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This study investigated the role of tolerance induction in an inflammatory setting in regard to the early growth response genes Egr2 and Egr3. T cells robustly respond to pathogenic antigens during infection, but are tolerant to stimulation by self-antigens. The intrinsic mechanisms for self-tolerance in the periphery are still not clear. Egr2 and 3 are induced in tolerant T cells in response to antigen stimulation by NFAT-medicated tolerant signalling; however, their function in tolerant T cells is still unknown. The study demonstrated that Egr2 and 3, induced in tolerant T cells, are not directly involved in defective proliferation and IL-2 production, the hallmarks of T cell tolerance. However, they are essential for preventing inflammatory response of tolerant T cells. In the absence of Egr2 and 3, tolerant T cells show impaired proliferation and production of IL-2, but produce high levels of IFN-γ, a key inflammatory cytokine. This phenotype resembles CD4 T cells from autoimmune diseases such as lupus which show poor proliferative response, but hyper-inflammation. Our study demonstrated, for the first time, a distinctive mechanism to control inflammation from proliferative tolerance regulated by Egr2 and 3, which may be an important mechanism for the control of autoimmune diseases.

Experimental autoimmune encephalomyelitis (EAE) is a CD4+ T cell mediated autoimmune disease of the central nervous system and shares many characteristics with multiple sclerosis (MS). Induction of EAE is mediated by myelin reactive CD4+ T helper (Th) cells, particularly Th1 and Th17 cells, which can be provoked by the immunization with myelin derived protein (or peptide) and Toll-like receptor (TLR) stimulus (eg, complete Freund¡s adjuvant, CFA). If given an injection of soluble peptide before immunization, mice do not develop EAE (they are tolerant). This approach has been widely applied, evoking tolerance in primary responses (i.e., in naive T cells). Therefore the first hypothesis of this thesis is that peptide induced protection from EAE is a result from T cell deletion or / and anergy. As MS patients have ongoing disease and over 85% of MS patients develop a relapsing-remitting course, memory T cells are key targets when considering peptide-induced tolerance as a therapeutic strategy. Thus, a model for ¡memory EAE¡ was established to test a second hypothesis that the myelin reactive memory T cells can be controlled by the administration of soluble peptide. Here, adoptive transfer of T cells from T cell receptor transgenic mice (2D2) recognizing myelin oligodendrocyte glycoprotein 35-55 (pMOG) was used to investigate the pMOG-reactive memory responses. Soluble pMOG administration could induce a transient expansion of 2D2 T cells followed by their loss through apoptosis. A model using double immunization was established by immunizing mice first with pMOG together with unmethylated CpG oligonucleotide (CpG) as an adjuvant, and subsequently immunizing with pMOG in CFA. This produced EAE with early onset and high incidence compared to mice which received pMOG/CFA only. Cells from mice that received the double immunization protocol produced high levels of IFN-γ, suggesting that memory T cell responses have been triggered in the mice. Administration of soluble peptide before secondary immunization could ameliorate EAE, indicating that memory T cells are susceptible to tolerance induction. pMOG-reactive memory T cells were further assessed by isolating CD4+ CD25- CD44high CD62Llow cells from pMOG-experienced 2D2 mice. These cells showed early and high production of IFN-γ, and early but transient production of IL-2, compared with naive population. These data provide basic information relevant to translating peptide-induced T cell tolerance from mice to humans.

Orientador: Priscila Lupino Gratão / Resumo: Devido às intensas mudanças climáticas globais e atividades antropogênicas, a salinidade tornou-se uma das principais problemáticas limitantes à produção agrícola. Para lidar com essa problemática, o estudo de genótipos e cultivares que sejam tolerantes ao sal, bem como alternativas através de inoculantes torna-se cada vez mais necessário. O objetivo deste trabalho foi caracterizar a resposta antioxidante através da inoculação de Azospirillum brasilense em milho, e correlacionar a atividade destas enzimas ao aumento na capacidade da planta em tolerar o estresse ocasionado pela salinidade. Os tratamentos foram formados pela combinação de cloreto de sódio (0 e 100 mM de NaCl) via água de irrigação e ausência e presença do inóculo de A. brasilense, sendo o experimento conduzido inteiramente casualizado, com quatro repetições Os resultados demonstraram diferentes respostas de acordo com as análises de peroxidação lipídica (MDA), quantificação de nitrogênio (N) e sódio (Na+), massa seca (MS) e atividades enzimáticas, como superóxido dismutase (SOD, EC 1.15.1.1), glutationa redutase (GR, EC 1.6.4.2), guaiacol peroxidase (GPOX, EC 1.11.1.7) e glutationa peroxidase (GSH-PX, EC. 1.11.1.9). Os resultados mostraram que 100 mM de NaCl ocasionou peroxidação lipídica, com consequente aumento do teor de MDA. Entretanto, com a presença da bactéria nesta condição, o teor de MDA foi reduzido, houve aumento do acúmulo de N e as enzimas apresentaram diferenças significativas entre si, com aument... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Due to intense global climate change and anthropogenic activities, salinity has become one of the main problems limiting agricultural production. To deal with this problem, the study of genotypes and cultivars that are salt tolerant and also alternatives through inoculants becomes increasingly necessary. The main of this work was to characterize an antioxidant response through the inoculation of Azospirillum brasilense in maize and to correlate the activity of the enzymes with the salt-stress tolerance. The experiment was carried out in a completely randomized design with four replications. The treatments were performed by combination of sodium chloride (0 and 100 mM NaCl) through irrigation water and absence and presence of A. brasilense inoculation. Overall results showed different responses according to lipid peroxidation (MDA), nitrogen (N) and Na+ contents, dry mass (DM) and enzymatic activities, such as superoxide dismutase (SOD, EC 1.15.1.1), glutathione reductase (GR, EC 1.6.4.2), guaiacol peroxidase (GPOX, EC 1.11.1.7) and glutathione peroxidase (GSH-PX, EC 1.11.1.9). The results showed were that 100 mM NaCl caused lipid peroxidation with consequent increases in MDA content. However, MDA content was reduced and antioxidant enzymes demonstrated significant differences in the presence of the bacteria. Our data suggest that A. brasilense may confer plant tolerance in maize to salt stress and acquired tolerance can be related to the antioxidant system, mainly GSH-PX and ... (Complete abstract click electronic access below) / Mestre

Background: Patients treated with an inhibitor of the mechanistic target of rapamycin (mTORI) in a calcineurin inhibitor (CNI)-free immunosuppressive regimen after heart transplantation (HTx) show a higher risk for transplant rejection. We developed an immunological monitoring tool that may improve the identification of mTORI-treated patients at risk for rejection. Methods: Circulating dendritic cells (DCs) and regulatory T cells (Tregs) were analysed in 19 mTORI- and 20 CNI-treated HTx patients by flow cytometry. Principal component and cluster analysis were used to identify patients at risk for transplant rejection. Results: The percentages of total Tregs (p = 0.02) and CD39+ Tregs (p = 0.05) were higher in mTORI-treated patients than in CNI-treated patients. The principal component analysis revealed that BDCA1+, BDCA2+ and BDCA4+ DCs as well as total Tregs could distinguish between non-rejecting and rejecting mTORI-treated patients. Most mTORI-treated rejectors showed higher levels of BDCA2+ and BDCA4+ plasmacytoid DCs and lower levels of BDCA1+ myeloid DCs and Tregs than mTORI non-rejectors. Conclusion: An mTORI-based immunosuppressive regimen induced a sufficient, tolerance-promoting reaction in Tregs, but an insufficient, adverse effect in DCs. On the basis of patient-specific immunological profiles, we established a flow cytometry-based monitoring tool that may be helpful in identifying patients at risk for rejection.

Le facteur VIII (FVIII) joue un rôle essentiel dans la coagulation sanguine. Lorsque le FVIII fait génétiquement défaut, une pathologie hémorragique grave survient: l'hémophilie A (HA) congénitale. La complication majeure de la prise en charge de ces patients est l'apparition d'allo-anticorps (alloAcs) dirigés contre le FVIII thérapeutique administré. Dès lors, la seule thérapeutique efficace est l'induction de tolérance immune (ITI) qui vise à les éradiquer. Cependant, ce traitement échoue dans 30% des cas, sans qu'aucun facteur ne permette actuellement de prédire l'échec de ce traitement contraignant et coûteux. des facteurs immunologiques prédictifs de l'efficacité de l'ITI ont été recherchés chez 25 patients par analyse du répertoire épitopique et isotypique des Acs anti-FVIII à l'aide de la technologie x-MAP. Des biomarqueurs individuels (Acs anti-A2 et -A1 du FVIII), et des combinaisons originales ont été identifiés (0,841 < AUC < 0,946). Des manifestations hémorragiques peuvent apparaitre chez des patients non hémophiles, dues à des autoAcs anti-FVIII (HA acquise). Dans certains cas, les autoAcs se développent au moment du postpartum. peu de données sont disponibles sur cette réponse immune. Dans une seconde étude portant sur 73 cas, nous avons découvert un profil immunologique (autoAcs anti-A1) différenciant les HA du postpartum et les autres HA acquises. Les profils d'IgG anti-FVIII que nous avons établis s'avèrent prometteurs pour prédire l'efficacité de l'ITI et engendrer une cartographie précise de la réponse autoimmune chez les patients atteints d'HA acquise. / Factor VIII (FVIII) plays a critical role in blood coagulation. When FVIII s genetically defective, a serious hemorrhagic disease occurs: congenital hemophilia A (HA). The main complication of the management of these patients is the appearance of alloantibodies (alloAbs) directed against administred therapeutic FVIII. therefore, the only effective treatment is the immune tolerance induction (ITI), which aims to eradicate these alloAbs. However, this treatment fails in up to 30% of cases, without any factor currently able to predict the failure of this constraining and expensive treatment. Immunological factors predictive to the efficacy of ITI were investigated in 25 patients by analysis of epitopic and isotypic IgG profile of anti-FVIII Abs using x-MAP technology. Individual biomarkers (anti-FVIII A1 and -A2 Abs), and original combinations were identified (0,841 < AUC < 0,946). Hemorrhagic manifestations can occur in non-hemophiliac patients, due to anti-FVIII autoAbs (acquired HA). In some patients, the autoAbs appear in postpartum period but few data are available on the immune response due to the rarity of the disease. In a second study of 73 cases, we found a different immunological profile between patients with postpartum HA and the other acuired HA patients. IgG profiles of anti-FVIII we have established are promising for predicting the effectiveness of ITI and generate an accurate mapping of autoimmune response in patients with acquired HA.

L'objectif de notre projet de recherche est l'exploration de la faisabilité de l'allogreffe des tissus composites (ATC) chez les nouveau-nés ayant des anomalies congénitales sévères de la main ou du visage. Dans la partie bibliographique, nous avons étudié les mécanismes de tolérance néonatale chez la souris, ainsi que la transplantation in utero des cellules souches hématopoïétiques avec des modèles animaux et humains. Ensuite, les propriétés du système immunitaire du nouveau-né humain ont été décrites avec étude des différents protocoles de conditionnement non-myéloablatifs utilisés pour induire une tolérance aux greffes d'organes solides, afin de trouver le type de conditionnement utilisable chez les nouveaux nés pour l'induction de tolérance. La greffe du thymus et de la moelle osseuse vascularisée avec l'ATC ont été également étudiés. Enfin, une revue exhaustive des différentes études d'ATC concernant l'induction de tolérance chez les humains et les larges animaux a été faite. Un premier modèle préclinique expérimental d'ATC a été élaboré chez le porcelet nouveau-né. Des études ultérieures ont par suite étudié les agents immunosuppresseurs ainsi que le régime de conditionnement avec l'administration de cyclosporine A., des thymo-globulines de lapin anti-porc et du mycophénolate mofétil. Un protocole d'induction de tolérance pour l'ATC chez les porcelets nouveau-nés a été rédigé et l'expérimentation sera réalisée courant 2014-2015. Si la tolérance d'ATC spécifique du donneur pourra être induite avec notre protocole, nous allons par la suite élaborer un protocole d'induction de tolérance et un programme d'allogreffe de main applicable chez les nouveau-nés humains / This present research is devoted to the exploration of performing vascularized composite allografts as a treatment for severe congenital hand or face anomalies in neonates or very young infants. The bibliographic studies at first revised the discovery and mechanisms of neonatal tolerance in mice, as well as in utero hematopoietic stem cells transplantation in large-animal models and human fetuses. Then the properties of human neonatal immune system were described; and the non-myeloablative or non-toxic conditioning regimens for solid organ transplant tolerance induction were also studied, in order to give the clue to a applicable conditioning regimen for tolerance induction in neonates. The potent thymus and vascularized bone marrow transplantation in neonatal VCA were considered as advantages. Finally, the researches concerning tolerance induction for VCA in large animal models and in human patients were reviewed. ln experimental studies, the preclinical VCA was firstly established in neonatal swines. Subsequent experiments thus studied the immunosuppressive agents, as well as conditioning regimen, including the administration of cyclosporine A, rabbit anti-pig thymocyte globulin and mycophenolate mofetil for VCA in pig neonates. The findings in these experiments were then concluded. Based on these finding, a general tolerance induction protocol for VCA in neonatal swines was designed and experiment will be performed in year 2014-2015. lf donor-specific tolerance for VCA could be induced with present protocol, we will subsequently elaborate an applicable tolerance induction protocol and hand allotransplantation program in human newborn infants