Diabetes mellitus (DM) is the 4th leading cause of mortality in Taiwan. Chronic persistent inflammation as demonstrated by higher proinflammatory mediators in blood has been correlated to cardiovascular complications of type 2 DM. The cellular and molecular mechanism of chronic inflammation in type 2 DM remains to be determined. This study was conducted to explore altered innate immunity in toll-like receptor (TLR) expression and signaling of monocytes from type 2 DM patients. Blood leukocytes from type 2 DM patients were counted and studied for TLR2 and TLR4 expression and signaling. Each experiment was run with 1 to 2 type 2 DM patients, simultaneously with 1 to 2 age-matched normal adults as controls. 31 type 2 DM patients and 37 normal age-matched controls completed the study. Results showed that blood monocytes from type 2 DM patients had a significantly higher TLR4 but not TLR2 expression. Using a TLR4 ligand, lipopolysaccharide (LPS), to trigger TNF£\ production, a significantly higher TNF£\ production by blood leukocytes from type 2 DM patients than age-matched controls was found. The higher TNF£\ production by blood leukocytes from type 2 DM patients was associated with down-regulation of suppressor of cytokine signaling 1and 3 (SOCS-1 and SOCS-3) expression. We have further postulated that increase of oxidative stress or decrease of IFN-£\ production in type 2 DM patients was related to the alteration of TLR-4 response. Correction of SOCS-1 expression by addition of antioxidant, superoxide dismutase (SOD), but not IFN-£\, significantly decreased TNF£\ production in blood leukocytes from type 2 DM patients. This study is the first in the literature to identify an alteration of TLR4 expression associated with depressed SOCS-1 expression in leukocytes of type 2 DM patients. Results from this study highlight a potential pathway to improve chronic inflammation of type 2 DM patients via modulation of TLR4 expression and SOCS-1 mRNA expression of leukocytes.

A paracoccidioidomicose é uma micose sistêmica de natureza profunda que afeta preferencialmente o tecido pulmonar podendo disseminar via linfo-hematogênica para outros órgãos e tecidos, sendo causada principalmente pelo Paracoccidioides brasiliensis, fungo que apresenta dimorfismo térmico. O sistema imune inato mediado por macrófagos é extremamente importante para o controle de infecções e está envolvido na indução e regulação da resposta imune/inflamatória. Estas células são capazes de reconhecer patógenos por meio de receptores de reconhecimento de padrões (PRRs), tais como receptores Toll-like (TLR). Além desses PRRs, recentemente, demonstrou-se a importância da via de sinalização Notch no sistema imune inato e na regulação da atividade dos macrófagos. Nossos dados demonstram que a cepa Pb18 do P. brasiliensis é capaz de ativar o receptor Notch1 em macrófagos J774. A ativação desse receptor concomitante com a ativação de TLR 4 (via LPS) induz a produção de IL-6, e apresenta elevada carga fúngica e menor fagocitose, o que favorece a patogenia. Ao utilizarmos um inibidor farmacológico da γ-secretase (DAPT) para inibir a ativação do receptor Notch1 em macrófagos, é possível observar diminuição da carga fúngica, diminuição de IL-6, aumento de TNF-α e aumento da fagocitose. Entretanto, a ausência do receptor TLR 4 em macrófagos derivados de medula óssea de camundongos TLR 4-/-, na presença de DAPT, percebe-se diminuição da capacidade fagocítica desses macrófagos e também diminuição da carga fúngica, evidenciando a relação entre TLR 4 e Notch1. Em adição, realizamos um tratamento em camundongos BALB/c com DAPT previamente à infecção com Pb18. Nossos resultados evidenciaram que animais com este tratamento apresentaram diminuição da carga fúngica dos pulmões, diminuição de IL-6, ativação de macrófagos e aumento de IgG, após 45 dias de infecção, indicando um perfil de cura desses animais. O mesmo tratamento foi realizado em camundongos BALB/c NUDE, seguido da infecção com Pb18. Nestes animais, verificamos que há maior produção de citocinas pró-inflamatórias no pulmão, aumento de células CD19+ e a carga fúngica dos animais tratados manteve-se similar ao dos animais não tratados, indicando que o perfil protetor observado em animais com DAPT é dependente da resposta das células T. Juntos, esses resultados evidenciam que o Pb18 é capaz de ativar o receptor Notch1 em macrófagos e utiliza a via de sinalização Notch-TLR 4 como um possível mecanismo de escape, podendo fornecer uma nova abordagem de estudo da imunidade envolvida na paracoccidioidomicose experimental. / Paracoccidioidomycosis is a systemic mycosis of deep nature that primarily affects the lung and can spread via lymphatic and hematogenous to other organs and tissues. It is mainly caused by Paracoccidioides brasiliensis fungus which exhibit thermal dimorphism. The innate immune system mediated by macrophages is extremely important for the control of infection and is involved in the induction and regulation of immune/inflammatory response. These cells are able to recognize pathogens through pattern recognition receptors (PRRs) such as Toll-like receptors (TLR). Beyond these PRRs, the importance of Notch signaling has recently been demonstrated in the innate immune system and the regulation of macrophage activity. Our data demonstrate that the Pb18 strain of P. brasiliensis is able to activate the Notch1 receptor in J774 macrophages. Activation of this receptor with also activation of TLR 4 (via LPS) induces IL-6 production, induces phagocytosis and decreases fungal burden, which favors the pathogenesis. By using a γ-secretase pharmacological inhibitor (DAPT) for inhibiting the activation of Notch1 receptor on macrophages, it is possible to observe decreased fungal burden, less production of IL-6, and increased TNF-α and phagocytosis. However, due to the absence of TLR 4 receptor in bone marrow derived macrophages from TLR 4-/- mice, these macrophages showed decreased phagocytic ability and also reduced fungal burden in the presence of DAPT, showing a relationship between TLR 4 and Notch1. In addition, we made a treatment with DAPT in BALB/c mice prior to infection with Pb18. And our results showed that DAPT-treated animals exhibited a decrease of fungal burden in the lungs, and a decrease of IL-6. Furthermore, we observed an increase of IgG after 45 days of infection, indicating probably a healing of these animals. Same treatment was made in BALB/c NUDE mice, followed by infection with Pb18. In these animals, we observed an increased production of proinflammatory cytokines in the lung and increased CD19+ cells, but fungal burden was similar in both group (treated and untreated), which indicates that treatment with DAPT is dependent on T cell response. Taken together, these results showed that Pb18 is able to activate the Notch 1 receptor on macrophages and uses the Notch-TLR 4 signaling pathway as a possible escape mechanism, and may provide a new immunity study approach in experimental paracoccidioidomycosis.

A paracoccidioidomicose é uma micose sistêmica de natureza profunda que afeta preferencialmente o tecido pulmonar podendo disseminar via linfo-hematogênica para outros órgãos e tecidos, sendo causada principalmente pelo Paracoccidioides brasiliensis, fungo que apresenta dimorfismo térmico. O sistema imune inato mediado por macrófagos é extremamente importante para o controle de infecções e está envolvido na indução e regulação da resposta imune/inflamatória. Estas células são capazes de reconhecer patógenos por meio de receptores de reconhecimento de padrões (PRRs), tais como receptores Toll-like (TLR). Além desses PRRs, recentemente, demonstrou-se a importância da via de sinalização Notch no sistema imune inato e na regulação da atividade dos macrófagos. Nossos dados demonstram que a cepa Pb18 do P. brasiliensis é capaz de ativar o receptor Notch1 em macrófagos J774. A ativação desse receptor concomitante com a ativação de TLR 4 (via LPS) induz a produção de IL-6, e apresenta elevada carga fúngica e menor fagocitose, o que favorece a patogenia. Ao utilizarmos um inibidor farmacológico da γ-secretase (DAPT) para inibir a ativação do receptor Notch1 em macrófagos, é possível observar diminuição da carga fúngica, diminuição de IL-6, aumento de TNF-α e aumento da fagocitose. Entretanto, a ausência do receptor TLR 4 em macrófagos derivados de medula óssea de camundongos TLR 4-/-, na presença de DAPT, percebe-se diminuição da capacidade fagocítica desses macrófagos e também diminuição da carga fúngica, evidenciando a relação entre TLR 4 e Notch1. Em adição, realizamos um tratamento em camundongos BALB/c com DAPT previamente à infecção com Pb18. Nossos resultados evidenciaram que animais com este tratamento apresentaram diminuição da carga fúngica dos pulmões, diminuição de IL-6, ativação de macrófagos e aumento de IgG, após 45 dias de infecção, indicando um perfil de cura desses animais. O mesmo tratamento foi realizado em camundongos BALB/c NUDE, seguido da infecção com Pb18. Nestes animais, verificamos que há maior produção de citocinas pró-inflamatórias no pulmão, aumento de células CD19+ e a carga fúngica dos animais tratados manteve-se similar ao dos animais não tratados, indicando que o perfil protetor observado em animais com DAPT é dependente da resposta das células T. Juntos, esses resultados evidenciam que o Pb18 é capaz de ativar o receptor Notch1 em macrófagos e utiliza a via de sinalização Notch-TLR 4 como um possível mecanismo de escape, podendo fornecer uma nova abordagem de estudo da imunidade envolvida na paracoccidioidomicose experimental. / Paracoccidioidomycosis is a systemic mycosis of deep nature that primarily affects the lung and can spread via lymphatic and hematogenous to other organs and tissues. It is mainly caused by Paracoccidioides brasiliensis fungus which exhibit thermal dimorphism. The innate immune system mediated by macrophages is extremely important for the control of infection and is involved in the induction and regulation of immune/inflammatory response. These cells are able to recognize pathogens through pattern recognition receptors (PRRs) such as Toll-like receptors (TLR). Beyond these PRRs, the importance of Notch signaling has recently been demonstrated in the innate immune system and the regulation of macrophage activity. Our data demonstrate that the Pb18 strain of P. brasiliensis is able to activate the Notch1 receptor in J774 macrophages. Activation of this receptor with also activation of TLR 4 (via LPS) induces IL-6 production, induces phagocytosis and decreases fungal burden, which favors the pathogenesis. By using a γ-secretase pharmacological inhibitor (DAPT) for inhibiting the activation of Notch1 receptor on macrophages, it is possible to observe decreased fungal burden, less production of IL-6, and increased TNF-α and phagocytosis. However, due to the absence of TLR 4 receptor in bone marrow derived macrophages from TLR 4-/- mice, these macrophages showed decreased phagocytic ability and also reduced fungal burden in the presence of DAPT, showing a relationship between TLR 4 and Notch1. In addition, we made a treatment with DAPT in BALB/c mice prior to infection with Pb18. And our results showed that DAPT-treated animals exhibited a decrease of fungal burden in the lungs, and a decrease of IL-6. Furthermore, we observed an increase of IgG after 45 days of infection, indicating probably a healing of these animals. Same treatment was made in BALB/c NUDE mice, followed by infection with Pb18. In these animals, we observed an increased production of proinflammatory cytokines in the lung and increased CD19+ cells, but fungal burden was similar in both group (treated and untreated), which indicates that treatment with DAPT is dependent on T cell response. Taken together, these results showed that Pb18 is able to activate the Notch 1 receptor on macrophages and uses the Notch-TLR 4 signaling pathway as a possible escape mechanism, and may provide a new immunity study approach in experimental paracoccidioidomycosis.

A esporotricose caracteriza-se como uma micose subcutânea causada por fungos dimórficos do gênero Sporothrix, capazes de acometer o homem e uma grande variedade de animais, dentre eles os felinos. A princípio, Sporothrix schenckii era a única espécie conhecida como responsável pela esporotricose. Após estudos genotípicos e fenotípicos de isolados ambientais, clínicos humanos e animais, verificou-se alta variabilidade entre os isolados e estabeleceu-se a existência de um Complexo Sporothrix. Dentro deste, a maior causadora de surtos epidêmicos, justificada por uma maior virulência e capacidade de evasão da resposta imune, é a espécie Sporothrix brasiliensis. Nesse sentido, dada a ausência de estudos direcionados a está espécie, objetivou-se avaliar a importância de receptores Toll like-2 (TLR-2) e Toll like-4 (TLR-4) na infecção por S. brasiliensis. Além disso, utilizando técnicas de proteômica, procurou-se elucidar proteínas diferencialmente expressas em S. brasiliensis quando comparado à espécie S. schenckii. Para avaliação da resposta imune utilizaram-se modelos in vitro e in vivo de infecção, e para a investigação das proteínas diferencialmente expressas, utilizou-se a técnica de proteômica Bottom-up. A investigação da resposta imune in vitro mostrou a dependência dos receptores TLR-2 e TLR-4 no desencadeamento da resposta imune. Os ensaios in vivo mostraram a importância desses receptores no controle da infecção e dependência dos mesmos na produção de citocinas, principalmente nos primeiros 14 dias de infecção. Na ausência do receptor TLR-2, houve a polarização de resposta Th17 na tentativa de controle da infecção. Quando avaliadas as diferenças entre as espécies S. brasiliensis e S. schenckii, em termos de proteínas expressas, verificou-se que S. brasiliensis expressa diferencialmente 60 proteínas. Dentre essas, 9 são relatadas na literatura, como importantes na virulência e escape imunológico dos principais fungos de importância médica. Os resultados encontrados no presente trabalho permitem concluir que reconhecimento de S. brasiliensis é dependente dos receptores TLR-2 e TLR-4. Estudos que investiguem a utilização de outras vias de sinalização como mecanismos compensatórios, bem como, o sinergismo desses receptores no contexto da infecção por S. brasiliensis são fundamentais na compreensão da fisiopatologia dessa doença. No que tange a caracterização proteica, estudos com mutantes para cada uma das proteínas descritas nesse trabalho devem ser avaliados. / Sporotrichosis is a subcutaneous mycosis caused by dimorphic fungi of the genus Sporothrix that affects humans and animals, predominantelly felines. Inicially, Sporothrix schenckii was the only specie associated to sporotrichosis. However, after genotypic and phenotypic studies of human and animal clinical isolates, a high variability among the isolates was found and was concluded the existence of a complex: the Sporothrix Complex. Inside the Sporothrix complex, the major cause of epidemic outbreaks, justified by a greater virulence and ability to evade the immune system, is Sporothrix brasiliensis. Concerning this, the absence of studies directed to this specific specie, the aim was to evaluate the importance of Toll like receptor-2 (TLR-2) and Toll like receptor-4 (TLR-4) during S. brasiliensis infection. In addition, was look using proteomics techniques, the proteins differentially expressed in S. brasiliensis when compared to S. schenckii. To evaluate the immune response, in vitro and in vivo tecniques were used, and for the investigation of differentially expressed proteins, the Bottom-up proteomics technique was used. The investigation of the in vitro immune response showed the dependence of TLR-2 and TLR-4 receptors on phagocytosis and the production of inflammatory mediators, such as cytokines and NO. In vivo assays showed the importance of these receptors to control the infection and their dependence on cytokine production during the first 14 days of infection. In the absence of the TLR-2 receptor, the Th17 response was polarized in an attempt to control the infection. Evaluating the differences between S. brasiliensis and S. schenckii, in terms of expressed proteins, it was verified that S. brasiliensis differentially expressed 60 proteins. Among these, 9 are reported in the literature, as important in the virulence and immune evasion among the most important medical fungi. The results found in the present study allow to conclude that S. brasiliensis recognition is dependent on TLR-2 and TLR-4 receptors. Studies investigating the use of other signaling pathways as compensatory mechanisms, as well as the synergism of these receptors in the context of S. brasiliensis infection, are fundamental to understand the pathophysiology of this disease. Regarding the protein characterization, studies with mutants for each of the proteins described in this work should be evaluated.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / nÃo hà / Paclitaxel (PCX) foi o 1 antineoplÃsico efetivo no tratamento de cÃnceres refratÃrios a quimioterapia convencional. Clinicamente, induz artralgias e mialgias de carÃter incapacitante que comprometem a qualidade de vida e limitam o tempo de tratamento antitumoral, acometendo atà 57% dos doentes. Apesar destas repercussÃes clÃnicas nada foi descrito atà o momento, visando elucidar o envolvimento de citocinas prÃ-inflamatÃrias, na gÃnese da atividade hipernociceptiva do PCX, jà que a droga compartilha com o LPS uma via de sinalizaÃÃo desencadeada por receptores Toll (TLR-4 e TLR-2) para geraÃÃo de genes que codificam TNF-&#945;. Dados da literatura sugerem que ocorra um âcross-talkâ entre esses dois membros da famÃlia Toll e, que agonistas diferentes de TLR-2 e TLR-4 sÃo capazes de induzir a ativaÃÃo de NF-&#945;B, AP1 e MAP kinase e a geraÃÃo de TNF-&#945;, citocina chave na cascata de liberaÃÃo de mediadores inflamatÃrios finais que atuam diretamente no nociceptor. Dados do laboratÃrio registraram que o zymosan (ZY) intrarticular em joelhos de ratos produz uma periartrite caracterÃstica da hipernocicepÃÃo no teste de incapacitaÃÃo articular (IA), e que PCX (8mg/kg) amplificou essa resposta quando se injetou  da dose do ZY. Tal amplificaÃÃo foi inibida com o prÃ-tratamento com inibidores de citocina e de prostanÃides. Objetivo. Investigar a participaÃÃo do TLR-4 e TNF-&#945; na gÃnese do efeito potencializador do PCX na artralgia experimental induzida por ZY. Material e MÃtodos. Ratos foram prÃ-tratados Sc com talidomida (TLD), pentoxifilina, dexametasona, indometacina ou celecoxib e estimulados com subdose de ZY (250&#956;g/animal; i-art). ApÃs a 1 medida do tempo de suspensÃo de pata (TSP) no teste de IA, os animais receberam PCX (8mg/kg; ip). Numa segunda etapa os ratos receberam durante trÃs dias consecutivos o prÃ-tratamento com atorvastatina (3, 10, 30mg/kg/dia; VO). Os seguintes parÃmetros foram avaliados: modulaÃÃo da hipernocicepÃÃo no teste de incapacitaÃÃo articular, dosagem de citocinas em lavado de joelho de ratos (TNF-&#945;, IL-1&#945;, Il-6, KC e CINC) e imunohistoquÃmica para TNF-&#945;, IL-1&#945; e TLR-4 no tecido sinovial. Resultados. Ficou demonstrado que PCX (8mg/kg) potencializa a artralgia experimental induzida por ZY em ratos avaliada pelo aumento significativo do TSP (p<0,001) na 4Âh de artrite em relaÃÃo ao controle no teste de IA. Tal efeito foi inibido de maneira significativa pelo prÃ-tratamento com TLD (45mg/kg) e essa inibiÃÃo foi associada à reduÃÃo dos nÃveis de TNF-&#945; produzido pelas cÃlulas do tecido sinovial no lavado articular e da marcaÃÃo imunohistoquÃmica para TNF-&#61537;. Da mesma forma a inibiÃÃo dessa resposta amplificadora do PCX foi ratificado pelo prÃ-tratamento com atorvastatina nas trÃs doses utilizadas no modelo, tambÃm sendo associado à diminuiÃÃo significativa dos nÃveis de TNF-&#61537; no lavado articular e visÃvel reduÃÃo na marcaÃÃo imunohistoquÃmica para TNF-&#61537;, IL-1&#61538; e TLR-4, nas trÃs doses utilizadas. ConclusÃes. PCX potencializa a hipernocicepÃÃo induzida por ZY por um mecanismo indireto sobre cÃlulas residentes da membrana sinovial que liberam TNF-&#61537; provavelmente pela ativaÃÃo da NF-&#61547;B via TLR-4/MD2, pois esse efeito potencializador foi inibido pela atorvastatina, um provÃvel antagonista de TLR-4. O TNF-&#61537; liberado age iniciando a cascata de mediadores envolvidos com a dor inflamatÃria, o que justifica em parte as artralgias dos pacientes em tratamento com PCX. / Paclitaxel (PCX) was the first effective antineoplastic medicine in the treatment of tumors that do not respond to conventional chemotherapy. Clinically, it induces incapacitating arthralgias and myalgias that interfere with the patient quality of life and limit the duration of the treatment. This is observed in up to 57% of the patients using the drug. Despite these clinical manifestations, nothing has been published that could explain the involvement of pro-inflammatory cytokines in the triggering of the hypernociceptive effect of PCX, even though it is known that the drug shares with LPS a signaling pathway started by Toll-like receptors (TLR-2 and TLR-4) that activates genes coding for TNF-&#945;. The literature suggests that there is a crosstalk between these two members of the Toll family and that different agonists of TLR-2 and TLR-4 are able to induce the activation of NF-kB, AP1 and MAP kinase in the generation of TNF-&#945;, a key cytokines in the cascade liberating the final inflammatory mediators that act directly on the nociceptor. Data obtained in laboratory show that the injection of zymozan into rat knee-joints produces a periarthritis characteristic of the hypernociception seen in the knee joint incapacitation test and that PCX (8mg/kg) amplified the response when  of the zymozan (ZY) doses was injected. The amplification was inhibited when animals were pre-treated with inhibitors of cytokines and prostanoids. Objective: To study the role of TNF-&#945; and TLR-4 on the initiation of the potentiating effect of PCX on the experimental arthralgia induced by ZY. Material and Methods: Rats were pre-treated Sc with thalidomide, pentoxifiline, dexametazone, indometacin and celecoxib and then stimulated with an intra-articular subdoses of ZY (250&#956;g/animal). After the first measurement of the paw elevation time in the knee joint incapacitation test, the animals were treated with PCX (8mg/kg ip). On a second trial, rats were treated for three consecutive days with atorvastatin (3, 10, 30mg/kg/day; VO). The following parameters were evaluated: modulation of the effect on the knee joint incapacitation test (JIT), amount of cytokines in the ratâs knee lavage (TNF-&#945;, IL-1 &#946;, IL-6, KC and CINC) and immunohistochemistry for TNF-&#945;, IL-1&#946; and TLR-4 on synovial tissue. Results: It was shown that PCX (8mg/kg) potentiates the experimental arthralgia induced by ZY in the rats as evaluated by the significant increase in paw elevation time (p<0.001) at the 4th h of arthritis in relations to controls. Such effect was significantly inhibited by pre-treatment with thalidomide (45mg/kg) and the inhibition was associated with a decrease in the amount of TNF-&#945; produced by synovial tissue cells and detected in the joint lavage and in the immunohistochemistry for TNF-&#945;. Likewise the inhibition of the amplifying response to PCX was seen with pre-treatment with atorvastatin at the three doses used in the experiment, which was also associated with a lower TNF-&#945; in the joint lavage and perceptible decrease in the immunohistochemistry for TNF-&#945;, IL-1&#946; and TLR-4. Conclusions: PCX potentiates the hypernociception induced by ZY through an indirect effect on synovial membrane resident cells that release TNF-&#945; probably through activation of the NF-kB pathway by TLR-4/MD2, since the potentiating effect was inhibited by atorvastatin, a TLR-4 antagonist. Released TNF-&#945; act starting the cascade of mediators involved in the inflammatory pain and this partially explains the arthralgia in patients treated with PCX.

A esporotricose caracteriza-se como uma micose subcutânea causada por fungos dimórficos do gênero Sporothrix, capazes de acometer o homem e uma grande variedade de animais, dentre eles os felinos. A princípio, Sporothrix schenckii era a única espécie conhecida como responsável pela esporotricose. Após estudos genotípicos e fenotípicos de isolados ambientais, clínicos humanos e animais, verificou-se alta variabilidade entre os isolados e estabeleceu-se a existência de um Complexo Sporothrix. Dentro deste, a maior causadora de surtos epidêmicos, justificada por uma maior virulência e capacidade de evasão da resposta imune, é a espécie Sporothrix brasiliensis. Nesse sentido, dada a ausência de estudos direcionados a está espécie, objetivou-se avaliar a importância de receptores Toll like-2 (TLR-2) e Toll like-4 (TLR-4) na infecção por S. brasiliensis. Além disso, utilizando técnicas de proteômica, procurou-se elucidar proteínas diferencialmente expressas em S. brasiliensis quando comparado à espécie S. schenckii. Para avaliação da resposta imune utilizaram-se modelos in vitro e in vivo de infecção, e para a investigação das proteínas diferencialmente expressas, utilizou-se a técnica de proteômica Bottom-up. A investigação da resposta imune in vitro mostrou a dependência dos receptores TLR-2 e TLR-4 no desencadeamento da resposta imune. Os ensaios in vivo mostraram a importância desses receptores no controle da infecção e dependência dos mesmos na produção de citocinas, principalmente nos primeiros 14 dias de infecção. Na ausência do receptor TLR-2, houve a polarização de resposta Th17 na tentativa de controle da infecção. Quando avaliadas as diferenças entre as espécies S. brasiliensis e S. schenckii, em termos de proteínas expressas, verificou-se que S. brasiliensis expressa diferencialmente 60 proteínas. Dentre essas, 9 são relatadas na literatura, como importantes na virulência e escape imunológico dos principais fungos de importância médica. Os resultados encontrados no presente trabalho permitem concluir que reconhecimento de S. brasiliensis é dependente dos receptores TLR-2 e TLR-4. Estudos que investiguem a utilização de outras vias de sinalização como mecanismos compensatórios, bem como, o sinergismo desses receptores no contexto da infecção por S. brasiliensis são fundamentais na compreensão da fisiopatologia dessa doença. No que tange a caracterização proteica, estudos com mutantes para cada uma das proteínas descritas nesse trabalho devem ser avaliados. / Sporotrichosis is a subcutaneous mycosis caused by dimorphic fungi of the genus Sporothrix that affects humans and animals, predominantelly felines. Inicially, Sporothrix schenckii was the only specie associated to sporotrichosis. However, after genotypic and phenotypic studies of human and animal clinical isolates, a high variability among the isolates was found and was concluded the existence of a complex: the Sporothrix Complex. Inside the Sporothrix complex, the major cause of epidemic outbreaks, justified by a greater virulence and ability to evade the immune system, is Sporothrix brasiliensis. Concerning this, the absence of studies directed to this specific specie, the aim was to evaluate the importance of Toll like receptor-2 (TLR-2) and Toll like receptor-4 (TLR-4) during S. brasiliensis infection. In addition, was look using proteomics techniques, the proteins differentially expressed in S. brasiliensis when compared to S. schenckii. To evaluate the immune response, in vitro and in vivo tecniques were used, and for the investigation of differentially expressed proteins, the Bottom-up proteomics technique was used. The investigation of the in vitro immune response showed the dependence of TLR-2 and TLR-4 receptors on phagocytosis and the production of inflammatory mediators, such as cytokines and NO. In vivo assays showed the importance of these receptors to control the infection and their dependence on cytokine production during the first 14 days of infection. In the absence of the TLR-2 receptor, the Th17 response was polarized in an attempt to control the infection. Evaluating the differences between S. brasiliensis and S. schenckii, in terms of expressed proteins, it was verified that S. brasiliensis differentially expressed 60 proteins. Among these, 9 are reported in the literature, as important in the virulence and immune evasion among the most important medical fungi. The results found in the present study allow to conclude that S. brasiliensis recognition is dependent on TLR-2 and TLR-4 receptors. Studies investigating the use of other signaling pathways as compensatory mechanisms, as well as the synergism of these receptors in the context of S. brasiliensis infection, are fundamental to understand the pathophysiology of this disease. Regarding the protein characterization, studies with mutants for each of the proteins described in this work should be evaluated.

Aim: The aim of this study is to determine the frequency of single nucleotide polymorphisms (SNPs) in various pattern recognition receptor (PRR) genes, including Toll like receptors (TLR) -2, -4, -9, and CD14 in chronic (CP), localized (LAP) and generalized aggressive (GAP) periodontitis and periodontally healthy (NP) patients in an African American population. Methods: A total of 205 subjects were involved in the study. The LAP group consists of 25 subjects, the GAP group 50 subjects, the CP group 73 subjects and the NP group 57subjects. Genotyping was performed in TLR2 (G2408A), TLR4 (A896G),TLR9 (T1486C) and CD14 (C260T) genes by TaqMan® allelic discrimination using Assay-by-DesignSM SNP Genotyping Assays (Applied Biosystems). Accuracy of genotyping was confirmed by known DNA samples of each genotype and by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analyses on selected samples. Fisher’s exact test and chi-square analyses were performed to compare genotype and allele frequencies. Within disease groups, we investigated whether SNPs were related to disease severity by step-wise logistic regression adjusted for age, gender, and smoking status. Results: There was a significant difference in the distribution of specific TLR9 (T1486C) genotypes between diseased-groups versus reference group. Expression of TT genotype was more prevelant in periodontally-diseased individuals compared to periodontally-healthy subjects (p<0.0001) whereas individuals expressing C allele of the TLR9 SNP (CC&CT) were more frequently found in healthy group after adjusting for age, gender, and smoking status (p<0.0001) There was no statistically significant difference in the distribution of genotypes between groups for any other TLRs or CD 14 polymorphism. Conclusion: Based on findings of this study, homozygocity for the T allele of TLR 9 polymorphism was related to the periodontal disease susceptibility in African Americans. Additionally, presence of C allele at TLR-9 appeared to confer resistance to periodontal destruction. Our results showed that specific SNPs in TLR-2, -4 and CD 14 genes are not related to periodontitis in African Americans. However, low copy number of certain alleles warrants further investigations with increased sample size to explore the role of SNPs in periodontal disease. This study was supported by the Alexander Fellowship.

Toll-like receptor (TLR)-4 is a pattern recognition receptor (PRR) that recognises the pathogen-associated molecular pattern (PAMP) lipopolysaccharide (LPS) produced by Gram-negative bacteria. LPS binds to Myeloid differentiation 2 (MD-2)/TLR-4 heterodimers, driving their dimerisation and inducing a conformational change of the intracellular TLR-4 toll/interleukin-1 receptor (TIR) domains. The adaptor protein Myeloid differentiation primary response gene 88 (MyD88)-adaptor-like (Mal)/TIR domain-containing adaptor protein (TIRAP) then binds to the TIR domains of TLR-4 and acts as a bridge for MyD88 which goes on to form the myddosome, a large protein complex of six to eight MyD88 molecules and four Interleukin-1 receptor- associated kinase (IRAK) 4 and four IRAK1/2 molecules. This triggers a signalling cascade which results in nuclear factor (NF)-κB transcription factor activation and production of pro-inflammatory effector molecules such as the cytokine Tumour Necrosis Factor (TNF)-α. Upon activation TLR-4 is also endocytosed where it interacts with a second set of adaptor proteins TIR-domain-containing adaptor- inducing interferon (IFN)-β (TRIF)-related adaptor molecule (TRAM) and TRIF to initiate the type I IFN response. How TLR-4 dimerisation results in the formation of the oligomeric myddosome is not fully understood, but it is possible that the stoichiometry of Mal/TIRAP may be important in the formation of this protein complex. The aim of my thesis was to determine the stoichiometry of Mal/TIRAP at the plasma membrane of immortalised bone marrow derived macrophages (iBMDMs) and whether this stoichiometry changes upon stimulation with different TLR-4 ligands. To investigate Mal/TIRAP stoichiometry I first developed a viral transduction experimental cell model to visualise fluorescently labelled Mal/TIRAP. Mal/TIRAP-/- iBMDMs were lentivirally transduced with a Mal/TIRAPHALO construct. The halotag was fluorescently labelled then the cells were stimulated with TLR-4 ligands, such as LPS, fixed at different time points, then imaged. Total internal reflection fluorescence (TIRF) microscopy was used to image the plasma membrane and photobleaching experiments performed to determine Mal/TIRAP stoichiometry. I developed a computer-based analysis pipeline to analyse the resulting photobleaching data. Under resting conditions, Mal/TIRAP is present at the plasma membrane in clusters of approximately ten Mal/TIRAP molecules per cluster. After five minutes of stimulation with 10 ng/ml LPS Mal/TIRAP redistributes into cluster sizes of approximately six, twelve and much larger. After ten and fifteen minutes stimulation with 10 ng/ml LPS the clusters return to the resting size of approximately ten Mal/TIRAP molecules per cluster with a few much larger clusters remaining present. This confirms the rapid time frame within which TLR-4 signalling occurs at the plasma membrane and is consistent with myddosome stoichiometry of six MyD88 molecules or proposed super myddosomes of twelve MyD88 molecules. The computer-based analysis pipeline developed can be used to analyse any protein of interest at the plasma membrane. Protein ligands have also been found to activate TLR-4; for example allergens, such as Fel d 1 and Der p 2, as well as endogenous damage associated molecular patterns (DAMPs), such as extracellular matrix (ECM) proteins, for example fragments of fibronectin and tenascin-C. The mechanism by which these proteins interact with TLR-4 and induce signalling is unclear. Proteins from the ECM (fragments FNIII1c, FNIII13-14, FNIII9-E and FNIII9-E-14 from fibronectin and the fibrinogen-like globe (FBG) domain of tenascin-C) were tested using a transient transfection assay in HEK293 cells and shown to activate TLR-4. In conclusion, I have developed new tools and methodology to investigate how TLR-4 signals in response to LPS and DAMPs in living cells. Whether DAMP- activated TLR-4 forms similar signalling complexes to those induced by LPS will form part of a future study.

Lipopolissacarídeos (LPS), pode tanto proteger quanto exacerbar o desenvolvimento da asma. LPS inicia a ativação da resposta imune via ligação da molécula Toll-like receptor 4 (TLR4) que sinaliza por duas vias distintas, as moléculas adaptadoras MyD88 e TRIF. LPS é um adjuvante que induz resposta do tipo Th1, enquanto que o hidróxido de alumínio (Alum) desperta respostas Th2, porém, a mistura de ambos adjuvantes na indução da resposta alérgica pulmonar ainda não foi investigada. No presente estudo, nós determinamos o efeito de dois agonistas de TLR4, um natural (LPS) e outro sintético (ER-803022) adsorvidos ao Alum sobre o desenvolvimento de doença alérgica pulmonar. Os animais foram sensibilizados pela via subcutânea com os antígenos, Ovoalbumina (OVA) ou Toxóide Tetânico (TT) na presença ou ausência de agonistas de TLR4 co-adsorvidos ao Alum e desafiados com os respectivos antígenos pela via intranasal. Nossos resultados mostraram que a sensibilização com OVA ou TT e LPS coadsorvidos ao Alum, impede o estabelecimento da resposta alérgica mediada por linfócitos Th2, tais como, influxo de eosinófilos, produção de citocinas do tipo 2, hiperreatividade brônquica, secreção de muco, e produção de IgE ou IgG1 anafilática. Apesar dos níveis de IgG2a, isotipo associado com as respostas Th1 estarem aumentados, análise da histopatologia pulmonar não revelou um desvio para o padrão Th1 de inflamação. Verificamos que a presença das moléculas TLR4, MyD88, IL-12/IFN-g mas não TRIF foram necessários para LPS exercer seu efeito inibitório. O agonista sintético de TLR4, menos tóxico que LPS, também protegeu contra o desenvolvimento de inflamação alérgica pulmonar. Em conclusão, nosso trabalho esclarece o efeito da sinalização do TLR4 na sensibilização alérgica e indica que agonista sintético de TLR4 com baixa toxicidade, pode ser utilizado para modular a capacidade adjuvante do Alum e conseqüentemente diminuir a indução de alergias. / Epidemiological and experimental data suggest that bacterial lipopolysaccharides (LPS) can either protect from or exacerbate allergic asthma. LPS triggers immune responses through Toll-like receptor (TLR) 4 that in turn activates two major signaling pathways via either MyD88 or TRIF adaptor proteins. LPS is a pro-Th1 adjuvant while aluminum hydroxide (Alum) is a strong Th2 adjuvant, but the effect of mixing both adjuvants on development of lung allergy has not been investigated. We determined whether natural (LPS) or synthetic (ER-803022) TLR4 agonists adsorbed onto alum adjuvant affect allergen sensitization and development of airway allergic disease. To dissect LPS-induced molecular pathways we used TLR4, MyD88, TRIF, or IL-12/IFN-g deficient mice. Mice were sensitized subcutaneously to allergens such as ovalbumin (OVA) or tetanus toxoid (TT) with or without TLR4 agonists coadsorbed onto Alum and challenged twice via intranasal route with the same allergens. The development of type 2 immunity was evaluated 24 h after last allergen challenge. We found that sensitization with OVA or TT plus LPS co-adsorbed onto Alum impaired allergeninduced Th2-mediated responses such as airway eosinophilia, type 2 cytokines secretion, airway hyperreactivity, mucus hyper production and serum levels of IgE or IgG1 anaphylactic antibodies. Although the levels of IgG2a, a Th1 affiliated isotype increased, investigation into the lung-specific effects revealed that LPS did not induce a Th1 pattern of inflammation. LPS impaired the development of Th2 immunity, signaling via TLR4 and MyD88 molecules via the IL-12/IFN-g axis, but not through TRIF pathway. Moreover, the synthetic TLR4 agonists that proved to have a less systemic inflammatory response than LPS also protected against allergic asthma development. TLR4 agonists co-adsorbed with allergen onto Alum down modulate Th2 immunity and prevent the development of polarized T cell-mediated airway inflammation. Thus, our work clarifies the effect of TLR4 signaling in allergic sensitization and indicates that TLR4 agonists with low toxicity might be useful for down regulating the pro-Th2 adjuvant activity of alum and consequently decrease the induction of allergy.

Lipopolissacarídeos (LPS), pode tanto proteger quanto exacerbar o desenvolvimento da asma. LPS inicia a ativação da resposta imune via ligação da molécula Toll-like receptor 4 (TLR4) que sinaliza por duas vias distintas, as moléculas adaptadoras MyD88 e TRIF. LPS é um adjuvante que induz resposta do tipo Th1, enquanto que o hidróxido de alumínio (Alum) desperta respostas Th2, porém, a mistura de ambos adjuvantes na indução da resposta alérgica pulmonar ainda não foi investigada. No presente estudo, nós determinamos o efeito de dois agonistas de TLR4, um natural (LPS) e outro sintético (ER-803022) adsorvidos ao Alum sobre o desenvolvimento de doença alérgica pulmonar. Os animais foram sensibilizados pela via subcutânea com os antígenos, Ovoalbumina (OVA) ou Toxóide Tetânico (TT) na presença ou ausência de agonistas de TLR4 co-adsorvidos ao Alum e desafiados com os respectivos antígenos pela via intranasal. Nossos resultados mostraram que a sensibilização com OVA ou TT e LPS coadsorvidos ao Alum, impede o estabelecimento da resposta alérgica mediada por linfócitos Th2, tais como, influxo de eosinófilos, produção de citocinas do tipo 2, hiperreatividade brônquica, secreção de muco, e produção de IgE ou IgG1 anafilática. Apesar dos níveis de IgG2a, isotipo associado com as respostas Th1 estarem aumentados, análise da histopatologia pulmonar não revelou um desvio para o padrão Th1 de inflamação. Verificamos que a presença das moléculas TLR4, MyD88, IL-12/IFN-g mas não TRIF foram necessários para LPS exercer seu efeito inibitório. O agonista sintético de TLR4, menos tóxico que LPS, também protegeu contra o desenvolvimento de inflamação alérgica pulmonar. Em conclusão, nosso trabalho esclarece o efeito da sinalização do TLR4 na sensibilização alérgica e indica que agonista sintético de TLR4 com baixa toxicidade, pode ser utilizado para modular a capacidade adjuvante do Alum e conseqüentemente diminuir a indução de alergias. / Epidemiological and experimental data suggest that bacterial lipopolysaccharides (LPS) can either protect from or exacerbate allergic asthma. LPS triggers immune responses through Toll-like receptor (TLR) 4 that in turn activates two major signaling pathways via either MyD88 or TRIF adaptor proteins. LPS is a pro-Th1 adjuvant while aluminum hydroxide (Alum) is a strong Th2 adjuvant, but the effect of mixing both adjuvants on development of lung allergy has not been investigated. We determined whether natural (LPS) or synthetic (ER-803022) TLR4 agonists adsorbed onto alum adjuvant affect allergen sensitization and development of airway allergic disease. To dissect LPS-induced molecular pathways we used TLR4, MyD88, TRIF, or IL-12/IFN-g deficient mice. Mice were sensitized subcutaneously to allergens such as ovalbumin (OVA) or tetanus toxoid (TT) with or without TLR4 agonists coadsorbed onto Alum and challenged twice via intranasal route with the same allergens. The development of type 2 immunity was evaluated 24 h after last allergen challenge. We found that sensitization with OVA or TT plus LPS co-adsorbed onto Alum impaired allergeninduced Th2-mediated responses such as airway eosinophilia, type 2 cytokines secretion, airway hyperreactivity, mucus hyper production and serum levels of IgE or IgG1 anaphylactic antibodies. Although the levels of IgG2a, a Th1 affiliated isotype increased, investigation into the lung-specific effects revealed that LPS did not induce a Th1 pattern of inflammation. LPS impaired the development of Th2 immunity, signaling via TLR4 and MyD88 molecules via the IL-12/IFN-g axis, but not through TRIF pathway. Moreover, the synthetic TLR4 agonists that proved to have a less systemic inflammatory response than LPS also protected against allergic asthma development. TLR4 agonists co-adsorbed with allergen onto Alum down modulate Th2 immunity and prevent the development of polarized T cell-mediated airway inflammation. Thus, our work clarifies the effect of TLR4 signaling in allergic sensitization and indicates that TLR4 agonists with low toxicity might be useful for down regulating the pro-Th2 adjuvant activity of alum and consequently decrease the induction of allergy.