A molécula adaptadora myeloid differentiation primary response gene 88 (MyD88) está envolvida na ativação de receptores Toll-like (TLRs), os quais são responsáveis pelo reconhecimento precoce pelas células do hospedeiro de patógenos invasores e pelo desencadeamento da resposta imunológica. O objetivo do presente estudo foi caracterizar o desenvolvimento e a progressão de lesões periapicais induzidas experimentalmente em dentes de camundongos knockout (KO) para a molécula MyD88 (MyD88 KO), comparados a animais wild-type (WT). Lesões periapicais foram induzidas nos primeiros molares inferiores de 30 camundongos WT e de 30 camundongos MyD88 KO. Decorridos 7, 21 e 42 dias, os animais foram submetidos à eutanásia em câmara de CO2 e as mandíbulas foram removidas e submetidas ao processamento histotécnico. A seguir, cortes representativos foram corados com hematoxilina e eosina (HE), para descrição das características do canal radicular e das regiões apical e periapical e para contagem do número de células inflamatórias (neutrófilos), em microscopia de luz, e para mensuração da área das lesões periapicais, em microscopia de fluorescência. Espécimes sequenciais foram analisados por meio de: histoenzimologia para a atividade da TRAP, para contagem de osteoclastos; coloração de Brown & Brenn, para localização de bactérias; e imunohistoquímica, para identificação de marcadores da osteoclastogênese (RANK, RANKL e OPG). Os dados foram submetidos à análise estatística por meio dos testes não-paramétricos de Mann-Whitney, Kruskal-Wallis e pós-teste de Dunn, utilizando o programa SPSS (Statistical Package for the Social Sciences) versão 17.O, com nível de significância de 5%. As demais análises foram expressas de maneira qualitativa. Com relação à extensão das lesões periapicais, o grupo MyD88 KO apresentou valores significantemente maiores do que o grupo WT nos períodos de 7 (p=0,001) e 21 dias (p=0,05), sendo que após 42 dias foi observada tendência de maiores valores, porém sem diferença significante (p=0,09). Foi observada maior quantidade de neutrófilos no grupo MyD88 KO, em comparação aos animais WT (p=0,01 em 7 dias; p=0,004 em 21 dias; e p<0,001 em 42 dias). Por outro lado, com relação à quantidade de osteoclastos, não foi observada diferença significante entre ambos os grupos, em todos os períodos experimentais (p=0,884 em 7 dias; p=0,506 em 21 dias; e p=0,211 em 42 dias). A análise microscópica descritiva do grupo MyD88 KO revelou um infiltrado inflamatório mais intenso, com presença abundante de células porlimorfonucleadas e mononucleadas e com grande destruição tecidual, após 7, 21 e 42 dias. A coloração de Brown e Brenn evidenciou uma maior disseminação bacteriana, inclusive nos tecidos periapicais, no grupo MyD88 KO, quando comparado aos animais WT. Com relação à imunohistoquímica, foram observadas marcações para RANK, RANKL e OPG de forma semelhante entre os dois grupos de animais. Com base nas metodologias e nos resultados obtidos no presente estudo pode-se concluir que na ausência da MyD88 os animais apresentaram lesões periapicais mais extensas, com um infiltrado inflamatório severo e com número significantemente maior de neutrófilos, quando comparados aos animais WT, sugerindo o importante papel desta molécula na resposta imune e inflamatória no combate à infecção de origem endodôntica. / The adaptor molecule myeloid differentiation primary response gene 88 (MyD88) is involved in the activation of toll-like receptors (TLRs), which are responsible for the early recognition by the host cells of invading pathogens and for triggering the immune response. The aim of the present study was to characterize the formation and progression of experimentally induced periapical lesions in teeth of MyD88 knockout (MyD88 KO) mice compared with wildtype (WT) mice. Periapical lesions were induced in the mandibular first molars of 30 WT and 30 MyD88 KO mice. After 7, 21 and 42 days, the animals were euthanized in a CO2 chamber and the mandibles were removed and subjected to histotechnical processing. Representative histological sections were stained with hematoxylin and eosin (HE) for description of the features of the root canal and the apical and periapical regions, and for counting of inflammatory cells (neutrophils) under conventional light microscopy and for determination of the size of the periapical lesions under fluorescence microscopy. Sequential specimens were evaluated by: TRAP histoenzymology, for osteoclast counting; Brown & Brenn staining, for localization of bacteria; and immunohistochemistry for identification of osteoclastogenesis markers (RANK, RANKL, OPG). Data were subjected to statistical analysis by the nonparametric Mann-Whitney and Kruskal-Wallis tests and the Dunn post-test, using the SPSS (Statistical Package for the Social Sciences) software, version 17.0. The significance level was set at 5%. The other analyzes were displayed qualitatively. Regarding the periapical lesion size, the MyD88 KO group presented significantly higher values than the WT group in the periods of 7 (p=0.001) and 21 days (p=0.05). Tendency for higher values was observed after 42 days, though without significant difference (p=0.09). A larger number of neutrophils in the MyD88 KO group were observed compared with the WT animals (p=0.01 at 7 days, p=0.004 at 21 days and p<0.001 at 42 days). On the other hand, regarding the number of osteoclasts, no statistically significant difference was observed between the groups at any of the experimental periods (p=0.884 at 7 days, p=0.506 at 21 days and p=0.211 at 42 days). Descriptive microscopic analysis of the MyD88 KO group revealed a more intense inflammatory infiltrate, with abundant presence of polymorphonuclear and mononuclear cells and wide tissue destruction, after 7, 21 and 42 days. Brown & Brenn staining showed an increased bacterial dissemination, including the periapical tissues in the MyD88 KO group, when compared with the WT animals. As for immunohistochemistry, RANK, RANKL and OPG immunostainings were similar between the two groups of animals. Based on the employed methodology and the obtained results, it may be concluded that in the absence of MyD88, the animals showed larger periapical lesions, with a severe inflammatory infiltrate and a significantly larger number of neutrophils, when compared with WT animals, suggesting the important role of this molecule during the immune and inflammatory response against infections of endodontic origin.

Paracoccina (PCN) é um constituinte de Paracoccidioides brasiliensis, um patógeno humano que causa a paracoccidioidomicose, micose sistêmica mais prevalente na América Latina. A PCN é uma proteína de função dual, com domínios de atividade lectínica e de N-acetilglicosaminidase. Análises proteômicas da preparação paracoccina revelaram a sua correspondência com uma proteína hipotética de P. brasiliensis do isolado 18 (Pb18), anotada como PADG-3347.1, que tem sequência polipeptídica semelhante a família das endoquitinases 18. Essas endoquitinases apresentam domínios distintos de atividade lectínica e enzimática. O conjunto de exons do gene correspondente, PADG-3347.1, foi clonado e expresso em E. coli, e as características físicas e biológicas da proteína recombinante foram comparadas com as da PCN. Além disso, a PADG-03347.1 recombinante (rPCN) foi avaliada por suas propriedades imunomoduladoras e sua capacidade em conferir proteção contra a infecção por P. brasiliensis. Nesse sentido, investigamos a interferência da administração profilática e terapêutica de rPCN no curso da infecção por P. brasiliensis em camundongos BALB/c. A histopatologia pulmonar dos camundongos tratados com a rPCN, mostrou menor ocorrência de granulomas, e estes também foram menores do que os observados nos animais controles. Consistente com a observação de poucas leveduras no centro dos granulomas, a contagem de UFC a partir do homogenato pulmonar dos camundongos tratados foi inferior ao observado nos animais controles. Além disso, a administração de rPCN, foi associada com altos níveis de IL-12, IFN-, TNF-, NO e IL-10 detectados no homogenato pulmonar. Os altos níveis de citocinas produzidos nos animais tratados com rPCN nos levou a investigar a ocorrência de interação da lectina com receptores presentes em células da imunidade inata, tais como TLR2 e TLR4. Verificamos que a rPCN ativa TLR2, nas formas homo ou heterodimérica, e TLR4, de modo independente dos correceptores CD14 e CD36. Estes dados revelam um possível mecanismo pelo qual rPCN gera proteção nos camundongos contra a PCM. rPCN, administrada terapêutica ou profilaticamente, induz a ativação de TLRs e imunidade Th1, conferindo proteção contra a infecção por P. brasiliensis. / Paracoccin is a constituent of Paracoccidioides brasiliensis, a human pathogen that causes paracoccidioidomycosis, the most prevalent systemic mycosis in Latin America. Paracoccin is a dual function protein exerting lectin and N-acetylglucosaminidase activities. Proteomic analysis of paracoccin preparation revealed its correspondence with a hypothetical protein from P. brasiliensis isolate Pb18 (Pb18), annotated as PADG-3347.1, which has a polypeptide sequence similar to the family 18 endochitinases. These endochitinases have distinct lectin and enzymatic domains. The multi-exon assembly of the correspondent gene (PADG-3347) was cloned and expressed in E. coli, and the physical and biological features of the recombinant protein were compared to those of the native paracoccin. Moreover, recombinant PADG-3347.1 (rPCN) was evaluated for its immunomodulatory properties and its ability to confer protection against murine P. brasiliensis infection. Thus, we investigated the interference of prophylactic and therapeutic administration of rPCN on the course of P. brasiliensis infection in BALB/c mice. The pulmonary histopathology of the treated mice showed lower incidence of granulomas, which were also smaller than those observed in the control animals. Consistently with the observation of few yeasts in the center of the granulomas, the CFU count provided by lung homogenates of treated mice was lower than the provided by control mice. Furthermore, administration of rPCN was associated with higher levels of IL-12, IFN-, TNF-, NO and IL-10, detected in the lung homogenates of animals. The high levels of cytokines produced in the rPCN treated mice prompted us to investigate the occurrence of interaction of the lectin with receptors present in innate immune cells, such as TLR2 and TLR4. We verified that rPCN activates TLR2,in homo or heterodimeric forms, and TLR4, in a manner that does not depend on CD14 and CD36 coreceptors. These data reveal a possible mechanism by which rPCN generates protection in mice against PCM. rPCN, administered therapeutic or prophylactically, induces TLRs activation and Th1 immunity, conferring protection against P. brasiliensis infection.

A molécula adaptadora myeloid differentiation primary response gene 88 (MyD88) está envolvida na ativação de receptores Toll-like (TLRs), os quais são responsáveis pelo reconhecimento precoce pelas células do hospedeiro de patógenos invasores e pelo desencadeamento da resposta imunológica. O objetivo do presente estudo foi caracterizar o desenvolvimento e a progressão de lesões periapicais induzidas experimentalmente em dentes de camundongos knockout (KO) para a molécula MyD88 (MyD88 KO), comparados a animais wild-type (WT). Lesões periapicais foram induzidas nos primeiros molares inferiores de 30 camundongos WT e de 30 camundongos MyD88 KO. Decorridos 7, 21 e 42 dias, os animais foram submetidos à eutanásia em câmara de CO2 e as mandíbulas foram removidas e submetidas ao processamento histotécnico. A seguir, cortes representativos foram corados com hematoxilina e eosina (HE), para descrição das características do canal radicular e das regiões apical e periapical e para contagem do número de células inflamatórias (neutrófilos), em microscopia de luz, e para mensuração da área das lesões periapicais, em microscopia de fluorescência. Espécimes sequenciais foram analisados por meio de: histoenzimologia para a atividade da TRAP, para contagem de osteoclastos; coloração de Brown & Brenn, para localização de bactérias; e imunohistoquímica, para identificação de marcadores da osteoclastogênese (RANK, RANKL e OPG). Os dados foram submetidos à análise estatística por meio dos testes não-paramétricos de Mann-Whitney, Kruskal-Wallis e pós-teste de Dunn, utilizando o programa SPSS (Statistical Package for the Social Sciences) versão 17.O, com nível de significância de 5%. As demais análises foram expressas de maneira qualitativa. Com relação à extensão das lesões periapicais, o grupo MyD88 KO apresentou valores significantemente maiores do que o grupo WT nos períodos de 7 (p=0,001) e 21 dias (p=0,05), sendo que após 42 dias foi observada tendência de maiores valores, porém sem diferença significante (p=0,09). Foi observada maior quantidade de neutrófilos no grupo MyD88 KO, em comparação aos animais WT (p=0,01 em 7 dias; p=0,004 em 21 dias; e p<0,001 em 42 dias). Por outro lado, com relação à quantidade de osteoclastos, não foi observada diferença significante entre ambos os grupos, em todos os períodos experimentais (p=0,884 em 7 dias; p=0,506 em 21 dias; e p=0,211 em 42 dias). A análise microscópica descritiva do grupo MyD88 KO revelou um infiltrado inflamatório mais intenso, com presença abundante de células porlimorfonucleadas e mononucleadas e com grande destruição tecidual, após 7, 21 e 42 dias. A coloração de Brown e Brenn evidenciou uma maior disseminação bacteriana, inclusive nos tecidos periapicais, no grupo MyD88 KO, quando comparado aos animais WT. Com relação à imunohistoquímica, foram observadas marcações para RANK, RANKL e OPG de forma semelhante entre os dois grupos de animais. Com base nas metodologias e nos resultados obtidos no presente estudo pode-se concluir que na ausência da MyD88 os animais apresentaram lesões periapicais mais extensas, com um infiltrado inflamatório severo e com número significantemente maior de neutrófilos, quando comparados aos animais WT, sugerindo o importante papel desta molécula na resposta imune e inflamatória no combate à infecção de origem endodôntica. / The adaptor molecule myeloid differentiation primary response gene 88 (MyD88) is involved in the activation of toll-like receptors (TLRs), which are responsible for the early recognition by the host cells of invading pathogens and for triggering the immune response. The aim of the present study was to characterize the formation and progression of experimentally induced periapical lesions in teeth of MyD88 knockout (MyD88 KO) mice compared with wildtype (WT) mice. Periapical lesions were induced in the mandibular first molars of 30 WT and 30 MyD88 KO mice. After 7, 21 and 42 days, the animals were euthanized in a CO2 chamber and the mandibles were removed and subjected to histotechnical processing. Representative histological sections were stained with hematoxylin and eosin (HE) for description of the features of the root canal and the apical and periapical regions, and for counting of inflammatory cells (neutrophils) under conventional light microscopy and for determination of the size of the periapical lesions under fluorescence microscopy. Sequential specimens were evaluated by: TRAP histoenzymology, for osteoclast counting; Brown & Brenn staining, for localization of bacteria; and immunohistochemistry for identification of osteoclastogenesis markers (RANK, RANKL, OPG). Data were subjected to statistical analysis by the nonparametric Mann-Whitney and Kruskal-Wallis tests and the Dunn post-test, using the SPSS (Statistical Package for the Social Sciences) software, version 17.0. The significance level was set at 5%. The other analyzes were displayed qualitatively. Regarding the periapical lesion size, the MyD88 KO group presented significantly higher values than the WT group in the periods of 7 (p=0.001) and 21 days (p=0.05). Tendency for higher values was observed after 42 days, though without significant difference (p=0.09). A larger number of neutrophils in the MyD88 KO group were observed compared with the WT animals (p=0.01 at 7 days, p=0.004 at 21 days and p<0.001 at 42 days). On the other hand, regarding the number of osteoclasts, no statistically significant difference was observed between the groups at any of the experimental periods (p=0.884 at 7 days, p=0.506 at 21 days and p=0.211 at 42 days). Descriptive microscopic analysis of the MyD88 KO group revealed a more intense inflammatory infiltrate, with abundant presence of polymorphonuclear and mononuclear cells and wide tissue destruction, after 7, 21 and 42 days. Brown & Brenn staining showed an increased bacterial dissemination, including the periapical tissues in the MyD88 KO group, when compared with the WT animals. As for immunohistochemistry, RANK, RANKL and OPG immunostainings were similar between the two groups of animals. Based on the employed methodology and the obtained results, it may be concluded that in the absence of MyD88, the animals showed larger periapical lesions, with a severe inflammatory infiltrate and a significantly larger number of neutrophils, when compared with WT animals, suggesting the important role of this molecule during the immune and inflammatory response against infections of endodontic origin.

O melanoma é a forma mais letal entre os cânceres de pele. Essa neoplasia freqüentemente apresenta-se resistente a abordagens terapêuticas. A angiogênese associada ao tumor representa um crítico passo da tumorigênese, resultado da ação de diferentes citocinas e fatores de crescimento como VEGF produzidos no microambiente tumoral. As galectinas extracelulares participam de múltiplos processos biológicos incluindo angiogênese tumoral e metástases, sua interação com as células presentes no microambiente tumoral pode ocorrer via receptores toll-like sugerindo seu envolvimento nos processos pro-inflamatórios e na secreção de citocinas. Recentemente mostramos que a ausência de gal-3 no estroma e parênquima tumoral diminui a angiogênese por interferir na resposta de macrófagos via VEGF e/ou TGFbeta1. Entretanto, o envolvimento de galectinas extracelulares na angiogênese e na modulação do sistema imune no microambiente tumoral ainda não está esclarecido. Assim, este estudo visa buscar respostas ao envolvimento das galectinas no crescimento tumoral e angiogênese contribuindo ao combate do melanoma maligno. Nossos resultados mostram a participação das galectinas 1 e 3 no crescimento tumoral e seu envolvimento com macrófagos via receptores toll-like, além de coordenarem a modulação do perfil de polarização de macrófagos derivados da medula óssea de camundongos wild-type. Dessa forma, podemos inferir que essas galectinas agem como coordenadoras de mudança de perfil dos macrófagos, uma vez que inibidas extracelularmente promovem uma diminuição do crescimento tumoral em camundongos wild-type, inoculados com células de melanoma murino e uma manutenção do perfil de macrófagos M1 in vitro. Assim, concluimos que as galectinas 1 e 3 extracelulares são importantes para o crescimento tumoral de melanomas murinos pois promovem o crescimento tumoral e são coordenadoras da mudança do perfil de macrófagos / Melanoma is the most aggressive form of skin cancer. This tumor often presents itself resistant to therapeutic approaches. The tumor-associated angiogenesis is a critical step in tumorigenesis and the result of the action of several cytokines and growth factors such as VEGF produced in the tumor microenvironment. The extracellular galectins participate in multiple biological processes including tumor angiogenesis and metastasis, their interaction with cells present in the tumor microenvironment may occur via toll-like receptors suggesting their involvement in pro-inflammatory processes and the secretion of cytokines. We have recently shown that the absence of Gal-3 the stroma and tumor parenchyma decreases angiogenesis by interfering with the macrophage response by VEGF and / or TGFbeta1. However, the involvement of extracellular galectins on angiogenesis modulation of the immune system in the tumor microenvironment is not yet clear. This study aims is to find answers to the involvement of galectins on tumor growth and angiogenesis contributing to the study of the malignant melanoma. Our results demonstrate the involvement of galectin 1 and 3 on tumor growth and its involvement in macrophage by toll-like receptors pathway, and coordinating the modulation of the polarization profile in wild-type mice bone marrow derived macrophages. Therefore, we show these galectins act as coordinators of macrophages profile change, since inhibited extracellularly promote a reduction in tumor growth in wild-type mice inoculated with murine melanoma cells and macrophages M1 maintenance of profile in vitro. Thus, we conclude that galectins 1 and 3 extracellular are important for tumor growth of murine melanomas because they promote tumor growth and are coordinators of change macrophages profile

INTRODUÇÃO: Os receptores Toll-like 2 e 4 (TLR-2 e TLR-4) são capazes de ativar células imunes inatas em resposta a bactérias Gram-positivas e Gram-negativas, respectivamente. Na artrite psoriásica (APs), doença articular inflamatória crônica, fatores genéticos, ambientais e infecciosos parecem estar envolvidos. OBJETIVO: Avaliar as expressões dos receptores: TLR-2; TLR-4; CD114 e do CD116 em monócitos e neutrófilos do sangue periférico de pacientes com APs e adicionalmente a prevalência do HLA-B27. MÉTODOS: Quarenta e cinco pacientes com diagnóstico de APs conforme os critérios CASPAR e 32 indivíduos saudáveis foram estudados. Dentre os 45 pacientes, 27 apresentavam APs ativa (DAS28 > 2,6) e 18 APs inativa (DAS28 < 2,5). A leitura das expressões do TLR-2, TLR-4, CD14, CD66, CD114, CD116 e do HLA-B27 foi realizada por citometria de fluxo no FACSCalibur da marca Becton-Dickson, utilizando anticorpos monoclonais da BD Biosciences, anti-humanos produzidos em murino. Os anticorpos monoclonais (AcMo) para marcar receptores de membrana empregados foram: CD14 conjugado com PerCP-Cy5.5 para marcar população de monócitos; CD66 conjugado com PE e FITC para população de neutrófilos; CD114 para marcar receptor de fator estimulatório de colônias de granulócitos e CD116 para marcar receptor de fator estimulatório de colônia de granulócitos-macrófagos. A análise estatística utilizou o teste U de Mann-Whitney e o teste exato de Fisher. Os valores obtidos em porcentagem foram expressos como média ± intervalo interquartil, de acordo com uma distribuição não-paramétrica, avaliados pelo teste de Shapiro-Wilk. RESULTADOS: Demonstramos aumento de expressão do TLR-2 em monócitos periféricos de pacientes com APs, APs ativa e APs inativa comparados aos controles (p < 0,002; p < 0,001 e p < 0,04, respectivamente). A expressão do TLR-4 foi similar nos pacientes com APs, APs ativa e APs inativa e controles (p < 0,23; p < 0,33 e p < 0,29, respectivamente). A expressão do receptor GCSF (CD114) e do receptor GM-CSF (CD116) foi similar nos pacientes e controles nas populações de monócitos e neutrófilos (p > 0,05). O HLA-B27 foi positivo em 1/3 dos pacientes com APs e 6% dos controles. Nos pacientes HLA-B27+ comparados aos controles HLA-B27+, a porcentagem de expressão do TLR-2 nos monócitos foi significantemente maior (p < 0,004). CONCLUSÃO: O aumento da expressão do TLR-2 em monócitos de pacientes com APs reforça o papel da imunidade inata e sugere que a exposição a bactérias Gram-positivas possa ter um papel na indução da resposta inflamatória nesta doença / INTRODUCTION: Toll-Like receptors 2 and 4 (TLR-2 and TLR-4) are able of activating innate immune cells in response to Gram-positive and Gram-negative bacteria, respectively. In Psoriatic Arthritis (APs), chronic inflammatory joint disease and genetic, environmental and infectious factors seems to be involved. OBJECTIVES: Evaluate expressions of TLR-2; TLR-4; CD114 and CD116 receptors in monocytes and neutrophils from peripheral blood patients with APs and additionally the prevalence of HLA-B27. METHODS: Forty five patients diagnosed with APs according with CASPAR criteria and 32 health individuals were studied. Among the 45 patients, 27 presented active APs (DAS28 > 2,6) and 18 inactive APs (DAS28 < 2,5). The evaluation of the TRL-2, TLR-4, CD14, CD66, CD114, CD116 and HLA-B27 expressions was held by flow cytometry in FACSCalibur from Becton-Dickson, utilizing BD Biosciences\' monoclonal antibodies, anti-human produced in mice. The monoclonal antibodies (AcMo) used to mark membrane receptors were: CD14 in conjunction with PerCP-Cy 5.5 to mark population of monocytes; CD66 in conjunction with PE and FITC for population of neutrophils; CD114 to mark stimulatory factor receptor for granulocyte colonies and CD116 to mark stimulatory factor receptor for granulocyte-macrophage colony. The statistical analysis utilized Mann-Whitney\'s U test and Fisher\'s exact test. The values obtained as percentages were expressed as median ± interquartile range, consistent with a non-parametrical distribution, assessed by Shapiro-Wilk\'s test. RESULTS: Increased expression of TLR-2 in peripheral monocytes of patients with APs, active APs and inactive APs compared to controls (p < 0.002; p < 0.001 and p < 0.04, respectively). TLR-4 expression was similar in patients with APs, active APs and inactive APs and controls (p < 0.23; p < 0.33 and p < 0.29 respectively). The expression of the G-CSF (CCD114) receptor and GM-CSF (CD116) receptor were similar in patients and controls in populations of monocytes and neutrophils (p > 0.05). HLA-B27 was positive in 1/3 of the patients with APs and 6% of the controls. The percentage of expression of TLR-2 in HLA-B27 + patients compared to HLA-B27 + controls was significantly higher (p < 0.004). CONCLUSION: Increased of TLR-2 receptors expression in patients with APs monocytes reinforces the role of innate immunity and suggests that the exposure to Gram-positive bacteria may have a role in the induction of the inflammatory response in this diseases

O melanoma é o mais agressivo tumor da pele, cuja resistência aos tratamentos quimioterápicos tem promovido a crescente utilização de imunoquimioterapia, como é o caso da utilização de agonistas dos receptores Toll-Like (TLRs). Nesse contexto, os compostos abreviados por P-MAPA e seu sintético estrutural MRB-CFI-1 com reconhecidas propriedades antitumorais e imunológicas, são fortes candidatos na terapia e prevenção desse tipo de câncer. Esse estudo visa determinar o potencial anticâncer do P-MAPA e de MRB-CFI-1 contra o melanoma murino em consequência ao padrão de resposta microambiental semelhante ao de morte imunogênica, em regimes de tratamento terapêutico ou vacinal, na vigência de quimioterapia com cisplatina e/ou em associação com antígenos de células tumorais totais. Após avaliação In vivo do crescimento de tumores B16F10 implantados em modelos murinos selvagem e nocaute para o gene Myd88, na vigência ou não do tratamento com cisplatina e/ou P-MAPA, nossos resultados mostraram que o P-MAPA apresentou atividade pró-tumoral e antagonizou a ação da cisplatina em inibir o crescimento dos tumores, de forma dependente de Myd88. Além disso, através de análises qualitativa e quantitativa pelo software ImageJ em fotomicrografias de secções tumorais coradas histologicamente, observamos que o P-MAPA promoveu mudanças microambientais nos tumores que podem impactar negativamente em seu desempenho. Como monoterapia em esquema de vacinação com lisado tumoral total em combinação com quimioterapia, o P-MAPA em dose baixa falhou em suprimir o crescimento de tumores B16F10, mas o seu sintético MRB-CFI-1 foi capaz de prevenir o crescimento desse tipo de melanoma num regime de vacinação profilática. Apesar do sucesso terapêutico desse imunomodulador em diversos modelos de câncer e de doenças infecciosas, o P-MAPA não foi eficaz em produz respostas microambientais contra o melanoma murino, dados esses que limitam a aplicabilidade clínica do composto. De outro modo, o composto fosfato inorgânico MRB-CFI-1 foi protetivo em retardar o aparecimento desse tipo de doença. Assim, o presente estudo foi importante por ampliar o entendimento funcional do P-MAPA numa abordagem imunoquimioterápica em modelos biológicos de tumores de melanoma, e representa uma importante mudança na utilização de constituintes individuais similares ao P-MAPA que sejam mais eficazes, de fácil obtenção, e de produção controlada e garantida / Melanoma is the most aggressive skin cancer, whose resistance to chemotherapeutic treatments has promoted the increasing use of immunochemotherapy, as is the case for the use of Toll-Like receptor agonists (TLRs). In this context, the compounds abbreviated by P-MAPA and its structural synthetic MRB-CFI-1 with recognized antitumor and immunological properties are strong candidates in the therapy and prevention of this type of cancer. This study aims to determine the anti-cancer potential of P-MAPA and MRB-CFI-1 against murine melanoma as a consequence of the microenvironmental response pattern similar to that of immunogenic death in therapeutic or vaccine treatment regimens when using chemotherapy with cisplatin alone or in combination with whole tumor cell antigens. After In vivo evaluation of the growth of B16F10 tumors implanted in wild-type and Myd88 gene knockout mice, under treatment or not with cisplatin and / or P-MAPA, our results showed that P-MAPA showed pro-tumor activity and antagonized the action of cisplatin in inhibiting the growth of tumors in a Myd88-dependent manner. In addition, using qualitative and quantitative analysis by ImageJ software in histological images of tumor sections, we observed that P-MAPA promoted microenvironmental changes in tumors that may negatively impact its performance. As monotherapy in vaccination schedule with total tumor lysate in combination with chemotherapy, low dose P-MAPA failed to suppress the growth of B16F10 tumors, but its synthetic MRB-CFI-1 was able to prevent the growth of this type of melanoma in prophylactic vaccination regimen. Despite the therapeutic success of this immunomodulator in various cancer models and infectious diseases, P-MAPA has not been effective in producing microenvironmental responses against murine melanoma, data that limit the clinical applicability of the compound. Otherwise, the inorganic phosphate compound MRB-CFI-1 was protective in delaying the onset of this type of disease. Thus, the present study was important because it broadened the functional understanding of P-MAPA in an immuno-chemotherapeutic approach in biological models of melanoma tumors and represents an important change in the use of individual constituents similar to P-MAPA that are more efficient, easily obtainable, and controlled and guaranteed production

INTRODUÇÃO: Os receptores Toll-like 2 e 4 (TLR-2 e TLR-4) são capazes de ativar células imunes inatas em resposta a bactérias Gram-positivas e Gram-negativas, respectivamente. Na artrite psoriásica (APs), doença articular inflamatória crônica, fatores genéticos, ambientais e infecciosos parecem estar envolvidos. OBJETIVO: Avaliar as expressões dos receptores: TLR-2; TLR-4; CD114 e do CD116 em monócitos e neutrófilos do sangue periférico de pacientes com APs e adicionalmente a prevalência do HLA-B27. MÉTODOS: Quarenta e cinco pacientes com diagnóstico de APs conforme os critérios CASPAR e 32 indivíduos saudáveis foram estudados. Dentre os 45 pacientes, 27 apresentavam APs ativa (DAS28 > 2,6) e 18 APs inativa (DAS28 < 2,5). A leitura das expressões do TLR-2, TLR-4, CD14, CD66, CD114, CD116 e do HLA-B27 foi realizada por citometria de fluxo no FACSCalibur da marca Becton-Dickson, utilizando anticorpos monoclonais da BD Biosciences, anti-humanos produzidos em murino. Os anticorpos monoclonais (AcMo) para marcar receptores de membrana empregados foram: CD14 conjugado com PerCP-Cy5.5 para marcar população de monócitos; CD66 conjugado com PE e FITC para população de neutrófilos; CD114 para marcar receptor de fator estimulatório de colônias de granulócitos e CD116 para marcar receptor de fator estimulatório de colônia de granulócitos-macrófagos. A análise estatística utilizou o teste U de Mann-Whitney e o teste exato de Fisher. Os valores obtidos em porcentagem foram expressos como média ± intervalo interquartil, de acordo com uma distribuição não-paramétrica, avaliados pelo teste de Shapiro-Wilk. RESULTADOS: Demonstramos aumento de expressão do TLR-2 em monócitos periféricos de pacientes com APs, APs ativa e APs inativa comparados aos controles (p < 0,002; p < 0,001 e p < 0,04, respectivamente). A expressão do TLR-4 foi similar nos pacientes com APs, APs ativa e APs inativa e controles (p < 0,23; p < 0,33 e p < 0,29, respectivamente). A expressão do receptor GCSF (CD114) e do receptor GM-CSF (CD116) foi similar nos pacientes e controles nas populações de monócitos e neutrófilos (p > 0,05). O HLA-B27 foi positivo em 1/3 dos pacientes com APs e 6% dos controles. Nos pacientes HLA-B27+ comparados aos controles HLA-B27+, a porcentagem de expressão do TLR-2 nos monócitos foi significantemente maior (p < 0,004). CONCLUSÃO: O aumento da expressão do TLR-2 em monócitos de pacientes com APs reforça o papel da imunidade inata e sugere que a exposição a bactérias Gram-positivas possa ter um papel na indução da resposta inflamatória nesta doença / INTRODUCTION: Toll-Like receptors 2 and 4 (TLR-2 and TLR-4) are able of activating innate immune cells in response to Gram-positive and Gram-negative bacteria, respectively. In Psoriatic Arthritis (APs), chronic inflammatory joint disease and genetic, environmental and infectious factors seems to be involved. OBJECTIVES: Evaluate expressions of TLR-2; TLR-4; CD114 and CD116 receptors in monocytes and neutrophils from peripheral blood patients with APs and additionally the prevalence of HLA-B27. METHODS: Forty five patients diagnosed with APs according with CASPAR criteria and 32 health individuals were studied. Among the 45 patients, 27 presented active APs (DAS28 > 2,6) and 18 inactive APs (DAS28 < 2,5). The evaluation of the TRL-2, TLR-4, CD14, CD66, CD114, CD116 and HLA-B27 expressions was held by flow cytometry in FACSCalibur from Becton-Dickson, utilizing BD Biosciences\' monoclonal antibodies, anti-human produced in mice. The monoclonal antibodies (AcMo) used to mark membrane receptors were: CD14 in conjunction with PerCP-Cy 5.5 to mark population of monocytes; CD66 in conjunction with PE and FITC for population of neutrophils; CD114 to mark stimulatory factor receptor for granulocyte colonies and CD116 to mark stimulatory factor receptor for granulocyte-macrophage colony. The statistical analysis utilized Mann-Whitney\'s U test and Fisher\'s exact test. The values obtained as percentages were expressed as median ± interquartile range, consistent with a non-parametrical distribution, assessed by Shapiro-Wilk\'s test. RESULTS: Increased expression of TLR-2 in peripheral monocytes of patients with APs, active APs and inactive APs compared to controls (p < 0.002; p < 0.001 and p < 0.04, respectively). TLR-4 expression was similar in patients with APs, active APs and inactive APs and controls (p < 0.23; p < 0.33 and p < 0.29 respectively). The expression of the G-CSF (CCD114) receptor and GM-CSF (CD116) receptor were similar in patients and controls in populations of monocytes and neutrophils (p > 0.05). HLA-B27 was positive in 1/3 of the patients with APs and 6% of the controls. The percentage of expression of TLR-2 in HLA-B27 + patients compared to HLA-B27 + controls was significantly higher (p < 0.004). CONCLUSION: Increased of TLR-2 receptors expression in patients with APs monocytes reinforces the role of innate immunity and suggests that the exposure to Gram-positive bacteria may have a role in the induction of the inflammatory response in this diseases

The events that trigger an autoimmune disease remain largely unknown. To study these events animal models are necessary because symptoms of autoimmune diseases are preceded by a long asymptomatic period in humans. Experimental autoimmune encephalomyelitis (EAE) is the best characterized model for cell mediated autoimmunity and an animal model for the human disease multiple sclerosis. EAE is induced in rodents by immunization with myelin antigens (Ags) together with adjuvants. After immunization, T cells are primed in the periphery by Ag presenting cells and subsequently invade the central nervous system where they mediate parenchymal inflammation, resulting in demyelination and clinical symptoms of an ascending paralysis. It is now generally recognised that the main cell type mediating EAE is the T helper type 17 (Th17) cell. Tolerance to EAE can be attained by DNA vaccination, but how the immune response against the myelin Ags is abrogated after DNA vaccination is not known. By employing short interfering RNA technology, induction of the innate immune signalling molecule interferon (IFN) -β was found to be necessary for the protective effect of DNA vaccination in EAE. In addition, DNA vaccination inhibited subsequent autoimmune Th17 cell responses. The Toll-like receptors (TLRs) of the innate immune system have evolved to recognise conserved molecular structures on microbes and signalling through them almost exclusively converge on the molecule MyD88. Signalling via MyD88 was found to be required for induction of EAE since mice deficient in this molecule did not develop disease. Upstream signalling via TLR4 and TLR9 had tolerogenic properties. In studies of Ag presentation in EAE, two major subtypes of dendritic cells (DCs) were examined. Plasmacytoid DCs were found to have a promoting role in the induction of EAE, partly via type 1 IFNs. Myeloid DCs had a redundant role in the induction phase of EAE, neither disease severity nor encephalitogenic Th17 responses were affected by their absence during priming. These studies further demonstrate that the cells and molecules of the innate immune system exhibit a crucial role in controlling the adaptive immune system which mediates tissue damage in autoimmune diseases.

The goal of the work presented was to further understand how both the body and dendritic cells (DCs) interact and respond to biomaterials through receptor-mediated mechanisms. The role of Toll-like receptor 4 (TLR4) was investigated in the host response to biomaterials, and it was found that TLR4-deficient mice (in comparison to wild-type) had a delayed acute inflammatory response as seen through an altered adherent leukocyte profile on implanted polymer discs. However, following a 2 week implantation, the response was resolved potentially through compensatory receptors. Therefore, TLR4 may aid in the initial response to a biomaterial through recognition of 'danger signal' molecules. An investigation into the role of TLR4 in the response of DCs to biomaterials was investigated using murine bone marrow-derived DCs (BMDC), and PLGA film or microparticle treatment of BMDCs resulted in TLR4-dependent signs of slight maturation in non/loosely adherent BMDCs. However, further investigation into BMDC populations within the culture system revealed that non/loosely adherent BMDCs took on an activated/mature phenotype while adherent BMDCs appeared to be less mature and more responsive to both LPS and biomaterial stimuli. Therefore, it was concluded that investigations into the responsiveness of BMDCs to stimuli in the future analyze both adherent and non/loosely adherent populations. Lastly, the role of integrin-mediated adhesion in biomaterial-induced DC maturation was investigated. Gene expression analysis revealed that PLGA treatment of human DCs increased adhesion molecule expression (including β1 and β2 integrin subunits), LPS treatment reduced adhesion molecule expression and agarose treatment did not alter their expression. Antibody blocking techniques pinpointed the role of β2 integrins (and not β1 integrins) in both the adhesion of DCs to TCPS or PLGA substrates and the regulation of a DC maturation marker (CD86). β2 (and not β1) was found co-localized with F-actin in podosomes of DCs adhering to PLGA, and the direct interaction of β2 (and not β1) to PLGA substrate was confirmed through crosslinking and immunofluorescence studies. Therefore, DCs utilized β2 integrins for both adhesion and maintenance of immunomodulatory status. This aids the field of tissue engineering and vaccine design by further developing the criteria for biomaterial-influenced immunomodulation.

Appropriate recognition of parasites is crucial for effective immune response, ensuring activation of adequate defence mechanisms. In vertebrates, it has frequently been demonstrated that genes encoding proteins involved in pathogen recognition by an adaptive immune system are often subject to intense selection pressures. On the contrary, much less information has been provided on the evolution of recognition mechanisms of innate immunity. The aim of this thesis is to describe the pattern of natural variation of innate immunity genes involved in pathogen recognition in rodents and to analyze the mechanisms of their evolution. We used murine rodents (subfamily Murinae) as a principal model group because they are potential reservoirs of various pathogens dangerous to humans. First, we studied the intraspecific variability of five bacterial sensing Toll-like receptors (TLR1, TLR2, TLR4, TLR5, and TLR6) in inbred strains derived from two subspecies of the house mouse (M. m. musculus, hereafter abbreviated as Mmm and Mus musculus domesticus, Mmd). Wild-derived inbred strains are suitable tools for studying variation of immunity genes because they provide information about alleles that occur in natural populations, and at the same time they occur at homozygous state. The most significant results include the findings of a stop codon in exon 2 of the Tlr5 gene in one Mmm strain and no variability in Tlr4 of Mmd. Following these results we decided to check whether the absence of Tlr4 polymorphism in Mmd reflects the pattern found in natural populations, or whether it is a consequence of insufficient sampling or subsequent breeding. We therefore sequenced Tlr4 in both subspecies across a large part of the Western Palearctic region (in total 39 Mmm and 62 Mmd individuals), then we compared these results with variability on mitochondrial DNA (cytochrome b). The result confirmed our prediction that observed variability in Mmd is strongly reduced also in free-living populations (compared to Mmm), probably due to strong purifying selection by pathogens with which they met during the westward colonization. However, the influence of random evolutionary processes (e.g. drift during bottlenecks) cannot be excluded based on our data. At the intraspecific level, we could not find any sign of positive selection. The last part of my dissertation is devoted to interspecific comparison of two receptors, TLR4 and TLR7. These two TLRs differ in the exposure and the ligands detection. TLR4 is an extracellular receptor detecting mainly bacterial ligands (especially lipopolysaccharides), while TLR7 is located inside the cell and detects ssRNA viruses. The aim of this part of the thesis was to describe variability of both receptors at the interspecific level and to reveal selection forces acting on TLRs in longer evolutionary time scale. In total we analyzed 23 rodent species of the subfamily Murinae in Europe, Asia and Africa. Our results suggest that purifying selection has been a dominant force in evolution of the Tlr4 and Tlr7 genes, but we also demonstrated that episodic diversifying selection has shaped the present species-specific variation in rodent Tlrs. Sites under positive selection were concentrated mainly in the extracellular domain of both receptors, which is responsible for ligand binding. The comparison between two TLRs lead us to the conclusion that the intracellular TLR7 is under much stronger negative selection pressure, presumably due to its interaction with viral nucleic acids.