expostos ao HIV durante a vida fetal não são contaminados. Neste sentido, seguindo as recomendações do consenso brasileiro (Ministério da Saúde), toda criança cuja transmissão vertical tenha sido descartada laboratorialmente não necessita de acompanhamento ambulatorial particularizado. Entretanto, resultados anteriores obtidos pelo nosso grupo demonstraram que, gestantes infectadas pelo HIV-1 que não controlam a carga viral plasmática (CVP), apresentam níveis elevados de citocinas inflamatórias e, no presente estudo, resultados revelam que neonatos não-infectados nascidos dessas gestantes apresentam anormalidades imunofuncionais no compartimento das células T do cordão umbilical quando expostos in vitro a ativadores policlonais, mas não aos antígenos do HIV-1. Ademais, quando comparada a neonatos não expostos, a ativação in vitro das células T de neonatos expostos ao HIV-1 com anti-CD3/anti-CD28 induziu a produção de níveis elevados de IL-17 e reduzidos de IL-10. Interessantemente, essa tendência das células T em secretar IL-17 parece estar relacionada à liberação de níveis elevados de IL-23 pelas células dendríticas derivadas de monócitos do sangue do cordão umbilical estimuladas com lipopolissacarídeo bacteriano. Uma ausência de sensibilização uterina aos antígenos do HIV-1 sugere que essas alterações possam traduzir um efeito adverso da produção elevada de citocinas inflamatórias maternas sobre o sistema imune do neonato, o que pode desequilibrar os eventos envolvidos na maturação e homeostasia imune fetal e neonatal, favorecendo o predomínio de fenótipos Th anômalos, tal como Th17. Essa hiper-responsividade das células Th17 pode vir a comprometer não apenas a capacidade da criança em responder de forma adequada a diferentes estímulos antigênicos ao longo de sua vida, como também pode torná-la mais suscetível a desordens imunomediadas / The acquired immunodeficiency syndrome (AIDS), caused by human immunodeficiency virus (HIV), is one of the most impelling epidemic in the world, and the HIV-infection in young women has been increasing fast in the recent times, mainly in developing countries where most of them become pregnant precociously. Although the great majority of the pediatric AIDS cases all over the world results from vertical transmission, approximately, two thirds of the children exposed to HIV during fetal life are not contaminated. In this context, following brazilian consensus recommendations (Health Ministry), every child whom vertical transmission had been laboratorialy discarded does not need a specific ambulatorial follow up. However, our previous results demonstrated that HIV-1-infected pregnant women who did not control their plasma viral load presented elevated levels of inflammatory cytokines, and in the present study our results revealed that non-infected neonates, born from these pregnant women display immune functional abnormalities in umbilical cord T cells compartment when exposed in vitro to policlonal activators, but not to HIV-1 antigens. Furthermore, when compared to non-exposed neonates, T cell in vitro activation with anti-CD3/anti-CD28 from neonates exposed to HIV-1 induced production of high IL-17 levels and decreased of IL-10. Interestingly, this T cell bias in secreting IL-17 seem to be related to liberation of high IL-23 levels by dendritic cells derived from umbilical cord blood monocytes following stimulation with bacterial lipopolysacharide (LPS). The lack of uterine sensitization to HIV-1 antigens suggests that, these alterations, may translate an adverse effect of a high level maternal inflammatory cytokines production on neonates immune system, which may unbalance events related to neonatal and fetal immune maturation and homeostasis, favoring Th anomalous phenotypes predominance, such as Th17. This Th17 hyper-responsiveness may then compromise not only the childs capacity to respond in an adequate way to different antigenic stimuli through life, as well as becoming them more susceptible to immune-mediated disorders

Plasmacytoid dendritic cells (pDC) constitute a very rare blood cell population and play a significant role in immune response and immune-mediated disorders. Investigations on primary pDCs are hindered not only due to their rarity but also because they represent a heterogeneous cell population which is difficult to culture ex vivo. We generated a conditionally immortalized pDC line (Dox-pDC) from mice with Doxycycline-inducible SV40 Large T Antigen with a comparable immune profile to primary pDCs. The Dox-pDC secrete pro- and anti-inflammatory cytokines upon Toll-like receptor 9 stimulation and upregulate their MHCI, MHCII and costimulatory molecules. Further, the Dox-pDC activate and polarize naïve T cells in vivo and in vitro in response to the model antigen Ovalbumin. Due to their long-term culture stability and their robust proliferation Dox-pDC represent a reliable alternative to primary mouse pDC.

Le virus de la rougeole (MV) provoque une maladie sévère chez les enfants qui induit une forte immunosuppression et peut dans certains cas infecter le système nerveux central (SNC). La protéine CD150, principal récepteur cellulaire du virus, permet l’entrée du MV en se liant à l’hémagglutinine (MV-H). L’altération fonctionnelle des cellules dendritiques (DC) étant considérée comme essentielle dans l’immunopathogénèse du MV, nous avons analysé les conséquences de l’interaction de MV-H avec les DC. Nous avons développé un modèle expérimental qui nous permet d’étudier l’interaction directe entre CD150 et MV-H hors contexte infectieux. Nos résultats montrent que cette interaction provoque une diminution de l’expression des molécules de surface CD80, CD83, CD86, et HLA-DR, de la production d’IL-12 par les DC, et de la capacité des DC à stimuler la prolifération des lymphocytes T. L’interaction CD150-MV-H a inhibé la phosphorylation des protéines kinases JNK1/2 dans les DC et les lymphocytes B (LB) induite par la stimulation de CD150 par un anticorps spécifique, mais pas celle des kinases ERK1/2. Par ailleurs MV-H seule induit la phosphorylation d’Akt via CD150 dans les DC et les LB. La liaison de CD150 par MV-H a réduit la réponse inflammatoire chez les souris transgéniques exprimant CD150 humain, ce qui confirme l’effet de l’interaction de CD150 et MV-H in vivo. Nos études ont révélé l’expression de CD150 dans les tumeurs du SNC et l’existence d’une nouvelle isoforme de CD150. Cette isoforme contient un exon supplémentaire de 83 pb et est exprimée dans les cellules lymphoïdes et les DC en plus des tumeurs du SNC. Bien que l’expression de CD150 soit uniquement intracellulaire dans les cellules tumorales, elle peut représenter un nouveau marqueur pour les tumeurs cérébrales humaines. Cette étude apporte un éclairage nouveau sur le rôle immunorégulateur de CD150 et sur la diversité de ses isoformes, et ouvre ainsi de nouvelles perspectives pour leurs applications thérapeutiques. / Measles virus (MV) causes an acute childhood disease, associated in certain cases with the infection of the central nervous system (CNS). MV induces a profound immunosuppression, resulting in high infant mortality. The major cellular receptor for MV is CD150, which binds MV hemagglutinin (MV-H). As dendritic cell (DC) dysfunction is considered to be essential for the MV immunopathogenesis, we analyzed consequences of MV-H interaction with DCs. We developed an experimental model allowing us to analyze the direct CD150-MV-H interaction in the absence of infectious context. This interaction caused the downregulation of surface expression of CD80, CD83, CD86 and HLA-DR molecules and inhibition of IL-12 production in DCs. DCs also failed to activate T cell proliferation. The CD150-MV-H interaction in DCs and B cells decreased the phosphorylation of JNK1/2, but not ERK1/2 kinases, after subsequent CD150 ligation with anti-CD150 antibodies. Moreover, MV-H by itself induced Akt phosphorylation via CD150 in DCs and B cells. Engagement of CD150 by MV-H in mice transgenic for human CD150 decreased the inflammatory reaction, contact hypersensitivity response, confirming the immunosuppressive effect of CD150-MV-H interaction in vivo. Furthermore, our studies revealed the CD150 expression in CNS tumors and identified the novel CD150 isoform, containing an additional 83bp exon expressed in lymphoid cells, DCs and CNS tumors. Although its isoforms remain intracellular in tumor cells, CD150 may represent a new marker for human brain tumors. Novel mechanism of CD150-induced immunosuppression and new CD150 isoform identified in these studies shed new light on its immunoregulatory role and CD150 isoform diversity and open perspectives for their clinical applications.

Stimulation of CD40 on APCs through CD40L expressed on helper CD4+ T cells activates and “licenses” the APCs to prime CD8+ T cell responses. While other stimuli, such as TLR agonists, can also activate APCs, it is unclear to what extent they can replace the signals provided by CD40-CD40L interactions. In this study, we used an adoptive transfer system to re-examine the role of CD40 in the priming of naïve CD8+ T cells. We find an approximately 50% reduction in expansion and cytokine production of TCR-transgenic T cells in the absence of CD40 on all APCs, and on dendritic cells in particular. Moreover, CD40-deficient and CD40L-deficient mice fail to develop endogenous CTL responses after immunization and are not protected from a tumor challenge. Surprisingly, the role for CD40 and CD40L are observed even in the absence of CD4+ T cells; in this situation, the CD8+T cell itself provides CD40L. Furthermore, we show that although TLR stimulation improves T cell responses, it cannot fully substitute for CD40. We also investigated whether CD40-CD40L interactions are involved in the generation, maintenance, and function of memory CD8+ T cells. Using a virus infection system as well as a dendritic cell immunization system, we show that the presence of CD40 on DCs and other host APCs influences the survival of activated effector cells and directly affects the number of memory CD8+ T cells that are formed. In addition, memory CD8+ T cell persistence is slightly impaired in the absence of CD40. However, CD40 is not required for reactivation of memory CD8+ T cells. It seems that CD40 signals during priming also contribute to memory CD8+ T cell programming but this function can be independent of CD4+T cells, similar to what we showed for primary responses. Altogether, these results reveal a direct and unique role for CD40L on CD8+ T cells interacting with CD40 on APCs that affects the magnitude and quality of primary as well as memory CD8+ T cell responses.

Les données de la littérature ont récemment souligné l’importance du dialogue réciproque qui s’instaure entre les cellules Natural Killer (NK) et les cellules dendritiques (DC) au cours des phases précoces de la réponse immune pour l’initiation des réponses T spécifiques. La présentation croisée d’antigènes (Ag), processus qui permet aux DC de présenter des Ag exprimés par d’autres cellules aux lymphocytes T CD8+, est requise pour le développement d’une immunité cellulaire spécifique dans la plupart des infections par des pathogènes intracellulaires et des tumeurs. L’étude des mécanismes physiologiques impliqués dans la régulation de cette fonction suscite donc un intérêt majeur. Ce travail a permis de mettre en lumière une coopération entre les cellules NK et les DC humaines pour la présentation croisée d’un Ag exprimé par des cibles tumorales. Dans ce contexte, nous avons montré que la lyse de ces cibles par les cellules NK n’est pas nécessaire à la capture de leurs Ag par les DC. Au contraire, la sécrétion d’IFN-γ et de TNF-α par les cellules NK activées au contact des cibles tumorales joue un rôle prépondérant dans l’induction de la présentation croisée d’Ag. Ainsi, nous avons identifié une nouvelle fonction « helper » des cellules NK à l’interface entre l’immunité innée et adaptative. Le ciblage de cette fonction pourrait offrir de nouvelles perspectives en immunothérapies anti-tumorales dont le but ultime est le développement d’une immunité cellulaire spécifique / Recent reports have demonstrated the importance of the reciprocal crosstalk between natural killer (NK) cells and dendritic cells (DC) occurring during early phase of immune response for shaping downstream T cell immunity. Antigen (Ag) cross-presentation, a process by which DC present Ag from neighboring cells to CD8+ T lymphocytes is a prerequisite for the developpment of specific cellular immunity against most intracellular pathogens and tumors. A more detailed understanding of the mechanisms that regulate this specific DC function is thus a major challenge for immunologists. Here, we highlight the cooperation between NK and DC for tumor cell-derived Ag cross-presentation. In this context, we show that the NK cell-mediated lysis of target cells is not required for Ag capture by DC. In contrast, both IFN-γ and TNF-α produced by NK cells upon recognition of tumor cells play a critical role in the induction of Ag cross-presentation. These findings define a novel « helper» function of NK cells bridging innate and adaptive immunity. This novel function could be harnessed in cancer immunotherapy for inducing Ag-specific cellular immunity

Alix (ALG-2 Interacting Protein X) est une protéine cytoplasmique impliquée dans divers processus cellulaires allant de l'apoptose à la cytocinèse en passant par le bourgeonnement des virus, la réparation membranaire et la régulation de la voie endosomale. Toutes ces fonctions sont étroitement associées à l'interaction d'Alix avec ses partenaires impliqués dans la déformation des membranes telles que les endophilines A, Tsg-101 et CHMP4B du complexe ESCRT (Endosomal Sorting Complex Required for Transport). Le but de ce projet est de caractériser le phénotype de la souris Alix ko récemment développée au laboratoire, dans l'espoir de mieux comprendre le rôle physiologique d'Alix. Ces souris, viables et fertiles, sont caractérisées par une microcéphalie apparaissant au cours de l'embryogenèse. Ce phénotype est accompagné d'une apoptose massive touchant les progéniteurs neuronaux durant la neurogenèse et d'une altération du développement de l'arborisation dendritique après la naissance. Les souris adultes présentent également des défauts de plasticité synaptique accompagnés d'une altération du recyclage des vésicules synaptiques. L'ensemble de ces processus repose sur la capacité d'Alix à contrôler le remodelage de la membrane plasmique. Au niveau moléculaire, nos travaux sur les neurones en cultures et sur les fibroblastes montrent une régulation de l'endocytose indépendante de la clathrine (CIE) par Alix et les endophilines A qui pourrait être à l'origine du phénotype neuronal de la souris. Cependant, l'association d'Alix avec CHMP4B du complexe ESCRT pourrait également être nécessaire au développement du système nerveux puisque l'interaction Alix-CHMP4B est nécessaire pour le contrôle de la CIE et de la mort neuronale.L'ensemble de ces résultats mets en évidence qu'à travers des mécanismes et des partenaires bien caractérisés, Alix est requise pour de nouvelles fonctions nécessaires au développement et au fonctionnement du système nerveux. / Alix (ALG-2 Interacting Protein X) is a cytoplasmic protein implicated in multiple processes including apoptosis, endosome function, membrane repair, viral budding and cytokinesis. Most of these involve modifications of plasma or endosomal membrane organization. Consistent with this, Alix is known to interact with diverse proteins modulating membrane deformation, such as endophilins or Tsg-101 and CHMP4B of the Endosomal Sorting Complex Required for Transport (ESCRT). By studying the phenotype of Alix ko mice that we recently developed, we aim to better understand the precise role of Alix in vivo. These mice are viable and fertile but develop microcephaly even at embryonic stages. This microcephaly is associated with an increase of apoptosis in neural progenitor cells in embryos and a defect in neurite outgrowth at post-natal stages. Later on, these mice also develop defects in synaptic plasticity related to an alteration of synaptic vesicle recycling. All of these processes are tightly dependent on membrane shaping and remodelling. These features of the phenotype can be related to a new function of Alix with endophilin A in the control of clathrin-independent endocytosis (CIE), as described in Alix ko fibroblasts and cultured neurons. However, these defects might also be related to the well-known Alix partner CHMP4B, since we now know that this interaction is needed for controlling CIE and neuronal cell death.All together, these results shed light on novel functions of Alix in the developing and adult central nervous system, all relying on well-known molecular mechanisms and partners.

Data de aprova??o ausente. / Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-02-13T18:51:06Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) tatiana_fernandes_araujo_almeida.pdf: 2531556 bytes, checksum: 11a2583c0414dc55d6f907a2566a4de5 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-03-06T12:21:52Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) tatiana_fernandes_araujo_almeida.pdf: 2531556 bytes, checksum: 11a2583c0414dc55d6f907a2566a4de5 (MD5) / Made available in DSpace on 2017-03-06T12:21:52Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) tatiana_fernandes_araujo_almeida.pdf: 2531556 bytes, checksum: 11a2583c0414dc55d6f907a2566a4de5 (MD5) Previous issue date: 2016 / O carcinoma espinocelular oral (CECO) ? uma neoplasia maligna que acomete principalmente idosos. O desencadeamento da doen?a ? relacionado ? exposi??o aos fatores de risco e ao decl?nio da fun??o imune associada ao envelhecimento cronol?gico. Alguns estudos t?m demonstrado um aumento na incid?ncia de CECO em adultos jovens, sugerindo envolvimento de outros fatores na etiologia da doen?a, como uma altera??o do sistema imunol?gico. As c?lulas dendr?ticas (CDs) s?o c?lulas apresentadoras de ant?geno profissionais e estimuladoras eficazes para a expans?o clonal de linf?citos. Acredita-se que uma altera??o da fun??o das CDs em pacientes com c?ncer contribui para a falha da resposta antitumoral, levando a uma consequente progress?o da doen?a. O objetivo deste estudo foi avaliar, atrav?s da imunoistoqu?mica, se h? diferen?a na quantifica??o tissular dos subgrupos de CDs, associada ? idade, em esp?cimes de bi?psias de CECO. Para isso, foram selecionados casos de CECO de pacientes em tr?s diferentes faixas et?rias: G1 (< 40 anos de idade, n = 12), G2 (? 40 at? < 60 anos de idade, n = 15) e G3 (? 60 anos de idade, n = 14). Os marcadores utilizados foram S100, CD1a, CD207 (para CDs imaturas, imCDs), CD83 e CD208 (para CDs maduras, mCDs). As imagens foram capturadas dos campos com maior intensidade de marca??o nas l?minas histol?gicas e a quantifica??o celular foi realizada com o aux?lio do software Image J. A frequ?ncia e localiza??o das CDs foram avaliadas e analizadas estatisticamente nas regi?es intratumoral (intertumoral e ou estromal) e extratumoral. No geral, imCDs foram significativamente mais frequentes que mCDs em todos os grupos. ImCDs e mCDs mostraram preferencialmente localiza??o intratumoral e extratumoral respectivamente. Comparando G1 versus G2 / G3 foi observada um significativo menor n?mero de mCDs em G1. Na compara??o de G1 em rela??o a G2 ou G3 houve um n?mero significativamente menor de ambas, imCDs e mCDs. Nossos resultados mostram que existe uma menor quantidade de imCDs e mCDs em CECO afetando pacientes jovens em compara??o com idosos, sugerindo um comprometimento da resposta imune antitumoral em G1e permitindo a progress?o do tumor. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Odontologia, Universidade Federal dos Vales do Jequitinhonha e Mucuri, [2016]. / Oral squamous cell carcinoma (OSCC) is a malignant neoplasm that mainly affects elderly people. The onset of the disease is related with exposure to risk factors and the decline of immune function associated with chronological aging. Previous studies have demonstrated an increased incidence of CECO in young adults, suggesting involvement of other factors in the etiology of the disease, such as a change in the immune system. Dendritic cells (DCs) are professional antigen presenting cells and effective for stimulating clonal expansion of lymphocytes. It is believed that a change in the function of DC in cancer patients contributes to the failure of the anti-tumor response, leading to a consequent progression of the disease. The aim of this study was to evaluate, by immunohistochemistry, if there were differences in tissue quantification of DCs subsets, associated with age in specimens of OSCC biopsies. For this OSCC cases were selected at three different ages: G1 (< 40 years old, n = 12), G2 (? 40 to <60 years old, n = 15) and G3 (? 60 years old, n = 14). The markers used were S100, CD1a, CD207 (for immature DCs, imDCs), CD83 and CD208 (for mature DCs, mDCs). Images were captured from fields with higher intensity staining in histological sections and cell quantification was performed with Image J software help. The frequency and localization of immunostained DCs were analyzed in intratumoral (intranestal and/or extranestal) and extratumoral areas and statistically compared. Overall, imDCs than mDCs were significantly more frequent in all groups. ImDCs and mDCs showed preferential intratumoral and extratumoral localization, respectivel. Comparing G1 than G2/G3 showed a significant lesser number of mDCs. G1 versus G2 or G3 there is a significantly lower number of both imDCs and mDCs. Our results show a lower number of both imDCs and mDCs in OSCC affecting younger than elderly patients, suggesting impairment of an effective antitumor immune response in G1 and enabling tumor progression, showing a gradual establishment of the antitumor immune response mediated by DCs according to the age, but with defects in quality.

La réponse humorale à immunoglobulines A (IgA) constitue un des principaux mécanismes immunologiques permettant de maintenir l'homéostasie intestinale. L'initiation de la réponse IgA se déroule dans les tissus lymphoides associés à l'intestin, où la reconnaissance des antigènes intestinaux entraine l'activation des lymphocytes B naïfs, la commutation isotypique vers IgA et leur différenciation en plasmocytes. Mon travail de thèse a consisté à étudier la contribution du foie et des cellules dendritiques plasmacytoides (pDC) dans la réponse IgA intestinale. L'utilisation de deux modèles murins permettant la déplétion sélective des pDC nous a permis de démontrer que, en dépit de données publiées montrant leur capacité à engager la réponse IgA in vitro, les pDC ne sont pas nécessaires in vivo pour l'induction ou le maintien de la réponse IgA homéostatique. Nous montrons ensuite que le foie abrite une population importante de plasmocytes à IgA. Chez la souris, nous montrons que ces cellules possèdent des caractéristiques phénotypiques distinctes des plasmocytes de l'intestin et proviennent de lymphocytes B récemment activés dans les plaques de Peyer. A l'homéostasie, ces plasmocytes hépatiques secrètent des IgA dirigées contre les bactéries de la flore intestinale. Enfin, dans un modèle murin de consommation chronique d'alcool, nous montrons une corrélation entre une augmentation de cette population cellulaire, une élévation sérique des IgA et des dépôts d'IgA hépatiques, deux désordres fréquemment observés chez les patients atteints d hépatopathies alcooliques. Nos données indiquent donc que le foie constitue un site effecteur alternatif de la réponse / IgA humoral response is one of the main mechanisms by which immune homeostasis is maintained in the intestine. The IgA response is initiated in gut-associated lymphoid tissues, where recognition of intestinal antigens drives naïve B cell activation, IgA class-switch recombination and plasma cell differentiation. My thesis work addressed the contribution of the liver and plasmacytoid dendritic cells (pDCs) in intestinal IgA response. By using two complementary mouse models allowing for selective depletion of pDCs, we have demonstrated that, in contrast to published work showing their ability to drive IgA response in vitro, pDCs are dispensable in vivo for the induction and the maintenance of homeostatic intestinal IgA responses.Then, we showed that the liver contains an important population of IgA plasma cells. In mice, we demonstrated that these cells harbor distinct phenotypic characteristics in comparison to intestinal IgA plasma cells, and are derived from B cells recently activated in Peyer’s patches. At homeostasis, hepatic IgA plasma cells secrete IgA directed against bacteria from intestinal flora. Finally, in a mouse model of chronic ethanol consumption, we found a correlation between an increase in hepatic igA plasma cell population, elevation of serum iGA and IgA deposits in liver sinusoids, two disorders frequently observed in alcoholic liver disease patients. Thus, our results indicate that the liver constitutes an alternative effector site for IgA response initiated in the intestine

As principais incapacidades funcionais que se observam após o Acidente Vascular Encefálico (AVE), sob o ponto de vista clínico, decorrem da hemiparesia, da incoordenação, da hipertonia espástica dos membros superior e inferior contralaterais à lesão e da fraqueza ipsilateral e contralateral ao hemisfério lesado. Evidências sugerem que o aprendizado e a realização de tarefas motoras de habilidade podem induzir mudanças comportamentais e neurofisiológicas, o que ocorre tanto em animais intactos quanto naqueles submetidos às lesões do SNC. Nesse sentido, alguns trabalhos evidenciam a participação da atividade dendrítica, observada pelo aumento da imunorreatividade a MAP2, induzida por experiências comportamentais. Sendo assim, o objetivo do presente estudo foi avaliar o desempenho motor e a expressão da MAP2 no córtex motor primário, em ratos sham ou submetidos à HIC e aos treinamentos de habilidade do alcance (TH) ou ao treinamento de não-habilidade (TNH). Para tanto, ratos Wistar adultos foram inicialmente adaptados às diferentes tarefas motoras empregadas ao longo de três semanas, sendo, posteriormente, submetidos à cirurgia de indução da hemorragia intracerebral (HIC) por meio da administração intra-estriatal de colagenase tipo IV ou de veículo (animais Sham) (S). Em seguida, os animais dos grupos S_TH e HIC_TH foram submetidos ao treinamento da tarefa de habilidade do alcance e preensão, os animais dos grupos S_TNH e HIC_TNH foram submetidos ao treinamento da tarefa de não-habilidade e os animais S_ST e HIC_ST não receberam nenhum tipo de treinamento durante 4 semanas. Ao longo desse período, os animais foram testados pelo teste do Staircase quanto ao desempenho motor ao final da 2° e 4° semana de treinamento. Encerrado o período de treinamento, os animais foram profundamente anestesiados, perfundidos e tiveram seus encéfalos processados para a análise imunoistoquímica. Os resultados mostram que a realização da tarefa de habilidade do alcance e preensão foi capaz de aumentar a imunorreatividade da MAP2 no córtex motor primário (M1) em ambos os hemisférios, tanto em animais lesados quanto em animais não-lesados. Além disso, os animais HIC e HIC_TNH apresentaram também aumento da imunorreatividade à MAP2 em ambos os hemisférios. Porém, apenas os animais HIC_TH apresentaram recuperação funcional dos movimentos do membro anterior afetado, avaliados pelo teste comportamental. Concluindo, o presente estudo demonstra que o treino de habilidade induz plasticidade dendrítica no M1 em condições normalidade e lesão e, como estratégia de reabilitação, mostra-se superior ao treino de não-habilidade, na recuperação funcional do membro anterior após a HIC experimental. / Under clinical view, the main functional impairment observed after stroke is resulting from the hemiparesis, incoordination, spastic hypertonia and from ipsilateral and contralateral weakness . Evidences suggest that learning and achievement of motor tasks ability may induce behavioral and neurophysiological changes, which occur in both intact and injured animals. Accordingly, some studies reveal the participation of dendritic activity, observed by increasing the immunoreactivity to MAP2, induced by behavioral experiences. Thus, the aim of this study was to evaluate the motor performance and the expression of MAP2 in primary motor cortex (M1), in rats submitted or not to the IHC and rehabilitation using skilled (SK) or unskilled (US) training. Animals were initially adapted to different motor tasks employed over three weeks, and, subsequently, submitted to surgery for the induction of intracerebral hemorrhage (IHC) by means of administration of bacterial collagenase type IV or vehicle (animals Sham) (S) into the striatum . Then, animals in groups S_SK and IHC_SK were submitted to the training skilled forelimb reaching, animals in groups S_US and IHC_US were submitted to the training unskilled and animals S and HIC received no type of training during 4 weeks. Throughout that period, the reaching ability was tested using the Staircase test at the end of 2nd and 4thweek of training. At the end of the rehabilitation period, animals were deeply anesthetized, perfused and the immunohistochemistry was processed. Results show that the achievement of the task skilled forelimb reaching was able to increase the MAP2 immunoreactivity in primary motor cortex (M1) in both hemispheres, both in injured animals as in intact animals. In addition, animals form IHC and IHC_US groups also presented increased immunoreactivity to MAP2 in both cerebral hemispheres. However, only IHC_SK animals presented functional recovery of movements of the forelimb, evaluated by test behavioral. In conclusion, this study shows that training of skills tasks can induce modifications in M1 under conditions of normality and lesion and, as a strategy of rehabilitation, induced higher plasticity than the unskilled training correlated with functional recovery of the forelimb after IHC experimental.

Made available in DSpace on 2015-07-22T20:50:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-11-25 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Células iNKT restritas a CD1d são protetoras contra o desenvolvimento de melanoma murino, B16F10-Nex2, subcutaneamente em animais singênicos. Essa proteção pode ser deduzida pelo desenvolvimento acelerado do tumor em animais geneticamente deficientes na expressão de CD1d (CD1d-KO), com sobrevida significantemente menor do que a observada com animais WT submetidos ao mesmo desafio de células tumorais. CD1d é uma família de glicoproteínas relacionadas a MHC classe I, envolvidas na apresentação, principalmente em células dendríticas, de antígenos lipídicos para células iNKT. No presente trabalho focalizamos a identificação do lipídeo endógeno expresso em células de melanoma capaz de induzir uma resposta de vigilância imune baseada em células iNKT. Verificamos também a possibilidade de proteger animais contra o desenvolvimento tumoral utilizando células dendríticas primadas com o lipídeo endógeno. A extração dos componentes lipídicos de melanoma foi realizada a partir de tumores crescidos in vivo, evitando-se assim artefatos do cultivo celular in vitro. Foram testados três diferentes protocolos de extração (A, B e C), obtendo-se 14 frações diferentes que foram testadas na ativação do hibridoma DN32.D3, uma linhagem de células NKT murinas imortalizadas. A fração F3 do protocolo A (F3A) ativou o hibridoma DN32.D3 medido pela produção de IL-2. Para uma eficiente apresentação dos lipídeos dessa fração utilizamos com sucesso células dendríticas de medula óssea (BMDC) nos ensaios in vitro e in vivo, para apresentação de F3A e glicolipídeos ativadores de células NKT, agalactosilceramida (a-GalCer) e isoglobotrihexosilceramida (iGb3). Na tentativa de isolar o composto estimulatório presente em F3A de melanoma, essa fração foi analisada por HPTLC revelada com diversos reagentes específicos para resíduos de ácido siálico, açúcares neutros, fosfato e lipídeos totais. A fração também foi submetida a separações em colunas de sílica, ensaio de “immunostaining” quimioluminescente com lectina de Bandeiraea simplicifolia e análises em espectrometria de massa, onde foram identificados gangliosídeos como GM3 bem como glicoesfingolipídeos neutros como iGb3, Gb3, iGb4 e Gb4 por ESI-LIT-MS (electrosptray ionization-linear íon trap-mass spectrometry). Quando iGb3 foi incubado com BMDC e testado com células DN32D3 essas foram ativadas produzindo IL-2. GM3 consistentemente era um inibidor dessa ativação de células iNKT. Ensaios de citotoxicidade foram então realizados e verificamos que células de NKT estimuladas por BMDC incubadas com a-GalCer ou iGb3 circundavam as células tumorais B16F10-Nex2 visualizadas em microscopia de fluorescência e, em ensaio in vitro, as células NKT promoviam lise de até 40% das células tumorais B16F10-Nex2. Realizamos então ensaios in vivo, onde camundongos foram inoculados endovenosamente com células do melanoma murino e tratados ou não com células dendríticas primadas com a-GalCer ou iGb3. Ao excisarmos os pulmões dos animais, notamos que os grupos tratados com lipídeos ativadores de células NKT tinha cerca de 4 vezes menos nódulos pulmonares que o grupo não tratado. Nossos resultados mostram que o melanoma murino B16F10-Nex2 possui a molécula iGb3 e sua precursora iGb4, capaz de ativar células NKT conferindo a essas capacidade citotóxica in vitro contra o melanoma. Esse lipídeo (iGb3) também mostrou um efeito protetor contra metástases pulmonares oriundas do melanoma murino quando apresentado por células dendríticas utilizadas em protocolo de terapia celular. Esse é o primeiro trabalho mostrando que efetivamente um glicolipídeo endógeno ligante de CD1d é capaz de ativar células NKT com efeito protetor antitumoral, através de terapia celular com células dendríticas. Palavras chave: Melanoma B16F10-Nex2, células iNKT, glicoesfingolipídeos iGb3 e iGb4 GM3, células dendríticas, imuno vigilância. / CD1d-restricted iNKT cells are protective against the murine melanoma B16F10- Nex2 growing subcutaneously in syngeneic animals. This is inferred from the fast tumor growth in animals genetically deficient in CD1d (CD1d-KO), which showed a survival time significantly shorter than that of WT animals equally challenged with tumor cells. CD1d belongs to a family of glycoproteins resembling MHC class I, involved in the presentation, chiefly in dendritic cells, of lipidic antigens to iNKT cells. In the present work we focus on the identification of an endogenous lipid component expressed in melanoma cells able to induce an immunosurveillance response based on iNKT. We also investigated the possibility of animal protection against tumor development by using dendritic cells primed with the endogenous lipid. The extraction of membrane lipid components was carried out from in vivo grown tumors, thus avoiding artifacts from the in vitro grown cultures. Three different extraction protocols were tested (A, B, C), and 14 different fractions were obtained and tested for the activation of hybridoma DN32.D3, a cell line of immortalized murine NKT cells. Fraction F3 of protocol A (F3A) activated hybridoma DN32.D3 to produce IL-2. For an efficient presentation of lipids from this fraction we successfully used bone marrow dendritic cells (BMDC) on in vitro and in vivo assays. F3A and NKT-cell activating glycolipids, agalactosylceramide (a-GalCer) and isoglobohexosylceramide (iGb3), were tested. In the attempt to isolate the stimulatory component present in the melanoma F3A fraction, HPTLC was used and revealed with several specific reagents for sialic acid residues, neutral sugars, phosphate and total lipids. The fraction was also separated in silica columns, immunostained with Bandeiraea simplicifolia BS-1 lectin and analyzed by mass spectrometry. Ganglioside GM3 and neutral glycosphingolipids iGb3, Gb3, iGb4 and Gb4 were identified by ESI-LIT-MS (electrospray ionization- linear ion trap- mass spectrometry). By incubation of iGb3 with BMDC and these with DN32.D3 cells, the latter were activated to produce IL-2. GM3 consistently inhibited the activation of iNKT cells. Cytotoxicity assays were then carried out, in which we found that NKT cells stimulated by BMDC, primed with a-GalCer or iGb3, encircled the B16F10-Nex2 tumor cells as visualized by fluorescent microscopy. NKT cells promoted lysis of up to 40% of tumor cells. In vivo tests showed that mice injected endovenously with murine melanoma cells and treated with dendritic cells primed with a-GalCer or iGb3, had lungs with 4-fold fewer nodules than an equally challenged but untreated group. The present results show that the murine melanoma B16F10-Nex2 expresses iGb3 and its precursor iGb4, being able to activate NKT cells and conferring them a cytotoxic activity in vitro against melanoma. Such lipid (iGb3) was also protective in vivo reducing the melanoma pulmonary metastases when presented by dendritic cells used in cellular therapy protocol. This is the fist work showing effectively that an endogenous CD1d-restricted glycolipid able to activate iNKT cells display a protective antitumor effect, using cellular therapy with dendritic cells. / TEDE / BV UNIFESP: Teses e dissertações