Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 89-102).

Plusieurs populations de cellules immunitaires possèdent une activité cytotoxique permettant l'élimination de cellules altérées. Cette fonction cellulaire est ainsi déterminante dans le contrôle des infections, des processus tumoraux, ou encore des maladies inflammatoires chroniques. Mon projet de thèse se concentre sur des aspects fondamentaux de l'activité lytique de deux populations de lymphocytes T cytotoxiques : les lymphocytes T CD8+ et les lymphocytes T CD4+ régulateurs de type 1. Pour cela, l'exploration des mécanismes de cette activité a été conduite au travers de deux modèles, pathologique et thérapeutique, à différentes échelles biologiques : au niveau de la population ou de la cellule individuelle, mais aussi différentes échelles d'organisations moléculaires : cellulaire et nanoscopique. Nous avons pu démontrer que l'activité de lyse de lymphocytes T CD8+ cytotoxiques face à un excès de cellules cibles est efficace sur des temps prolongés, reposant sur une capacité individuelle fortement hétérogène à effectuer une lyse multiple. L'importance de cette activité de lyse soutenue a été renforcée par l'identification d'un défaut lytique particulièrement prononcé sur le long- terme chez des lymphocytes T CD8+ issus de patients atteints du syndrome de Wiskott-Aldrich. Ce défaut est lié à une activation réduite de l'intégrine LFA-1 et un délai dans la délivrance du coup létal. De plus, la protéine WASP permet de restreindre LFA-1 de haute-affinité en nanoclusters denses ainsi que de permettre l'organisation en un ring de LFA-1 et la localisation des granules lytiques à l'intérieur de celui-ci. Par ailleurs, les lymphocytes T CD4+ régulateurs de type 1 développés dans le cadre d'une thérapie cellulaire (Ovasave(r)) démontrent une capacité de lyse envers les cellules myéloïdes, en complément d'une activité immunosuppressive sur les lymphocytes T conventionnels. Cette activité est mise en place sur du long-terme, jusqu'à atteindre une efficacité optimale, lié à un délai dans la délivrance du coup létal. De manière surprenante, malgré une spécificité pour l'ovalbumine, l'activité cytotoxique semble être indépendante de l'activation du TCR. En outre, la lyse est granzyme-dépendante mais perforine-indépendante. Ainsi ces lymphocytes T thérapeutiques manifestent une activité cytotoxique alternative. Pour conclure, mon projet de thèse a permis de caractériser une activité de lyse soutenue basée sur une capacité individuelle hétérogène. Cette habilité à soutenir une lyse sur du long-terme implique une stabilité de la synapse, où WASP joue notamment un rôle clé pour l'activation et l'organisation de LFA-1. Les lymphocytes T régulateurs thérapeutiques démontrent aussi une activité de lyse soutenue, cependant les acteurs moléculaires sont non conventionnels. De manière générale, une activité de lyse soutenue permettrait de calibrer une réponse cytotoxique prolongée en rapport à la taille de la population cible, ainsi que le partage avec d'autres fonctions cellulaires comme la sécrétion de cytokines. / During different pathological conditions such as infections, tumoral processes or chronic inflammation diseases, altered cells are eliminated through a cytotoxic activity mediated by several immune cell populations. This cellular function is therefore crucial for carrying out the action of the immune system. My thesis project focuses on fundamental aspects of the lytic activity of two cytotoxic lymphocyte populations: CD8+ T cells and type-1 CD4+ regulatory T cells. To explore the mechanisms of this activity, this study has been driven on two cases, pathological and therapeutic models, at the population and single-cell levels and also at the cellular and nanoscopic scales of the molecular organisation. We have been able to demonstrate that the CD8+ T cell lysis activity against an excess of target cells is effective over prolonged periods, relying on a highly heterogeneous individual capacity to perform multiple lysis. The importance of this sustained cytotoxic activity was reinforced by the identification of a lytic defect, particularly pronounced on a long time period, of CD8+ T cells from Wiskott-Aldrich syndrome patients. This defect is related to a reduced activation of the LFA-1 integrin and delay in the lethal hit delivery. In addition, the WASP protein allows to restrict high affinity LFA-1 to dense nanoclusters as well as the assembly of LFA- 1 ring and the localization of the lytic granules inside this ring. Moreover, type-1 CD4+ regulatory T cells from a cellular therapy (Ovasave(r)) demonstrated a cytotoxic activity toward myeloid cells, additionally to an immunosuppressive activity on conventional T cells. This activity is implemented over long time periods, until reaching optimal efficiency, and is related to a delay in the lethal hit delivery. Surprisingly, despite a specificity for ovalbumin, the cytotoxic activity measured in absence of the antigen suggests a TCR independence. In addition, lysis is not mediated by perforin but is exclusively granzyme-dependent. Thus, these therapeutic T cells exhibit an alternative cytotoxic activity. To conclude, my thesis project permits to characterize a sustained lysis activity relying on a heterogeneous individual capacity. This ability to sustain a lytic activity involves stability of the synapse, where WASP plays a key role towards the activation and organization of LFA-1. The therapeutic regulatory T lymphocytes also demonstrated a sustained cytotoxic activity, however the molecular actors are unconventional. On the whole, sustained lytic activity would be key to the calibration of cytotoxic responses in relation to the size of the target population, as well as sharing with other cellular functions such as cytokine secretion.

Professional antigen presenting cells (APCs) represent an important link between the innate and adaptive immune system. Macrophages (MΦs) and dendritic cells (DCs) serve as sentinels in the periphery collecting samples from their environment and processing this information. These cells then present antigenic fragments to T cells in the context of self-MHC molecules. Although a clear role for both of these APCs in the stimulation of already activated or memory T cells has been established, the ability of MΦs to activate naive T cells is still unknown. In this thesis the ability of bone marrow-derived MΦs and DCs to prime naive CD8+ and CD4+ T cells was investigated. Using adoptively transferred transgenic CFSE-Iabeled P-14 T cells, specific for gp33 from lymphocytic choriomeningitis virus in the context of Db, we were able to demonstrate the ability of both MΦs and DCs to induce naive CD8+ T cells proliferation. Once primed by MΦs these T cells gained effector function as shown by interferon- γ (IFN-γ) production and in vivo cytolysis. In addition, immunization of wild type animals with gp33-pulsed MΦs, as well as DCs, led to greater than a 95% reduction in lymphocytic choriomeningitis virus titers. To rule out the role of cross-presentation in the observed priming, two models were used. In the first model, lethally irradiated F1 bxs chimeras reconstituted with either H-2s or H-2b bone marrow were used as host for the adoptive transfer experiments. Since the gp33 peptide binds to Db, the H-2s reconstituted animals should be unable to cross-present the peptide to the P-14 T cells. Using this model, we were able to clearly demonstrate the ability of MΦs to activate naive P-14 T cells to undergo division. Additional experiments, demonstrated that these MΦ primed T cells went on to develop into effector cells. Finally, the ability of the MΦ primed T cells to develop into functional memory cells was demonstrated. To confirm the chimera results, these experiments were repeated using β2 microglobulin deficient animals (whose cells don't express MHC I) as host in adoptive experiments. MΦs were able to stimulate the naive P-14 T cells to divide and gain effector function as demonstrated by the ability to produce IFN-γ. In contrast to the CD8 system, MΦ were poor stimulators of D011.10 CD4+ T cell proliferation. Additionally, D011.10 T cells stimulated by DCs were able to produce interleukin-2 (IL-2), IL-4, tumor necrosis factor and granulocyte-macrophage colony stimulating factor where as MΦ stimulated D011.10 T cells were only able to produce IL-2. In conclusion this body of work clearly demonstrates the in vivo ability of MΦ to stimulate CD8+ T cell proliferation, effector function, as well as the formation of functional CD8+ T cell memory. Whether or not the nature of the memory pools stimulated by the two APCs is exactly the same is still unknown and needs further investigation. The ability of APCs other than DCs to stimulate functional protective memory needs to be considered in the quest to design vaccines that offer broad-spectrum protection.

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

No description available.

Each individual experiences many sequential infections throughout the lifetime. An increasing body of work indicates that prior exposure to unrelated pathogens can greatly alter the disease course during a later infection. This can be a consequence of a phenomenon known as heterologous immunity. Most viruses invade the host through the mucosa of a variety of organs and tissues. Using the intranasal mucosal route of infection, the thesis focused on studying modulation of lymphocytic choriomeningitis virus (LCMV)-specific memory CD8 T cells upon respiratory vaccinia virus (VV) infection and the role of these memory CD8 T cells in heterologous immunity against VV and altered immunopathology in the lung. The VV infection had a profound impact on memory T cells specific for LCMV. The impact included the up-regulation of CD69 expression on LCMV-specific CD8 memory T cells and the activation of their in vivoIFN-γ production and cytotoxic function. Some of these antigen-specific memory T cells selectively expanded in number, resulting in modulation of the original LCMV-specific T cell repertoire. In addition, there was a selective organ-dependent redistribution of these LCMV-specific memory T cell populations in secondary lymphoid tissue (the mediastinal lymph node and spleen) and the non-lymphoid peripheral (the lung) organs. The presence of these LCMV-specific memory T cells correlated with IFN-γ-dependent enhanced VV clearance, decreased mortality and marked changes in lung immunopathology. Thus, the participation of pre-existing memory T cells specific for unrelated agents can alter the dynamics of mucosal immunity. This is associated with an altered disease course in response to a pathogen. The roles for T cell cross-reactivity and cytokines in the modulation of memory CD8 T cells during heterologous memory CD8 T cell-mediated immunity and immunopathology were investigated. Upon VV challenge, there were preferential expansions of several LCMV-specific memory CD8 T cell populations. This selectivity suggested that cross-reactive responses played a role in this expansion. Moreover, a VV peptide, partially homologous to LCMV NP 205, stimulated LCMV-NP205 specific CD8 T cells, suggesting that NP205 may be a cross-reactive epitope. Poly I:C treatment of LCMV-immune mice resulted in a transient increase but no repertoire alteration of LCMV-epitope-specific CD8 T cells. These T cells did not produce IFN-γ in vivo. These results imply that poly I:C, presumably through its induced cytokines, was assisting in initial recruitment of LCMV-specific memory CD8 T cells in a nonspecific manner. VV challenge of LCMV-immune IL-12KO mice resulted in activation and slightly decreased accumulation of LCMV-specific CD8 T cells. Moreover, there was a dramatic reduction of in vivoIFN-γ production by LCMV-specific IL-12KO CD8 T cells in the lung. I interpreted this to mean that IL-12 was important to augment IFN-γ production by memory CD8 T cells upon TCR engagement by antigens and to induce further accumulation of activated memory CD8 T cells during the heterologous viral infection. This thesis also systematically examined what effect the sequence of two heterologous virus challenges had on viral clearance, early cytokine profiles and immunopathology in the lung after infecting mice immune to one virus with another unrelated viruses. Four unrelated viruses, [LCMV, VV, influenza A virus or murine cytomegalovirus (MCMV)], were used. There were many common changes observed in the acute response to VV as a consequence of prior immunity to any of three viruses, LCMV, MCMV or influenza A virus. These included the enhanced clearance of VV in the lung, associated with enhanced TH1 type responses with increased IFN-γ and suppressed pro-inflammatory responses. However, immunity to the three different viruses resulted in unique pathologies in the VV-infected lungs, but with one common feature, the substitution of lymphocytic and chronic mononuclear infiltrates for the usual acute polymorphonuclear response seen in non-immune mice. Immunity to influenza A virus appeared to influence the outcome of subsequent acute infections with any of the three viruses, VV, LCMV and MCMV. Most notably, influenza A virus-immunity protected against VV but it actually enhanced LCMV and MCMV titers. This enhanced MCMV replication was associated with enhanced TH1 type response and pro-inflammatory cytokine responses. Immunity to influenza A virus appeared to dramatically enhance the mild lymphocytic and chronic mononuclear response usually observed during acute infection with either LCMV or MCMV in non-immune mice, but LCMV infection and MCM infection of influenza A virus-immune mice each had its own unique features. Thus, the specific sequence of virus infections controls the outcome of disease.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-07-18T21:46:22Z No. of bitstreams: 1 Fernanda Oliveira Novais Avaliacao do papel....pdf: 2271339 bytes, checksum: eee5aa084065678cf55651296f356919 (MD5) / Made available in DSpace on 2012-07-18T21:46:22Z (GMT). No. of bitstreams: 1 Fernanda Oliveira Novais Avaliacao do papel....pdf: 2271339 bytes, checksum: eee5aa084065678cf55651296f356919 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / Linfócitos T CD8+ são células do sistema imune adaptativo capazes de induzir a morte de células infectadas através de mecanismos citotóxicos. No modelo de infecção intradérmica por Leishmania revelou-se que os linfócitos T CD8+ são responsáveis tanto pela indução de patogênese bem como pela imunidade contra a infecção primária por L. major. Até o momento, o papel dos linfócitos T CD8+ não foi estudado na infecção experimental por L. braziliensis. Neste estudo, investigamos o recrutamento dos linfócitos T CD8+ para o sítio de infecção e determinamos a sua função. Cinco semanas após a infecção intradérmica por L. braziliensis, camundongos BALB/c apresentaram um aumento na porcentagem de linfócitos T CD8+ presentes na orelha infectada e estes produziram, principalmente, IFN-g e Granzima B. Já no linfonodo de drenagem, estas células não produzem granzima mas, sim, IFN-g e TNF-a. Utilizando o mesmo modelo de infecção, camundongos BALB/c ou C57BL/6 depletados de linfócitos T CD8+ ou camundongos deficientes em b2-microglobulina ou em CD8 apresentaram redução no tamanho da lesão ao longo da infecção e menor carga parasitária cinco semanas após a infecção. A depleção de linfócitos T CD8+ não induziu qualquer alteração no recrutamento e produção de IFN-g, TNF-a e IL-10 pelos linfócitos T CD4+ no sítio de infecção ou no linfonodo de drenagem. Além disso, a capacidade proliferativa ou a produção de citocinas específicas in vitro após estímulo com células dendríticas infectadas por L. braziliensis não sofreram alteração. A ausência de linfócitos T CD8+ após a 8 infecção por L. braziliensis também não alterou o recrutamento de monócitos inflamatórios nem a sua diferenciação em células dendríticas. Por último, a análise histológica e de citometria de fluxo mostrou aumento no recrutamento de neutrófilos para o sítio de infecção e este resultado pode ser correlacionado com o controle da doença. Para confirmar o envolvimento dos linfócitos T CD8+ no desenvolvimento de lesão por L. braziliensis, transferimos linfócitos T CD8+ de camundongos naïve ou imunes, bem como linfócitos T CD4+ somente ou linfócitos T CD8+ e T CD4+ para camundongos RAG-/-. Neste contexto, a transferência de linfócitos T CD8+ naïve ou imunes induziu uma intensa patologia no sítio de infecção bem como a disseminação de parasitas para outros sítios, como a orelha não infectada, pata e nariz. Camundongos RAG-/- controle e aqueles que receberam linfócitos T CD8+ naïve ou imunes apresentam a mesma quantidade de parasitas no sítio de infecção, embora o aspecto da lesão tenha sido muito diferente. A transferência de linfócitos T CD4+ foi capaz de controlar a carga parasitária nestes animais e o mesmo foi observado após transferência de linfócitos T CD4+ e T CD8+ em conjunto. Nestes animais não observamos lesões em outros sítios, indicando que os linfócitos T CD8+ contribuem para a disseminação dos parasitas. Por último, transferimos linfócitos T CD8+ provenientes de camundongos selvagens ou deficientes de IFN-g e perforina e observamos que, na ausência de perforina, a patologia e a disseminação parasitária são controladas. Portanto, este estudo sugere envolvimento dos linfócitos T CD8+ na patogênese induzida por L. braziliensis devido ao seu potencial citotóxico e, em paralelo, inibindo o recrutamento de neutrófilos para o sítio de infecção. / CD8+ T lymphocytes are part of the adaptive immune system and are considered cytotoxic because of their ability to induce death in infected cells. Using the intradermal model of Leishmania infection, it has been shown that CD8+ T cells play an essential role in both pathogenesis and immunity to primary infection with L. major in the skin. So far, the role of these lymphocytes in the experimental model of infection using L. braziliensis has not been evaluated. In this study we determined the recruitment and function of these cells upon infection with L. braziliensis. Five weeks after infection, the frequency of CD8+ T cells was increased in the dermal site and these cells produced mainly IFN-g and granzyme B in infected mice. In the draining lymph nodes, these cells produced high levels of IFN-g and TNF-a, but not granzyme B. Using the same intradermal model of infection, we analysed the outcome of infection in the absence of CD8+ T lymphocytes using both antibody depletion in BALB/c and C57BL/6 mice and mice deficient in b2- microglobulin and CD8. In all groups, the absence of CD8+ T cells was correlated with better control of lesion development and parasite load in both depleted BALB/c and in b2-microglobulin deficient mice. In the absence of CD8+ T cells, CD4+ T lymphocytes were recruited to the same extension and produced same levels of IFN-g, TNF-a and IL-10 both in the infected ear and in draining lymph nodes when compared to infected mice that were not depleted. Also, there was no change in the proliferative potential and in IFN-g production by these cells after re-stimulation with infected dendritic cells. Analysis of inflammatory monocyte recruitment and differentiation of these cells into dendritic cells were similar in both depleted and non-depleted mice. On the other hand, histological and flow cytometric analyses showed increased neutrophil recruitment to the site of infection and this can be correlated with disease control. To confirm the role of CD8+ T cells in the lesion development of L. braziliensis infected mice, we then transferred CD8+ T cells from naïve or immune mice, as well as CD4+ T cells alone or together with T CD8+ to RAG deficient mice. The transfer of CD8+ T cells from immune or naïve mice into RAG recipients induced an intense pathology upon infection with L. braziliensis in the infection site, but also in uninfected tissues such as the uninfected ear, nose and footpad. Evaluation of parasite numbers in the infected ear showed that RAG deficient mice without T cells and those transferred with CD8+ T cells from naïve or immune mice had similar number of parasites although the pathology was very different. The transfer of CD4+ T cells alone or in association with CD8+ T cells induced parasite control in the infection site. In these mice, we could not detect lesions in other sites and we concluded that the transfer of CD8+ T cells alone induces parasite dissemination in RAG deficient mice. Finally, the transfer of CD8+ T cells from perforin deficient mice led to control in lesion development and in parasite dissemination. In this study we can conclude that CD8+ T cells are involved in the pathogenesis of L. braziliensis due to their cytotoxic potential and by inhibiting neutrophil recruitment to the infection site.

Malgré les programmes de dépistage mis en place et le vaste arsenal thérapeutique disponible, 8,2 millions de décès dans le monde ont été attribués au cancer pour l'année 2012 (données Globocan 2012, OMS). L'immunothérapie antitumorale est en plein essor et consiste notamment à exploiter le système immunitaire de l'hôte pour obtenir une réponse contre la tumeur. L'utilisation de vecteurs bactériens, capables de délivrer un message antigénique et de stimuler de manière concomittante l'immunité innée, fait partie des approches de vaccination antitumorale prometteuses. Parmi ces vecteurs, une bactérie Pseudomonas aeruginosa mise au point par notre laboratoire présente l'intérêt de pouvoir injecter in vivo des antigènes de tumeur, via son système de sécrétion de type III (SST3), directement dans le compartiment intracellulaire des cellules présentatrices d'antigènes. La voie de présentation du CMH I est ainsi favorisée et permet la génération d'une réponse des lymphocytes T cytotoxiques vis-à-vis de la tumeur exprimant l'antigène. Cependant, la poursuite des études précliniques et cliniques paraît délicate, en raison du risque infectieux lié à une bactérie pathogène, quand bien même atténuée. Lors de ce travail, nous avons donc développé une nouvelle souche de P. aeruginosa Killed But Metabolically Active (KBMA), incapable de se répliquer, mais toujours apte à jouer son rôle de vecteur. Une analyse de la réponse immune antitumorale, suite à l'immunisation par différents vecteurs, a permis de mettre en évidence une forte infiltration de la tumeur par des lymphocytes T CD8+ spécifiques de l'antigène, mais également une protection à long terme liée à la présence d'un pool majoritaire de lymphocytes T CD8+ spécifiques effecteurs mémoires. Enfin nous avons cherché à appliquer cette technologie à l'antigène de tumeur anhydrase carbonique 9 (AC9), exprimé par de nombreuses tumeurs solides chez l'homme. / Despite cancer screening programs and the available therapeutic armamentarium, 8.2 million deaths worldwide were due to cancer in 2012 (data Globocan 2012, WHO). The antitumor immunotherapy is booming and aims at using the immune system of the host as a response against the tumor. The use of bacterial vectors, able to deliver an antigenic message and concomitantly stimulate innate immunity, is one of the most promising approaches to antitumor vaccination. Among these vectors, the bacterium Pseudomonas aeruginosa developed by our laboratory has the advantage of being able to inject in vivo tumor antigens via its type III secretion system (T3SS) directly in the intracellular compartment of antigen-presenting cells. The MHC I presentation pathway is thus favored and allows the generation of a cytotoxic T lymphocytes response against antigen-expressing tumors. However, further preclinical and clinical studies remain difficult, because of the risk of infection related to a bacterial pathogen, even if attenuated. In this work, we have developed a new strain of P. aeruginosa Killed But Metabolically Active (KBMA) unable to replicate, but still able to play its role as a vector. An analysis of the antitumor immune response following immunization with different vectors, allowed to demonstrate a strong tumor infiltration by antigen-specific CD8+ T lymphocytes, but also a long-term protection related to the presence of a major pool of antigen-specific effector memory CD8+ T cells. Finally we are seeking to apply this technology to the tumor antigen carbonic anhydrase 9 (CA9), expressed by many solid tumors in humans.

Submitted by ACACIO DUARTE PACHECO (acacio@fmva.unesp.br) on 2016-09-01T16:16:58Z No. of bitstreams: 1 Acácio Impressão Tese Final.pdf: 1381975 bytes, checksum: 5982f80f260a714c02111156f309cccb (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-09-02T18:54:19Z (GMT) No. of bitstreams: 1 pacheco_ad_dr_araca.pdf: 1381975 bytes, checksum: 5982f80f260a714c02111156f309cccb (MD5) / Made available in DSpace on 2016-09-02T18:54:19Z (GMT). No. of bitstreams: 1 pacheco_ad_dr_araca.pdf: 1381975 bytes, checksum: 5982f80f260a714c02111156f309cccb (MD5) Previous issue date: 2016-07-19 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A ocorrência de miocardite em cães naturalmente acometidos por Leishmania infantum chagasi tem sido demonstrada em estudos recentes, contudo o tipo de resposta imune incitada no tecido cardíaco de cães com leishmaniose visceral (LV) ainda não foi determinada. Sendo assim, foram avaliados fragmentos da: parede livre de átrio direito (AD), parede livre de ventrículo direito (VD), parede livre de ventrículo esquerdo (VE) e septo interventricular (SIV) de 20 cães naturalmente acometidos por LV para determinação das alterações histopatológicas e da resposta imune no tecido cardíaco. A presença de miocardite foi observada em 75% dos animais avaliados, sendo o infiltrado inflamatório do tipo linfoplasmocitário a alteração mais comum nesses fragmentos. A imunomarcação de Leishmania spp. foi observada em 60% dos animais. Não houve correlação entre a intensidade do infiltrado inflamatório e a presença de Leishmania sp. no miocárdio (p=0,0682). A imunomarcação do parasito apresentou correlação com a presença de linfócitos T CD4+ (p<0,0001) e T CD8+ (p=0,0002), linfócitos B CD79+ (p=0,046) e IgG (p=0,040). Os níveis de IL-4 (p=0,030), IL-12 (p=0,002), IFN- (p<0,0001) foram menores em cães infectados em relação ao grupo controle, independente da presença ou não do parasito no miocárdio (p>0,05). No presente estudo, a presença de alterações miocárdicas, associadas a resposta imunológica mista foram comuns em cães com LV. Novos estudos avaliando paralelamente, a presença de inflamação miocárdica e o perfil de citocinas no tecido cardíaco, à resposta imunológica sistêmica são indicados para confirmação da resposta imunológica presente no miocárdio de cães acometidos por LV. / Recent studies have demonstrated the occurrence of myocarditis in dogs naturally infected with Leishmania infantum chagasi, however the type of immune response in cardiac tissue of dogs with visceral leishmaniasis (VL) has not been determined. For this purpose, fragments from right atrial free wall (AD), right ventricular free wall (RV), left ventricular free wall (LV) and interventricular septum (IVS) of 20 dogs naturally affected by VL were evaluated to determine the histopathological changes and the immune response in heart tissue. The presence of myocarditis has been reported in 75% of the evaluated animals, and an lymphoplasmocytic inflammatory infiltrate was the most common change in these fragments. The immunostaining of Leishmania sp. was observed in 60% of the evaluated animals. There was no correlation between the intensity of the inflammatory infiltrate and the presence of Leishmania sp. (p = 0.0682). The immunostaining of the parasite correlated with the presence of CD4 + (p <0.0001) and CD8 + T lymphocytes (p = 0.0002), CD79 + B lymphocytes (p = 0.046) and IgG (p = 0.040). IL-4 (p = 0.030), IL-12 (p = 0.002) and IFN- levels (p <0.0001) were lower in the infected dogs when compared to control group, regardless of the presence or absence parasite in the myocardium (p> 0.05). The results obtained in this study suggest that the presence of myocardial changes is common in dogs with VL, associated with a mixed immune response in heart tissue. However, the local inflammatory process seems not enough to trigger the production of cytokines normally involved in VL, possibly due to a low parasite load in dogs myocardium, especially when compared to other organs such as spleen, liver and bone marrow. Further studies evaluating at the same time the presence of myocardial inflammation and the behavior of these circulating cytokines in target organs with immune activity are indicated to confirm the immune response present in the myocardium of dogs affected by VL. / FAPESP: 2013/12129-8 / CNPq: 141536/2013-6