1,25-dihydroxyvitamin D3 (Vitamin D) is a potent immunomodulator capable of generating regulatory T cells (Tregs) and contributing to immune tolerance. Additionally, vitamin D has been shown to promote mucosal immunity when used as a vaccine adjuvant. We show here that pretreatment of an area of skin with the synthetic vitamin D analog calcipotriol combined with transcutaneous immunization results in the induction of CD4⁺CD25⁺ Tregs capable of inhibiting the elicitation of a contact hypersensitivity response. We also demonstrate that topical calcipotriol has significant effects on the immune response to subcutaneously injected vaccines, and compare it with another common topical immunosuppressant, the corticosteroid betamethasone-17-valerate (BMV). Functionally, calcipotriol and BMV treatment both result in the suppression of CD8⁺ T cell priming in response to subcutaneous vaccination, despite the topical co-administration of the potent Th1 inducing TLR9 agonist unmethylated CpG DNA. The effects of calcipotriol on the humoral response are subtler as we observe marginally increased production of antigen-specific IgG1 immunoglobulins along with a strong suppression of the IgG2a isotype. This is in contrast to pretreatment with BMV, which instead suppresses the production of IgG1 and IgA antibodies. In the draining lymph nodes of calcipotriol treated animals, we see no change in the percentage of Foxp3⁺ CD4⁺ T cells post-immunization, but show that tolerance is transferable with the adoptive transfer of CD4⁺CD25⁺ cells. Despite a decrease in the percentage of antigen-bearing APCs in the DLN of calcipotriol treated animals, the DCs maintain high expression of co-stimulatory markers and can induce CD4⁺ T cell proliferation ex vivo. Our data indicate that calcipotriol has distinct effects on immune responses to subcutaneous vaccines consistent with its role as an immunomodulator, although the mechanism(s) through which it is acting remain unclear. We believe that further research is warranted into its potential use as part of a treatment modality for allergy and autoimmune disorders. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Mansoa hirsute (Bignoniaceae), popularly known as Cipo d'álho, is chemically little known, although it is popularly used in the treatment of many disesases. The present studiy aimed to study the biological potential of the extracts and fractions obtained from leaves of M. hirsuta, to isolate and purify the active principles and to study the metabolomics of the plant. Plant materials were collected in five different regions from Semi-Arid in northeastern Brazil and were submitted to classic phytochemistry and analysis of the metabolomics. For that, the brute extract of the plants of Gentio do Ouro was obtained by extraction with ethanol 96% and was submitted to partitioning with different solvents, while the volatile were extracted by Clavenger apparatus. The isolated compounds were identified by GC-MS and NMR. Extract and subfractions were submitted to biological tests to clarify their possible biological activities. The brute extracts of the plants provenientes of the five regions was submitted to NMR analysis RMN to adquire 1D 1H and 2D COSY and J-resolved spectra. NMR data were submitted to chemiometry (HCA, PC and PSL-of the) to classify the species according to their metabolome. Pentacyclic triterpenes ursólico and oleanolic acids, and the steroids stigmasterol, ß-stitosterol and lupeol were isolated, together with dialyl sulfide and trialyl sulfide. The ethyl acetate fraction was able to inhibit the production of NO (57.92%) and proliferation of linfocites (99.92%), while the acid ursólico and oleanolic presented inhibition values greater than 99%. Metabolic analysis allowed to establish that plants of Gentio do Ouro are rich in pentacyclic triterpens, while plants from Boninal contains mostly flavonoids in its chemical composition. The results also suggest that protoantocianidinas are the main constituents of the plants from Caetité, while saponinas are found in the plants from Campo Alegre de Lourdes. Plants of Morro do Chapéu were distinguished form the other regions for being rich in oligosaccharides and aminoacids. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A espécie Mansoa hirsuta (Bignoniaceae), popularmente conhecida como Cipó d'alho, é quimicamente pouco conhecida, embora seja popularmente utilizada no tratamento de muitas infecções. O presente trabalho objetivou estudar o potencial biológico dos extratos e frações obtidos das folhas de M. hirsuta, isolar e purificar os possíveis princípios ativos e estudar o perfil metabolômico da planta. O material vegetal foi coletado em cinco regiões diferentes do Semi-Árido nordestino e foi submetido à fitoquímica clássica e análise metabolômica. Para tanto, o extrato bruto das plantas de Gentio de Ouro foi obtido pela extração com etanol 96% e submetido a fracionamento com diferentes solventes, enquanto os voláteis foram extraídos por Clavenger. Os compostos isolados foram identificados por GC-MS e dados de RMN. O extrato e as subfrações foram submetidos a testes biológicos para esclarecer suas possíveis atividades biológicas. Os extratos brutos das plantas provenientes das cinco regiões foi submetido a análise por RMN para obtenção de espectros 1D 1H e 2D COSY e J-resolvido. Os dados provenientes do RMN foram então submetidos à métodos quimiométricos (HCA, PCA e PSL-DA) com vistas a classificar as espécies de acordo com seu metaboloma. Os triterpenos pentacíclicos ácidos ursólico e oleanólico e os esteróides estigmasterol, β-stitosterol e lupeol foram isolados, juntamente com o dissulfeto de dialila e trissulfeto de dialila. A fração acetato de etila inbiu a produção de NO (57,92%) e a linfoproliferação (99.92%), enquanto os ácidos ursólico e oleanólico apresentaram valores de inibição maiores que 99%. A análise metabólica permitiu estabelecer que as plantas oriundas de Gentio de Ouro são ricas em triterpernos pentacíclicos, enquanto as plantas de Boninal contêm principalmente flavonóides em sua composição química. Os resultados também sugerem que as protoantocianidinas são os principais constituintes das plantas oriundas de Caetité, enquanto saponinas são encontradas na plantas de Campo Alegre de Lourdes. As plantas de Morro do Chapéu diferem daquelas das demais regiões por serem ricas em oligossacarídeos e aminoácidos.

Orientador: Liana Maria Cardoso Verinaud / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T08:18:08Z (GMT). No. of bitstreams: 1 Zanucoli_FabiodosSantosMachado_M.pdf: 3072241 bytes, checksum: 66a7959825671f33da79f21f4dbeda55 (MD5) Previous issue date: 2015 / Resumo: A esclerose múltipla (EM) é uma doença inflamatória crônica de origem autoimune que afeta o sistema nervoso central (SNC), provocando disfunções neurológicas em decorrência de desmielinização axonal, principalmente na substância branca dos órgãos do SNC. A doença acomete mais de 2 milhões de pessoas no mundo todo e não tem cura. As abordagens terapêuticas utilizadas com sucesso consistem na aplicação de anticorpos monoclonais, citocinas imunomoduladoras e fármacos antiinflamatórios. Ainda assim, tais estratégias são dependentes de produtos de alto custo e toxicidade. A utilização de fármacos antimaláricos no tratamento da Encefalomielite Autoimune Experimental (EAE, modelo experimental de EM), como cloroquina e diidroartemisinina, tem apresentado resultados promissores na atenuação do quadro clínico dos camundongos; entretanto, os efeitos tóxicos dessas substâncias constituem um importante fator limitante para a utilização clínica. Neste sentido, o presente trabalho teve por objetivo caracterizar os efeitos terapêuticos da primaquina (PQ), um fármaco antimalárico análogo à cloroquina mas com toxicidade reduzida, na EAE emvcamundongos C57BL/6. Para tanto, camundongos foram tratados com PQ por via intraperitoneal (i.p.) por cinco dias consecutivos e os efeitos da administração direta foram avaliados sobre as subpopulações de linfócitos T esplênicos. Semelhantemente, camundongos portadores de EAE foram tratados com PQ como acima e o desenvolvimento da doença foi avaliado. Os resultados obtidos mostraram uma redução significativa na severidade da EAE em camundongos tratados com primaquina em comparação com animais pertencentes ao grupo controle. A melhora no quadro clínico se correlacionou com (i) aumento na expressão de genes de citocinas anti-inflamatórias e decréscimo na expressão de genes de citocinas inflamatórias, (ii) redução na ativação de micróglia e (iii) diminuição da reatividade glial no SNC de camundongos tratados com PQ em relação aos não tratados. Também se verificou um aumento na frequência de células T reguladoras em baços de camundongos tratados com PQ. Posteriormente, células T reguladoras (Treg, CD4+CD25+) e células T efetoras (CD4+CD25- ) foram isoladas de baços de camundongos tratados com PQ e transferidas adotivamente a camundongos portadores de EAE. Foi observada uma redução na severidade da EAE em camundongos recipientes de células Treg em comparação com camundongos recipientes de células T efetoras. De forma semelhante ao observado em camundongos tratados diretamente com PQ, a menor severidade da EAE se correlacionou com redução na expressão de mediadores inflamatórios e de reatividade glial no SNC. A capacidade imunossupressora de PQ foi avaliada também. Neste sentido, células dendríticas (DCs) diferenciadas a partir de precursores de medula óssea foram tratadas com PQ e avaliadas quanto ao seu perfil fenotípico e funcional. Observou-se uma redução na expressão de marcadores de maturação de DCs tratadas com PQ (DC-PQ) em comparação às células não tratadas (DC-PBS). A transferência adotiva de DC-PQ resultou na redução do quadro clínico de EAE. Os resultados obtidos nesse trabalho permitem concluir que a primaquina é um fármaco eficaz na atenuação dos sintomas da EAE por meio da modulação de componentes celulares e moleculares do sistema imune / Abstract: Multiple sclerosis (MS) is an autoimmune chronic inflammatory disease that affects the central nervous system (CNS) causing neurological disorders arised from axonal demyelination, particularly in the white matter of the CNS organs. The disease affects more than 2 million people worldwide and still has no cure. The successfully used therapeutic approaches consist in the application of monoclonal antibodies, immunomodulatory cytokines and anti-inflammatory drugs. Still, such strategies are dependent on expensive and toxic drugs. The use of antimalarial drugs in the treatment of experimental autoimmune encephalomyelitis (EAE, experimental model of MS), such as chloroquine and dihydroartemisinin, has shown promising results in attenuation of the clinical signs of mice; however, the toxic effects of these substances are an important limiting factor for clinical use. In this sense, this study aimed to characterize the therapeutic effects of primaquine (PQ), an antimalarial drug analog to chloroquine but with reduced toxicity, in EAE in C57BL / 6 mice. To this end, mice were treated with PQ intraperitoneally (i.p.) for five consecutive days, and the effects of direct administration were evaluated on spleen T lymphocyte subpopulations. Similarly, mice bearing EAE were treated with PQ as above, and disease development was evaluated. The results showed a significant reduction in the severity of EAE in mice treated with primaquine compared to animals in the control group. Amelioration of the clinical signs was correlated with (i) an increase in gene expression of anti-inflammatory cytokines and decrease in gene expression of inflammatory cytokines, (ii) reduction in microglia activation and (iii) the decreased CNS glial reactivity in mice treated with PQ compared to untreated group. There was also an increase in the frequency of regulatory T cells in the spleens of mice treated with PQ. Subsequently, regulatory T cells (Tregs, CD4+CD25+ ) and effector T cells (CD4+CD25-) were isolated from spleens of mice treated with PQ, and adoptively transferred to mice with EAE. It was observed a reduction in the severity of EAE in mice recipients of Treg cells compared to mice recipients of effector T cells. Similarly to what was observed in mice directly treated with PQ, the lower severity of EAE was correlated with a reduction in the expression of inflammatory mediators and glial reactivity in the CNS. The immunosuppressive capacity of PQ was evaluated as well. In this regard, dendritic cells (DCs) differentiated from bone marrow precursors were treated with PQ and assessed for phenotypic and functional profile. It was observed a reduction in the expression of DC maturation markers treated with PQ (DC-PQ) compared to untreated cells (DC-PBS). The adoptive transfer of DC-PQ resulted in the reduction of the clinical signs of EAE. The results of this work have shown that primaquine is an effective drug in ameliorating the symptoms of EAE through modulation of cellular and molecular components of the immune system / Mestrado / Imunologia / Mestre em Genética e Biologia Molecular

As Doenças inflamatórias intestinais (DII) são multifatoriais e sua etiologia envolve susceptibilidade genética, fatores ambientais, disbiose e ativação exacerbada do sistema imunológico no intestino. Essas doenças também tem sido relacionadas a baixos níveis de dehidroepiandrosterona (DHEA), um hormônio precursor de diversos esteroides e relacionado à modulação das respostas imunes. Porém, os mecanismos precisos que relacionam as ações deste hormônio com a proteção ou susceptibilidade à doença de Crohn ou colite ulcerativa ainda não são totalmente conhecidos. Sendo assim, este projeto buscou entender o papel imunomodulador do DHEA exógeno in vitro e in vivo durante a inflamação intestinal experimental induzida por dextran sulfato de sódio (DSS) em camundongos C57BL/6. Inicialmente, in vitro, DHEA inibiu a proliferação de células do baço de forma dose dependente nas concentrações de 5?M, 50?M ou 100?M, com diminuição da produção de IFN-?. Este hormônio não foi tóxico para células de linhagem mieloide, embora tenha causado necrose em leucócitos nas doses mais elevada (50 ?M e 100?M), o que pode ter influenciado a diminuição das citocinas in vitro. Nos ensaios in vivo, os camundongos tratados com DHEA (40 mg/Kg) foram avaliados na fase de indução da doença (dia 6) e durante o reparo tecidual, quando os animais expostos ao DSS e ao DHEA por 9 dias foram mantidos na ausência destas drogas até o dia 15. Houve diminuição do escore pós-morte, melhora no peso e nos sinais clínicos da inflamação intestinal, com redução de monócitos no sangue periférico com 6 dias e aumento de neutrófilos circulantes na fase de reparo tecidual (15 dias). Ainda, a suplementação com DHEA levou à redução da celularidade da lâmina própria (LP) e ao restabelecimento do comprimento normal do intestino. O uso deste hormônio também diminuiu a expressão do RNAm de IL-6 e TGF-?, enquanto aumentou a expressão de IL-13 no colón dos animais durante a fase de indução da doença, o que provavelmente ajudou na atenuação da inflamação intestinal. Além disso, houve acúmulo de linfócitos CD4+ e CD8+ no baço e diminuição apenas de linfócitos CD4+ nos linfonodos mesentéricos (LNM), indicando retenção das células CD4+ no baço após uso do DHEA. O tratamento foi também capaz de aumentar a frequência de células CD4 produtoras de IL-4 e diminuir CD4+IFN-?+ no baço, além de reduzir a frequência de CD4+IL-17+ nos LNM, sugerindo efeito do DHEA no balanço das respostas Th1/Th2/Th17 relacionadas à colite. Em adição, as células de baço dos animais tratados com DHEA e expostos ao DSS se tornaram hiporresponsivas, como visto pela diminuição da proliferação após re-estímulos in vitro. Finalmente, DHEA foi capaz de atuar no metabolismo dos camundongos tratados, levando à diminuição de colesterol total e da fração LDL no soro durante a fase de indução da doença, sem gerar quaisquer disfunções hepáticas. Com isso, podemos concluir que o DHEA atua por meio do balanço das respostas imunes exacerbadas, minimizando os danos locais e sistêmicos causados pela inflamação intestinal induzida por DSS. / Inflammatory bowel diseases (IBD) are multifactorial diseases whose etiology involves genetic susceptibility, environmental factors, dysbiosis and exacerbated activation of the immune system in the gut. These diseases have also been associated to lower levels of dehydroepiandrosterone (DHEA), a precursor of various steroid hormones, related to modulation of immune responses. However, the precise mechanisms that link the actions of this hormone with protection or susceptibility to Crohn\'s disease or ulcerative colitis are still not fully understood. Thus, this project aimed to understand the immunomodulatory role of exogenous DHEA in vitro and in vivo in experimental intestinal inflammation induced by dextran sodium sulfate (DSS) in C57BL/6 mice. Initially, in vitro, DHEA inhibited the proliferation of spleen cells in a dose dependent way on the concentrations of 5?M, 50?M and 100?M, with decreased production of IFN-?. This hormone was not toxic to myeloid lineage cells, although it caused necrosis of leukocytes at the highest doses (50?M and 100?M), which may have influenced the decrease of the cytokines in vitro. Mice treated with DHEA (40 mg / kg) were evaluated at the induction phase of the disease (day 6) and during tissue repair, when animals exposed to DSS and DHEA for 9 days were maintained in the absence these drugs until the day 15. There was decrease of postmortem score, improved weight and clinical signs of intestinal inflammation, besides reduced peripheral blood monocytes on day 6, together with an increase in circulating neutrophils in tissue repair phase (15 days). Supplementation with DHEA also led to a reduction in cellularity of the lamina propria (LP) and to the restoration of normal length of the gut. The use of this hormone also decreased the expression of of IL-6 and TGF-? mRNA, while IL-13 was augmented in the colon of mice during the induction phase of the disease, a fact probably related to attenuation of intestinal inflammation. Furthermore, there was accumulation of CD4+ and CD8+ cells in the spleen along with decreased CD4+ leukocytes in mesenteric lymph nodes (MLN), indicating retention of CD4+ cells in the spleen after use of DHEA. The treatment was also able to increase the frequency of CD4+ cells producing IL-4 and decrease CD4+IFN-?+ in spleen, with reduced frequency of CD4+IL-17+ in the MLN, suggesting a role for DHEA on the balance of Th1/Th2/Th17 responses related colitis. In addition, splenocytes of mice treated with DHEA and exposed to DSS became hiporresponsives as seen by decreased proliferation after re-stimulation in vitro. Finally, DHEA was able to act on the metabolism of treated mice, leading to decreased total cholesterol and LDL cholesterol in serum during the induction phase of the disease, without generating any liver dysfunction. Thus, we concluded that DHEA acts by balancing the exacerbated immune responses, minimizing local and systemic damages caused by intestinal inflammation induced by DSS.

Os alimentos desempenham papel fundamental na nutrição e também na imunidade, pois podem conter substâncias que interagem direta ou indiretamente com o sistema imune, principalmente o de mucosas. Alguns polissacarídeos não-amido (PNAs) originados de plantas, algas e fungos comestíveis podem modular a função imune, contribuindo para a manutenção da saúde. A banana apresenta composição monossacarídica da parede celular similar à de plantas com efeitos imunomoduladores, permitindo formular a hipótese de que os PNAs dessa fruta também tenham essas propriedades. Assim, o objetivo foi extrair, purificar e caracterizar PNAs hidrossolúveis da polpa de banana das cultivares Nanicão e Thap Maeo e testá-los em cultivos de macrófagos RAW 267.4, células THP-1, macrófagos diferenciados por PMA THP-1 e células HL-60 in vitro avaliando a ação imunomoduladora através da dosagem de citocinas, NO e ensaios de fagocitose. As frações hidrossolúveis foram caracterizadas quanto ao seu conteúdo monossacarídico e ligações por hidrólise enzimática, identificando homogalacturonanos, mananos, arabinogalactanos, xilogalacturonanos e galactoglucomananos. As frações foram testadas para ausência de endotoxinas e viabilidade celular por MTT. Foram estabelecidas as doses não-tóxicas e ensaiadas as concentrações de 10, 50 e 200 µg.ml-1 das frações polissacarídicas para os testes in vitro. Não houve toxicidade para macrófagos e monócitos, enquanto que para a linhagem de células pro-mielóciticas de leucemia humana HL-60, as frações se mostraram citotóxicas. Houve aumento de atividade fagocítica nas frações WSP, UFP e HTP quando comparadas ao controle negativo de células, assim como a produção de citocinas inflamatórias como IL-1β, TNF-α, IL-6, além da quimiocina IL-8 nas células oriundas de THP-1 e diminuição nos marcadores TLR-2 com aumento de CD14 nas células THP-1, além do aumento do tamanho celular promovido pelas frações polissacarídicas mostrando diferenciação para série granulocítica. Portanto, PNAs de ambas as cultivares de banana possuem potencial imunomodulador seja inflamatório, anti-inflamatório ou de diferenciação de células THP-1 imaturas in vitro. / Food plays a major role in nutrition as in immunity, as they may contain substances that interact directly or indirectly with the immune system, especially those located on mucous membranes. Some non-starch polysaccharides (NSPs) originate from plants, algae, and edible fungi can modulate immune function, contributing to the maintenance of health. The banana presents cell wall monosaccharide composition like the other plant polysaccharides with immunomodulatory effects allowing to formulate the hypothesis that this fruit NSPs also have these properties. The objective was to extract, purify and characterize water-soluble banana pulp NSPs of Nanicão and Thap Maeo cultivars and test them in vitro on cell lineages of RAW 267.4 macrophages, PMA differentiated THP-1 macrophages, THP-1 monocytes, and HL-60, evaluating their immunomodulatory action by the dosage of cytokines, NO and phagocytosis assays. Water-soluble fractions were characterized as to their content and monosaccharide linkages by enzymatic hydrolysis, identifying homogalacturonans, mannans, arabinogalactans, xylogalacturonan, and galactoglucomannans in its composition. Fractions were tested for the absence of endotoxins and cell viability by MTT. Non-toxic concentrations doses of 10, 50 and 200 µg.ml-1 of the polysaccharide fractions were established for in vitro testing. The NSPs fractions WSP, UFP, and HTP tested promoted an increase in phagocytic activity in THP-1 PMA derived macrophages compared to negative control cells, as well as the production of inflammatory cytokines such as IL-1β, TNF-α, IL-6. An increase in the THP-1 CD14 cell receptor and decrease in CD33, CD123, CD11c and TLR-2 receptors were promoted by the banana polysaccharides. Besides, there was an increase in cell size showing cell differentiation towards granulocyte line and demonstrating that NSPs from both cultivars were immunomodulatory with a capacity of immature cell differentiation and proving that in vitro screening test is an efficient method to test other foodborne substances with immunomodulatory potential.

La grande originalité de mon projet réside dans la génération de cellules stromales mésenchymateuses (MSCs) à partir de cellules souches pluripotentes induites humaines (iPS). Je rappellerai les propriétés phénotypiques, de multipotence et immunosuppressives des MSCs et m’attarderai sur leurs différents mécanismes immunomodulateurs. Cependant, leur nombre limité et leur isolation difficile limitent leur utilisation thérapeutique nécessitant une autre source de cellules.Mon travail a donc été de générer et de caractériser des MSCs issues d’iPS (huiPS-MSCs). L'avantage des huiPS-MSCs réside dans leur plus grande disponibilité et la possibilité d'en avoir à volonté. Encore faut-il valider l’intérêt thérapeutique potentiel de ces huiPS-MSCs. Premièrement, mes résultats in vitro montrent que les huiPS-MSCs présentent une activité immunosuppressive sur les lymphocytes T (LT) activés conduisant à une induction de LT régulateurs FoxP3+ fonctionnels. Deuxièmement, dans une approche plus axée sur la thérapie, j’ai analysé in vivo l’activité́ immunosuppressive des huiPS-MSCs dans un modèle de réaction xénogénique de greffon contre l'hôte (souris immunodéficientes NSG injectées avec des LT humains). Je montre clairement, après traitement avec les huiPS-MSCs, une réduction de la proportion de LT humains producteurs de cytokines inflammatoires (IFNγ et TNFα) typiques de la pathologie et l’apparition concomitante de LT présentant un phénotype régulateur (production d’IL10 et expression de FoxP3). La fin de mon travail a été de caractériser moléculairement la régulation de l’expression de PD-L1, une molécule immuno-régulatrice puissante, entre les MSCs issues de la moelle osseuse (BM-MSCs) de donneurs sains et nos huiPS- MSCs. Les huiPS-MSCs ont une expression constitutive de PD-L1, qui est absente sur les BM-MSCs. J’ai analysé les microARNs susceptibles de limiter l’expression de PD-L1, j’ai pu en identifier plusieurs. En mesurant leur expression dans les différentes MSCs à notre disposition, je montre que cette expression est inverse par rapport à celle de PD-L1. J’ai ainsi pu démontrer l’activité immunosuppressive de nos huiPS-MSCs in vitro et in vivo avec une perspective d’induction de tolérance immune, et caractériser la régulation de l’expression de PD-L1, molécule immunosuppressive exprimée par les huiPS-MSCs. / The mesenchymal stromal cells (MSCs) present many features that render attractive as therapeutic cells. Their phenotype, multipotency and immunosuppressive properties are well described. Nevertheless, major restriction for their clinical use is due to the limited in vitro expansion and low quantity of cells that can be collected from adult tissues. The originality of my project consisted in the generation of mesenchymal stromal cells (MSCs) from human induced pluripotent stem cells (iPS). These huiPS-MSCs could fulfill some of the specification required to improve MSCs use in therapeutic approaches: welldefined and unlimited number of cells with reproducible functional characteristics. In a first approach, I characterized the huiPS-MSCs generated in the laboratory. My results highlight the immunosuppressive activity in vitro of the huiPS-MSCs on T-cell stimulation that induces a switch in T-cell cytokine polarization toward the generation of Treg cells. Secondly, in a more therapy-oriented approach, I analyzed in vivo immunosuppressive activity of huiPS-MSCs in a xenogeneic graft versus host model (NSG immunodeficient mice injected with human T lymphocytes). My data showed significantly reduced percentages of human-differentiated T cells producing Th1 inflammatory cytokines (IFNγ and TNFα). By contrast, T cells producing IL-10 and FoxP3+ Treg cells, absent in nontreated animals, were detected in huiPS-MSCs treated mice, confirming the in vitro results of a tolerizing process. The end of my work was to characterize the molecular regulation of the expression of PDL1, an immunoregulatory molecule expressed by the MSCs. Comparing bone marrow MSCs (BM-MSCs) from healthy donors and our huiPS-MSCs, I showed that the huiPSMSCs have a constitutive expression of PD-L1, which is absent on BM-MSCs. Analysing microRNAs that could limit the expression of PD-L1, I could identify several microRNAs which expression is inverse to the expression of PD-L1. For the first time, my results highlight the immunosuppressive activity of huiPS-MSCs on human T-cell stimulation with a concomitant generation of human Treg cells in vivo and characterize the regulation of PD-L1 expression, an immunosuppressive molecule expressed by the MSCs. They may favor the development of new tools and strategies based on the use of huiPS cells and their derivatives for the induction of immune tolerance.

A microencapsulação tem sido utilizada promissoramente para melhorar a viabilidade de probióticos. Porém, pouco se sabe sobre o impacto desta na manutenção da funcionalidade do probiótico in vivo. Este trabalho teve como objetivo avaliar duas cepas de lactobacilos, encapsular essas cepas por um sistema envolvendo partículas lipídicas recobertas por interação eletrostática de polímeros e avaliar o efeito da encapsulação na manutenção da capacidade imunomoduladora das cepas. Na primeira etapa do estudo L. rhamnosus 64 e L. paracasei BGP1 foram avaliados quanto à resistência à lisozima e aos fluidos gastrointestinais simulados, perfil de hidrofobicidade da parede celular, susceptibilidade a antibióticos, atividade antagonista contra patógenos e capacidade de utilização de prebióticos. Em etapa seguinte as cepas foram encapsuladas e as microcápsulas avaliadas quanto à morfologia, tamanho e distribuição de partículas, umidade, atividade de água e efeito do pH e temperatura em sua estabilidade. Para avaliar a susceptibilidade dos microrganismos ao processo de encapsulação e estresse tecnológico, investigou-se o impacto do efeito da homogeneização com Ultra-Turrax, tolerância à temperatura, salinidade, diferentes pH e fluidos gastrointestinais simulados na viabilidade das bactérias. A viabilidade dos microrganismos durante a estocagem também foi estudada. Por fim, avaliou-se a manutenção da capacidade imunomoduladora dos microrganismos microencapsulados por meio da dosagem de citocinas pró e anti-inflamatórias e determinação da capacidade protetora contra infecção de Salmonella entérica sorovar Typhimurium em modelo animal. Os microrganismos demonstraram resistência à lisozima, com taxas de sobrevivência superiores a 80%. O perfil de hidrofobicidade da parede celular, foi baixo, entre 8,47 e 19,19%. Demonstraram resistência apenas à vancomicina (35 µg) e eritromicina (15 µg). As duas cepas foram capazes de antagonizar o crescimento de Escherichia coli V517, Salmonella enteritidis OMS-Ca, Staphylococcus aureus 76 e Listeria monocytogenes ATCC 15313. Quanto à capacidade de utilização de prebióticos, os microrganismos apresentaram comportamentos inversos, utilizaram preferencialmente inulina, raftilose 95 e lactulose. Nos testes de resistência aos fluidos gastrointestinais simulados constatou-se declínio significativo de células viáveis, com subtração de até 3,37 log UFC/mL. As cápsulas obtidas apresentaram formato típico e tamanhos médios de 80,12 ± 1,89 e 83,92 ± 1,70 µm. Condições de pH extremos (1,5 e 9,0) e temperatura superior a 50 °C comprometeram a estabilidade das cápsulas. A encapsulação melhorou significativamente a tolerância dos microrganismos à altas concentrações de sal e elevação de temperatura. Além disso, favoreceu a resistência dos microrganismos frente aos fluidos gastrointestinais simulados. A estabilidade dos microrganismos durante o período de estocagem também foi favorecida, após 120 dias de estocagem a 7 e 25 °C a concentração de microrganismos viáveis permaneceu superior a 7,0 log UFC/g. Nos testes in vivo para avaliação da manutenção da capacidade de imunomodulação constatou-se através de dosagem de citocinas (IL-2, IL-6, IL-10 e TNF-α) e IgA secretora, que a encapsulação não alterou a resposta imunológica provocada pelas cepas estudas. Concluiu-se que os microrganismos apresentaram comportamento in vitro de acordo com o desejado para candidatos ao uso de probióticos. A microencapsulação foi bem-sucedida, proporcionando as duas cepas maior resistência frente às condições adversas e de estresse tecnológico. / Microencapsulation has been used successfully to improve the viability of probiotics microorganisms. The aim of this work was to evaluate two strains of lactobacilli, to encapsulate these strains by a system involving lipid particles coated by electrostatic interaction of polymers and to evaluate the effect of encapsulation in the maintenance of immunomodulatory capacity of these strains. In the first stage of the study L. rhamnosus 64 and L. paracasei BGP1 were evaluated for resistance to lysozyme and simulated gastrointestinal fluids, cell wall hydrophobicity profile, susceptibility to antibiotics, antagonist activity against pathogens and prebiotic utilization capacity. In the next step, the strains were encapsulated and the microcapsules evaluated regarding morphology, particle size and distribution, moisture, water activity and pH and temperature. The tolerance to temperature, salinity, different pH and simulated gastrointestinal fluids in the viability of the bacteria were also evaluated. The probiotics viability during the storage period was also studied. Finally, the maintenance of the immunomodulatory capacity of the encapsulated microorganisms was evaluated by means of the dosage of pro and anti-inflammatory cytokines and IgA. L. rhamnosus 64 and L. paracasei BGP1 demonstrated resistance to lysozyme, with survival rates above 80%. The hydrophobicity profiles of the cell wall were from 8.47 to 19.19%. Susceptibility to antibiotics also corroborated the literature, demonstrating resistance only to vancomycin (35 µg) and erythromycin (15 µg). The two strains were able to antagonize the growth of Escherichia coli V517, Salmonella enteritidis OMS-Ca, Staphylococcus aureus 76 and Listeria monocytogenes ATCC 15313. As far as the capacity of using prebiotics the two strains of lactobacilli presented inverse behaviors, they used preferably inulin, raftilose 95 and lactulose. In the tests of resistance to the simulated gastrointestinal fluids it was verified a significant decline of viable cells, with subtraction of up to 3.37 log CFU / mL, justifying the application of encapsulation technology. In the encapsulation step the capsules were produced with gum arabic, porcine gelatin and vegetable fat. The obtained capsules presented a typical format and average sizes of 80.12 ± 1.89 and 83.92 ± 1.70 µm. Extreme pH conditions (1.5 and 9.0) and temperature above 50 ° C compromised the stability of the capsules. The encapsulation significantly improved the tolerance of microorganisms to high salt concentrations and elevation of temperature. In addition, it favored the resistance of the microorganisms to the simulated gastrointestinal fluids. The stability of the microorganisms during the storage period was also favored, after 120 days of storage at 7 and 25 ° C the concentration of viable microorganisms remained higher than 7.0 log CFU / g. In the in vivo tests for evaluation of the maintenance of the immunomodulation capacity, the cytokines (IL-2, IL-6, IL-10 and TNF-α) and secretory IgA were determined, that encapsulation did not alter the immunological response by the strains of lactobacilli studied. It was concluded that the microorganisms presented in vitro behavior in accordance with the one desired for probiotic candidates. Microencapsulation was successful, giving both strains greater resistance to adverse conditions and technological stress.

Due to their immunomodulatory and regenerative potential, mesenchymal stem cells (MSCs) represent a promising therapeutic tool for cell-based therapy, organ transplantation or tissue engineering. To improve clinical applicability of MSCs, new methods to increase their delivery and efficacy have been tested in the latest years but the mechanism of observed alterations has not yet been described. In the present project we focused on studying the effect of several factors that can significantly affect the therapeutic success of MSC-based treatment. Initially, we analysed the therapeutic effect of MSCs applied locally on nanofiber scaffold with incorporated cyclosporine A (CsA) in a mouse model of allogeneic skin transplantation. Our results indicate that application of MSCs in the presence of CsA direct M1/M2 macrophage polarization towards regulatory phenotype. This phenotype switching is accompanied by decreased production of nitric oxide (NO) and interferon  (IFN-) and increase production of interleukin 10 (IL-10), and may result in suppression of the local inflammatory reaction. The next goal of proposed study was to analyse the effect of the treatment based on MSCs combined with immunosuppressive drugs with different mechanism of action on the balance among distinct T cell subpopulations. We...

β-Glucans are known for their ability to trigger both protective and damaging immune responses. Here we have explored the role of the beta-glucan receptor Dectin-1 in archetypical models of protective and non-protective immunomodulation induced by beta-glucan rich ligands. In the first model, we explored the role of Dectin-1 in the ability of soluble purified β-glucans to mediate protection against systemic Staphylococcus aureus infection in mice. In the second model, we explored the role of Dectin-1 in zymosan induced multiple organ dysfunction syndrome. In both cases, these β-glucan rich compounds had marked effects invivo which were unaltered by Dectin-1 deficiency, suggesting that this receptor has a redundant role in these murine models.

β-Glucans are known for their ability to trigger both protective and damaging immune responses. Here we have explored the role of the beta-glucan receptor Dectin-1 in archetypical models of protective and non-protective immunomodulation induced by beta-glucan rich ligands. In the first model, we explored the role of Dectin-1 in the ability of soluble purified β-glucans to mediate protection against systemic Staphylococcus aureus infection in mice. In the second model, we explored the role of Dectin-1 in zymosan induced multiple organ dysfunction syndrome. In both cases, these β-glucan rich compounds had marked effects invivo which were unaltered by Dectin-1 deficiency, suggesting that this receptor has a redundant role in these murine models.