O escorpião Tityus serrulatus (Ts) é o responsável pela maioria casos de envenenamento escorpiônicos do Brasil. Embora sua peçonha seja constituída de inúmeros componentes, as neurotoxinas apresentam maior relevância por interagirem especificamente com canais para sódio (Nav) ou potássio (Kv) dependentes de voltagem. Até o momento, já foram descritas 20 neurotoxinas (Ts1 -> Ts20) na peçonha do Ts. No entanto, através de análises ômicas, estima-se que este número seja bem superior. Toxinas que interagem seletivamente com canais iônicos são utilizadas como ferramentas farmacológicas, pois permitem a identificação de canais específicos e a determinação de seus papéis fisiológicos. Adicionalmente, estas toxinas podem ser utilizadas para o desenvolvimento de novos medicamentos para tratar doenças relacionadas a canais iônicos. Considerando o elevado potencial biotecnológicos desta classe de moléculas, o presente trabalho isolou (através de 3 etapas cromatográficas) e caracterizou uma nova toxina da peçonha de Ts, denominada Ts19 Frag-II. A nova toxina demonstrou possuir 49 resíduos de aminoácidos e massa molecular de 5534,54012 Da. Classificada como ?-KTx, especula-se que a Ts19 Frag-II seja produzida a partir de uma modificação pós-transducional, denominada neste estudo de post-splitting, de um transcrito da Ts19. A caracterização funcional da Ts19 Frag-II foi realizada utilizando diferentes ensaios de atividade biológica. Uma extensa avaliação eletrofisiológica em canais iônicos (16 Kvs e 5 Navs) expressos em oócitos de X. leavis demonstrou que a toxina é capaz de bloquear seletivamente canais para potássio do tipo Kv1.2 (IC50 = 544 ± 32 nM). Ensaios in vivo (camundongos C57BL/6) de dor revelaram que a Ts19 Frag-II (2 e 4?g) não é capaz de induzir comportamento nociceptivo espontâneo ou mecânico em camundongos, tanto pela administração intraplantar quanto pela via intratecal. Ensaios in vivo (camundongos BALB/c) também demonstraram que a nova toxina (4 e 8?g) induz aumento dos níveis séricos de ureia, ALT, ?-globulina, IL-6, TNF-?, IL-17A e NO, além de diminuir a quantidade de ?-globulinas. Adicionalmente, utilizando ensaios in vitro de cultura celular de linfócitos T CD4+, demonstrou-se que a Ts19 Frag-II (2 ?g) foi capaz de diminuir a diferenciação de células Th17, assim como suprimir sua função (diminuiu a produção de IL-17 e IL-22). Assim, no presente estudo foi realizado o isolamento e a caracterização molecular e funcional de uma nova toxina de Ts, a qual apresentou atividade neurotóxica e pró-inflamatória, podendo contribuir significativamente para a gravidade do quadro de envenenamento ocasionado pelo escorpião Ts. Adicionalmente, sua seletividade para canais Kv1.2 faz com que Ts19 Frag-II possa ser utilizada como ferramenta de estudo deste canal iônico / The scorpion Tityus serrulatus (Ts) is responsible for most cases of scorpion envenomations in Brazil. Although its venom consist of many components, neurotoxins present major relevance because their specific interaction with voltage-gated sodium (Nav) or potassium (Kv) channels. So far, 20 neurotoxins have been described (Ts1 -> Ts20) in the Ts venom. However, omics analysis indicate that this number is much higher. Toxins that interact selectively with ion channels are used as pharmacological tools, as they allow the identification of specific channels and the determination of their physiological roles. Additionally, these toxins may be used for the development of new drugs for treating disorders related to ion channels. Considering the high biotechnology potential of this class of molecules, this study isolated (by 3 chromatographic steps) and characterized a new toxin from Ts venom, named Ts19 Frag-II. The novel toxin presented 49 amino acid residues and a molecular mass of 5534.54012 Da. Classified as ? - KTx, it is speculated that the Ts19 Frag-II is produced from a post- translational modification, referred in this study as post- splitting, a transcript of Ts19. The functional characterization of Ts19 Frag-II was performed using different biological assays. The extensive electrophysiological study on ion channels (16 Kvs and 5 Navs) expressed in oocytes of X. leavis showed that the toxin is able to selectively block the potassium channel Kv1.2 (IC50 = 544 ± 32 nM). In vivo assays (C57BL/6 mice) of nociception showed that Ts19 Frag-II (2 and 4?g) is not able to induce spontaneous or mechanical nociceptive behavior in mice, both by intraplantar as by intrathecal administration. In vivo assays (BALB/c mice) also demonstrated that the novel toxin (4 and 8?g) increased serum levels of urea, ALT, ?-globulin, IL-6, TNF-?, IL-17A and NO, besides decreasing ?-globulins. In addition, using in vitro assays of CD4+ T-lymphocyte cell culture, it was demonstrated that Ts19 Frag-II (2 ug) was able to decrease the differentiation of Th17 cells, as well as it suppresses Th17 function (decreased IL-17 and IL-22 production). Therefore, the present study isolated and performed the molecular and functional characterization of a new toxin from Ts, which presented neurotoxic and pro-inflammatory activity and could contribute significantly to the severity of the envenoming caused by the Ts scorpion. Moreover, based on its selectivity for Kv1.2 channels, this toxin could be used as a tool to study this type of ion channel

Paracoccidioides brasiliensis é um fungo dimórfico patogênico agente etiológico da paracoccidioidomicose (PCM), uma micose endêmica no Brasil. A busca por alternativas para reduzir o tempo de tratamento da PCM levou ao desenvolvimento de uma vacina de DNA contendo a sequência do peptídeo P10 de P. brasiliensis. Neste trabalho, avaliamos a geração de células T de memória e células T reguladoras em camundongos imunizados com esta vacina de DNA antes e após o desafio com o fungo, através da análise de seus esplenócitos e linfócitos pulmonares por citometria de fluxo. Os resultados mostram um aumento no percentual de células T reguladoras e de memória no baço e pulmões dos animais imunizados antes e depois de 30, 60 e 120 dias do desafio em comparação com os grupos controle e não imunizado. Outro experimento revelou que o modelo experimental da PCM in vivo é capaz de induzir a expressão de ROR&#947t. Este estudo mostra que nossa vacina de DNA contra a PCM gera células com fenótipo de reguladoras e de memória, caracterizando seu potencial para o tratamento desta micose. / Paracoccidioides brasiliensis is a dimorphic fungal pathogen that is the etiological agent of paracoccidioidomycosis (PCM), a mycosis endemic in Brazil. The search for new alternatives to reduce the duration of PCM treatment led to the development of a DNA vaccine encoding the peptide P10 of P. brasiliensis. Presently, we evaluated the generation of memory and regulatory T cells in mice immunized with this DNA vaccine, before and after the challenge with the fungus by analizing their splenocytes and pulmonary lymphocytes by flow cytometry. The results show an increase in the percentage of regulatory and memory T cells on spleens and lungs of immunized mice before and after 30, 60 and 120 days of challenge compared with the control and untreated groups. Another experiment revealed that the PCM in vivo infection model is capable of inducing ROR&#947t expression. This study demonstrates that our DNA vaccine against PCM generates cells with a regulatory and memory phenotype, which shows its potencial in the treatment of this mycosis.

A cromoblastomicose é uma infecção fúngica crônica que acomete pele e tecido subcutâneo. As lesões podem ser classificadas em tumoral, verrucosa, cicatricial e do tipo placa. A resposta imune é principalmente celular e a forma grave da doença correlaciona-se com citocinas de perfil Th2. Nós exploramos populações celulares do tipo T reguladoras e Th17. Foram utilizadas vinte e três biópsias da forma verrucosa obtidas de pacientes com diagnóstico clínico e histopatológico de cromoblastomicose, sem tratamento. Foi realizado o método de imunohistoquímica para detectar Foxp3, CD25, TGF-beta, IL-6, IL-17 e IL-23. A IL-17 predominou sobre os outros marcadores, embora haja número regular de Foxp3. TGF-beta, IL-6 e IL-23 raramente foram visualizados. A constituição de uma resposta imune local com alta expressão de IL-17 e baixa expressão de outras citocinas pode ser, ao menos em parte, uma tentativa de ajudar o sistema imunológico contra infecções fúngicas. Células Foxp3 poderiam ser capazes de interferir na resposta imune eficiente contra fungos, mas também beneficiar o hospedeiro, através da capacidade de reduzir os danos do tecido que seguem uma resposta imune local. Esses elementos celulares podem contribuir para a cronicidade que caracteriza esta doença / Chromoblastomycosis is a chronic fungal infection that affects skin and subcutaneous tissue. Lesions can be classified in tumorous, verrucous, cicatricial and plaque type. The immune response is primarily cellular and the severe form of the disease correlates with a Th2 pattern of cytokines. We intended to explore the populations of regulatory T cells and the Th17 pattern. Twenty-three biopsies of verrucous form were obtained from patients with clinical and histopathological diagnostic of chromoblastomycosis, without treatment. It was performed an immunohistochemistry method to detect Foxp3, CD25, TGF-beta, IL-6, IL-17 and IL-23. IL-17 predominated over the other markers in chromoblastomycosis, although there was a regular number of Foxp3. TGF- beta, IL-6 and IL-23 were rarely visualized. The constitution of a local immune response with high expression of IL-17 and low expression of other cytokines could be at least in part, an attempt to help the immune system against fungal infection. Foxp3 cells could be able to interfere with the efficient immune response against fungi, but also benefit the host, through the ability to reduce the tissue damage that follows a local immune response. They could play a role in chronicity that characterizes this disease

Avaliou-se a capacidade dos cimentos endodônticos: Sealapex®, Activ-GP® e AH-Plus® de alterarem o perfil das citocinas nas respostas Th1, Th2 e Th17, após a implantação destes em subcutâneo de camundongos. A quantificação das citocinas IL-2, IL-6, TNF-α, IFN-γ, IL-4, IL-10 e IL-17 foi realizada in vivo, no tecido reacional circundante aos implantes, os quais foram confeccionados a partir de sondas nasogástricas estéreis e apirogênicas de cloreto de polivinila preenchidas com os cimentos, tendo um grupo controle com sondas vazias. Utilizou-se camundongos isogênicos da linhagem C57BL/6 machos de 6/8 semanas de vida sendo que cada um recebeu 2 implantes na região dorsal (lado direito e esquerdo). Após os períodos experimentais de 7, 21 e 63 dias, com os camundongos anestesiados, os implantes foram removidos juntamente com o tecido circundante, e os animais sacrificados em seguida por deslocamento cervical. As amostras de cada grupo foram divididas sendo: duas, contendo implante/tecido, processadas histotecnicamente e as demais com apenas tecido (sem implante) foram homogeneizadas e centrifugadas com solução formada por tampão RIPA e inibidor de protease. O sobrenadante, fruto deste processo, foi coletado e a dosagem das citocinas realizada através do kit CBA mouse-Th1/Th2/Th17 Cytokine Kit (BD Cytometric Bead Array, San Jose, CA, USA) em análise por citometria de fluxo. Os parâmetros avaliados foram a concentração da citocina em função do cimento testado em cada período experimental. Submeteu-se os resultados à análise estatística por meio do teste t com a correção de Welch\'s. Para todos os testes o nível de significância foi de 5%. Com relação à IL-2 houve diferenças estatísticas significantes entre os grupos Activ-GP® e AH-Plus® (p=0,0391). No período de 21 dias foram detectadas diferenças entre o grupo controle e AH-Plus® (p=0,0402) e entre o grupo Sealapex® e AH-Plus® (p=0,0244). O AH-Plus® induziou um maior aumento na IL-6, aos 7 dias em relação ao Activ-GP® (p=0,0286) e aos 21 dias entre o grupo controle (p=0.0402) e Activ-GP® (p=0.0244). Os níveis de TNF-α foram estatisticamente superiores após 7 dias quando comparou-se o grupo AH-Plus® com os demais. Observou-se que no grupo controle aos 7 e 21 dias ocorreram diferenças estatísticas em relação ao Sealapex® e AH-Plus® respectivamente quando avaliadas as concentrações de IFN-γ. Houve também diferenças estatísticas entre o grupo controle e Sealapex® (p=0,0158) no período de 7 dias para a citocina IL-4. Os valores de IL-10 foram estatisticamente superiores para o grupo controle em relação ao Activ-GP® no período de 21 dias (p=0,0471). Com relação a IL-17 no período de 21 dias, observou-se os maiores valores para o grupo controle, seguido pelo Sealapex®, Activ-GP® e AH-Plus®. Foram detectadas diferenças entre os grupos controle e AH-Plus® (p=0,0121), controle e Activ-GP® (p=0,0262) e entre Sealapex® e Activ GP® (p=0,0314). Baseado nesses resultados podê-se concluir que: os cimentos endodônticos são capazes de modular as respostas Th1, Th2 e Th17 através da inibição ou estimulação da liberação das citocinas testadas. O cimento AH-Plus® promoveu as maiores diferenças no perfil de resposta Th1. / It was evaluated the capacity of the following endodontic sealers: Sealapex, Activ-GP and AH-Plus to modify the cytokine profile in Th1, Th2 and Th17 responses, after their implantation in the subcutaneous tissue of mice. Quantification of IL-2, IL-6, TNF-α, IFN-γ, IL-4, IL-10 and IL-17 was performed in vivo, in the reactional tissue surrounding the implants, which were made from sterile nasogastric probes and apyrogenic of polyvinyl chloride filled with sealer, and a control group of empty probes. It was used isogenic mice of C57BL/6 lineage, 6/8 weeks old males, each of which received two implants in the dorsal region (left and right). After the experimental time of 7, 21 and 63 days, the mice were anesthetized and the implants were removed along with the surrounding tissue, the animals were then sacrificed by cervical dislocation. Samples from each group were divided as follows: two containing implant / tissue processed histologically and with only the remaining tissue (without implant) were mixed and centrifuged with a solution formed by RIPA buffer and protease inhibitors. The supernatant result of this process was collected and cytokine dosage accomplished by mouse-Th1/Th2/Th17 Cytokine CBA Kit Kit (BD cytometric Bead Array, San Jose, CA, USA) for flow cytometry analysis. The evaluated parameters were the cytokine concentration in function of sealer tested in each trial. The results were submitted to statistical analysis using the t test with Welch\'s correction. For all tests the significance level was 5%. With respect to IL-2 there were significant statistical differences between groups-Activ GP and AH-Plus (p=0.0391). In the period of 21 days differences were found between the control group and AH-Plus (p=0.0402) and between the group Sealapex and AH-Plus (p=0.0244). The AH-Plus induced a greater increase in IL-6, at 7 days compared to Activ-GP (p=0.0286) and at 21 days between the control group (p=0.0402) and Activ-GP (p=0.0244). The levels of TNF-α were significantly higher after 7 days when the AH-Plus group was compared with others. It was observed that in the control group at 7 and 21 days there were statistical differences in relation to Sealapex and AH-Plus respectively when evaluated concentrations of IFN-γ. There were also significant differences between the control group and Sealapex (p=0.0158) within 7 days for the cytokine IL-4. The amounts of IL-10 were statistically higher in the control group compared to the Activ GP in a period of 21 days (p=0.0471). With respect to IL-17 in a period of 21 days, it was observed the highest values for the control group, followed by Sealapex, Activ-GP and AH-Plus. Differences were found between the control groups and AH-Plus (p=0.0121), control and Activ-GP (p=0.0262) and between Sealapex and Activ-GP (p=0.0314). Based on the presented results theendodontic sealers are able to promote changes in the response cytokine profile Th1, Th2 and Th17; Sealer AH-Plus produced the greatest changes, in the Th1 response profile.

En condition d'obésité, le tissu adipeux est le théâtre d'infiltration et d'accumulation de cellules immunitaires dont les lymphocytes Th17, impliqués dans de nombreuses maladies inflammatoires chroniques et auto-immunes. Les mécanismes d'activation et de prolifération des Th17 au sein du tissu adipeux ne sont pas connus. Nous avons suggéré un rôle des cellules souches adipocytaires (CSA) dans l'induction de l'inflammation médiée par les Th17. Nos résultats montrent, pour la première fois, et grâce à un modèle de co-culture combinant les CSA du tissu adipeux obèse avec des cellules mononucléées du sang circulant, que seules les CSA issues de sujets obèses, et non pas de sujets minces, sont capables d'induire l'activation des Th17 et des monocytes, qui en retour activent la sécrétion d'IL-6 par les CSA. L'environnement inflammatoire, induit par cette interaction, est à l'origine de l'inhibition de l'adipogenèse et de la réduction de la sensibilité à l'insuline des adipocytes. De plus, tout comme les CSA, les adipocytes issus de sujets obèses induisent le même phénotype inflammatoire. Enfin, une étude en cours nous a permis de montrer que les acides gras polyinsaturés de type -3, inhibent spécifiquement l'activation des Th17 mais n'ont aucun impact sur les monocytes inflammatoires, dans notre modèle, possiblement lié à une inhibition de l'expression d'ICAM-1. En conclusion, une triade inflammatoire constituée de CSA ou d'adipocytes issus de sujets obèses combinés avec des monocytes et des Th17, forme un cercle vicieux où l'inflammation est maintenue et amplifiée / In obesity, adipose tissue is massively infiltrated by a panel of inflammatory immune cells such as IL-17-secreting Th17 lymphocytes. The mechanisms by which Th17 cells are induced are less understood. We postulated that adipose stem cells (ASC), which are known to play immunomodulatory functions in contact with immune cells, might be involved in Th17 activation. By using an in-vitro co-culture model, we demonstrated for the first time, that ASC issued from obese , but not lean AT were able to activate IL-17 secretion from Th17 cells, and IL-1 secretion from monocytes. In turn, such an interaction led to increased secretion of IL-6 from ASC. The inflammatory environment generated from this interaction was then able to inhibit adipogenesis and adipocyte insulin-sensitivity. We also showed that adipocytes harvested from obese adipose tissue were able to promote Th17 cell and monocyte activation. Finally, we demonstrated that polyunsaturated fatty acids omega-3 presented anti-inflammatory properties towards Th17 cells, while they had no effect on monocytes in our co-culture model (manuscript in preparation), which was related to decrease of ICAM-1 expression. In conclusion, we highlight herein a crosstalk between ASC, monocytes and Th17 cells in obese adipose tissue, which leads to a vicious circle of pro-inflammatory cytokine secretion

Les chimiothérapies conventionnelles anticancéreuses ont été développées dans le but de traiter le cancer par élimination directe et/ou par inhibition de croissance les cellules tumorales en division. Les cellules endothéliales en prolifération à l’origine de la vascularisation intra-tumorale sont également connues pour être sensibles aux effets cytotoxiques des agents anticancéreux. Depuis, de nombreuses études ont montré que certaines thérapies conventionnelles peuvent être exploitées pour leurs capacités anti-angiogéniques (Browder et al. Cancer Research 2000). La stratégie mise en place consiste à suivre des protocoles où la thérapie est administrée à des doses faibles non myéloablatives et plus fréquemment que les thérapies conventionnelles, appelés dosages métronomiques (Hanahan et al. JCI 2000, Gasparini et al. Lancet Oncology 2001). Le cyclophosphamide (CTX) est un agent alkylant communément utilisé en chimiothérapie dans des protocoles à dosage métronomique. Dans les années 1980, 2 études ont montré que le CTX utilisé à dose métronomique pouvait avoir aussi un rôle sur l’immunité en réduisant la fonction suppressive d’une population de lymphocytes T CD4+ dans un modèle expérimental de tumeur (Awwad et al. Cancer Research 1989) et chez des patients atteints de cancer (Berd et al. Cancer Research 1987). Depuis, les connaissances ont progressé et à présent le CTX métronomique est reconnu pour pouvoir limiter l’expansion et les fonctions des lymphocytes T régulateurs (Treg) (Ghiringhelli et al. EJI 2004, Lutsiak et al. Blood 2005) conduisant à une polarisation des cellules T auxiliaires vers un profil Th1 (Matar et al. Eur J cancer 2000 et Cancer Immunol Immunother 2002). Utilisé en association, le CTX métronomique s’avère donc être un outil intéressant dans le traitement anticancéreux (Hermans et al. Cancer Research 2003, Taieb et al. JI 2006). Nos résultats montrent l’importance des lymphocytes T CD4+ sécréteurs d’IL-17 et d’IFNg dans les effets du CTX. / Conventional cancer chemotherapies were developed to target cancer cells either by directly eliminating them or by inhibiting the growth of dividing tumor cells. Proliferating endothelial cells at the origin of intratumoral vascularization are known to be sensitive to the cytotoxic effects of antineoplastic agents. Many studies have shown that some conventional therapies can be exploited for their anti-angiogenic capabilities (Browder et al. Cancer Research 2000). The adopted strategy, called metronomic chemotherapy, consists of administering low doses of drug that do not induce myelosuppression, on a more frequent schedule as compared to conventional therapies (Hanahan et al. JCI 2000, Gasparini et al. Lancet Oncology 2001). Cyclophosphamide (CTX) is an alkylating agent commonly used as a metronomic chemotherapy. In the 1980s, two studies demonstrated that when used at a metronomic dosing, CTX could impact the immune response particularly in reducing the suppressive function of a CD4+ T lymphocyte population in an experimental tumour model (Awwad et al. Cancer Research 1989) and in cancer patients (Berd et al. Cancer Research 1987). Since then, knowledge has evolved and now CTX used as a metronomic or low-dose therapy is administered to limit expansion and functions of regulatory T cells (Treg) (Ghiringhelli et al. EJI 2004, Lutsiak et al. Blood 2005), leading to a helper T cell polarization toward a Th1 profile (Matar et al. Eur J cancer 2000 et Cancer Immunol Immunother 2002). When used in combination, CTX turns out to be a potent drug in the antineoplastic treatments armamentarium (Hermans et al. Cancer Research 2003, Taieb et al. JI 2006). Our results demonstrate the importance of CTX effects on IL-17 and IFNg secreting CD4+ T lymphocytes.

Avaliou-se a capacidade dos cimentos endodônticos: Sealapex®, Activ-GP® e AH-Plus® de alterarem o perfil das citocinas nas respostas Th1, Th2 e Th17, após a implantação destes em subcutâneo de camundongos. A quantificação das citocinas IL-2, IL-6, TNF-α, IFN-γ, IL-4, IL-10 e IL-17 foi realizada in vivo, no tecido reacional circundante aos implantes, os quais foram confeccionados a partir de sondas nasogástricas estéreis e apirogênicas de cloreto de polivinila preenchidas com os cimentos, tendo um grupo controle com sondas vazias. Utilizou-se camundongos isogênicos da linhagem C57BL/6 machos de 6/8 semanas de vida sendo que cada um recebeu 2 implantes na região dorsal (lado direito e esquerdo). Após os períodos experimentais de 7, 21 e 63 dias, com os camundongos anestesiados, os implantes foram removidos juntamente com o tecido circundante, e os animais sacrificados em seguida por deslocamento cervical. As amostras de cada grupo foram divididas sendo: duas, contendo implante/tecido, processadas histotecnicamente e as demais com apenas tecido (sem implante) foram homogeneizadas e centrifugadas com solução formada por tampão RIPA e inibidor de protease. O sobrenadante, fruto deste processo, foi coletado e a dosagem das citocinas realizada através do kit CBA mouse-Th1/Th2/Th17 Cytokine Kit (BD Cytometric Bead Array, San Jose, CA, USA) em análise por citometria de fluxo. Os parâmetros avaliados foram a concentração da citocina em função do cimento testado em cada período experimental. Submeteu-se os resultados à análise estatística por meio do teste t com a correção de Welch\'s. Para todos os testes o nível de significância foi de 5%. Com relação à IL-2 houve diferenças estatísticas significantes entre os grupos Activ-GP® e AH-Plus® (p=0,0391). No período de 21 dias foram detectadas diferenças entre o grupo controle e AH-Plus® (p=0,0402) e entre o grupo Sealapex® e AH-Plus® (p=0,0244). O AH-Plus® induziou um maior aumento na IL-6, aos 7 dias em relação ao Activ-GP® (p=0,0286) e aos 21 dias entre o grupo controle (p=0.0402) e Activ-GP® (p=0.0244). Os níveis de TNF-α foram estatisticamente superiores após 7 dias quando comparou-se o grupo AH-Plus® com os demais. Observou-se que no grupo controle aos 7 e 21 dias ocorreram diferenças estatísticas em relação ao Sealapex® e AH-Plus® respectivamente quando avaliadas as concentrações de IFN-γ. Houve também diferenças estatísticas entre o grupo controle e Sealapex® (p=0,0158) no período de 7 dias para a citocina IL-4. Os valores de IL-10 foram estatisticamente superiores para o grupo controle em relação ao Activ-GP® no período de 21 dias (p=0,0471). Com relação a IL-17 no período de 21 dias, observou-se os maiores valores para o grupo controle, seguido pelo Sealapex®, Activ-GP® e AH-Plus®. Foram detectadas diferenças entre os grupos controle e AH-Plus® (p=0,0121), controle e Activ-GP® (p=0,0262) e entre Sealapex® e Activ GP® (p=0,0314). Baseado nesses resultados podê-se concluir que: os cimentos endodônticos são capazes de modular as respostas Th1, Th2 e Th17 através da inibição ou estimulação da liberação das citocinas testadas. O cimento AH-Plus® promoveu as maiores diferenças no perfil de resposta Th1. / It was evaluated the capacity of the following endodontic sealers: Sealapex, Activ-GP and AH-Plus to modify the cytokine profile in Th1, Th2 and Th17 responses, after their implantation in the subcutaneous tissue of mice. Quantification of IL-2, IL-6, TNF-α, IFN-γ, IL-4, IL-10 and IL-17 was performed in vivo, in the reactional tissue surrounding the implants, which were made from sterile nasogastric probes and apyrogenic of polyvinyl chloride filled with sealer, and a control group of empty probes. It was used isogenic mice of C57BL/6 lineage, 6/8 weeks old males, each of which received two implants in the dorsal region (left and right). After the experimental time of 7, 21 and 63 days, the mice were anesthetized and the implants were removed along with the surrounding tissue, the animals were then sacrificed by cervical dislocation. Samples from each group were divided as follows: two containing implant / tissue processed histologically and with only the remaining tissue (without implant) were mixed and centrifuged with a solution formed by RIPA buffer and protease inhibitors. The supernatant result of this process was collected and cytokine dosage accomplished by mouse-Th1/Th2/Th17 Cytokine CBA Kit Kit (BD cytometric Bead Array, San Jose, CA, USA) for flow cytometry analysis. The evaluated parameters were the cytokine concentration in function of sealer tested in each trial. The results were submitted to statistical analysis using the t test with Welch\'s correction. For all tests the significance level was 5%. With respect to IL-2 there were significant statistical differences between groups-Activ GP and AH-Plus (p=0.0391). In the period of 21 days differences were found between the control group and AH-Plus (p=0.0402) and between the group Sealapex and AH-Plus (p=0.0244). The AH-Plus induced a greater increase in IL-6, at 7 days compared to Activ-GP (p=0.0286) and at 21 days between the control group (p=0.0402) and Activ-GP (p=0.0244). The levels of TNF-α were significantly higher after 7 days when the AH-Plus group was compared with others. It was observed that in the control group at 7 and 21 days there were statistical differences in relation to Sealapex and AH-Plus respectively when evaluated concentrations of IFN-γ. There were also significant differences between the control group and Sealapex (p=0.0158) within 7 days for the cytokine IL-4. The amounts of IL-10 were statistically higher in the control group compared to the Activ GP in a period of 21 days (p=0.0471). With respect to IL-17 in a period of 21 days, it was observed the highest values for the control group, followed by Sealapex, Activ-GP and AH-Plus. Differences were found between the control groups and AH-Plus (p=0.0121), control and Activ-GP (p=0.0262) and between Sealapex and Activ-GP (p=0.0314). Based on the presented results theendodontic sealers are able to promote changes in the response cytokine profile Th1, Th2 and Th17; Sealer AH-Plus produced the greatest changes, in the Th1 response profile.

Produzida por macrófagos e células dendríticas, a IL-27 é uma citocina heterodimérica capaz de induzir células Tr1 produtoras de IL-10 e consequentemente regular linfócitos Th1, Th2 e Th17, dependendo da doença envolvida. Partindo-se do pressuposto de que a infecção causada por Trypanosoma cruzi normalmente induz miocardite refletida pela migração intensa de linfócitos Th1 para o tecido cardíaco, nós analisamos o papel regulador da IL-27 nesta condição inflamatória. Nós inicialmente verificamos que a IL-27 foi prontamente induzida in vitro em células infectadas com T. cruzi. Para gerar miocardite intensa coordenada por linfócitos Th1, nós polarizamos linfócitos T naïves para o padrão Th1 na ausência de moléculas relacionadas ao perfil Th17 (camundongos IL-17R-/-, IL-23-/- e IL-6-/-). Como esperado, a inflamação cardíaca intensa e o dano tecidual foram observados na ausência das moléculas do padrão Th17, o que contribuiu para a morte prematura dos animais IL-17R-/-, IL-23-/- e IL-6-/-, precisa e notoriamente pela indução da migração excessiva de linfócitos Th1 para o tecido cardíaco via CXCL-9 e CXCL-10. Para explorar os mecanismos pelos quais a IL-27 controla a miocardite induzida pelo T. cruzi, nós encontramos um recrutamento substancial de macrófagos produtores de IL-27 para o tecido cardíaco, o qual foi mediado pelas quimiocinas CCL3 e CCL4 na ausência de moléculas do padrão Th17. Para determinar quais os receptores necessários para a produção de IL-27, nós observamos que macrófagos derivados da medula óssea de camundongos deficientes de TLR4-/-, TLR9-/- e NLRP3-/- aboliram completamente a produção desta citocina após a infecção in vitro com T. cruzi, enquanto o receptor TLR2 foi dispensável. Nós também verificamos que macrófagos produtores de IL-27 suprimiram linfócitos Th1 através da indução de células Tr1 produtoras de IL-10 após a infecção com T. cruzi. Em seguida, nós avaliamos se a IL-27 foi correlacionada com a proteção cardíaca durante a doença de Chagas. Nós observamos níveis séricos elevados de IL-27 tanto em pacientes com a forma clínica indeterminada ou cardíaca leve, enquanto pacientes com cardiomiopatia moderada ou grave produziram níveis reduzidos de IL-27. Neste estudo, nós descrevemos um novo mecanismo regulador desempenhado por macrófagos produtores de IL-27 no controle da miocardite induzida por T. cruzi. Macrófagos produtores de IL-27 podem suprimir processos inflamatórios desencadeados por linfócitos Th1, os principais vilões na doença de Chagas. / IL-27 is a heterodimeric cytokine produced by macrophages and dendritic cells known to induce IL-10-producing Tr1 cells and to regulate Th1, Th2, and Th17 lymphocytes, depending on the underlying disease. Because the infection caused by Trypanosoma cruzi normally induces myocarditis mirrored by an outstanding migration of Th1 cells to the heart tissue, we analyzed the regulatory role of IL-27 in this inflammatory condition. We firstly verified that IL-27 was promptly induced by in vitro T. cruzi-infected spleen cells. To generate a robust myocarditis coordinated by Th1 lymphocytes, we polarized lymphocytes to a Th1 pattern by infecting mice in the absence of Th17-related molecules (IL-17R-/-, IL-23-/-, and IL-6-/- mice). As expected, an impressive cardiac inflammation and damage was observed in the absence of Th17-related molecules, leading IL-17R-/-, IL-23-/-, and IL-6-/- mice to the premature death, precisely and notably by inducing an exuberant Th1 migration to the heart tissue via CXCL9 and CXCL10 chemokines. To explore the mechanisms by which IL-27 controls T. cruzi-induced myocarditis, we found a striking recruitment of IL-27-producing macrophages to the heart tissue mediated by increased levels of CCL3 and CCL4 chemokines in the absence of Th17-associated molecules. To gain further insights into the receptors required to IL-27 production, we observed that bone marrow-derived macrophages from TLR4-/-, TLR9-/-, and NLRP3-/- mice completely abolished IL-27 production after in vitro T. cruzi infection, while TLR2 was dispensable. We also verified that IL-27-producing macrophages supressed Th1 lymphocytes by inducing IL-10-producing Tr1 cells after T. cruzi infection. We next assessed whether IL-27 was correlated to cardiac protection during Chagas Disease. We observed augmented serum levels of IL-27 in either patients with indeterminate (asymptomatic) form or mild cardiac form, whereas patients with moderate or severe cardiomyopathy were poor producers of IL-27. Here, we described a novel regulatory mechanism developed by IL-27-producing macrophages in the control of T. cruzi-induced myocarditis. IL-27-producing macrophages can suppress inflammatory processes caused by Th1 lymphocytes, the bona fide culprits of Chagas Disease.

Diabetes tipo 1 (DM1) e uma doenca autoimune que se inicia devido a defeitos na tolerancia imunologica a auto-antigenos, resultando na destruicao autoimune das celulas pancreaticas em individuos geneticamente suscetiveis. Os receptores NOD-like (NLRs) sao receptores intracelulares responsaveis pelo reconhecimento de padroes moleculares associados a patogenos (PAMPs) e padroes moleculares associados ao dano (DAMPs). Estudos recentes tem demonstrado que os receptores NOD1 e NOD2 desempenham um importante papel na ativacao da imunidade inata contra patogenos e na regulacao da imunidade adaptativa, uma vez que sua ativacao leva a producao de citocinas relacionadas a diferenciacao de linfocitos T auxiliares produtores de IL-17 (Th17). Porem, a importancia desses receptores no DM1 ainda e incerto. Nesse sentido, investigamos o papel dos receptores NOD1 e NOD2 na patogenese do DM1, com enfoque na diferenciacao de linfocitos Treg/Th17/Th1 e na plasticidade desses subtipos celulares. Nossos resultados mostram que camundongos deficientes de NOD2, mas nao NOD1 ou RIP2, sao resistentes ao DM1, como comprovado por menor incidencia, hiperglicemia, diminuicao do infiltrado inflamatorio e normalizacao dos niveis de insulina quando comparado aos controles. Foi observado tambem que animais NOD2-/- tiveram uma reducao da populacao de linfocitos Th17, Tc17, Th1 e T citotoxicos nos linfonodos pancreaticos, o que correlaciona com a inibicao da producao de IL-23p19 e IFN- no pancreas. Em paralelo, foi evidenciado o aumento do numero de celulas T reguladoras, macrofagos do perfil M2 nos linfonodos pancreaticos e elevada producao de IL-10 no pancreas de animais NOD2-/-. Alem disso, foi observado que animais NOD2-/- apresentaram uma menor populacao de linfocitos T duplo-positivos (Foxp3+RORt+ e IL-17+IFN+). Posteriormente, foi detectado menor producao de IL- 1, IL-6, IL-23p19 e IL-12p40 por celulas dendriticas de animais deficientes de NOD2. De forma interessante, foi observada a translocacao de bacterias para os linfonodos pancreaticos de animais diabeticos. Adicionalmente, animais tratados com antibioticos tornaram-se resistentes ao DM1, o que nos fornece indicios da contribuicao da microbiota intestinal na inducao da doenca. Por fim, comprovamos alta expressao genica de NOD2 nos linfonodos pancreaticos e no pancreas na fase inicial (pre-diabetica) em outro modelo de DM1, utilizando camundongos NOD (nonobese diabetic mice). Portanto, nossos dados indicam que a ativacao do receptor NOD2 por componentes bacterianos da microbiota intestinal induz a producao de citocinas pro-inflamatorias com subsequente diferenciacao/conversao de linfocitos do perfil Th17/Th1 e progressao do DM1. Dessa forma, estes dados apontam o bloqueio do receptor NOD2 como uma potencial terapia imunomoduladora para o DM1 em humanos. / Type 1 diabetes is an autoimmune disease that precipitates due to defects in the self tolerance to auto- antigens, resulting in the autoimmune destruction of the pancreatic cells in genetically susceptible individuals. NOD-like (NLRs) receptors are intracellular receptors responsible for the recognition of pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs). Recent studies have shown a role of NOD1 and NOD2 receptors in the innate immune response against pathogens and in the adaptive immune response, since its activation leads to the generation of cytokines related to the differentiation of IL-17-producing T helper cells (Th17). However, the role of these receptors in T1D remains elusive. Therefore, we investigated the role of NOD1 and NOD2 receptors in the pathogenesis of T1D, focusing on the differentiation of Treg/Th1/Th17 lymphocytes and in the plasticity of these subtypes. Our data demonstrate that NOD2-/- mice, but not NOD1-/- or RIP2-/-, are resistant to T1D, as shown by the lower incidence, hyperglycemia, less insulitis and normal insulin production when compared to wild type mice. It was also observed that NOD2-/- mice have a reduction in the Th17, Tc17, Th1 and cytotoxic T lymphocyte population within the pancreatic lymph nodes (PLNs), which correlates with the inhibition of IL-23p19 and IFN production in the pancreas. In parallel, there was an increase in Treg cells, M2 macrophages in the PLNs and IL-10 production in the pancreatic tissue of NOD2-/- mice. Also, NOD2-/- mice presented a downregulation of Foxp3+RORt+ and IL-17+IFN+ double-positive T cells. Later, it was shown that IL-1, IL-6, IL-23p19 and IL-12p40 production was downregulated in mice deficient to the NOD2 receptor. Interestingly, we observed a bacterial translocation to the pancreatic lymph nodes in diabetic mice, what could be triggering NOD2 activation, thus contributing to T1D development. As expected, mice pre-treated with antibiotics failed to become diabetic, suggesting a possible role of the gut microbiota in the development of the disease. Lastly, we observed a higher relative expression of NOD2 in the PLNs and pancreas of pre-diabetic mice, using another mouse model of the disease, the nonobese diabetic (NOD) mouse. Collectively, our data suggest that components from the gut microbiota are capable of translocating to the PLNs, thus triggering the activation of NOD2, which in turn induces the production of proinflammatory cytokines related to the differentiation of Th1/Th17 cells, thus contributing to T1D development in a mouse model of the disease. Therefore, the blockade of NOD2 appears as an interesting therapeutical target in the treatment of type 1 diabetes in humans.

L’asthme est une maladie multifactorielle hétérogène qui engendre une inflammation pulmonaire associée à une variété de manifestations cliniques, dont des difficultés respiratoires graves. Globalement, l’asthme touche environ une personne sur 6 et présente actuellement un sérieux problème de santé publique. Bien que de nombreux traitements soient disponibles pour soulager les symptômes de la maladie, aucun traitement curatif n’est actuellement disponible. La compréhension des mécanismes qui régissent l’état inflammatoire au cours de la maladie est primordiale à la découverte de nouvelles cibles thérapeutiques efficaces. Les cellules dendritiques captent les allergènes dans les poumons et migrent vers les ganglions drainants pour les présenter aux cellules T et engendrer la réponse inflammatoire pathogénique chez les asthmatiques. Nous avons contribué à l’avancement des connaissances mécanistiques de l’asthme en identifiant chez la souris la sous-population de cellules dendritiques responsable de l’initiation et du maintien de la réponse inflammatoire locale et systémique associée à l’asthme. En effet, nous avons démontré que le SIRPα, récepteur extracellulaire impliqué dans la régulation de la réponse immune, est sélectivement exprimé à la surface des cellules dendritiques immunogéniques. L’interruption de la liaison entre le SIRPα et son ligand, le CD47, interfère avec la migration des cellules dendritiques SIRPα+ et renverse la réponse inflammatoire allergique. Ce mécanisme constitue une avenue thérapeutique prometteuse. D’ailleurs, les molécules de fusion CD47-Fc et SIRPα-Fc se sont avérées efficaces pour inhiber l’asthme allergique dans le modèle murin. Nous avons également démontré l’implication des cellules dendritiques SIRPα dans un modèle d’inflammation pulmonaire sévère. L’administration répétée de ces cellules, localement par la voie intra-trachéale et systémiquement par la voie intra-veineuse, mène au développement d’une réponse inflammatoire mixte, de type Th2-Th17, similaire à celle observée chez les patients atteints d’asthme sévère. La présence de cellules T exprimant à la fois l’IL-17, l’IL-4, l’IL-13 et le GATA3 a été mise en évidence pour la première fois in vitro et in vivo dans les poumons et les ganglions médiastinaux grâce à ce modèle. Nos expériences suggèrent que ces cellules Th2-Th17 exploitent la plasticité des cellules T et sont générées à partir de la conversion de cellules Th17 qui acquièrent un phénotype Th2, et non l’inverse. Ces résultats approfondissent la compréhension des mécanismes impliqués dans l’initiation et le maintien de l’asthme allergique et non allergique, en plus d’ouvrir la voie à l’élaboration d’un traitement spécifique pour les patients asthmatiques, particulièrement ceux pour qui aucun traitement efficace n’est actuellement disponible. / Asthma is a heterogeneous multifactorial disease resulting in airway inflammation associated with a variety of clinical manifestations, which include severe breathing difficulties. Asthma affects approximately one out of six people and is currently a serious public health problem. As of now, many treatments are available to relieve the symptoms of the disease, but no definitive cure is available. Understanding the mechanisms that regulate the inflammatory condition during the disease is essential to the discovery of effective new therapeutic targets. Dendritic cells capture allergens in the lungs, migrate to the draining lymph nodes where they activate cognate T cells, which cause the pathogenic inflammatory response. My work help defined and deepened the mechanistic understanding of asthma by identifying the subpopulation of dendritic cells responsible for the initiation and maintenance of local and systemic inflammatory response. We demonstrated that SIRPα is selectively expressed on the surface of immunogenic dendritic cells. Indeed, the interruption of the ligation between SIRPα and its ligand, CD47, interferes with the migration of SIRPα+ dendritic cells and reverses the allergic inflammatory response. This mechanism is a promising new therapeutic avenue. Moreover, we showed that the soluble fusion molecules CD47-Fc and SIRPα-Fc are potent inhibitors of the allergic asthma in a mouse model. In addition, we demonstrated the involvement of SIRPα+ dendritic cells in a model of severe airway inflammation induced upon local and systemic repeated administration of those cells. Either treatment led to the development of a mixed Th2-Th17 inflammation, a phenotype recently described in patients with severe asthma. This model allowed us to show the presence of T cells expressing at once IL-17, IL-4, IL-13 and GATA3 in vitro and in vivo in the lungs and in the mediastinal lymph nodes. Our results suggest that these Th2-Th17 cells are generated from the conversion of Th17 cells acquiring a Th2 phenotype, and not the other way around, a hallmark of Th17 cells plasticity. These results deepen the understanding of the mechanisms involved in the initiation and maintenance of allergic and non-allergic asthma. Besides, we open a way to the development of a specific treatment for asthmatic patients, particularly those for whom no effective treatment is currently available.