Cancer, historically considered a genetic disease, is currently acknowledged to affect the whole body. Our immune system is one key player that can elicit a response against malignant cells but can also promote tumorigenesis. Tumors avoid immune recognition by creating a suppressive microenvironment and inducing tolerance. T-cells are regarded a major effector cell type in tumor immunotherapy. An important ”switch” needed for T-cell activation involves so-called costimulatory and coinhibitory receptors. In this thesis, experimental tumor models were used to investigate the potential of immunomodulatory antibodies to stimulate immune cells and subsequently eliminate tumors. First, systemic antibody blockade of two negative checkpoint regulators (CTLA-4 and PD-1) present on T-cells was evaluated in combination with local CpG therapy or standard BCG treatment. Indeed, this combinatorial therapy with CpG augmented anti-tumor effects with increased levels of tumor-directed T-cells and reduced tumor-infiltrating Tregs. Secondly, as these immunomodulatory antibodies elicit severe side effects in patients, a local low-dose delivery regimen was explored as an alternative to systemic bolus treatment. Our results demonstrated that an approximately seven times lower dose of aCTLA-4, compared to systemic delivery, could eradicate both primary and distant tumors. CD40-expressing APCs are another potential target in antibody-mediated cancer therapy. CD40-stimulated dendritic cells (DCs) have the capability to activate tumor-directed T-cells to kill tumor cells. We next sought to investigate agonistic CD40 antibody efficacy and in vivo biodistribution when delivered locally compared to the equivalent systemic dose. Anti-tumor effects were dependent on CD8+ T-cells, host CD40 expression and the presence of tumor antigen at the injection site. CD40 antibodies were cleared from the circulation and accumulated in lymphoid organs, where, upon repeated aCD40 dosing, target APC populations increased in numbers and upregulated their surface CD40 expression. Lastly, CD40 agonist antibodies were mixed with nanoparticles to enhance their stimulatory properties. B-cells demonstrated increased proliferative capacity and DCs became more activated when exposed to the cocktail. Further, this combination reduced serum levels of pro-inflammatory cytokines compared to plain antibodies.       The results herein advocate further exploratory studies of the delivery of monoclonal antibodies at the tumor site in order to improve anti-tumor effects and reduce toxicity.

INTRODUÇÃO: Toxocaríase é uma infecção parasitária de distribuição global, causada pela fase larval de Toxocara spp. Os hospedeiros naturais são cães e gatos, nos quais o parasita completa o ciclo chegando a fase adulta. Outros hospedeiros podem ser infectados pela fase larval do parasita, após ingestão de ovos embrionados do solo, mãos contaminadas, fomites, ou ingestão de carne ou vísceras de animais infectados. Em hospedeiros paratênicos o parasita não completa o ciclo, invadindo em estágio larval vísceras ou outros tecidos, onde podem sobreviver e induzir a patologia. O presente estudo teve como objetivo caracterizar o hamster (Mesocricetus auratus), como modelo experimental de toxocaríase, inicialmente através do estudo das lesões histopatológicas em fígado, pulmão e rim. A caracterização da resposta imunológica do modelo, foi feita através do estudo de citocinas envolvidas nas respostas Th1 e Th2, e foi sugerida uma correlação entre alterações glomerulares e depósitos de complexos antígenos-anticorpo pré-formados na circulação. MÉTODOS: Hamsters foram inoculados com ovos embrionados de Toxocara canis, e mantidos no biotério do Instituto de Medicina Tropical de São Paulo. O estudo histopatológico foi desenvolvido utilizando-se cortes parafinados corados por hematoxilina e eosina. Para detecção de antígenos nos tecidos foram realizadas reações imunohistoquímicas, utilizando-se anticorpo monoclonal e policlonal anti- Toxocara canis. Utilizando-se o soro dos animais infectados e animais controle, foi realizada pesquisa de antígeno e anticorpo por ELISA. Para pesquisa de imunoglobulinas IgG e IgM e complemento, foram utilizados cortes congelados de rins para realização de reação de Imunofluorescência. Fragmentos de rins foram incluídos para utilização em microscopia eletrônica, para detecção de antígenos de toxocara e de imune complexos. Para caracterização de resposta imunológica foram estudadas citocinas envolvidas na resposta Th1 e Th2 por técnica de RT-PCR. RESULTADOS: Os achados histopatológicos demonstraram desde o início da infecção, presença de larvas em maior número no fígado, seguido de pulmão e raramente rins. Em fígado remanescentes larvares foram visualizados cercados por reação inflamatória granulomatosa. Logo no início da infecção foi encontrado pneumonite intersticial e intraalveolar focal, e lesão renal com glomérulo apresentando hiperplasia focal de células mesangiais (glomerulite mesangio-proliferativa). Houve marcação de antígenos em todos os grupos de animais infectados, tanto pelo anticorpo monoclonal, como pelo policlonal. Depósitos de imunoglobulinas e complemento foram marcados em glomérulo por imunofluorescência A análise dos soros por ELISA, demonstrou na pesquisa de anticorpos aumento gradativo no decorrer da infecção, acompanhado de diminuição de antígenos. Depósitos de antígenos em glomérulos foram detectados por microscopia imonoeletrônica. No RT-PCR foi detectado aumento significativo do nível de IL-4, com tendência de elevação de IL-10 e IFN-?. CONCLUSÃO: O hamster demonstrou ser um modelo experimental eficiente para toxocaríase. Entretanto este modelo é mais adequado para infecções de curto prazo. A resposta imunológica avaliada por RT-PCR, com elevado nível da expressão de IL-4, sugere uma resposta Th2, mas a tendência de aumento de IL-10 e IFN-? poderia sinalizar uma resposta mista Th1 e Th2. Achados de depósitos de imunoglobulinas no glomérulo sugerem a possibilidade de que as manifestações renais com síndrome nefrótica em humanos possa vir a ter como base a toxocaríase / INTRODUCTION: Toxocariasis is a parasitic infection of global distribution, caused by the larval stage of Toxocara spp. The natural hosts are dogs and cats, in which the parasite completes the cycle reaching adulthood. Other hosts can be infected with the larval stage of the parasite, after ingestion of embryonated eggs from the soil, contaminated hands, fomites, or ingestion of meat or viscera of infected animals. In paratenics hosts the parasite not complete the cycle, encroaching on larval stage in viscera or other tissues where they can survive and induce pathology. The present study aimed to characterize the hamster, Mesocricetus auratus, as experimental model of toxocariasis, initially through the study of histopathological lesions in the liver, lung and kidney. The characterization of immune response model, was made through the study of cytokines Th1 and Th2 responses involved, and a correlation was suggested between glomerular changes and antibody-antigen complexes deposits preformed in the circulation. METHODS: Hamsters were inoculated with embryonated eggs of Toxocara canis, and kept in the bioterium of the Institute of Tropical Medicine of the São Paulo. The histopathologic study was developed using paraffin slides stained by hematoxylin and eosin. For detection of antigens in tissues immunohistochemistry reactions were performed using monoclonal and polyclonal anti-Toxocara canis sera. Using the serum of infected and control animals, search has been carried out of antigen and antibody by ELISA. For the search of immunoglobulins IgG, IgM and complement, were used slides prepared from frozen fragments of kidneys and a immunofluorescence reaction. Fragments of kidneys were included for electron microscopy to detect antigens of Toxocara and immune complexes. For characterization of Th1 and Th2 response cytokines involved were detected by RT-PCR technique. RESULTS: Histopathological findings demonstrated since the beginning of the infection the presence of larvae in greater numbers in the liver, followed by lung and rarely kidneys. In the liver larval remnants were surrounded by a granulomatous inflammatory reaction. Early in the infection was found interstitial pneumonitis with intraalveolar focal inflammatory infiltrate and renal injury with glomerulus showing mesangial cell focal hyperplasia (mesangioproliferative glomerulonephritis). There were the presence of antigens in all groups of animals infected detected by both the monoclonal and polyclonal antibodies. Deposits of immunoglobulin and complement were present in glomerulus by immunofluorescence analysis. ELISA, showed that the presence of antibodies increased gradually in the course of infection, accompanied by progressive diminution of antigens. Clusters of antigen/s were detected by immunoelectron microscopy. RT-PCR showed a significant increase of IL-4, with a tendency of increase of IL- 10 and IFN-?. CONCLUSION: The hamster has proved to be an efficient experimental model for toxocariasis. However this model is best suited for short-term infections. The immune response evaluated by RT-PCR, with high level of expression of IL-4, suggests a Th2 response, but the trend of increase of IL-10 and IFN-? might suggest a Th1 and Th2 mixed response. Findings of immunoglobulin deposits in glomeruli suggests the possibility that the renal manifestations with nephrotic syndrome in humans might have, in certain circunstances, as a basis the toxocariasis

Introdução: A fibrose pulmonar idiopática (FPI) é definida como um tipo de doença fibrosante intersticial crônica de etiologia desconhecida limitada aos pulmões e que apresenta padrão histológico de pneumonia intersticial usual. A prevalência de FPI é estimada em, aproximadamente, 20/100.000 em homens e em 13/100.000 em mulheres, sendo que a idade média do diagnóstico é 67 anos e a sobrevivência média é 2 a 5 anos. Estima-se que 5 milhões de pessoas sejam afetadas em todo o mundo. O tratamento clínico atual está associado com melhora parcial e transitória, com resultados duvidosos ou insatisfatórios. Na abordagem cirúrgica da FPI, tem destaque o transplante pulmonar, cuja realização é rara, devido à escassez de doadores e à limitação das equipes capazes de realizar tais procedimentos. A terapia celular é uma alternativa terapêutica com grande potencial de aplicabilidade na fibrose pulmonar. Objetivos: Utilizar um modelo de fibrose pulmonar induzida pela bleomicina em ratos para investigar os efeitos da terapia com células-tronco mesenquimais (CTM) e o meio condicionado no remodelamento pulmonar com objetivo de elucidar o mecanismo de ação das CTM e meio condicionado, na reversão da fibrose pulmonar. Para tanto, buscamos: 1) padronizar a cultura de células-tronco mesenquimais e meio de cultura; 2) caracterizar o modelo experimental de fibrose pulmonar induzida pela bleomicina por microscopia óptica antes e após o tratamento com CTM e meio de cultura; 3) avaliar a expressão de biomarcadores sorológicos (Fibrinogênio, Fator von Willebrand e PDGF); 4) quantificar a expressão de proteínas relacionadas ao estresse oxidativo (NOS), citocinas pró-inflamatórias e pró-fibróticas (IL-17 e TGF-beta) e pró-angiogênicas (VEGF e endotelina) por imuno-histoquímica; e 5) quantificar a deposição de fibras do colágeno I e V por imunofluorescência. Materiais e Métodos: Utilizou-se um total de 44 ratos Wistar machos albinos com peso médio de 250-300g e 8 semanas de idade. Quatro grupos experimentais foram compostos de 10 animais, que participaram do experimento divididos em três momentos: D0, D10 e eutanásia (D14 ou D21). Em D0, foi realizada a instilação orotraqueal de bleomicina na dose de 1,5 U/kg; em D10, foi realizada a infusão em veia caudal de células-tronco mesenquimais na dose de 106 CTM/Kg ou 200 ?l de meio condicionado. Para o preparo das CTM, foi obtido, em média, 1,2 g de tecido adiposo, procedida a dissociação com colagenase tipo I, sendo que a contagem média de células foi de 3,05 x 106 células linfomononucleares/g de tecido adiposo. Estas células foram cultivadas durante 21 dias em meio Knockout DMEM-F12 suplementado com 10% de soro fetal bovino. Os seguintes três critérios foram utilizados para comprovar o perfil das células-tronco mesenquimais: aderência plástica, expressão de CD90 por citometria de fluxo ( > 90%) e capacidade de diferenciação em três linhagens mesodérmicas. Em D10, um pool destas células alogênicas foi infundido intravenosamente, na veia caudal, a uma concentração de 1 x 106 células/ animal num volume de 200 ul de solução salina. Em D14 ou D21, os animais foram eutanasiados e analisados quanto ao peso e conforme análises microscópico-laboratoriais. As análises histológicas foram realizadas por dois especialistas diferentes em estudo duplo-cego. Os parâmetros de ganho de peso e recuperação microscópica do tecido pulmonar foram analisados em cada grupo. Resultados: Nossos dados mostram que as células-tronco mesenquimais oriundas do tecido adiposo abdominal de ratos Wistar tiveram seu perfil fenotípico, capacidade de adesão plástica e diferenciação em 3 linhagens mesodérmicas estabelecidas inequivocamente, conforme estabelecido internacionalmente pela ISSCR. As células-tronco mesenquimais e o meio condicionado induziram: a recuperação clínica após tratamento; a reversão da inflamação e de fibrose pulmonar induzida pela bleomicina; a reversão da arteriopatia no parênquima pulmonar distal; a redução das concentrações de fibrinogênio, fator von Willebrand e PDGF (marcadores sorológicos); a diminuição da expressão de enzimas oxidantes; a diminuição da expressão de endotelina e a modulação da expressão da proteína de remodelamento (IL-17), da ativação dos fibroblastos TGF-B e da síntese de colágeno. Conclusão: Neste estudo, comprovamos que as terapias com células-tronco mesenquimais derivadas do tecido adiposo e com o meio condicionado foram eficazes na modulação dos processos inflamatórios e fibrogênicos no modelo induzido por bleomicina, agindo na ativação miofibroblástica, e, também, na restauração tecidual e endotelial. Além disso, o meio condicionado se mostrou tão eficiente quanto as células-tronco propriamente ditas, no efetivo remodelamento pulmonar, devendo ser considerado como uma proposta terapêutica viável e inovadora / Introduction: Idiopathic pulmonary fibrosis (IPF) is defined as a type of chronic interstitial fibrosing disease of unknown etiology limited to the lungs and presenting a histological pattern of usual interstitial pneumonia. The prevalence of IPF is estimated at approximately 20/100,000 in men and 13/100,000 in women, with the mean age of diagnosis being 67 years and the average survival is 2 to 5 years. It is estimated that 5 million people are affected worldwide. The current clinical treatment is associated with partial and transient improvement, with dubious or unsatisfactory results. In the surgical approach to IPF, pulmonary transplantation is a prominent feature, which is rare because of the scarcity of donors and the limitation of the teams capable of performing such procedures. Cell therapy is a therapeutic alternative with great potential for applicability in pulmonary fibrosis. Objectives: To use a model of bleomycin-induced lung fibrosis in rats to investigate the effects of mesenchymal stem cell (MSC) therapy and conditioned medium on lung remodeling in order to elucidate the mechanism of action of MSC and conditioned medium, in the reversion of pulmonary fibrosis. To do so, we aim to: 1) standardize the culture of mesenchymal stem cells and conditioned medium; 2) characterize the experimental model of lung fibrosis induced by bleomycin by optical microscopy before and after treatment with MSC and conditioned medium; 3) to evaluate the expression of serological biomarkers (Fibrinogen, Factor von Willebrand and PDGF); 4) to quantify the expression of proteins related to oxidative stress (NOS), pro-inflammatory and pro-fibrotic (IL-17 and TGF-beta) and pro-angiogenic cytokines (VEGF and endothelin) by immunohistochemistry; and 5) to quantify the deposition of collagen I and V fibers by immunofluorescence. Materials and methods: A total of 44 male albino Wistar rats weighing 250-300g and 8 weeks of age were used. Four experimental groups were composed of 10 animals, which participated in the experiment divided in three moments: D0, D10 and euthanasia (D14 or D21). In D0, orotracheal instillation of bleomycin at the dose of 1.5 U/ kg was performed; in D10 caudal vein infusion of mesenchymal stem cells at the dose of 106 MSC/ kg or 200 ul of conditioned medium was performed. To prepare the MSC, a mean of 1.2 g of adipose tissue was obtained, dissociated with type I collagenase and the mean cell count was 3.05 x 106 lymphomonuclear cells / g of adipose tissue. These cells were cultured for 21 days in DMEM-F12 Knockout medium supplemented with 10% fetal bovine serum. The following three criteria were used to prove the profile of mesenchymal stem cells: plastic adherence, expression of CD90 by flow cytometry ( > 90%) and differentiation capacity in three mesodermal lines. In D10, a pool of these allogeneic cells was infused intravenously into the caudal vein at a concentration of 1 x 106 cells / animal in a volume of 200 ul of saline. In D14 or D21 the animals were euthanized and analyzed for weight and microscopic-laboratory analyzes. Histological analyzes were performed by two different specialists in a double-blind study. The parameters of weight gain and microscopic recovery of lung tissue were analyzed in each group. Results: Our data show that the mesenchymal stem cells derived from the abdominal adipose tissue of Wistar rats had their phenotypic profile, plastic adhesion capacity and differentiation in 3 mesodermal lines established unequivocally, as established internationally by ISSCR. Mesenchymal stem cells and conditioned medium induced: clinical recovery after treatment; bleomycin-induced reversal of inflammation and pulmonary fibrosis; the reversal of arteriopathy in the distal pulmonary parenchyma; the reduction of fibrinogen, von Willebrand factor and PDGF concentrations (serologic markers); decreased expression of oxidizing enzymes; the reduction of endothelin expression and the modulation of the expression of the remodeling protein (IL-17), the activation of TGF-B fibroblasts and the synthesis of collagen. Conclusion: In this study we demonstrated that therapies with mesenchymal stem cells derived from adipose tissue and conditioned medium were effective in the modulation of inflammatory and fibrogenic processes in the bleomycin-induced model, acting to modulate myofibroblastic activation and also in tissue restoration and endothelial cells. In addition, the conditioned medium proved to be as efficient as the stem cells themselves, in effective pulmonary remodeling, and should be considered as a viable and innovative therapeutic proposal

Made available in DSpace on 2016-04-29T13:17:49Z (GMT). No. of bitstreams: 1 Najara Karine Salomao Pereira Almeida.pdf: 2355640 bytes, checksum: 39d43e135617768e1e42e6084f16eca9 (MD5) Previous issue date: 2013-05-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Chronic Mild Stress (SMS) is an anhedonia experimental animal model, induced by the chronic exposition of rats to a mild stressors protocol, and measured by the intake of sucrose, intracranial stimulation and/or site preference conditioning. Aside from anhedonia, this model is also recognized by the production of losses in the body weight, independently of specific feeding regimes and other characteristics analogous to the conditions that compose the depression diagnosis. The objective of the present study was to investigate if the exposition of the subjects to a water VT concurrent scheme water with sucrose 8% VT, of same value, before and after the stressors protocol, would produce disturbances: (1) in the body weight of the subjects; (2) in the diary consumption of food and water; (3) in the consumption and preference of liquids; (4) in the time during which the subjects remained close to the stimulus water and to the stimulus water with sucrose in the operant conditioning box, and (5) in the time in which the subjects have emitted responses in the site of the water fountain and of the water with sucrose 8% fountain. The design was composed by three experimental conditions: (1) exposition of the subjects VTP3, VTP4, VTP7, VTP8, P5, P6, P13 and P15 to the stressors protocol; (2) submission of the subjects VTP3, VTP4, VTP7 and VTP8 to the concurrent 20s VT sessions and (3) application of consumption and liquids´ preference tests in all of the experimental subjects, including the subject C10. The main disturbances noticed were: (a) decrease in the weight losses during the exposition to the protocol and decrease in weight variations during the role experiment; (b) diary water consumption similar to the ones of the subjects submitted to the FR and VI sessions; (c) increase in the diary food consumption, mainly during the exposition to the protocol; (d) constant consumption and preference by sucrose; (e) preference by sucrose in the 20s VT sessions after the exposition to the protocol, and (f) increase in the subjects´ general activity along the submission to the 20s VT sessions. The subjects exposed only to the protocol and to the liquid consumption tests didn´t present decrease in the sensibility to the reinforcer stimulus water with sucrose, what is commonly observed in other studies / Chronic Mild Stress (CMS) é um modelo animal experimental de anedonia, induzida através da exposição crônica de ratos a um protocolo de estressores pouco severos, e medida a partir de ingestão de sacarose, estimulação intracraniana e/ou condicionamento de preferência de lugar. Além de anedonia, este modelo também é reconhecido por produzir perda de peso corporal, independente de regime alimentar específico, e outras características análogas ao que compõe o diagnóstico de depressão. O objetivo do presente estudo foi investigar se a exposição de sujeitos a um esquema concorrente VT água VT água com sacarose a 8%, de mesmo valor, antes e após o protocolo de estressores, produzia alterações: (1) no peso corporal dos sujeitos; (2) no consumo diário de ração e água; (3) no consumo e preferência de líquidos; (4) no tempo em que os sujeitos permaneceram do lado do estímulo água ou do estímulo água com sacarose na caixa de condicionamento operante, e (5) no tempo em que os sujeitos emitiram respostas na região do bebedouro água ou do bebedouro água com sacarose a 8%. O delineamento foi composto por três condições experimentais: (1) exposição dos sujeitos VTP3, VTP4, VTP7, VTP8, P5, P6, P13 e P15 ao protocolo de estressores; (2) submissão dos sujeitos VTP3, VTP4, VTP7 e VTP8 as sessões concorrentes VT 20s e (3) aplicação dos testes de consumo e preferência de líquidos a todos os sujeitos da pesquisa, incluindo o sujeito C10. As principais alterações observadas foram: (a) menor diminuição de peso durante a exposição ao protocolo e menor variação de peso durante todo o experimento (b) consumo diário de água semelhante aos dos sujeitos submetidos às sessões de FR e VI; (c) aumento no consumo diário de ração, principalmente durante a exposição ao protocolo; (d) consumo de líquidos e preferência por sacarose constantes; (e) preferência por sacarose nas sessões VT 20s após a exposição dos sujeitos ao protocolo, e (f) aumento da atividade geral dos sujeitos ao longo da submissão às sessões VT 20s. Os sujeitos expostos somente ao protocolo e aos testes de consumo de líquido não apresentaram diminuição de sensibilidade ao estímulo reforçador água com sacarose comumente observado nos outros estudos

Introdução: A fibrose pulmonar idiopática (FPI) é definida como um tipo de doença fibrosante intersticial crônica de etiologia desconhecida limitada aos pulmões e que apresenta padrão histológico de pneumonia intersticial usual. A prevalência de FPI é estimada em, aproximadamente, 20/100.000 em homens e em 13/100.000 em mulheres, sendo que a idade média do diagnóstico é 67 anos e a sobrevivência média é 2 a 5 anos. Estima-se que 5 milhões de pessoas sejam afetadas em todo o mundo. O tratamento clínico atual está associado com melhora parcial e transitória, com resultados duvidosos ou insatisfatórios. Na abordagem cirúrgica da FPI, tem destaque o transplante pulmonar, cuja realização é rara, devido à escassez de doadores e à limitação das equipes capazes de realizar tais procedimentos. A terapia celular é uma alternativa terapêutica com grande potencial de aplicabilidade na fibrose pulmonar. Objetivos: Utilizar um modelo de fibrose pulmonar induzida pela bleomicina em ratos para investigar os efeitos da terapia com células-tronco mesenquimais (CTM) e o meio condicionado no remodelamento pulmonar com objetivo de elucidar o mecanismo de ação das CTM e meio condicionado, na reversão da fibrose pulmonar. Para tanto, buscamos: 1) padronizar a cultura de células-tronco mesenquimais e meio de cultura; 2) caracterizar o modelo experimental de fibrose pulmonar induzida pela bleomicina por microscopia óptica antes e após o tratamento com CTM e meio de cultura; 3) avaliar a expressão de biomarcadores sorológicos (Fibrinogênio, Fator von Willebrand e PDGF); 4) quantificar a expressão de proteínas relacionadas ao estresse oxidativo (NOS), citocinas pró-inflamatórias e pró-fibróticas (IL-17 e TGF-beta) e pró-angiogênicas (VEGF e endotelina) por imuno-histoquímica; e 5) quantificar a deposição de fibras do colágeno I e V por imunofluorescência. Materiais e Métodos: Utilizou-se um total de 44 ratos Wistar machos albinos com peso médio de 250-300g e 8 semanas de idade. Quatro grupos experimentais foram compostos de 10 animais, que participaram do experimento divididos em três momentos: D0, D10 e eutanásia (D14 ou D21). Em D0, foi realizada a instilação orotraqueal de bleomicina na dose de 1,5 U/kg; em D10, foi realizada a infusão em veia caudal de células-tronco mesenquimais na dose de 106 CTM/Kg ou 200 ?l de meio condicionado. Para o preparo das CTM, foi obtido, em média, 1,2 g de tecido adiposo, procedida a dissociação com colagenase tipo I, sendo que a contagem média de células foi de 3,05 x 106 células linfomononucleares/g de tecido adiposo. Estas células foram cultivadas durante 21 dias em meio Knockout DMEM-F12 suplementado com 10% de soro fetal bovino. Os seguintes três critérios foram utilizados para comprovar o perfil das células-tronco mesenquimais: aderência plástica, expressão de CD90 por citometria de fluxo ( > 90%) e capacidade de diferenciação em três linhagens mesodérmicas. Em D10, um pool destas células alogênicas foi infundido intravenosamente, na veia caudal, a uma concentração de 1 x 106 células/ animal num volume de 200 ul de solução salina. Em D14 ou D21, os animais foram eutanasiados e analisados quanto ao peso e conforme análises microscópico-laboratoriais. As análises histológicas foram realizadas por dois especialistas diferentes em estudo duplo-cego. Os parâmetros de ganho de peso e recuperação microscópica do tecido pulmonar foram analisados em cada grupo. Resultados: Nossos dados mostram que as células-tronco mesenquimais oriundas do tecido adiposo abdominal de ratos Wistar tiveram seu perfil fenotípico, capacidade de adesão plástica e diferenciação em 3 linhagens mesodérmicas estabelecidas inequivocamente, conforme estabelecido internacionalmente pela ISSCR. As células-tronco mesenquimais e o meio condicionado induziram: a recuperação clínica após tratamento; a reversão da inflamação e de fibrose pulmonar induzida pela bleomicina; a reversão da arteriopatia no parênquima pulmonar distal; a redução das concentrações de fibrinogênio, fator von Willebrand e PDGF (marcadores sorológicos); a diminuição da expressão de enzimas oxidantes; a diminuição da expressão de endotelina e a modulação da expressão da proteína de remodelamento (IL-17), da ativação dos fibroblastos TGF-B e da síntese de colágeno. Conclusão: Neste estudo, comprovamos que as terapias com células-tronco mesenquimais derivadas do tecido adiposo e com o meio condicionado foram eficazes na modulação dos processos inflamatórios e fibrogênicos no modelo induzido por bleomicina, agindo na ativação miofibroblástica, e, também, na restauração tecidual e endotelial. Além disso, o meio condicionado se mostrou tão eficiente quanto as células-tronco propriamente ditas, no efetivo remodelamento pulmonar, devendo ser considerado como uma proposta terapêutica viável e inovadora / Introduction: Idiopathic pulmonary fibrosis (IPF) is defined as a type of chronic interstitial fibrosing disease of unknown etiology limited to the lungs and presenting a histological pattern of usual interstitial pneumonia. The prevalence of IPF is estimated at approximately 20/100,000 in men and 13/100,000 in women, with the mean age of diagnosis being 67 years and the average survival is 2 to 5 years. It is estimated that 5 million people are affected worldwide. The current clinical treatment is associated with partial and transient improvement, with dubious or unsatisfactory results. In the surgical approach to IPF, pulmonary transplantation is a prominent feature, which is rare because of the scarcity of donors and the limitation of the teams capable of performing such procedures. Cell therapy is a therapeutic alternative with great potential for applicability in pulmonary fibrosis. Objectives: To use a model of bleomycin-induced lung fibrosis in rats to investigate the effects of mesenchymal stem cell (MSC) therapy and conditioned medium on lung remodeling in order to elucidate the mechanism of action of MSC and conditioned medium, in the reversion of pulmonary fibrosis. To do so, we aim to: 1) standardize the culture of mesenchymal stem cells and conditioned medium; 2) characterize the experimental model of lung fibrosis induced by bleomycin by optical microscopy before and after treatment with MSC and conditioned medium; 3) to evaluate the expression of serological biomarkers (Fibrinogen, Factor von Willebrand and PDGF); 4) to quantify the expression of proteins related to oxidative stress (NOS), pro-inflammatory and pro-fibrotic (IL-17 and TGF-beta) and pro-angiogenic cytokines (VEGF and endothelin) by immunohistochemistry; and 5) to quantify the deposition of collagen I and V fibers by immunofluorescence. Materials and methods: A total of 44 male albino Wistar rats weighing 250-300g and 8 weeks of age were used. Four experimental groups were composed of 10 animals, which participated in the experiment divided in three moments: D0, D10 and euthanasia (D14 or D21). In D0, orotracheal instillation of bleomycin at the dose of 1.5 U/ kg was performed; in D10 caudal vein infusion of mesenchymal stem cells at the dose of 106 MSC/ kg or 200 ul of conditioned medium was performed. To prepare the MSC, a mean of 1.2 g of adipose tissue was obtained, dissociated with type I collagenase and the mean cell count was 3.05 x 106 lymphomonuclear cells / g of adipose tissue. These cells were cultured for 21 days in DMEM-F12 Knockout medium supplemented with 10% fetal bovine serum. The following three criteria were used to prove the profile of mesenchymal stem cells: plastic adherence, expression of CD90 by flow cytometry ( > 90%) and differentiation capacity in three mesodermal lines. In D10, a pool of these allogeneic cells was infused intravenously into the caudal vein at a concentration of 1 x 106 cells / animal in a volume of 200 ul of saline. In D14 or D21 the animals were euthanized and analyzed for weight and microscopic-laboratory analyzes. Histological analyzes were performed by two different specialists in a double-blind study. The parameters of weight gain and microscopic recovery of lung tissue were analyzed in each group. Results: Our data show that the mesenchymal stem cells derived from the abdominal adipose tissue of Wistar rats had their phenotypic profile, plastic adhesion capacity and differentiation in 3 mesodermal lines established unequivocally, as established internationally by ISSCR. Mesenchymal stem cells and conditioned medium induced: clinical recovery after treatment; bleomycin-induced reversal of inflammation and pulmonary fibrosis; the reversal of arteriopathy in the distal pulmonary parenchyma; the reduction of fibrinogen, von Willebrand factor and PDGF concentrations (serologic markers); decreased expression of oxidizing enzymes; the reduction of endothelin expression and the modulation of the expression of the remodeling protein (IL-17), the activation of TGF-B fibroblasts and the synthesis of collagen. Conclusion: In this study we demonstrated that therapies with mesenchymal stem cells derived from adipose tissue and conditioned medium were effective in the modulation of inflammatory and fibrogenic processes in the bleomycin-induced model, acting to modulate myofibroblastic activation and also in tissue restoration and endothelial cells. In addition, the conditioned medium proved to be as efficient as the stem cells themselves, in effective pulmonary remodeling, and should be considered as a viable and innovative therapeutic proposal

INTRODUÇÃO: Toxocaríase é uma infecção parasitária de distribuição global, causada pela fase larval de Toxocara spp. Os hospedeiros naturais são cães e gatos, nos quais o parasita completa o ciclo chegando a fase adulta. Outros hospedeiros podem ser infectados pela fase larval do parasita, após ingestão de ovos embrionados do solo, mãos contaminadas, fomites, ou ingestão de carne ou vísceras de animais infectados. Em hospedeiros paratênicos o parasita não completa o ciclo, invadindo em estágio larval vísceras ou outros tecidos, onde podem sobreviver e induzir a patologia. O presente estudo teve como objetivo caracterizar o hamster (Mesocricetus auratus), como modelo experimental de toxocaríase, inicialmente através do estudo das lesões histopatológicas em fígado, pulmão e rim. A caracterização da resposta imunológica do modelo, foi feita através do estudo de citocinas envolvidas nas respostas Th1 e Th2, e foi sugerida uma correlação entre alterações glomerulares e depósitos de complexos antígenos-anticorpo pré-formados na circulação. MÉTODOS: Hamsters foram inoculados com ovos embrionados de Toxocara canis, e mantidos no biotério do Instituto de Medicina Tropical de São Paulo. O estudo histopatológico foi desenvolvido utilizando-se cortes parafinados corados por hematoxilina e eosina. Para detecção de antígenos nos tecidos foram realizadas reações imunohistoquímicas, utilizando-se anticorpo monoclonal e policlonal anti- Toxocara canis. Utilizando-se o soro dos animais infectados e animais controle, foi realizada pesquisa de antígeno e anticorpo por ELISA. Para pesquisa de imunoglobulinas IgG e IgM e complemento, foram utilizados cortes congelados de rins para realização de reação de Imunofluorescência. Fragmentos de rins foram incluídos para utilização em microscopia eletrônica, para detecção de antígenos de toxocara e de imune complexos. Para caracterização de resposta imunológica foram estudadas citocinas envolvidas na resposta Th1 e Th2 por técnica de RT-PCR. RESULTADOS: Os achados histopatológicos demonstraram desde o início da infecção, presença de larvas em maior número no fígado, seguido de pulmão e raramente rins. Em fígado remanescentes larvares foram visualizados cercados por reação inflamatória granulomatosa. Logo no início da infecção foi encontrado pneumonite intersticial e intraalveolar focal, e lesão renal com glomérulo apresentando hiperplasia focal de células mesangiais (glomerulite mesangio-proliferativa). Houve marcação de antígenos em todos os grupos de animais infectados, tanto pelo anticorpo monoclonal, como pelo policlonal. Depósitos de imunoglobulinas e complemento foram marcados em glomérulo por imunofluorescência A análise dos soros por ELISA, demonstrou na pesquisa de anticorpos aumento gradativo no decorrer da infecção, acompanhado de diminuição de antígenos. Depósitos de antígenos em glomérulos foram detectados por microscopia imonoeletrônica. No RT-PCR foi detectado aumento significativo do nível de IL-4, com tendência de elevação de IL-10 e IFN-?. CONCLUSÃO: O hamster demonstrou ser um modelo experimental eficiente para toxocaríase. Entretanto este modelo é mais adequado para infecções de curto prazo. A resposta imunológica avaliada por RT-PCR, com elevado nível da expressão de IL-4, sugere uma resposta Th2, mas a tendência de aumento de IL-10 e IFN-? poderia sinalizar uma resposta mista Th1 e Th2. Achados de depósitos de imunoglobulinas no glomérulo sugerem a possibilidade de que as manifestações renais com síndrome nefrótica em humanos possa vir a ter como base a toxocaríase / INTRODUCTION: Toxocariasis is a parasitic infection of global distribution, caused by the larval stage of Toxocara spp. The natural hosts are dogs and cats, in which the parasite completes the cycle reaching adulthood. Other hosts can be infected with the larval stage of the parasite, after ingestion of embryonated eggs from the soil, contaminated hands, fomites, or ingestion of meat or viscera of infected animals. In paratenics hosts the parasite not complete the cycle, encroaching on larval stage in viscera or other tissues where they can survive and induce pathology. The present study aimed to characterize the hamster, Mesocricetus auratus, as experimental model of toxocariasis, initially through the study of histopathological lesions in the liver, lung and kidney. The characterization of immune response model, was made through the study of cytokines Th1 and Th2 responses involved, and a correlation was suggested between glomerular changes and antibody-antigen complexes deposits preformed in the circulation. METHODS: Hamsters were inoculated with embryonated eggs of Toxocara canis, and kept in the bioterium of the Institute of Tropical Medicine of the São Paulo. The histopathologic study was developed using paraffin slides stained by hematoxylin and eosin. For detection of antigens in tissues immunohistochemistry reactions were performed using monoclonal and polyclonal anti-Toxocara canis sera. Using the serum of infected and control animals, search has been carried out of antigen and antibody by ELISA. For the search of immunoglobulins IgG, IgM and complement, were used slides prepared from frozen fragments of kidneys and a immunofluorescence reaction. Fragments of kidneys were included for electron microscopy to detect antigens of Toxocara and immune complexes. For characterization of Th1 and Th2 response cytokines involved were detected by RT-PCR technique. RESULTS: Histopathological findings demonstrated since the beginning of the infection the presence of larvae in greater numbers in the liver, followed by lung and rarely kidneys. In the liver larval remnants were surrounded by a granulomatous inflammatory reaction. Early in the infection was found interstitial pneumonitis with intraalveolar focal inflammatory infiltrate and renal injury with glomerulus showing mesangial cell focal hyperplasia (mesangioproliferative glomerulonephritis). There were the presence of antigens in all groups of animals infected detected by both the monoclonal and polyclonal antibodies. Deposits of immunoglobulin and complement were present in glomerulus by immunofluorescence analysis. ELISA, showed that the presence of antibodies increased gradually in the course of infection, accompanied by progressive diminution of antigens. Clusters of antigen/s were detected by immunoelectron microscopy. RT-PCR showed a significant increase of IL-4, with a tendency of increase of IL- 10 and IFN-?. CONCLUSION: The hamster has proved to be an efficient experimental model for toxocariasis. However this model is best suited for short-term infections. The immune response evaluated by RT-PCR, with high level of expression of IL-4, suggests a Th2 response, but the trend of increase of IL-10 and IFN-? might suggest a Th1 and Th2 mixed response. Findings of immunoglobulin deposits in glomeruli suggests the possibility that the renal manifestations with nephrotic syndrome in humans might have, in certain circunstances, as a basis the toxocariasis

L’Hypertensions Artérielle Pulmonaire (HTAP) résulte de l’obstruction progressive des artères pulmonaires de petits calibres due à un remodelage de la paroi vasculaire ainsi qu’à une vasoconstriction. Cette thèse repose sur 3 piliers de la physiopathologie de l’HTAP : l’inflammation, l’épigénétique et la susceptibilité génétique de la maladie.Le premier projet démontre que les cellules dendritiques des patients atteints d’HTAP sont moins sensibles à l’action immunomodulatrice des glucocorticoïdes et orientent la réponse des lymphocytes T vers une réponse Th17, souvent impliquée dans les maladies autoimmunes.Le second projet a pour but de comparer le profil de méthylation des cellules endothéliales d’artères pulmonaires (CE-AP) de patients atteints d’HTAP comparé aux CE-AP de patients contrôles. Nous avons mis en évidence des clusters de gènes ayant un profil de méthylation différents entre les CE-HTAP comparé aux CE-CTR. Un gène différentiellement méthylé s’est dégagé durant l’étude bioinformatique : ABCA1 (ATP binding cassette 1). L’altération de son expression prédite par l’étude bioinformatique a été validée chez l’homme et le modèle de rat monocrotaline.Enfin, le dernier projet décrit la caractérisation d’un nouveau modèle animal de susceptibilité génétique à l’HTAP liée à des mutations dans le gène Bmpr2 chez le rat, au niveau hémodynamique, histologique, vasculaire, moléculaire et électrophysiologique. Nous avons démontré que ce modèle reproduit des schémas physiopathologiques pertinents vis-à-vis de la maladie humaine, ces résultats font de ce modèle un nouveau outil d’étude de la physiopathologie de l’HTAP. / Pulmonary arterial hypertension (PAH) is characterized by a progressive pulmonary arterial obstruction due to pulmonary vascular remodeling of distal arterioles as well as abnormal vasoconstriction. This project allowed to study three biological process clearly establish to be implicated in physiopathology of PAH.The first project demonstrated a dendritic cells dysfunction and a Th17 immune polarization of idiopathic PAH patients.The objective of the second project was to study the epigenetic variations in pulmonary endothelial cells (PEC) through a specific pattern of DNA methylation. We identified clusters of probes that discriminates controls and PAH patients. During bioinformatics study, ABCA1 (ATP binding cassette 1) gene was emphasized. Alteration of ABCA1 expression predicted during bioinformatics study has been validated in human and in monocrotaline rat model.The last project described the validation of a new PAH model by a hemodynamic, histological, vascular, molecular and electrophysiological characterization of heterozygous rat mutated to Bmpr2 gene. Whole functional and molecular dysregulation define this animal model like a useful tool in the study of BMPRII signaling alteration in PAH physiopathology.

La contribution de l’inhalation et de la voie percutanée à l’exposition totale à des composés organiques volatils (COV) présents dans l’eau potable est une problématique qui suscite un intérêt grandissant en santé publique et au niveau réglementaire. Jusqu’à tout récemment, seule l’ingestion était considérée dans l’évaluation du risque des contaminants de l’eau. L’objectif de ce projet était de caractériser l’impact de l’exposition multivoie sur la pharmacocinétique et la dose interne de trois COV : le toluène (TOL), le n-hexane (HEX) et le cyclohexane (CYCLO). À cette fin, un modèle expérimental animal a été utilisé et un modèle toxicocinétique à base physiologique (TCBP) a été adapté pour le TOL. Des rats Sprague-Dawley ont été exposés par voies uniques (inhalation, orale et percutanée) ou simultanées (multivoie) aux solvants. Pour le TOL, les trois voies ont été expérimentées, alors que la voie percutanée n’a pas été retenue pour le HEX et le CYCLO. Des prélèvements sanguins ont permis de caractériser les cinétiques sanguines. Les niveaux sanguins, obtenus lors des expositions multivoies, étaient généralement plus élevés que la somme des niveaux associés aux expositions par voies uniques, fait illustré par le rapport des surfaces sous la courbe expérimentale versus les prédictions (TOL : 1,30 et 2,19 ; HEX : 1,55 ; CYCLO : 0,98 et 0,99). Le modèle TCBP prédit bien la cinétique du TOL lors d’expositions par voies uniques et par multivoies. Les données expérimentales obtenues suggèrent que la dose interne résultant d’une exposition multivoie ne peut pas toujours être prédite par la somme des doses internes obtenues lors d’expositions par voies uniques. Ce phénomène serait explicable par la saturation du métabolisme. La modélisation TCBP est un outil efficace pour l’estimation du risque relatif à l’exposition multivoie aux COV présents dans l’eau potable. / The contribution of dermal and inhalation routes of exposure to volatile organic chemicals (VOCs) in drinking water is increasingly drawing attention. Until recently, ingestion was the only route considered in risk assessment of drinking water contaminants. The general objective of this study was to characterize multi-route exposures on the pharmacokinetics and internal dose of three VOCs: toluene (TOL), n-hexane (HEX) and cyclohexane (CYCLO). Towards this goal, an experimental animal model was developed and a physiologically-based toxicokinetic (PBTK) model was adapted in order to take account route-specific absorption parameters of TOL. Sprague-Dawley rats were given a single dose of VOCs by oral ingestion, inhalation or dermal route. Additional groups of rat were exposed by the three routes simultaneously. In the case of HEX and CYCLO, dermal route was not considered. Blood samples were collected in order to compare kinetics following simple and multiroute exposures. Blood levels obtained following multiroute exposures were generally higher than predictions (i.e., the sum of the blood levels obtained for single route exposures) (as revealed by area under curve ratio: TOL :1,30 et 2,19; HEX : 1,55; CYCLO : 0,98 et 0,99). The PBTK model described adequately the kinetics of TOL in rats following single and multiroute exposures. The results suggest that internal dose arising from multiple routes is not necessarely comparable to the sum of the blood levels obtained for single route exposures. This phenomenon would be attributable to degree of saturation during aggregate exposures. Furthermore, the present study demonstrated that PBTK model can be used to predict/interpret blood levels associated with multiroute exposures to VOCs in drinking water, using toluene as a model substance.

This thesis concerns the investigation of novel immunotherapies for cancer eradication. CpG therapy was used in order to target antigen-presenting cells (APCs), facilitating antigen presentation and activation of T cells. Blockade of the two major immune checkpoint regulators (CTLA-4 and PD-1) was also studied to ensure proper and sustained T cell activation. The therapies were investigated alone and compared to BCG, the standard immunotherapy in the clinic today for bladder cancer. In addition, CpG as well as BCG was combined with CTLA-4 or PD-1 blockade to examine if the combination could improve therapy. Single and combination strategies were assessed in an experimental bladder cancer model. In addition, one of the therapies (local aCTLA-4 administration) was evaluated in an experimental pancreatic cancer model. To be able to study the effects of CpG in humans, a human whole blood loop system has been used. This allowed us to dissect the potential interplay between CpG and complement. CpG was found to be superior to the conventional therapy, BCG, in our experimental model and T cells were required in order for effective therapy to occur. Used as a monotherapy, CTLA-4 blockade but not PD-1 blockade, prolonged survival of mice. When CTLA-4 or PD-1 blockade was combined with CpG, survival was enhanced and elevated levels of activated T cells were found in treated mice. In addition, Treg levels were decreased in the tumor area compared to tumors in control treated mice. CTLA-4 blockade was also effective when administrated locally, in proximity to the tumor. Compared to systemic CTLA-4 blockade, local administration gave less adverse events and sustained therapeutic success. When CpG was investigated in a human whole blood loop system it was found to tightly interact with complement proteins. This is an interesting finding which warrants further investigation into the role of TLRs in complement biology. Tumor therapy could be affected either negatively or positively by this interaction. The results presented herein are a foundation for incorporating these combination therapies into the clinic, specifically for bladder cancer but in a broader perspective, also for other solid tumors such as pancreatic cancer.

La contribution de l’inhalation et de la voie percutanée à l’exposition totale à des composés organiques volatils (COV) présents dans l’eau potable est une problématique qui suscite un intérêt grandissant en santé publique et au niveau réglementaire. Jusqu’à tout récemment, seule l’ingestion était considérée dans l’évaluation du risque des contaminants de l’eau. L’objectif de ce projet était de caractériser l’impact de l’exposition multivoie sur la pharmacocinétique et la dose interne de trois COV : le toluène (TOL), le n-hexane (HEX) et le cyclohexane (CYCLO). À cette fin, un modèle expérimental animal a été utilisé et un modèle toxicocinétique à base physiologique (TCBP) a été adapté pour le TOL. Des rats Sprague-Dawley ont été exposés par voies uniques (inhalation, orale et percutanée) ou simultanées (multivoie) aux solvants. Pour le TOL, les trois voies ont été expérimentées, alors que la voie percutanée n’a pas été retenue pour le HEX et le CYCLO. Des prélèvements sanguins ont permis de caractériser les cinétiques sanguines. Les niveaux sanguins, obtenus lors des expositions multivoies, étaient généralement plus élevés que la somme des niveaux associés aux expositions par voies uniques, fait illustré par le rapport des surfaces sous la courbe expérimentale versus les prédictions (TOL : 1,30 et 2,19 ; HEX : 1,55 ; CYCLO : 0,98 et 0,99). Le modèle TCBP prédit bien la cinétique du TOL lors d’expositions par voies uniques et par multivoies. Les données expérimentales obtenues suggèrent que la dose interne résultant d’une exposition multivoie ne peut pas toujours être prédite par la somme des doses internes obtenues lors d’expositions par voies uniques. Ce phénomène serait explicable par la saturation du métabolisme. La modélisation TCBP est un outil efficace pour l’estimation du risque relatif à l’exposition multivoie aux COV présents dans l’eau potable. / The contribution of dermal and inhalation routes of exposure to volatile organic chemicals (VOCs) in drinking water is increasingly drawing attention. Until recently, ingestion was the only route considered in risk assessment of drinking water contaminants. The general objective of this study was to characterize multi-route exposures on the pharmacokinetics and internal dose of three VOCs: toluene (TOL), n-hexane (HEX) and cyclohexane (CYCLO). Towards this goal, an experimental animal model was developed and a physiologically-based toxicokinetic (PBTK) model was adapted in order to take account route-specific absorption parameters of TOL. Sprague-Dawley rats were given a single dose of VOCs by oral ingestion, inhalation or dermal route. Additional groups of rat were exposed by the three routes simultaneously. In the case of HEX and CYCLO, dermal route was not considered. Blood samples were collected in order to compare kinetics following simple and multiroute exposures. Blood levels obtained following multiroute exposures were generally higher than predictions (i.e., the sum of the blood levels obtained for single route exposures) (as revealed by area under curve ratio: TOL :1,30 et 2,19; HEX : 1,55; CYCLO : 0,98 et 0,99). The PBTK model described adequately the kinetics of TOL in rats following single and multiroute exposures. The results suggest that internal dose arising from multiple routes is not necessarely comparable to the sum of the blood levels obtained for single route exposures. This phenomenon would be attributable to degree of saturation during aggregate exposures. Furthermore, the present study demonstrated that PBTK model can be used to predict/interpret blood levels associated with multiroute exposures to VOCs in drinking water, using toluene as a model substance.