Les adénovirus humains (HAdVs) provoquent généralement une infection bénigne chez l'hôte sain. En revanche, chez les patients immunodéprimés et immunocompétents, ils peuvent causer des infections sévères à létales. Les vecteurs HAdVs sont couramment utilisés dans les domaines de la thérapie génique et de la vaccination. L'immunité pré-existante de l’hôte protège généralement contre les infections de type sauvage mais peut entraîner des effets indésirables lors du relargage des virus tel que l’induction de processus inflammatoire locale ou général. Après l'infection, l'inflammation va principalement entraîner le recrutement de cellules dendritiques (DCs), de monocytes et de neutrophiles. Les DCs ont la capacité unique de présenter l'antigène et d’activer les cellules T, qui, par la suite, aideront les cellules B à produire des anticorps. En plus de leurs activités phagocytaires, des peptides antimicrobiens dérivés des neutrophiles (AMPs) jouent un rôle central dans l'immunité innée. Les AMPs peuvent neutraliser les microbes infectieux et / ou activer différents types de cellules immunitaires. Dans ce contexte, nous avons étudié l'interaction ex vivo entre le facteur hôte (anticorps anti-HADV et AMPs) contre les HAdVs dans les DCs ainsi que le rôle des DCs activés indirectement (indir-DCs) lors de la réponse immunitaire. Nous avons caractérisé le profil des cytokines et des chimiokines sécrétées par les DCs stimulées par différents HAdVs, AMPs et les combinaisons qui en découlent. Enfin, nous avons constaté que l’opsonisation d’HAdV5 par les IgG accroît la capacité de capture antigénique des MoDCs (cellules dendritiques dérivées de monocytes) et induit la mort cellulaire par pyroptose. Je me suis donc concentrée sur les caractéristiques et la fonction des indir-DCs dans l’immunité contre les HAdVs. Afin de mieux comprendre les propriétés et les phénotypes des indir-DCs et DCs activés directement (dir-DCs), nous avons caractérisé leurs profils de maturation, les facteurs influençant cette maturation, et leur capacité fonctionnelle de recruter les leucocytes. Ainsi nous avons pu mettre en évidence que les dir-DCs empêchent le recrutement leucocytaire tandis que les indir-DCs favorisent la migration des monocytes. L’ensemble de ces données contribue à comprendre comment l'immunité pré-existante contre les HAdVs peut impacter l’efficacité des traitements contre les maladies HAdVs ainsi que la conception de vaccins. / Human adenoviruses (HAdV) generally cause mild infection in healthy host, but in immunocompromised and immunocompetent patients, they cause severe on lethal infections. HAdV vector are also commonly used for gene transfer and vaccination. We know that pre-existing immunity can protect from wild type infection and cause adverse effects during vector delivery including local and system inflammation. Following HAdV infection on vector use, inflammation leads to the recruitment of dendritic cells (DCs), monocyte and neutrophils. DCs have the unique ability to present antigens and activate T cells, that subsequently aid B cells to produce antibodies. In addition to their phagocytic activities, neutrophil-derived antimicrobial peptides (AMPs) play a central role in innate immunity. AMPs can kill invading microbes and/or activate various cell types. Here I studied the ex vivo interaction between host factor (anti-HAdV antibodies and AMPs) to HAdV in DCs and the role of indirect-activated DC (indir-DCs) in immune response. I characterized the profile of cytokines and chemokines of DC stimulated with different HAdV, AMPs and their combination. We recently found that IgG-oposonization of HAdV5 increase the update by MoDCs (monoctyes-derived dendritic cells) and induced pyroptotic cell death. I therefore focused on the characteristic and function of indir-DCs in anti-HAdV immunity. To better understand the properties and phenotypes of indir-DCs and direct-activated DCs (dir-DCs), we characterize their maturation profile, the factors influencing their maturation, and the functional ability to recruit leukocyte. We found that dir-DC prevent leucocyte recruitment while indir-DC increases monocyte migration. These data contribute to understand how the pre-existing immunity to HAdV impacts treatment for HAdV diseases and vaccine design.

No description available.

Chaque cadre doit contenir un résumé de 1700 caractères maximum, espaces compris. En cas de dépassement, la coupure sera automatique. Le doctorant adresse son texte sous forme électronique selon les recommandations de la bibliothèqueLes "Liver X receptors " (LXR) sont des récepteurs nucléaires impliqués dans Phoméostasie du cholestérol. Dans les macrophages, la stimulation de la voie LXR accroît la clairance des corps apoptotiques et réprime la réponse inflammatoire. Les LXR inhibent également la prolifération et la survie de cellules malignes.L'activation des LXR dans les cellules dendritiques plasmocytoïdes (PDG) augmentent la clairance des microparticules (MP), via l'induction du récepteur au phosphatidylsérines BAIL L'internalisation des MP active la voie NF-KB ou la voie LXR pour des MP dérivées respectivement, de cellules endothéliales (EMP) ou plaquettaires (PMP). Ces deux voies de signalisation se réprimaient mutuellement, déterminant la réponse inflammatoire des PDG.La contrepartie leucémique des PDC (LPDC) est à l'origine d'une leucémie aiguë agressive, la BPDCN. Nous avons observé une dérégulation de Phoméostasie du cholestérol dans ces cellules. L'activation de la voie LXR entraine un efflux du cholestérol associé à un effet cytotoxique et antiprolifératif. Ils peuvent impliquer : la répression de NF-KB ; ainsi que l'inhibition de la signalisation induite par le facteur de survie IL-3 (incluant STAT5 et Akt). L'utilisation d'un modèle xénogénique murin de BPDCN traitée par agoniste LXR montre une diminution de la cytopénie induite par les LPDC et des infiltrats spléniques et médullaires.Ces travaux démontrent la fonctionnalité de la voie LXR dans les PDC et LPDC, ainsi qu'une régulation croisée avec NF-KB. L'activation de cette voie a démontré son implication dans la clairance des MP et la régulation de la réponse inflammatoire des PDC, ainsi qu'un effet anti-leucémique sur les LPDC. / Nuclear Liver X Receptors (LXR) are involved in cholesterol homeostasis. In macrophages, LXR promote apoptotic body/cell clearance and repress inflammatory responses. LXR are also shown to inhibit proliferation and survival of malignant cells.In plasmacytoid dendritic cells (PDC), LXR stimulation increases microparticle (MP) engulfment via the increased expression of the PS receptor, BAIL MP engulfment induced NF-icB or LXR activation, depending on the endothelial (EMP) or platelet (PMP) origin of MP, respectively. Overall, we show a crosstalk involving LXR and NF-KB, which dictates the inflammatory fate of PDC engulfing MP.The leukemic PDC counterpart (LPDC) is responsible of an aggressive hematologic malignancy, called blastic plasmacytoid dendritic cell neoplasm (BPDCN). In contrast to healthy PDC and other acute leukemias (including lymphoid and myeloid acute leukemias), we report here a specific downregulation of cholesterol homeostasis-related genes in LPDC. LXR pathway activation increases cholesterol efflux and inhibits cell proliferation and survival. This may involve: inhibition of NF-KB signaling pathway and of signaling pathways induced by the survival factor IL-3 (involving Akt and STAT5). Using a xenogeneic mouse model of BPDCN, LXR agonist treatment reduces BPDCN-induced cytopenia as well as bone marrow and spleen LPDC infiltration.Overall, we demonstrate that LXR receptors are functional in PDC and LPDC and are involved in a cross-regulation mechanism with NF-KB. LXR receptors promote MP clearance and control inflammatory responses in PDC, as well as exert an anti-leukemic therapeutic effect in BPDCN via several mechanisms, including cholesterol efflux.

Stimuler le système immunitaire est un enjeu majeur dans le traitement des leucémies aigües. Nous avons centré notre étude sur la leucémie aigüe myéloïde (LAM) et la leucémie à cellules dendritiques plasmacytoïdes (LAPDC). Dans une première partie, nous avons étudié les médicaments immunomodulateurs (IMiDs) utilisés dans le traitement du myélome multiple et des syndromes myélodysplasiques à délétion 5q. Les IMiDs présentent des propriétés anti-angiogéniques, anti-prolifératives, pro-apoptotiques, et immunomodulatrices en particulier sur les cellules NK (Natural Killer). Nous avons évalué les effets anti-leucémiques des IMiDs (lenalidomide et pomalidomide) dans le but d’améliorer l’activité cytotoxique des NK dans la LAM. Nous avons mis en évidence une altération de la survie des blastes de LAM par les IMiDs in vitro, et dans un modèle in vivo de greffe dans les souris immunodéficientes NOD/SCID/IL2rg-/- (NSG). Nous avons également montré une sensibilisation par les IMiDs des blastes de LAM à la lyse par les NK allogéniques, indépendamment de la cible moléculaire connue, le cereblon. Le traitement des blastes de LAM par IMiDs stimule les fonctions NK. Enfin, nous avons décrit des modifications phénotypiques induites par les IMiDs sur les récepteurs NK, et une diminution d’expression de HLA-classe I sur les cellules de LAM. Ces résultats encouragent la poursuite du développement des IMiDs dans la LAM, en particulier les associations stimulant les fonctions NK. Dans une seconde partie, nous avons développé un modèle murin de LAPDC dans la souris NSG. Cet outil préclinique est indispensable dans l’élaboration de stratégies d’immunothérapie dans les leucémies aigües. / Boosting the Immune System is a major challenge in the treatment of acute leukemias. We focused our study on acute myeloid leukemia (AML) and plasmacytoid dendritic cell leukemia (BPDCN). In the first part, we studied immunomodulatory drugs (IMiDs) that are currently used in the treatment of patients with myeloma and myelodysplastic syndrome with 5q deletion. IMiDs exhibit anti-angiogenesis, anti-proliferative, pro-apoptotic, and immunomodulatory properties especially on NK cells and T lymphocytes. We investigated the anti-leukemic effects of two IMiDs (lenalidomide and pomalidomide) in order to improve NK cell cytotoxic activity in AML. We have shown that IMiDs impaired survival of AML blasts in vitro, and in vivo in NOD/SCID/IL2rg-/- (NSG) murine model. In addition, IMiDs treatment sensitized AML blasts to allogeneic NK cell mediated lysis, independently of Cereblon, the known molecular target of IMiDs. IMiDs treatment of AML blasts enhanced NK cell functions such as degranulation and cytokine production. Finally, we have described phenotypic changes induced by IMiDs on NK receptors, and a down-regulation of HLA-class I on AML blasts. These results encourage continuing investigation for the use of IMiDs in AML, especially in combination with immunotherapies based on NK cells. In a second part, we have developed a murine model of plasmacytoid dendritic cell leukemia (BPDCN) in NSG mice. Murine model of leukemia are essential preclinical tools in the development of new immunotherapies in acute leukemias.

INTRODUÇÃO: Líquen plano (LP) é uma doença mucocutânea de natureza inflamatória crônica de etiologia ainda desconhecida. A estimulação da imunidade inata via os receptores Toll-like (TLRs) podem influenciar as células dendríticas e direcionar a resposta de células T CD4+ e CD8+ efetoras, assim como também favorecer o estado inflamatório do LP. OBJETIVOS: Avaliar o perfil fenotípico de células dendríticas mielóides (mDCs) e plasmocitóides (pDCs) e de linfócitos T CD4+ e CD8+ após estímulo com agonistas de TLRs no sangue periférico de pacientes com LP. Além disto, avaliar a frequência, perfil de maturação e os subtipos de células T CD4+ e TCD8+ reguladores. MÉTODOS: Foram selecionados 18 pacientes com LP (15 mulheres, 3 homens), com 41,57 ± 4,73 anos de idade e um grupo controle com 22 indivíduos sadios (18 mulheres, 4 homens), com 43,92 ± 7,83 anos de idade. As células mononucleares (CMNs) de sangue periférico foram avaliadas por citometria de fluxo quanto à: 1) Produção de TNF-? em mDCs e de IFN-? em pDCs em CMNs ativadas por agonistas de TLR 4, 7, 7/8 e 9; 2) Análise de células T CD4+ e CD8+ monofuncionais e polifuncionais após estímulo com agonistas de TLR 4, 7/8, 9 e enterotoxina B de Staphylococcus aureus (SEB); 3) Avaliação de células Th17 e Th22/Tc22 em CMNs após estímulo com SEB; 4) Frequência, perfil de maturação e subtipos de células T CD4+ e CD8+ reguladoras. RESULTADOS: 1) Nos pacientes com LP foi demonstrado um aumento na frequência de mDCs TNF-alfa+ após estímulo com agonistas de TLR4/LPS e TLR7-8/CL097, mas com imiquimode/TLR7 houve diminuição da expressão de CD83. Já nas pDCs do grupo LP, o imiquimode foi capaz de diminuir a expressão de CD80 e o CpG/TLR9 diminuiu a expressão de CD83 no LP. 2) As células T CD4+ secretoras de IL-10 mostraram aumento da frequência nos níveis basais, que diminuiu após estímulo com LPS e SEB. Em contraste, a produção de IFN-y aumentou em resposta ao LPS enquanto diminuiu para CpG. As células T CD4+ polifuncionais, secretoras de 5 citocinas simultâneas (CD4+IL-17+IL-22+TNF+IL-10+IFN-y+) diminuíram no LP após estímulo com CL097 e CpG. Entretanto, na ausência de IL-10, houve aumento da frequência de células CD4+IL-17+IL-22+TNF+IFN-y+ em resposta ao LPS. Um aumento na polifuncionalidade foi observado em células TCD4+ que expressam CD38, marcador de ativação crônica e na ausência de IL-10. Similarmente, às TCD4+, uma diminuição de células T CD8+ IFN-y+ e TNF+ foram detectadas após estímulo com CpG. 3) As células Th22/Tc22 nos níveis basais e após estímulo com SEB se mostraram aumentadas. As células Th17 não mostraram diferenças entre os grupos. 4) A frequência das células T CD4+ e CD8+ reg totais (CD25+Foxp3+CD127low/-) está elevada no LP. Quanto aos perfis de maturação, há aumento na frequência de células TCD4+ de memória efetora enquanto que para as células T CD8+ há predomínio das células de memória central. Quanto aos subtipos, há aumento nas células T CD4+ regs periféricas (pT reg). CONCLUSÕES: O estado de ativação das mDCs após ativação das vias de TLRs 4 e 7/8 pode influenciar na geração de resposta T efetoras no LP. O perfil de resposta monofuncional e polifuncional aos estímulos TLRs reflete a ativação destas células no sangue periférico. Além disso, o aumento de Th22/Tc22 e das células T regs indicam uma relação entre regulação e células efetoras no sangue periférico evidenciando que existem alterações extracutâneas no LP / BACKGROUND: Lichen planus (LP) is a mucocutaneous disease of chronic inflammatory course of unknown etiology. Stimulation of the innate immune system via Toll-like receptors (TLRs) may influence the dendritic cells and targeting the CD4+ and effector CD8+ T cell responses, as well as promoting inflammatory status of the LP. OBJECTIVES: To evaluate the phenotypic profile of myeloid dendritic cells (mDCs), plasmacytoid (pDCs) and CD4+ and CD8+ T lymphocytes after stimulation with TLR agonists in peripheral blood of patients with LP. Moreover, to evaluate the frequency, maturation profile and subtypes of CD4+ and CD8+ T regulators cells. METHODS: We selected 18 patients with LP (15 women, 3 men) with 41.57 ± 4.73 years old and a control group of 22 healthy subjects (18 women, 4 men), with 43.92 ± 7, 83 years old. Mononuclear cells from peripheral blood (PBMCs) were assessed by flow cytometry for: 1) mDC TNF-alfa production and pDCs IFN-alfa production in PBMCs activated by agonists of TLR 4, 7, 7/8 and 9; 2) Analysis of monofunctional and polyfunctional CD4+ and CD8+ T cells after stimulation with TLR 4 agonists, 7/8, 9 and Staphylococcus aureus enterotoxin B (SEB); 3) Evaluation of Th17 and Th22/ Tc22 cells in PBMCs after stimulation with SEB; 4) Frequency, maturation profile and subtypes of regulatory CD4+ and CD8+ T cells. RESULTS: 1) Patients with LP showed an increased frequency of TNF-alfa+ mDCs after stimulation with agonists of TLR4/LPS and TLR7-8 /CL097, whereas with imiquimod /TLR7 induced a decreased CD83 expression. Already in the pDCs of LP group the imiquimod was able to decrease the CD80 expression and CpG/TLR9 decreased CD83 expression. 2) CD4+ T cells secreting IL-10 demonstrated an increased frequency at the baseline levels, which decreased after stimulation with LPS and SEB. In contrast, the production of IFN-y increased in response to LPS while decreased to CpG. Polyfunctional CD4+ T cells secreting simultaneously 5 cytokines (CD4+IL-17+IL-22+TNF+IL-10+IFN-y+) decreased in the LP after stimulation with CpG and CL097. However, in the absence of IL-10, occurred an increased frequency of CD4+IL-17+IL-22+IFN-y+TNF+ in response to LPS. An increase in the polyfunctional response was seems in CD4+ T cells expressing CD38, a chronic activation marker, in the absence of IL-10. Similarly to the CD4+ T cells, a decreased CD8+ T cells secreting IFN-? and TNF was observed in LP, after stimulation with CpG. Polyfunctional CD8+ T cells from LP group showed decreased response with 3 and 4 cytokines at baseline condition, and upon SEB and CL097 stimulations, occurred an increased frequency of these cells. In LP group, T cells CD8+CD38+ polyfunctional showed low capacity, such as CD4+CD38+ cells. 3) The Th22/Tc22 cells already at baseline and after stimulation with SEB showed increased frequency. The Th17 cells showed no differences between groups. 4) The frequency of CD4+ and CD8+ total reg (CD25+Foxp3+CD127low/-) is increased in LP. The profiles of maturity, an increase in the frequency of CD4+ effector cells whereas for memory CD8+ T cells there is a predominance of central memory cells. As for the subtypes, there is an increase in CD4+ peripheral T regs cells (pT reg). CONCLUSIONS: The activation state of mDCs after activation of the pathways of TLRs 4 and 7/8 can influence the response and generation of effector T cells in the LP. The monofunctional and polyfunctional response profile for TLRs stimuli reflects the activation of these cells in peripheral blood. Furthermore, the increase of Th22/Tc22 and T regs cells indicate a relationship between regulatory and effector cells in peripheral blood showing that there are extra-cutaneous alterations in the LP

A paracoccidioidomicose (PCM) é uma doença sistêmica causada pelos fungos Paracoccidioides brasiliensis e Paracoccidioideslutzii. O comprometimento da pele e mucosa oral são frequentes na PCM. As células dendríticas e queratinócitos do tegumento, devido à sua função como células apresentadoras de antígenos, atuam na resposta imune inata e adaptativa contra agentes patogênicos. Com o objetivo de verificar a interação do P. brasiliensis com essas células, estudamos 47 biopsias de mucosa oral e 52 de pele de lesões de doentes com diagnóstico comprovado de PCM. As biopsias foram submetidas à técnica de imuno-histoquímica de dupla-marcação com os anticorpos anti-fator XIIIa (marcador de dendrócitos dérmicos), anti-CD207 (marcador de células de Langerhans maduras), anti-pancitoqueratinas (AE1-AE3) e anti-P. brasiliensis. Fez-se também a reação de dupla marcação por técnica de imunofluorescência, com análise por microscopia confocal a laser, para a melhor visualização da interação entre queratinócitos e os fungos. Quarenta e dois por cento das amostras de mucosa oral exibiram formas fúngicas no citoplasma de dendrócitos dérmicos. As células de Langerhans, tanto nas biopsias de mucosa oral como de pele, não mostraram leveduras ou antígenos do fungo no seu citoplasma. Cinquenta e quatro por cento das biopsias de pele e sessenta por cento das amostras de mucosa exibiram leveduras no citoplasma de queratinócitos. Os resultados obtidos permitem concluir que o parasitismo de queratinócitos pode representar possível mecanismo de evasão do fungo aos mecanismos imunes locais. Os dendrócitos dérmicos fator XIIIa positivos e queratinócitos podem estar a atuar como células apresentadoras de antígenos para suprir a função, provavelmente prejudicada, das células de Langerhans nas lesões de pele e mucosa oral da PCM humana / Paracoccidioidomycosis (PCM) is a systemic disease caused by the fungus Paracoccidioides brasiliensis, which compromises various organs, mainly the lungs. The skin and oral mucosa are often affected. Dendritic cells and keratinocytes of the integument play a role in innate and adaptive immune response against pathogens, due to their function as antigen presenting cells. Aiming to verify the interaction of P. brasiliensis with these cell populations, we studied 52 biopsies of skin and 47 oral mucosa samples taken from patients with proven diagnosis of PCM. The biopsies were subjected to double immune staining technique with anti-factor XIIIa (marker of dermal dendrocytes), anti- CD207 (marker of mature Langerhans cells), anti-pan cytokeratins (AE1-AE3) and anti P. brasiliensis antibodies. Analyses with confocal laser microscopy were also performed to better visualization of the interaction between keratinocytes and the fungi. Factor XIIIa + dermal dendrocytes of 42% samples of oral mucosa displayed yeast forms in their cytoplasm. We did not observe yeast cells in the cytoplasm of Langerhans cells in both skin and oral mucosa samples. Fifty -four percent of skin and 60% of mucosal samples displayed yeast cells in the cytoplasm of keratinocytes. The parasitism of keratinocytes may represent a possible mechanism of evasion of the fungus to local immune mechanisms, or even as a result of keratinocytes ability to antigen presentation in PCM. Factor XIIIa dendrocytes and keratinocytes may be acting as antigenpresenting cells to fulfill the function of Langerhans cells, probably impaired, in skin and mucosa of human PCM

A forma indeterminada é a fase inicial da hanseníase, que se caracteriza histologicamente pelo infiltrado inflamatório leve, não granulomatoso, de linfócitos e histiócitos ao redor de vasos, anexos e nervos. No local de entrada do M. leprae, as células apresentadoras de antígeno do tipo células dendríticas são as primeiras a encontrar o bacilo. Este, no interior de células dendríticas, desencadeia a produção local de citocinas e quimiocinas, que resultam em proliferação de linfócitos T helper 1 ou T helper 2, assim definindo uma resposta imune celular ou humoral, respectivamente. As lesões tuberculoides mostram predominância das citocinas de padrão Th1 como IL-2, TNF-alfa, IFN-y, IL-12 e IL-18, enquanto que nas lesões virchowianas predominam citocinas de padrão Th2, como IL-4, IL-5, IL-10 e TGF-beta. Na pele, as principais células dendríticas são células dendríticas mieloides, células de Langerhans e alguns dendrócitos dérmicos. São identificadas respectivamente pela expressão imuno-histoquímica de S100, CD1a e Fator XIIIa. Células de Langerhans e dendrócitos dérmicos Fator XIIIa positivos estão aumentados em quantidade nas lesões tuberculoides quando comparadas com lesões virchowianas. Os objetivos do presente estudo foram: 1) caracterizar a inflamação \"in situ\" na hanseníase indeterminada através da quantificação das marcações imuno-histoquímicas de: CD57, CD4, CD8, CD1a, S100, FXIIIa, CD68, Foxp3, CD123, IL-1, IL-2r, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, IFN-, TNF-alfa, TGF-beta, iNOS, granzima B, receptor Toll-like 2/4, e antígeno BCG, 2) comparar o perfil fenotípico e citocínico das lesões na hanseníase indeterminada entre grupos de reação de Mitsuda positiva e negativa, a fim de investigar se existem padrões que possam prever para qual forma a doença evoluiria, e 3) revisar a histopatologia da forma indeterminada através da análise semiquantitativa das alterações vistas à coloração de hematoxilina/eosina. Foram selecionadas 15 lesões de pacientes com hanseníase indeterminada. Foram usados grupos controles de expressão de Fator XIIIa e CD1a em 10 casos de pele normal. A histopatologia mostrou discretas alterações epidérmicas, como alteração vacuolar e exocitose de linfócitos (33% dos casos cada), apoptose de queratinócitos (26%), atrofia e acantose (06% dos casos cada); infiltrado inflamatório linfomononuclear neural (100%), perivascular superficial (100%), perivascular profundo (93%), peri-écrino (40%) e peri-folículo pilossebáceo (20%), além de melanófagos em 93% dos casos. Esses achados mostraram que nem sempre todos os ambientes estão acometidos por inflamação na histopatologia. Em 66% dos pacientes foi encontrado antígeno bacilar (por Fite-Faraco ou técnica imuno-histoquímica anti-BCG), portanto a forma indeterminada não deve ser considerada sistematicamente como paucibacilar. Não houve diferença significativa de padrões de marcadores entre os grupos Mitsuda positivo e negativo. No microambiente inflamatório das lesões houve expressões significativas de TLR 2/4, Fator XIIIa, CD4, CD8, IL-2r, IL-4, IL-10, iNOS e TGF-beta. A expressão importante de IL-4, IL-10 e TGF- beta nas lesões de hanseníase indeterminada significaram tendência de resposta imune para o polo Th2, um ambiente de tolerância à permanência do bacilo. A baixa expressão de IFN-y colaborou para a inexpresiva resposta Th1. Não houve diferença significativa na expressão de CD1a entre as lesões e pele normal. Fator XIIIa foi expresso em mais que 50 células/mm2 em todos os casos, com quantidades significativamente maiores que outras células dendríticas nas lesões (S100, CD68, CD123) e que a pele normal. Estes achados demonstraram a importância dos dendrócitos dérmicos Fator XIIIa positivos na apresentação de antígeno na fase inicial da hanseníase / The indeterminate form is the initial stage of leprosy, which is characterized histologically by mild inflammatory infiltrate, non granulomatous, with lymphocytes and histiocytes around vessels, nerves and adnexals. When M. leprae enter the host, antigen-presenting cells of dendritic type are the first cells to find the bacillus. Once inside dendritic cells, the bacillus elicits local production of cytokines and chemokines, which result in proliferation of T lymphocytes helper 1 or T helper 2, thereby defining a cellular or humoral immune response, respectively. The tuberculoid lesions show predominance of Th1 cytokines such as IL-2, TNF-alfa, IFN-y, IL-12 and IL-18, whereas in the lepromatous lesions predominate cytokines of Th2 pattern such as IL-4, IL-5 IL-10 and TGF-beta. In the skin, main dendritic cells are myeloid dendritic cells, Langerhans cells, and some dermal dendrocytes. They are identified respectively by immunohistochemical expression of S100, CD1a and Factor XIIIa. Langerhans cells and dermal dendrocytes Factor XIIIa positive are increased in number in tuberculoid lesions compared with lepromatous lesions. The objectives of this study were: 1) to characterize \"in situ\" inflammation in indeterminate leprosy through the quantification of immunohistochemical markers: CD57, CD4, CD8, CD1a, S100, FXIIIa, CD68, Foxp3, CD123, IL-1, IL-2r, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, IFN-y, TNF-alfa, TGF-beta, iNOS, granzyme B, Toll-like receptor 2/4, and BCG antigen, 2) compare the phenotypic and cytokinic profile of indeterminate leprosy lesions between positive and negative Mitsuda reaction groups in order to investigate if there are patterns that can predict which way the disease may evolve, and 3 ) review the histopathology of the indetermate form by semi-quantitative analysis of changes seen in hematoxylin / eosin. Fifteen lesions of patients with indeterminate leprosy were selected. There was used control groups of Factor XIIIa and CD1a expression in 10 cases of normal skin. Histopathology showed discrete epidermal changes, such as vacuolar changes and lymphocyte exocytosis (in 33% of cases each), keratinocyte apoptosis (26%), atrophy and acanthosis (in 06% of cases each); neural lymphocytic inflammatory infiltrate (100%), superficial perivascular (100%), deep perivascular (93%), peri-eccrine (40%), peri-pilosebaceous follicle (20%), and melanophages in 93% of cases. These findings showed that not always all environments are affected by inflammation in histopathology. In 66% of patients it was found bacterial antigen (by Fite-Faraco or immunohistochemical technique anti-BCG), so the indeterminate form should not be systematically considered as paucibacillary. There was no significant difference in phenotypic and cytokinic patterns between the positive and negative Mitsuda groups. In the microenvironment of inflammatory lesions there was significant expression of TLR 2/4, Factor XIIIa, CD4, CD8, IL-2r, IL-4, IL-10, TGF-beta and iNOS. The important expression of IL-4, IL-10 and TGF-beta in indeterminate leprosy meant tendency to Th2 immune response pole, an environment of tolerance to permanence of bacillus. Low IFN-? expression contributed to the negligible Th1 response. There was no significant difference in the expression of CD1a between the lesions and normal skin. Factor XIIIa was expressed as greater than 50 cells / mm2 in all cases, with significantly larger quantities than other dendritic cells in lesions (S100, CD68, CD123) and than normal skin. These findings demonstrate the importance of Factor XIIIa positive dermal dendrocytes in antigen presentation at the initial stage of leprosy

Em todas as formas clínicas da leishmaniose tegumentar americana os macrófagos são as células efetoras mais importantes na destruição do parasita intracelular. As células dendríticas são células apresentadoras de antígeno localizadas nos sítios de inoculação, como pele e mucosa. Os dendrócitos dérmicos Fator XIIIa positivos são células derivadas de linhagem mielomonocítica e consideradas complementares às células de Langerhans no processo de apresentação de antígenos e indução da resposta imune. As células dendríticas plasmocitoides representam um subgrupo de células dendríticas precursoras presentes no sangue periférico e órgãos linfoides. Estas células são identificadas pela alta expressão de receptor da cadeia alfa da interleucina-3 (CD123) e são fortes produtoras de interferon tipo I. Elas são raramente observadas na pele humana normal, e foram demonstradas em dermatoses inflamatórias e virais. O óxido nítrico e seus derivados atuam como moléculas efetoras da citotoxicidade macrofágica contra parasitas. A expressão da enzima óxido nítrico sintase induzida (iNOS) e a geração de óxido nítrico é importante no controle da infecção por diferentes espécies de Leishmania. Cinqüenta e dois espécimes de biópsias cutâneas de pacientes diagnosticados com leishmaniose tegumentar americana foram classificados histologicamente de acordo com o padrão de resposta tecidual, se granulomatoso ou inflamatório difuso não específico. O objetivo deste estudo foi demonstrar e quantificar a presença de células dendríticas plasmocitoides em 36 das biópsias, através de estudo imuno-histoquímico com anticorpo anti-CD123, comparando os achados entre os diferentes tipos de resposta tecidual; verificar a expressão de iNOS por dendrócitos dérmicos Fator XIIIa positivos e comparar com a expressão de iNOS por macrófagos nas lesões cutâneas, através de estudo imuno-histoquímico com dupla marcação pelos anticorpos antiCD68 e antiiNOS em 43 biópsias cutâneas, e pelos anticorpos anti-Fator XIIIa e antiiNOS em 34 amostras, comparando os achados entre os diferentes padrões de resposta tecidual. Foram evidenciadas células dendríticas CD123+ em todos espécimes de lesões cutâneas de leishmaniose tegumentar americana estudados. Em 22/36 amostras, as células dendríticas plasmocitoides estavam dispostas isoladamente entre outras células inflamatórias; em 14/36 amostras estavam agrupadas, pelo menos focalmente, principalmente no grupo granulomatoso (13 amostras) e em um caso do grupo não específico; dez amostras exibiram células na junção dermoepidérmica, sendo oito no grupo granulomatoso e duas no grupo não específico. Entretanto, não houve diferença no número de células CD123+/mm2 entre os dois grupos estudados. Esses resultados sugerem que as células dendríticas plasmocitoides participam da resposta imune nas lesões cutâneas de leishmaniose tegumentar americana. A expressão de iNOS por dendrócitos dérmicos Fator XIIIa positivos foi evidenciada em todos os espécimes estudados, sendo que a maioria dos macrófagos expressou iNOS. Não houve diferença estatisticamente significativa entre o número de células CD68+/mm2 e CD68+iNOS+/mm2 nos diferentes padrões de resposta tecidual, tampouco no número de células Fator XIIIa+/mm2, mas o número de células FatorXIIIa+iNOS+/mm2 foi maior no grupo granulomatoso. Quando comparadas 34 amostras, todas elas submetidas a estudos com anticorpos anti-Fator XIIIa, anti-CD68, anti- FatorXIIIa/iNOS e anti-CD68/iNOS, foi maior o número total de macrófagos que dendrócitos dérmicos Fator XIIIa positivos, expressando ou não iNOS, e a porcentagem de macrófagos coexpressando iNOS foi maior que a coexpressão de iNOS por dendrócitos dérmicos Fator XIIIa positivos, mas esta diferença não foi estatisticamente significativa quando comparados os grupos histológicos separadamente. Os resultados demonstram que os dendrócitos dérmicos Fator XIIIa positivos expressam iNOS, menos que os macrófagos, mas proeminentemente no grupo granulomatoso, sugerindo a sua participação na patogênese de lesões cutâneas de leishmaniose tegumentar americana, como células com capacidade leishmanicida e/ou apresentadoras de antígeno / In all forms of American tegumentary leishmaniasis lesions, macrophages are the most important effector cells involved in intracellular parasite destruction. Dendritic cells are antigen-presenting cells that are localized at the entry sites, such as skin and mucosa. Factor XIIIa+ dermal dendrocytes are bone marrow-monocytic lineagederived cells and considered complementary cells to Langerhans cells in the process of antigen presentation and inducing immune response. Plasmacytoid dendritic cells constitute a subset of dendritic cells precursors in peripheral blood and organized lymphoid tissue. These cells are identified by their high levels of interleukin-3 receptor alpha chain (CD123) and are vigorous type I interferon producing cells. They are rarely present in normal human skin, and have been demonstrated in inflammatory and viral dermatoses. Nitric oxide radical and derivatives act as effector molecules of macrophage cytotoxicity against invading parasites. Expression of inducible nitric oxide synthase (iNOS) and generation of nitric oxide is important in control of infection in different Leishmania species. Fifty-two samples of skin biopsies obtained from American tegumentary leishmaniasis patients were histologically classified as granulomatous reaction or non specific diffuse inflammatory reaction. The aim of the study was to demonstrate and quantify the presence of plasmacytoid dendritic cells in thirty-six skin biopsies, by immunohistochemistry with anti-CD123, comparing findings in both patterns of tissue response; to verify the expression of iNOS by Factor XIIIa+ dermal dendrocytes and compare to the expression of iNOS by macrophages in cutaneous lesions, by doublestaining technique with antiCD68 and antiiNOS antibodies in forty-three skin biopsies and anti-factor XIIIa and antiiNOS antibodies in thirty-four biopsies, comparing findings between different tissue response patterns. Dendritic CD123+ cells were demonstrated in all specimens of American tegumentary leishmaniasis lesions. The number of CD123+ cells/mm2 in the two groups did not differ. In 22/36 samples, plasmacytoid dendritic cells were intermingled with other inflammatory cells, and were grouped, at least focally, in 14/36 samples. Thirteen cases from the granulomatous group and one non specific case showed clusters of cells in the dermal inflammatory infiltrate. Ten biopsies displayed plasmacytoid dendritic cells at the dermoepidermal junction, two in the non specific group and eight in the granulomatous group. The findings suggest that plasmacytoid dendritic cells participate in the immune response of American tegumentary leishmaniasis skin lesions. Expression of iNOS by Factor XIIIa+ dermal dendrocytes was shown in all specimens, and most of the macrophages expressed iNOS. The total number of CD68+ cells/mm2 and CD68+iNOS+ cells/mm2 in the two groups did not differ, nor the total number of FactorXIIIa+ cells/mm2, but the number of FactorXIIIa+iNOS+ cells/mm2 was higher in the granulomatous group. When comparing thirty-four samples that were all tested to anti-Factor XIIIa, anti-CD68, anti-FactorXIIIa/anti-iNOS and anti-CD68/anti-iNOS, it was higher the total number of macrophages, either non-expressing or expressing iNOS than iNOS-expressing Factor XIIIa+ dermal dendrocytes, and the total percentage of iNOS-expressing macrophages was higher than iNOSexpressing Factor XIIIa+ dermal dendrocytes, but this percentage was not significant when granulomatous and non specific groups were separately analyzed. The results demonstrate that FactorXIIIa+ dermal dendrocytes express iNOS, less than macrophages, but prominently in the granulomatous group, suggesting they play a role in the pathogenesis of American tegumentary leishmaniasis skin lesions as immune effectors and/or antigenpresenting cells

Aplicando tecnologia de microarray, objetivamos traçar o perfil do programa de maturação das Mo-DC pulsadas com HIV autólogo inativado por AT-2, a fim de identificar marcadores específicos de ativação funcional e sugerir um perfil de expressão de genes úteis na identificação de respostas ao modelo in vitro das Mo-vacina DC. Essas informações podem ajudar a estabelecer assinaturas moleculares das funções celulares mais relevante para a melhoria das vacinas terapêuticas. O perfil transcricional foi analisado com base das vias celulares moduladas das Mo-DCs no estado imaturo, transitório e maduro. O HIV-1 inativado por AT-2 induz ativação de genes associados à apresentação de antígenos. Os conjuntos de genes do citoesqueleto podem influenciar a mudança de comportamento migratório das Mo-DCs ativadas. O aumento na expressão dos receptores celulares contribuem para o recrutamento de monócitos, DCs e macrófagos para o local da infecção. Além disso, modulam a resposta imune inata e adaptativa, incluindo a polarização das células Th e sub-regulação da resposta inflamatória, que pode interferir significativamente com a resposta imune. Coletivamente, o perfil transcricional das Mo-DCs induzido pelo HIV-1 inativado com AT-2 reflete uma significativa reprogramação imunológica e celular das células envolvidas na resposta imune do hospedeiro. Os resultados deste estudo focaram na interpretação de genes específicos dos perfis de transcrição das Mo-DCs como modelo terapêutico utilizado na vacina anti-HIV. As análises de assinaturas gene associado e sua correlação as respostas funcionais simplificam a identificação de indivíduos susceptíveis a vacina e a compreensão de eventuais falhas em ensaios clínicos. Microarray permitiu a análise quantitativa e simultânea da expressão de um elevado número de genes. Os estudos do perfil de expressão foram extremamente úteis para identificar os eventos moleculares e vias envolvidas nas funções de celular induzida por estímulos específicos. Em particular, os resultados sobre o padrão global da expressão dos genes subjacentes as modificações induzidas pelo HIV-1 inativado por AT-2, na fase inicial da administração do antígeno, pôde ser extremamente útil para a identificarmos marcadores de ativação e avaliar os efeitos biológicos que poderiam estar envolvidos para modificação e otimização de estratégias vacinação com Mo-DC / Applying microarray technology, we intend to profile the program to mature Mo-DC pulsed with autologous inactivated HIV by AT-2, in order to identify specific markers of functional activation and suggest a profile of expression of specific genes, useful identification of responders to in vitro model of Mo-DC vaccine. Such information may help to establish detailed molecular signatures of cellular functions most relevant to improving the therapeutic vaccines. The transcriptional profile was analyzed on the basis of the cellular pathways modulated in immature MoDC, transitional MoDC and mature MoDC. The AT-2-inactivated HIV-1 induction of MoDC results in the activation of genes associated with antigen presentation functions. A set of cytoskeletal genes that may potentially mediate shape change and migratory behavior of activated MoDC is also observed. The increase in the expression of immune receptors contribute to the recruitment of monocytes, DCs, and macrophages to the site of infection. Moreover, they modulate both innate and adaptive immune response, including the polarization of Th cells, and the down-regulation of the inflammatory response, which may significantly interfere with the immune response. Collectively, the transcriptional profile induced by AT-2-inactivated HIV-1 in MoDc reflects a significant cellular and immunological reprogramming of cells directly involved in the host immune response. The results of this study focused on the interpretation of specific genes of transcription profile of MoDC used in therapeutic HIV vaccine model. Supplementing the analyses with examination of associated gene signatures and their correlation to functional responses will simplify the identification of responsive vaccine individuals and the understanding of eventual failures in individuals enrolled in clinical trials. Microarray approach allows quantitative and simultaneous analysis of gene expression of a large amount of genes and the systematic studies of expression patterns are extremely useful for identify molecular events and key pathways involved in cellular functions induced by specific stimuli. In particular, data on the global pattern of gene expression underlying the modifications induced by AT-2-inactivated HIV-1 in MoDC, at early stages of antigen administration, may be extremely helpful for the identification of exclusive activation markers to trace the biological effects of modifications/optimizations of the MoDc vaccination strategy

Imunização terapêutica utilizando células dendríticas derivadas de monócitos (MoDCs) pulsadas com antígenos de HIV constitui um meio promissor de potencializar a resposta imune específica anti-HIV em pacientes infectados. Neste contexto, é importante ressaltar que células dendríticas além de estimular a resposta imune específica, podem ser capazes de promover a tolerância periférica em linfócitos T CD4+ e T CD8+ ao induzir deleção, anergia ou através da expansão de células T reguladoras (T regs). Experimentos in vitro foram conduzidos para avaliar a capacidade de MoDCs pulsadas com HIV autólogo inativado em induzir apoptose celular, respostas celulares específicas e a geração de T regs. Os pacientes avaliados neste estudo foram indivíduos infectados pelo HIV, sem uso de tratamento antirretroviral (n = 14) com número de células T CD4+ acima de 350 células/L. MoDCs foram geradas a partir de células mononucleares de sangue periférico e em seguida foram pulsadas com vírus autólogo inativado por Aldrithiol-2, tratadas com estímulo para maturação e então cultivadas com linfócitos autólogos. A apoptose de linfócitos T e MoDCs e a frequência de células efetoras e reguladoras foram avaliadas por citometria de fluxo. Os resultados obtidos mostraram que não houve diferença nos níveis de apoptose de células T CD4+, T CD8+ ou MoDCs entre os grupos pulsadas e não pulsadas com HIV inativado. Foi observado que tanto MoDCs pulsadas quanto aquelas não pulsadas com o vírus autólogo inativado foram capazes de induzir células T CD4+ secretoras de IFN-, enquanto que apenas MoDCs pulsadas levou a um aumento no percentual de células T CD8+ efetoras. Pacientes com contagem de células T CD4+ acima de 500 células/L apresentaram um percentual maior de células T CD4+ secretoras de IFN após estimulo de MoDCs pulsadas. Esta diferença não foi observada em células T CD8 +. T regs também foram induzidas in vitro após cocultivo com MoDCs. Níveis basais mais elevados de T regs foram encontrados em pacientes com carga viral plasmática baixa. Em conjunto, os resultados indicam que MoDCs pulsadas com HIV-1 são capazes de induzir linfócitos T efetores, mas também aumentam a frequência de T regs in vitro. Além disto, pacientes com maior contagem de células T CD4 + foram capazes de responder de forma mais eficiente ao estímulo com MoDCs pulsadas. Viremia persistente na infecção crônica pelo HIV pode estar associada significativamente à perda de T reg / Therapeutic immunization using inactivated autologous HIVpulsed dendritic cells (DCs) is a promising strategy to enhance specific anti-HIV immune responses in infected patients. In this context, it is important to note that DC besides stimulate a specific immune response, may be able to promote tolerance in peripheral CD4 + and CD8 + T cells inducing deletion, anergy or through expansion of regulatory T cells (T reg). In vitro experiments were conducted to evaluate the capacity of autologous HIVstimulated DC to induce apoptosis, effector cellular T cell responses and T reg generation. For these purposes, we used peripheral blood from HAARTnaïve HIVinfected patients (n=14) with CD4+ T cell counts above 350 cell/L for generation of monocytederived DC (MoDC). MoDC were pulsed with aldrithiol-2 (AT-2)-inactivated autologous virus and matured. MoDC were then cocultured with autologous lymphocytes and the apoptosis, production of IFN- and T reg cell frequency were evaluated by flow cytometry. There was no difference in the rate of apoptosis of CD4, CD8 T cells or MoDC between the groups pulsed and not pulsed with inactivated HIV. MoDC pulsed or not with inactivated autologous virus induced IFN- +CD4+ T cells, whereas only pulsed MoDC were able to increase effector CD8+ T cells percentage along the time culture. Patients with CD4+ T cell counts above 500 had an increased percentage of CD4 + T cells secreting IFN- upon DC pulsed with HIV. This difference was not observed in CD8 + T cells. Interestingly, T reg were also induced in vitro after MoDC cocultivation. Higher baseline T reg counts were found in patients with lower plasma viral loads. These results show that MoDC pulsed with HIV-1 are able to induce effector lymphocyte but also elevate the frequency of T reg in vitro. Patients with higher CD4+ T cell counts are able to respond more efficiently to the stimulation with pulsed MoDC, and persistent viremia in chronic HIV infection is associated with significant loss of T reg