As células dendríticas (DCs) são células do sistema imune inato que são especializadas na instrução de repostas imunes adaptativas No baço murinho, as DCs convencionais podem ser classificadas em CD8α+ DEC205+ e CD8α- DCIR2+. Anticorpos monoclonais (mAbs) αDEC205 e αDCIR2 (33D1) conjugados a proteínas antigênicas e são utilizados como estratégia de direcionamento de antígenos para as DCs CD8α+ e CD8α-. Foi utilizado o antígeno quimérico MSP119PADRE conjugado aos mAbs αDEC205 e αDCIR2 e o Poly I:C como adjuvante. A montagem da resposta celular e humoral foi analisada 3, 4, 5 e 6 dias após primeira e a segunda As DCS CD8α- são especializadas na instrução de células TFH. Porém, num segundo dose de mAbs há a instrução de uma resposta Th2/Th17. E, o direcionamento de antígenos para DCS CD8α+ induz uma resposta Th1, indicando que essas células são especializadas na instrução desse perfil de resposta auxiliar. Apesar disso, na segunda imunização há a diferenciação de células TFH. / As dendritic cells (DC) are innate immune cells that are specialized to prime adaptive immune cells. In the murine spleen, conventional DCs can be classified into CD8α+ DEC205+ and CD8α-DCIR2+. Monoclonal antibodies (mAbs) αDEC205 and αDCIR2 (33D1) conjugated to antigenic proteins have benn used as antigen targeting strategy to CD8α+ and CD8α- DCs. MSP119PADRE chimeric antigen conjugated to mAbs αDEC205 and αDCIR2 and Poly I: C as adjuvant was used. The assembly of the cellular and humoral response was analyzed 3, 4, 5 and 6 days after the first and second doses of imnunization. DCs CD8α- are specialized in the instruction of TFH cells. However, there is an a promotion of Th1 response by CD8α+, indicating that these cells are specialized in the instruction of the helper response profile. Nevertheless, in the second immunization there is a differentiation of TFH cells.

L'objectif de cette thèse a été de caractériser les patrons spatiaux de diversité inter- et intraspécifique au sein des réseaux dendritiques, d'expliciter les processus évolutifs et écologiques qui les sous-tendent, et d'isoler les possibles covariations spatiales et interactions existant entre ces différentes facettes de biodiversité. Pour cela, j'ai tout d'abord développé de nouvelles méthodes statistiques permettant l'analyse, par des modèles causaux, de données sous la forme de matrices de distances, afin de pouvoir analyser plusieurs facettes de biodiversité dans un unique cadre statistique au niveau alpha et bêta. J'ai par la suite étudié de manière intégrative les patrons de diversité interspécifique et intraspécifique génétique d'une part, et intraspécifique génétique et intraspécifique phénotypique d'autre part, au sein du bassin versant Garonne- Dordogne. Enfin, j'ai utilisé un modèle de dynamique éco-évolutive afin d'étudier l'impact de la structure et des gradients environnementaux caractérisant les réseaux dendritiques sur l'adaptation locale au sein de ces réseaux. / The aim of this thesis was to characterized the spatial patterns of inter- and intraspecific diversity within riverine networks, to better understand the ecological and evolutionary processes underlying them and to explore how the different facets of biodiversity interact with one another. First, I developed novel statistical approaches allowing the application of causal modeling to data in the form of pairwise matrices, thus allowing the study within integrative frameworks of several biodiversity facets at the alpha and beta levels. I then studied integratively the patterns of interspecific and intraspecific genetic diversity and of intraspecific genetic and intraspecific phenotypic diversity within the Garonne-Dordogne river basin. Finally, I used an eco-evolutionary metapopulation dynamics model to assess the impacts of the structure and environmental gradients that characterize riverine networks on local adaptation.

Chemoattractant agents play a crucial role in the initiation of immune responses, by regulating the traffic and function of leucocyte populations. Their receptors are therefore considered as potential targets for the development of new therapies in the fields of cancer and inflammatory diseases. ChemR23, a previously orphan receptor discovered in the laboratory, is structurally related to receptors for chemoattractant agents. It is expressed on immature myeloid and plasmacytoid dendritic cells (mDCs and pDCs respectively), as well as on adipocytes, macrophages, NK and endothelial cells. Chemerin, the endogenous ligand of ChemR23, is abundant in various human samples originating from inflammatory diseases, including pleural effusions. Chemerin is secreted as an inactive precursor, prochemerin, and is activated by the removal of six or seven amino-acids from its carboxy-terminus by serine proteases, such as as cathepsin G and elastase. Chemerin acts as a chemoattractant agent of low nanomolar potency for macrophages, immature mDCs and pDCs. It is however more active on pDCs, in line with the higher expression of ChemR23 on these cells. pDCs possess important immunoregulatory properties in lung diseases, and their ability to secrete large amounts of type I interferon (IFN) upon viral infection makes them crucial players in anti-viral immunity.<p>According to these elements, and to the role of neutrophils in the physiopathology of many inflammatory lung diseases and in the generation of active chemerin, we began in 2007 to study the role of chemerin and its receptor ChemR23 in inflammatory lung diseases. We first characterized the mouse chemerin/ChemR23 system, and described that this system was very similar to the human one, in terms of distribution, pharmacology and functional properties. We then used wild type mice (WT) and mice invalidated for the receptor (ChemR23-/-) in various models of inflammatory lung diseases, including asthma, lung fibrosis, viral pneumonia, and acute lung injury. <p>Whereas the asthma and lung fibrosis models did not allow to demonstrate a significant role of the chemerin/ChemR23 system (possibly as a result of the lack of production of active chemerin in these models), infection by either the Pneumonia Virus of Mice (PVM), the mouse counterpart of human RSV, or by a murinized H1N1 influenza strain resulted in a significantly higher mortality rate in ChemR23-/- mice as compared to their WT counterparts. Using the PVM-induced pneumonia model, we observed that the excessive mortality of knock-out mice is caused by an inadequate and excessive innate immune response characterized by a massive recruitment of neutrophils to the lungs, associated with a delayed viral clearance and lower type I IFN synthesis. This latter observation suggested an impairment of pDC recruitment, according to the important contribution of pDCs to the production of type I IFNs in viral diseases, and the role of chemerin in the recruitment of these cells. We indeed confirmed a lower recruitment of pDCs in the lung of infected ChemR23-/- mice, as compared to WT mice. However, experiments of adoptive transfert and depletion of pDCs failed to proof a link between impaired pDC recruitment and the excessive morbidity and mortality observed in ChemR23-invalidated mice. <p>In parallel, we studied the role of the chemerin/ChemR23 system in the control of innate immune responses, by using a model of acute lung injury caused by the intra-tracheal instillation of bacterial lipopolysaccharide (LPS). In this model, administration of recombinant chemerin together with LPS in WT mice resulted in a significant (about 50%) reduction of neutrophil recruitment to both lung parenchyma and airways. Assessment of pro-inflammatory cytokines and chemokines in broncho-alveolar lavage fluids confirmed this anti-inflammatory effect of chemerin, which was ChemR23-dependent, as the inflammatory response of ChemR23-/- mice was unaffected by chemerin. In our hands, chemerin does not modulate macrophage functions, in contrast to data recently published by other groups, attributing anti-inflammatory effects of chemerin or chemerin-derived peptide to the modulation of macrophage activation and phagocytosis. Other hypotheses that could take our observations into account are presently investigated, including an immunomodulatory role of chemerin on lung epithelial or endothelial cells, and/or the ChemR23-dependent recruitment of subtypes of macrophages or other myeloid cells endowed with immunosuppressive properties. <p>In conclusion, our studies characterized the mouse chemerin/ChemR23 system and highlighted the role of this system in the physiopathology of some inflammatory lung diseases. Our results suggest that the chemerin/ChemR23 system might be considered as a potential therapeutic target for the development of future anti-infectious and anti-inflammatory therapies, particularly for viral pneumonia, which represent a major public health problem, as well as for acute respiratory distress syndrome (ARDS) following severe acute lung injuries.<p> <p><p>Les agents chimioattractants jouent un rôle fondamental dans l’initiation des réponses immunes en régulant le trafic et la fonction des populations leucocytaires. Leurs récepteurs constituent dès lors des cibles d’intérêt pour le développement de traitements contre les maladies inflammatoires et le cancer. Le laboratoire d’accueil a identifié le récepteur ChemR23, exprimé à la surface des cellules dendritiques myéloïdes (mDCs) et plasmacytoïdes (pDCs) immatures, des macrophages, des cellules NK, des adipocytes, et des cellules endothéliales. Le ligand endogène du récepteur ChemR23, la chémérine, est présent en abondance dans divers échantillons pathologiques d’origine inflammatoire. La chémérine est produite sous la forme d'un précurseur inactif, la prochémérine, qui nécessite pour devenir active le clivage protéolytique de six ou sept acides aminés à son extrémité carboxy-terminale. La chémérine induit le chimiotactisme des macrophages et des DCs immatures, et en particulier des pDCs immatures en accord avec l’expression plus importante de ChemR23 par les pDCs. Les pDCs jouent un rôle immunorégulateur important en pathologie pulmonaire, en particulier dans la physiopathologie des pneumonies virales, par leur capacité à produire d’importantes quantités d’interféron (IFN) de type I.<p>Compte tenu de ces éléments et du rôle des polynucléaires neutrophiles dans de nombreuses pathologies pulmonaires, ainsi que dans la génération de chémérine active à partir de son précurseur, nous avons débuté en octobre 2007, l’étude du rôle de la chémérine et de son récepteur ChemR23 dans le contrôle des pathologies pulmonaires inflammatoires. Nous avons tout d’abord caractérisé le système chémérine/ChemR23 chez la souris et avons montré que ce système présentait des caractéristiques similaires à celles décrites chez l’homme, en termes de distribution, de pharmacologie et de propriétés fonctionnelles. <p>Ensuite, nous avons comparé des souris sauvages et invalidées pour le récepteur ChemR23 (ChemR23-/-) dans divers modèles de pathologies pulmonaires. Les modèles d’asthme et de fibrose pulmonaire induite par instillation de bléomycine ou de silice n’ont pas permis de mettre en évidence un rôle important du couple chémérine/ChemR23, peut-être en raison de l’absence de génération de forme active de chémérine dans ces modèles. En revanche, l’administration de deux agents viraux différents, le PVM (Pneumonia Virus of Mice), l’équivalent murin du RSV humain, et un virus de l’influenza H1N1 murinisé, a résulté en un taux de mortalité 40% plus élevé pour les souris ChemR23-/- par rapport à leurs homologues sauvages. En utilisant le modèle de pneumonie induite par le PVM, nous avons montré que cette différence de mortalité est causée par une réponse immune inappropriée et excessive, associée à une réduction de l’élimination du virus, ainsi qu’à un déficit de synthèse d’IFN de type I. Les pDCs, dans un contexte d’infection virale, sont capables de synthétiser d’importantes quantités d’IFN de type I, et nous avons mis en évidence un déficit relatif de recrutement en pDCs chez les souris ChemR23-/- infectées. Néanmoins, les expériences de transfert adoptif et de déplétion de pDCs n’ont pas permis de lier ce défaut de recrutement à l’excès de morbidité et de mortalité observé chez les souris ChemR23-/- infectées. <p>En parallèle, le rôle de ce couple ligand-récepteur dans le contrôle des réponses immunitaires innées a été étudié dans un modèle de pneumopathie aiguë induite par instillation intra-trachéale de lipopolysaccharide (LPS). Dans ce modèle, l’administration simultanée de chémérine recombinante avec le LPS entraîne chez les souris sauvages une diminution significative (environ 50%) du nombre de polynucléaires neutrophiles recrutés dans les voies aériennes et dans le parenchyme pulmonaire, ainsi qu’une importante diminution de synthèse de cytokines pro-inflammatoires. Cet effet anti-inflammatoire de la chémérine est dépendant de ChemR23, et ne semble pas être secondaire à un effet de la chémérine sur l’activation des macrophages, contrairement à certaines données publiées récemment par d’autres groupes. D’autres hypothèses permettraient cependant de prendre en compte ces observations, notamment un effet de la chémérine sur les cellules épithéliales et/ou endothéliales pulmonaires, ainsi que sur le recrutement de sous-populations de macrophages ou d’autres cellules myéloïdes possédant des propriétés immunosuppressives. Des expériences complémentaires ont été initiées afin de tester ces hypothèses. <p>En conclusion, après avoir caractérisé le système chémérine/ChemR23 chez la souris, nos études ont permis de mettre en évidence le rôle de ce couple ligand/récepteur dans la physiopathologie de certaines pneumopathies inflammatoires, ouvrant ainsi de nouvelles perspectives thérapeutiques, en particulier pour le traitement des pneumopathies virales, qui constituent un problème de santé publique majeur, ainsi que des syndromes de détresse respiratoire aiguë (ARDS). / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

A cromoblastomicose (CBM) é uma micose crônica cosmopolita muito comum em regiões rurais de países subdesenvolvidos, que compõe o grupo de doenças tropicais negligenciadas. Apesar de seu conhecimento centenário muitas são as questões a serem aclaradas, especialmente do ponto de vista imunológico. Em um estudo transversal retrospectivo, avaliamos por imuno-histoquímica células dendríticas, macrófagos polarizados (M1 e M2) e marcadores de matriz extracelular e os comparamos com espécimes hígidos, além de avaliação intragrupal separando as espécies F. pedrosoi e F. nubica; duas das tantas causadoras de CBM. Os achados evidenciaram participação de CD206+, células CD207/IL-17+, predomínio de macrófagos M2, degradação da matriz extracelular através de alta expressão de MMP-2, MMP-9, Fibronectina e Laminina, entretanto, os níveis de colágeno IV estão preservados. O compilado de dados nos leva a crer que mediante ao invasor as células de Langerhans e CD207/IL-17+ possivelmente se mobilizam para sinalizar a infecção, enquanto macrófagos polarizados contribuem para a captura do patógeno e juntos, células dendríticas e macrófagos, orquestram o estabelecimento da resposta de defesa. Ademais, as incessantes tentativas de contenção da infecção não só não conseguem eliminar o fungo como provocam a degradação da matriz extracelular, contribuindo para a formação de lesões exuberantes e a cronicidade da doença, mantendo tal padrão de resposta quer o patógeno seja a espécie F. pedrosoi quanto a F. nubica. O presente estudo espera contribuir com a melhor compreensão da imunopatologia da CBM e fomentar a discussão sobre opções terapêuticas mais efetivas em um futuro próximo / The Chromoblastomycosis (CBM) is a chronic and cosmopolitan mycosis very common to rural regions in underdeveloped countries, which makes up the group of neglected tropical diseases. Despite the centennial discovery of the disease, there are many issues to be clarified, especially from the immunological point of view. We evaluated by immunohistochemistry dendritic cells, polarized macrophages (M1 and M2), markers of extracellular matrix from CBM human skin lesions and compared with healthy specimens. In addition, the group of lesions was compared between F. pedrosoi and F. nubica; two of the many cause of CBM. The findings showed participation of the CD206+, cells co-expressing CD207/IL-17+, predominance of macrophages M2, degradation of the extracellular matrix through the high expression of MMP-2, MMP-9, Fibronectin and Laminin. However, there is preservation of the levels of Collagen IV. The compiled data leads us to believe that, in contact with the invader, Langerhans cells and CD207/IL-17+ cells possibly mobilized to signal infection, while macrophages polarized contribute to the capture of the pathogen and together, dendritic cells and macrophages, orchestrate the establishment of the defense response. Moreover, the incessant attempts to containment of infection not only is unable to eliminate the fungus but also cause the degradation of the extracellular matrix, contributing to the formation of exuberant lesions and the chronicity of the disease, maintaining this pattern of response against both F. pedrosoi or F. nubica. We believe that our study can contribute to the better understanding of the immunopathology of CBM and encourage the discussion on more effective therapeutic options in the near future

Os ecossistemas aquáticos são afetados por processos que ocorrem em escalas finas (locais) e amplas (regionais). Os processos locais incluem, por exemplo, filtros ambientais e interações interespecíficas, enquanto que os regionais abrangem principalmente questões relacionadas à dispersão de indivíduos. O entendimento de como esses processos atuam sobre comunidades, populações e interações em peixes de riachos é fundamental para a conservação dos ecossistemas aquáticos, pois permite predizer as consequências de alterações antrópicas e fornece subsídios para ações de manejo e políticas de conservação. Na presente tese, eu desenvolvi cinco estudos em distintas escalas espaciais. Cada um é apresentado em capítulos distintos. Nos capítulos 1 e 2, eu abordei questões relacionadas à compreensão de como alterações antrópicas feitas em distintas escalas espaciais influenciam as diversidades alfa e beta de comunidades de peixes de riachos. No capítulo 3, eu procurei entender como processos previstos na teoria de metacomunidades influenciam mudanças temporais na composição e abundâncias de espécies em comunidades locais. No capítulo 4, eu estudei como os impactos antrópicos levam a alterações no papel trófico e no intestino de populações de uma espécie generalista e persistente. Por fim, no capítulo 5, eu usei uma abordagem de aninhamento, desenvolvida inicialmente na Ecologia de Comunidades, para avaliar a ocupação de larvas de uma espécie de quironomídeo (Diptera) sobre o corpo de seu hospedeiro (uma espécie de peixe da família Loricaridae). / Aquatic ecosystems are influenced by processes that occur at fine (local) and broad (regional) scales. Local processes include, for example, environmental filters and interspecific interactions, whereas regional processes encompass mainly questions regarding individual dispersion. Knowledge on how these processes affect communities, populations and interactions in stream fish is essential for conservation of aquatic ecosystems, as it allows predicting consequences of human-alterations and provides subsidy for management actions and conservation policies. In this dissertation, I developed five studies using distinct spatial scales. I presented each one in a separate chapter. In the 1st and 2nd chapters, I addressed questions concerned with the understanding of how human alterations at different spatial scales influence alpha and beta diversity of stream fish communities. In the 3rd chapter I looked for understanding how processes predicted in metacommunity theory influence mid- to long-term changes in composition and species abundances of local communities. In the 4th chapter, I studied how anthropic impact drives modification in the trophic role and intestine of a generalist and persistent fish species. Lastly, in the 5th chapter, I employed nestedness approach previously developed for Community Ecology to evaluate occupation of chironomid species larvae (Diptera) on the body of its host (an armored catfish species of the family Loricariidae).

Une cellule dendritique (CD) qui exprime le facteur de transcription GILZ durant l’apprêtement de l’antigène et sa présentation aux cellules effectrices, génère des lymphocytes T régulateurs (LTregs) CD25high Foxp3+ sécréteurs d’IL-10. La production de GILZ est dépendante de l’action des glucocorticoïdes, de l’IL-10 et du TGF-.Nous avons mis en évidence chez l’homme qu’une corticothérapie orale de 48h induit l’expression de GILZ dans les cellules présentatrices de l’antigène circulantes (CPAs) de sujets allergiques. Les CPAs isolées après la corticothérapie génèrent des LTregs CD25high Foxp3+ IL-10+ spécifiques de l’allergène.Nous également constaté in vitro que les mastocytes participent à l’activation des CDs au cours des réactions allergiques en régulant l’expression de GILZ. Les médiateurs d’origine mastocytaire, dont l’histamine, diminuent l’expression de GILZ dans les CDs et altèrent ainsi leur capacité à activer des LTregs. Nous avons identifié le mécanisme par lequel l’histamine diminue l’expression de GILZ dans les CDs humaines. L’histamine inhibe l’activité transcriptionnelle de Foxo3, un facteur de transcription régulant l’expression de GILZ. Enfin, nous avons démontré que des souris transgéniques qui surexpriment GILZ constitutivement dans les CDs sont protégées contre le développement de l’asthme allergique. L’ensemble de ces résultats permet d’envisager de nouvelles stratégies d’immunomodulation dans l’allergie, centrée sur la régulation de l’expression de GILZ dans les CDs. / We previously showed in vitro that DCs with a high level of GILZ activate regulatory T cells (Tregs) whereas DCs with low level of GILZ trigger effector T lymphocytes. Glucocorticoids (GCs), IL-10 and TGF- are potent inducers of GILZ expression. The aim of this thesis is to extend the above findings to induction of tolerance to allergens. Modulation of GILZ expression by DCs should induce allergen-specific Tregs able to inhibit the activation and proliferation of allergen specific T cell clones. In order to validate this concept we demonstrated that:- allergen-specific tolerance can be achieved in allergic patients treated with oral GC through the induction of GILZ expression in their antigen-presenting cells, and the role of allergen-specific Tregs in this effect,- mast cells play a role in the activation of DCs by inhibiting their expression of GILZ and thus their ability to stimulate Tregs against harmless environmental allergens,- GILZ-expressing DCs protect against allergic asthma in a model of transgenic mice over-expressing GILZ in their DCs.The present study supports the concept of an immune regulation of allergic responses through the modulation of GILZ expression by DCs and opens new perspectives in the development of innovative immunotherapies in the treatment of allergic diseases.

Les SIgA possèdent la capacité de pouvoir adhérer spécifiquement à la membrane apicale des cellules M présentes au niveau des muqueuses monostratifiées. La capacité des cellules M à transporter les SIgA de la lumière intestinale jusqu'au GALT par un mécanisme de transcytose inverse a également été décrite. J'ai donc souhaité évaluer la capacité des SIgA à transporter efficacement un antigène vaccinal, à travers la barrière épithéliale par l'intermédiaire des cellules M vers les DCs présentes dans le MALT. Le mécanisme exact et notamment la structure moléculaire du récepteur permettant la transcytose inverse des IgA n'a pas été identifiée. Il m’a donc paru intéressant d'améliorer la compréhension des mécanismes physiologiques impliqués dans le transport d'une SIgA de la lumière intestinale jusqu’aux cellules immunitaires sous-muqueuses. Cette étude a permis de démontrer le rôle majeur de deux nouveaux récepteurs présents à la surface des cellules M, la dectine-1 et le siglec-5, dans l'activité physiologique rétrograde des SIgA. Cette étude a permis d'identifier les domaines des SIgA impliqués dans ce mécanisme. J'ai ensuite utilisé les SIgA comme vecteur vaccinal permettant le ciblage des cellules M. Les applications de cette approche à la vaccination par voie orale et nasale sont décrites dans la publication 4 en cours de rédaction. Durant ma thèse, j'ai pu démontrer que la transcytose inverse des SIgA est un mécanisme physiologique dépendant par exemple de récepteurs aux sucres. J'ai pu également démontrer que leur utilisation dans des approches de vaccination muqueuse peuvent être une voie très prometteuse notamment contre le VIH ou d'autres pathogènes muqueux / Secretory IgA (SIgA) are the main effectors of the mucosal immune response. More, SIgA have the capacity to adhere to the apical membrane of M cells present in the intestinal and nasal mucosa. After binding to M cells, SIgA are transported from the intestinal lumen to the GALT by a reverse transcytosis mechanism. In this work, I have assessed the capacity of SIgA to effectively deliver a vaccine antigen through the epithelial barrier via M cells to sub-mucosal dendritic cells (DCs). Precise mechanisms and the IgA-specific receptor(s) for reverse transcytosis have not yet been identified. In this work, I identified the receptors involved in SIgA reverse transcytosis. Both dectin-1 and siglec-5 allow the transport of the Cα1 domain of SIgA by murine an human M cells in vitro and also in vivo. This work is currently undergoing to immunity (publication 1) and should also be patented. Next, I tried to use the reverse transcytosis mechanism mediated by M cells to efficiently deliver an HIV-1 antigen by mucosal routes. We applied results obtained using SIgA as a vaccine vector for M cells targeting. This approach should help to protect antigen in the mucosal environment. Applications of this approach to oral and nasal immunisation are described in the incomplete publication 4. During any PhD, I was able to demonstrate that SIgA reverse transcytosis is a physiological mechanism depending on sugar receptors. I was also able to demonstrate that their use could be a very promising vaccine approach especially for mucosa] diseases or pathogens as HIV

L. (L.) amazonensis (La) e L. (V.) braziliensis (Lb) podem causar um espectro de manifestações clínicas e imunopatológicas no homem, sendo La responsável pela forma anérgica difusa e Lb pela forma mucocutânea da doença, formas polares e de elevada gravidade. Neste sentido, o objetivo do presente estudo foi avaliar os aspectos da resposta imune celular no ponto de inoculação e no linfonodo de drenagem de camundongos BALB/c inoculados no coxim plantar com 106 promastigotas de La e Lb. A evolução da lesão foi avaliada semanalmente sendo que na 4ª e 8ª semana PI biópsias do ponto de inoculação foram coletadas para determinação da densidade de células dendríticas (CD207+ e CD11c+), linfócitos T CD4+ e CD8+ e células iNOS+ por imunoistoquímica, e o linfonodo de drenagem para caracterização de subpopulações de células dendríticas e de linfócitos T CD4 e CD8 por citometria de fluxo. Células de linfonodo de drenagem foram cultivadas, com estímulo homólogo, para quantificação de citocinas (IL-4, IL- 10 e IFN-g) e nitrito nos sobrenadantes. A infecção por La levou à progressão da doença, com aumento do tamanho da lesão e da carga parasitária tanto na pele quanto no linfonodo de drenagem, enquanto que a infecção por Lb mostrou um discreto aumento da lesão entre a 6ª e 7ª semana PI com posterior regressão e redução da carga parasitária na pele e no linfonodo de drenagem. Aumento do número de células dendríticas dérmicas e de Langerhans foi observado na pele de camundongos inoculados com La na 4ª semana PI, juntamente com o aumento no número de células de Langerhans nos linfonodos de drenagem. Resposta imune celular preferencial de células T CD4+ foi observada tanto na pele quanto no linfonodo de camundongos inoculados com La, que mostrou ser predominantemente do tipo Th2 com a produção aumentada de IL-10 e IL-4. Já a infecção por Lb levou ao aumento na expressão de células dendríticas dérmicas e Langerhans na pele dos animais inoculados com Lb somente na 8ª semana PI, assim como aumento do número de células dendríticas dérmicas no linfonodo. A resposta imune celular foi caracterizada por células T CD4+ e CD8+ em pele e linfonodo dos animais infectados com Lb, vinculada a um perfil Th1 com a produção preferencial de IFN-g e altos níveis de NO. Aumento no número de células T regulatórias foi observado na infecção por Lb, que mostrou correlação direta com o número de linfócitos T CD4+ produtores de IL-10. Assim, a infecção por La foi relacionada à suscetibilidade, enquanto que a infecção por Lb foi relacionada à resistência do hospedeiro vertebrado. Estes resultados evidenciam não só o papel do parasita na modulação da resposta imune do hospedeiro como também das células dendríticas à infecção por Leishmania / L. (L.) amazonensis (La) and L. (V.) braziliensis (Lb) are responsible for a spectrum of clinical and immunopathological manifestations in humans, La is able to cause anergic diffuse leishmaniasis and Lb mucocutaneous leishmaniasis, polar forms with high severity. In this way, the aim of the present study was to evaluate aspects of the cellular immune response in the site of infection and in the draining lymph node of BALB/c mice inoculated in the hind footpad with 106 promastigotes of La and Lb. The evolution of the lesion size was evaluated weekly and in the 4th and 8th week PI biopsies from the site of infection were collected to determine the density of dendritic cells (CD207+ and CD11c+), CD4+ and CD8+ T cells and iNOS+ cells by immunohistochemistry, and the draining lymph node to characterize subsets of dendritic cells and CD4+ and CD8+ T cells by flow citometry. The draining lymph nodes cells were cultured with specific antigen to determine the cytokines (IL-4, IL-10 e IFN-g), and the nitric oxide in the supernatants. The infection caused by La led to the progression of disease with increase on lesion size and parasite load in the skin and draining lymph node, while Lb infection showed a discrete increase on the lesion size between 6th and 7th week PI with late regression and reduction in the skin as well as lymph node parasite load. An increase on the number of dermal dendritic and Langerhans cells were observed in the skin of BALB/c mice infected with La at 4th week PI together with an increase of Langerhans cells in the draining lymph node. The preferential CD4+ T cell immune response was observed in skin and lymph node of mice infected with La, which showed to be rather Th2 with an increase on the levels of IL-4 and IL-10. However, Lb infection led an increase of dermal dendritic cells and Langerhans cells in the skin only at 8th week PI, as well as an increase in the dermal dendritic cells in lymph node. The cellular immune response were characterized by CD4+ and CD8+ T cells in the skin and draining lymph node of mice infected with Lb, which was related to a Th1 immune response with production of high levels of IFN-g and nitric oxide. An increase of Regulatory T cells was observed in Lb infection, which showed the positive correlation with the IL-10 producing CD4+ T cells. So, the La infection was related to the susceptibility, while Lb infection was related to the resistance in the vertebrate host. These results emphasize the role of the parasite in the modulation of the host immune response to Leishmania infection

Desde o início da pandemia de HIV/aids, a imunoterapia vem sendo utilizada como alternativa terapêutica numa tentativa de estimular uma resposta do sistema imunológico contra o agente agressor. Esta abordagem tem sido considerada promissora para obtenção do controle da infecção em longo prazo. A administração de células apresentadoras de antígeno, em especial células dendríticas, é fundamentada pelos conceitos de imunoterapia passiva e de terapia celular ativa (vacina terapêutica) além da perspectiva da eliminação dos chamados reservatórios virais, unindo assim estas diversas estratégias de intervenção. Dentre os diferentes antígenos do HIV utilizados para pulsar as células dendríticas, alguns dos melhores resultados foram obtidos com a inativação química do vírus, preservando a integridade da estrutura da sua superfície.Há alguns anos nosso grupo vem trabalhando com terapia celular baseada em células dendríticas derivadas de monócitos autólogos, pulsadas com HIV inativado quimicamente, seguindo o protocolo descrito por Lu e colaboradores em 2004. Aqui, nós apresentamos os resultados de um ensaio clínico de fase I/II que teve como objetivos avaliar a tolerância, segurança e impacto imunovirológico, de diferentes formulações do produto em pacientes cronicamente infectados pelo HIV, sem uso de antirretrovirais. Os participantes foram alocados em três braços para receber composições distintas: 3x107 células dendríticas sem pulso adicional de HIV inativado (Braço A), 3x106 células dendríticas com pulso adicional de HIV inativado (Braço B) ou 3x107 células dendríticas com pulso adicional de HIV inativado (Braço C). O número de participantes no braço A evoluiu com uma considerável diminuição ao longo do período de observação do estudo, prejudicando as avaliações. As análises dos outros braços mostraram que as preparações foram seguras, não se observando eventos adversos relacionados à intervenção. Os resultados sugeriram um aumento na carga viral plasmática associados a uma redução das sub-populações de linfócitos TCD4+ e TCD8+ nos pacientes do braço C além de uma redução na quantidade de linfócitos T reguladores nos indivíduos do braço B / Since the beginning of the HIV / aids pandemic, immunotherapy is being used as an alternative therapy in attempt to stimulate an immune response against the pathogenic agent. This approach has been considered as promising for achieving the control of infection in the long term. Administration of antigen presenting cells, particularly dendritic cells is based on the concepts of passive immunotherapy and active cellular therapy (therapeutic vaccine), with the perspective of eliminating the so-called viral reservoirs, thus joining different intervention strategies. From different antigens tested for loading DCs, some of the best results were obtained with chemically inactivated virus, which preserves its surface proteins.Our group has been working with cellular therapy based on dendritic cells derived from autologous monocytes pulsed with chemicallyinactivatedHIV, following the protocol described by Lu et al in 2004.Here, we present the results of a phase I / II clinical trial aimed to evaluate tolerance, safety and immunovirological impact of different product formulations in chronically HIV-infected individuals, naïve for antiretroviral treatment.Participants were allocated to receive: 3x107 un-pulsed DCs (Arm A), 3x106 HIV-pulsed DCs (Arm B) or 3x107 HIV-pulsed DCs (Arm C). The number of participants in the arm A evolved with a considerable decrease over the study, so any considerations about effect of DCs without antigen overload were difficult to carry out. Outcomes in other arms showedthat they were safe, with no adverse events related to the products. The results suggested an increase in plasma viral load and decline in CD4+ and CD8+ T cells subpopulations after intervention in arm C. Additionally, we observed a decrease in percentage of regulatory T cells in arm B patients

A paracoccidioidomicose (PCM) é uma micose sistêmica e endêmica na América Latina com maior prevalência no Brasil, Colômbia e Venezuela. A doença é causada pelos fungos P. brasiliensis e P. lutzii. As células dendríticas são eficientes apresentadoras de antígenos e quando utilizadas como adjuvante podem ser de 100 a 1000 vezes mais efetiva nesta função. O peptídeo P10 corresponde a um trecho específico de 15 aminoácidos derivado da gp43, principal antígeno diagnóstico, e é reconhecido pelos linfócitos T CD4+ capaz de induzir resposta preferencialmentedo tipo Th1 e conferindo proteção no modelo experimental. Células indiferenciadas foram obtidas a partir de medula óssea de camundongos machos BALB/c B10.A, cultivadas na presença de GM-CSF e IL-4 por 9 dias, para a diferenciação de células dendríticas (DC). As células foram incubadas na presença do P10 por 2 horas, em estufa com 5% de CO2 a 37°C, e foram utilizadas nas imunizações. A maturação das células foi observada por citometria de fluxo com os marcadores CD11c, MHC-II, CD80 e CD86. Camundongos da linhagem BALB/c e B10.A foram submetidos à imunossupressão com dexametasona, por 20 dias, seguido pela infecção intratraqueal com o isolado Pb18. Após 30 dias, os grupos foram tratados com DCs ou DCs pulsadas com P10 em associação ou não com sulfametoxazol/Trimetoprim, por um período de 15 dias. O sacrifício ocorreu 45 dias após a infecção e foram retirados os pulmões, baço e fígado para quantificação de carga fúngica, análise histológica e dosagem de citocinas. Observamos diminuição significativa da carga fúngica nos pulmões dos animais que receberam DCs pulsadas com P10 associada ou não ao tratamento medicamentoso. Não foi observado crescimento fúngico em outros órgãos como fígado e baço em ambos os grupos. A análise histológica revelou redução da carga fúngica e preservação do parênquima pulmonar nos grupos tratados com Dcs pulsadas com P10. As dosagens das citocinas indicaram uma resposta imune mista Th1/Th2. Inicialmente reportamos que as DCs pulsadas com P10 reduz a carga fúngica em camundongos infectados. Neste trabalho, reportamos que as DCs pulsadas com P10 podem também reduzir a carga fúngica em animais anérgicos, mimetizando pacientes com a forma aguda/subaguda da doença / Paracoccidioidomycosis (PCM) is a systemic and endemic mycosis in South America, with higher prevalence in Brazil, Colombia and Venezuela. This disease is caused by fungi P. brasiliensis and P. lutzii. The peptide P10 matches a specific path of 15 amonoacids which is derived from gp43, main diagnostic antigen. This peptide is recognized by the T CD4+ lymphocytes and induces a response type Th1, giving protection at an experimental mode. Some dendritic cells (DC) have an efficient antigen and, when used as adjuvant, they can be 100 to 1000 times more effective. Undifferentiated cells were obtained from bone marrow of male mice type BALB/c and B10.A and cultivated in the presence of GM-CSF and IL-4 for 9 days, so that DCs would be differentiated. The cells were incubated in the presence of P10 for 2 hours in incubator at 37°C with 5% of CO2, and later utilized in the immunizations. Maturation of the cells was observed by flow cytometry with CD11c, MHC-II, CD80 and CD86 markers. Mice types BALB/c and B10.A had been submitted to an immunosuppression with dexamethasone for 20 days, before being intratracheally infected with isolate Pb18. After 30 days, the group of animals received immunizations with DCs or DCs pulsed with P10 associated or not with the treatment by Sulfametoxazol/Trimetoprim, this for a period of 15 days. The sacrifice occurred 45 day after the animals had been infected, and their lungs, spleen and liver were taken out for quantification of fungal burden, histology and cytokine assay. We observed a significant decrease of fungal burden in the lungs of animal that received DC pulsed with P10, associated or not with the drug treatment. In both groups we didn\'t observe fungal growth in organs such as liver and spleen. The histological analyses showed reduced fungal burden and preservation of lung parenchyma. The dosages of cytokines showed an immune response mixed Th1/Th2 type. At first we reported that the DCs pulsed with P10 were responsible for reducing the fungal burden in infected mice. In this paper we report that the DCs pulsed with P10 may also be responsible for reducing fungal burden in anergic animals, mimicking patients with the acute/subacute form of the disease