Estima-se que mais de 800.000 cirurgias cardíacas de revascularização do miocárdio por ano são realizadas no mundo. Nesta intervenção terapêutica uma das complicações mais comuns é a fibrilação atrial, que esta intimamente relacionada com aumento de risco de morbidade e mortalidade pós-operatória. Os fatores determinantes desta manifestação podem ser pré, intra e pós-operatórios que possivelmente se relaciona com as proteínas pró-inflamatórias, canais iônicos e sistema renina-angiotensina. Portanto, os polimorfismos nos genes que codificam essas proteínas podem ter importante papel no desenvolvimento da FAPo. O objetivo deste trabalho foi de avaliar a associação entre os polimorfismos dos genes IL6 (G-174C), KCNE5 (C97T) e AGT (A-217G) e a incidência da FAPo e seus efeitos na expressão de RNAm, em apêndice atrial direito e em sangue periférico. Para o estudo foram selecionados 76 indivíduos portadores de insuficiência coronariana obstrutiva com indicação de intervenção cirúrgica de revascularização do miocárdio. Desses, 16 com FAPo, 52 sem FAPo e 8 pacientes foram excluídos por motivo de óbito. Amostras de sangue periférico foram obtidas para análise de parâmetros bioquímicos e para extração de DNA genômico e RNA total, antes e 48 horas após a cirurgia cardíaca. Os polimorfismos de nucleotídeo único (SNP) dos genes da IL-6, KCNE5 e do AGT foram detectados pela PCR-RFLP e confirmados por sequenciamento de DNA. A expressão de RNAm em leucócitos totais de sangue periférico e de tecido foi analisada pela PCR em tempo real, utilizando o gene GAPDH como referência endógena. A freqüência do alelo -174G foi de 75 % no GFA e 69,2% no GC e não foi associada ao desenvolvimento de FAPo. A freqüência do alelo 97T foi de 0% no GFA e 10,8% no GC e não foi associada a menor incidência de FAPo. A freqüência do alelo -217A foi de 12,5% no GFA e 15,9% no GC e também não foi associado ao desenvolvimento de FAPo. A expressão de RNAm da IL-6 em LTSP foi reduzida do pré para o pós-operatório de cirurgia de RM em ambos os grupos e não houve correlação entre a expressão de RNAm da IL-6 em LTSP e apêndice auricular direito. A expressão de RNAm do KCNE5 em LTSP foi reduzida do pré para o pós-operatório de cirurgia de RM, exceto em indivíduos do gênero masculino do GC, sugerindo a influência do gênero na expressão desse gene. Não houve correlação entre a expressão de RNAm do KCNE5 em LTSP e apêndice auricular direito. Não se detectou expressão de RNAm do AGT em LTSP e sua expressão em apêndice auricular direito foi extremamente baixa, portanto não está associada ao desenvolvimento de FAPo. / It is estimated that more than 800,000 heart surgery coronary artery bypass grafting are performed annually in the world. This therapeutic intervention of the most common complication was atrial fibrillation, which is closely related to increased risk of morbidity and postoperative mortality. The determining factors of this event can be pre, intra and postoperative possibly relates to the pro-inflammatory proteins, ion channels and renin-angiotensin system. Therefore, polymorphisms in the genes encoding these proteins may play an important role in the development of PoAF. The objective of this study was to evaluate the association between polymorphisms of IL6 gene (G-174C), KCNE5 (C97T) and AGT (A-217G) and the incidence of PoAF and its effects on mRNA expression in right atrial appendage and peripheral blood. For the study we selected 76 individuals with obstructive coronary artery disease with indication for surgical myocardial revascularization. Of these, 16 with PoAF, 52 without PoAF and 8 patients were excluded because of death. Blood samples were obtained for biochemical analysis and extraction of genomic DNA and total RNA before and 48 hours after cardiac surgery. The single nucleotide polymorphisms (SNPs) of IL-6, KCNE5 and AGT were detected by PCR-RFLP and confirmed by DNA sequencing. mRNA expression in total leukocytes in peripheral blood and tissue were analyzed by real-time PCR, using GAPDH gene as endogenous reference. The frequency of-174G allele was 75% in the GFA and 69.2% in GC and was not associated with the development of FAPO. The 97T allele frequency was 0% in GFA and 10.8% in GC and was not associated with a lower incidence of PoAF. The frequency of allele-217A was 12.5% in the GFA and 15.9% in GC and was not associated with the development of PoAF. The mRNA expression of IL-6 on LTSP was reduced from preoperative to the postoperative period of CABG in both groups and no correlation between mRNA expression of IL-6 on LTSP and right atrial appendage. The mRNA expression of KCNE5 LTSP was reduced in the pre and postoperative CABG, except among males in the CG, suggesting the influence of gender on expression of this gene. There was no correlation between the expression of mRNA KCNE5 in LTSP and right auricular appendage. There was no mRNA expression of AGT in LTSP and its expression in right atrial appendage was extremely low, so it is associated with the development of PoFA.

O diabetes mellitus tipo 1A (DM1A), doença autoimune órgão-específica, resulta da destruição seletiva das células pancreáticas produtoras de insulina pela infiltração progressiva de células inflamatórias, particularmente linfócitos T auto-reativos. O DM1A tem etiologia complexa, resultante da interação de fatores ambientais e vários genes, particularmente os do sistema HLA (alelos -DR3 e -DR4). Paralelamente, genes que codificam outros componentes da resposta imune, como as citocinas, também são fortes candidatos à predisposição à autoimunidade. Sabe-se que a intensidade da resposta imunológica tem relação com a ativação e recrutamento de linfócitos T e B, produção de citocinas e autoanticorpos, associados às respostas imunológicas TH1 e TH2. Recentemente, uma nova sub-população de células T, a TH17, de intenso poder inflamatório, tem sido implicada em doenças autoimunes. O desenvolvimento das células TH17 sofre influência da citocina heterodimérica IL-27, composta pelas subunidades p28 e EBI3 (proteína do gene 3 induzida pelo vírus Epstein Barr), expressa predominantemente em macrófagos ou células dendríticas. A IL-27 tem sido associada à doença de Crohn, encefalomielite experimental autoimune e diabetes autoimune em camundongos. O seu bloqueio retarda o aparecimento do diabetes nestes animais. A implicação da IL-27 no DM1A em humanos é pouco conhecida. O presente estudo tem como objetivo pesquisar mutações ou polimorfismos na região 5 proximal e regiões codificadoras do gene da IL-27p28 e sua possível associação com predisposição ao DM1A. Estas regiões foram amplificadas pelo método de Reação em Cadeia da Polimerase (PCR) e submetido ao seqüenciamento automático e PCR-RFLP (Restriction Fragments Lenght Polymorphisms) utilizando DNA genômico obtido de leucócitos de sangue periférico. A casuística envolveu 614 indivíduos, sendo 318 pacientes portadores de DM1A (idade 19,6 ± 11,2 anos, 129M/189F), caracterizados por apresentarem hiperglicemia e necessidade precoce de insulinoterapia e um grupo controle, com 296 indivíduos saudáveis (idade 30,3±13,2 anos, 131M/165F), com glicemia de jejum e HbA1c normais. Na análise dos resultados do sequenciamento, observamos oito variantes alélicas, seis delas já descritas na literatura. As duas novas variantes alélicas causaram a substituição de uma citosina por uma timina na posição c.-347 C>T na região 5 proximal e a substituição de uma guanina por uma citosina no exon 5, na posição c.498 G>C, levando à mudança do aminoácido, de ácido glutâmico para ácido aspártico no resíduo 166 da seqüência da proteína. A distribuição dos genótipos na população estudada foi consistente com o equilíbrio de Hardy-Weinberg. As freqüências genotípicas e alélicas destas variantes não diferiram entre portadores de DM1 A e controles Não encontramos associação destas variantes com sexo e grupo étnico nos dois grupos e, com idade de diagnóstico do diabetes, presença de auto- anticorpos pancreáticos e extra-pancreáticos no grupo com DM1A. Não houve diferença na freqüência dos haplótipos estimados entre os pacientes DM1A e grupo controle. Sendo assim, nossos resultados sugerem que variantes alélicas no gene da IL-27p28 não estão implicadas na susceptibilidade ao DM1A na nossa população / Type 1A diabetes mellitus (T1D), an organ-specific autoimmune disease, results from the selective destruction of insulin-producing pancreatic cells by progressive infiltration of inflammatory cells, particularly autoreactive T lymphocytes. The complex etiology of T1D includes the interaction of environmental factors and multiple genes, particularly those of the HLA system (DR3 and DR4 alleles). In parallel, genes encoding other components of the immune response, such as cytokines, are also strong candidates for predisposition to autoimmunity. It is known that the intensity of immune response depends on the activation and recruitment of T and B lymphocytes, production of cytokines and autoantibodies, related to TH1 and TH2 immune responses. Recently, a new subpopulation of T cells exhibiting intense inflammatory activity, the TH17 subset, has been implicated in autoimmune diseases. The development of TH17 cells is influenced by IL-27, a heterodimeric cytokine composed of p28 and EBI3 (Epstein-Barr Virus- induced gene 3 protein) subunits, expressed predominantly in macrophages or dendritic cells. IL-27 has been associated with Crohn\'s disease, experimental autoimmune encephalomyelitis and autoimmune diabetes in mice. IL-27 blockage delays the onset of diabetes in these animals, but the role and involvement of IL-27 in T1D in humans has not yet been reported. The aim of this study was identify mutations or polymorphisms in the coding regions and boundary intron sequences of IL-27p28, including the 5 proximal region, and their possible association with the disease. Those regions were amplified by Polymerase Chain Reaction (PCR) and the polymorphisms determined by automatic sequencing and PCR-RFLP (Restriction Fragment length polymorphisms) The cohort involved 614 individuals - 318 patients with T1D ( 19.6 ± 11.2 years of age, 129M/189F) and 296 control subjects (30.3 ± 13.2 years of age, 131M/165F) with normal fasting glucose. We identified eight allelic variants in the 5 proximal and coding regions of IL- 27p28, six of them already described in database repositories. The two new allelic variants were: the substitution of a cytosine by a thymine at position c.- 347 C>T of the 5 proximal region and the substitution of a guanine by a cytosine in exon 5, at position c.498 G>C, determining a change in the aminoacid sequence of the protein at residue 166 from glutamic acid to aspartic acid. The genotypic frequencies of these variants were in Hardy- Weinberg equilibrium in both groups. The frequency of the alleles and genotypes did not differ between T1D patients and controls. There was no association between IL-27p28 variants with gender or ethnical origin in the population analyzed . Also, no association was found between these variants with age at diagnosis of diabetes nor with the presence of pancreatic and extrapancreatic autoantibodies in T1D patients The frequency of the estimated haplotypes were similar between groups. Our results suggest that allelic variants in the IL-27p28 gene are not involved in susceptibility to T1D in our population

Além das células T reguladoras clássicas (Treg), fenotipicamente definidas como CD4+CD25highCD127-FOXP3+, outros subtipos celulares que expressam moléculas reguladoras e produzem IL-10 podem desempenhar papel imunomodulador na malária humana. Caracterizamos subpopulações celulares que expressam marcadores de ativação celular e de atividade reguladora em indivíduos infectados por P. falciparum, P. vivax ou co-infectados com ambas as espécies, bem como em controles saudáveis de uma área endêmica de malária na Amazônia Brasileira. Mostramos que a malária induz duas subpopulações principais de células T reguladoras, uma expressando CTLA-4 (uma molécula de superfície que inibe a ativação de células T) e outra expressando OX40, uma molécula co-estimulatória que pode suprimir a atividade supressora da Treg. Mostramos que diferentes populações de PBMCs, provenientes de pacientes com malária e controles saudáveis, produzem IL-10 após a estimulação ex vivo com phorbol-12-myristate-13-acetate (PMA) e ionomicina em comparação com o estímulo com lipopolissacarídeo (LPS). / Besides classical regulatory T cells (Treg), which are phenotypically defined as CD4+CD25highCD127-FOXP3+, other cell subtypes expressing regulatory molecules and producing IL-10 may play an immunomodulatory role in human malaria. We characterized cell subpopulations that express markers of cell activation and regulatory activity in individuals infected with P. falciparum, P. vivax, or co-infected with both species, as well as in healthy controls from a malária-endemic area in the Brazilian Amazon. We show that malaria induces two major subpopulations of regulatory T cells, expressing CTLA-4 (a surface molecule that inhibits the activation of T cells) and another expressing OX40 (a co-stimulatory molecule that can suppress the suppressive activity of Treg). We showed that different populations of PBMCs from malaria patients and healthy controls, produce IL-10 after ex-vivo stimulation with phorbol-12-myristate-13-acetate (PMA) and ionomycin, compared to stimulation with lipopolysaccharide (LPS).

Notre équipe a précédemment démontré que l'Interleukine (IL)-24 une cytokine de la famille de l'IL-10 a un effet cytostatique voire cytotoxique sur des cellules B normales ou leucémiques mises préalablement en cycle mais non sur des cellules quiescentes. L'IL-24 inhibe également la différenciation plasmocytaire des cellules B humaines du centre germinatif dans un modèle de différentiation in vitro. Nous avons utilisé ce modèle pour analyser pour la première fois le transcriptome de cellules B stimulées ou non par IL-24 au bout de 6 et 36 h. Plusieurs transcrits impliqués dans le métabolisme et la réplication de l'ADN sont inhibés précocement à l'exception d'IGF-1 qui est stimulé. L'IGF1 ayant été décrit comme une molécule de survie des cellules B ou pré-B, nous avons analysé son effet biologique et démontré qu'il a au contraire un rôle proapoptotique à doses physiologiques. En revanche, l'IL-24 induit l'expression plus tardive des gènes de la voie mitochondriale de la mort cellulaire programmée (MCP). Cet effet est également retrouvé dans des LLC « IgVH mutées » ou non mais avec une cinétique distincte des cellules B normales. Au total, dans des cellules activées au préalable, l'IL-24 induit séquentiellement un blocage précoce du cycle cellulaire suivi d'une apoptose. D'autres gènes potentiellement importants dans la synapse immune ainsi que dans la régulation de l'immunité innée sont décrits et suggèrent que l'IL-24 a un rôle immunologique particulier au-delà de son effet cytostatique et potentiellement anti-tumoral / We have previously shown that Interleukin(IL)-24 a class-II cytokine of the IL-10 family has cytostatic and cytotoxic properties on normal and malignant human B-cells previously engaged into the cell cycle, but not on quiescent B-cells. IL-24 also inhibits the differentiation of germinal center B-cells in plasma cells in an in vitro model; the later was used to compare for the first time the transcriptome of B-cells cultured or not with IL-24 for 6 and 36h. Several "early" transcripts involved in DNA metabolism and replication were inhibited whereas that of Igf1 a molecule described as a B-cell growth factor was induced. We show herein that IgF1 has instead a proapoptotic role on B-cells at physiological concentrations. In contrast, several genes of the intrinsic apoptotic pathway were stimulated after 36h. This expression pattern was also found in CLL cells whether they were "IgVH mutated" or "unmutated", albeit with distinct kinetics from normal B-cells. In addition several genes belonging to the immune synapse and innate immunity were regulated by IL-24. These results disclose additional, possibly immunoregulatory properties, for IL-24 than its already described cytostatic and potentially anti-tumoral effects

Une des difficultés majeures dans le traitement des cancers est l’acquisition de résistance par les cellules tumorales vis-à-vis de la mort induite par les différentes chimiothérapies. Au sein du laboratoire, nous nous intéressons aux propriétés anti-tumorales d’un donneur de monoxyde d’azote (NO), le Glycéryl TriNitrate (GTN), fréquemment utilisé dans le traitement de l’angine de poitrine. Au cours de ce travail, nous avons étudié les mécanismes moléculaires par lesquels le GTN sensibilise les cellules tumorales de plusieurs types de cancer (colique, mammaire, prostatique) à la mort impliquant des voies de signalisation régulées par des cytokines telles que le TNFα, l’IL-6 ou encore le GDF-15.Une meilleure compréhension des mécanismes sous-jacents à l’action anti-tumorale du GTN permettrait de potentialiser son utilisation comme nouvelle thérapie anti-cancéreuse.Modèle colique : le GTN, en présence de la cytokine pro-inflammatoire TNFα, sensibilise les cellules cancéreuses coliques et mammaires à l’apoptose. Du point de vue mécanistique, le GTN induit la S-nitrosylation de cIAP1, inhibant ainsi son activité ubiquitine E3 ligase. Ce qui abroge la voie de signalisation classique NF-кB de survie cellulaire activée par la voie TNFα/TNFR1 en faveur d’une voie de signalisation pro-apoptotique.Modèle mammaire : le GTN intervient au niveau de la migration cellulaire en altérant la voie de signalisation Jak2/STAT3 activée par la cytokine pro-inflammatoire IL-6, dans un modèle de cancer du sein triple négatif. En présence de dérivés du platine (carboplatine) générant de l’IL-6, le GTN freine la migration des cellules en induisant la S-nitrosylation, et probablement l’inactivation, de la kinase Jak-2, indispensable pour l’activation de la voie.Modèle prostatique : le GTN sensibilise à la mort les cellules cancéreuses prostatiques résistantes au docétaxel en modulant le taux de deux marqueurs de résistance à cette chimiothérapie : la clusterine (CLU) et le growth differentiation factor 15 (GDF-15). Au niveau moléculaire, le GTN diminue le taux de l'isoforme cytoprotectrice soluble de la CLU (sCLU) et augmente le taux de l'isoforme cytotoxique nucléaire (nCLU) dans les cellules prostatiques résistantes au docétaxel. Plus particulièrement, en présence de GTN, nous avons établi un lien entre le GDF-15 et la modulation du taux des isoformes de la CLU. / One of the main difficulties in the treatment of cancers is the acquisition of resistance by the tumor cells vis-à-vis the death induced by the different chemotherapies. In the laboratory, we are interested in the anti-tumor properties of a nitric oxide (NO) donor, Glyceryl TriNitrate (GTN), frequently used in the treatment of angina pectoris. In this work, we investigated the molecular mechanisms by which GTN sensitizes tumor cells of several types of cancer (colonic, mammary, prostate) to death involving signaling pathways regulated by cytokines such as TNFα, IL-6 or GDF-15.A better understanding of the mechanisms underlying the GTN's anti-tumor action would make it possible to use it as a new anti-cancer therapy.Colon model: GTN, in the presence of the pro-inflammatory cytokine TNFα, sensitizes colon and mammary cancer cells to apoptosis. From a mechanistic point of view, GTN induces S-nitrosylation of cIAP1, thus inhibiting its ubiquitin E3 ligase activity. This abrogates the classical NF-κB signaling pathway of TNFα / TNFR1 activated cell survival in favor of a pro-apoptotic signaling pathway.Mammary model: GTN intervenes at the level of cell migration by altering the Jak2 / STAT3 signaling pathway activated by the pro-inflammatory cytokine IL-6, in a model of triple negative breast cancer. In the presence of platinum (carboplatin) derivatives generating IL-6, GTN inhibits cell migration by inducing S-nitrosylation, and probably inactivation, of Jak-2 kinase, essential for the activation of the way.Prostate model: GTN sensitizes prostatic prostate cancer cells to death by modulating the level of two markers of resistance to this chemotherapy: clusterin (CLU) and growth differentiation factor 15 (GDF-15). At the molecular level, GTN decreases the level of the soluble cytoprotective isoform of CLU (sCLU) and increases the level of nuclear cytotoxic isoform (nCLU) in prostatic cells resistant to docetaxel. More particularly, in the presence of GTN, we have established a link between GDF-15 and the modulation of the isoform rate of the CLU.

L’avènement des multi-thérapies antirétrovirales a permis une réduction importante de la mortalité associée au VIH en induisant notamment la chute de la charge virale à moins de 50 copies/mL et une récupération progressive du nombre de lymphocytes T CD4+ (LT-CD4). Cependant, certains patients définis comme mauvais répondeurs immunologiques (MRI) ne parviennent pas à récupérer un taux de CD4 généralement considéré comme « protecteur » (>500cellules/µL). L’interleukine-7 (IL-7), cytokine essentielle à la thymopoïèse et à l’homéostasie lymphocytaire T, a été utilisée en étude clinique afin de restaurer et maintenir le taux de LT-CD4 chez les patients MRI. La première partie de mon travail de thèse visait à évaluer l’impact d’une telle thérapie sur le réservoir viral circulant. Dans l’essai clinique sur lequel nous avons travaillé (INSPIRE 3, Cytheris), des cycles d’administration d’IL-7 ont induit une augmentation significative du nombre de LT-CD4 et CD8 circulants, avec une expansion majoritaire des populations naïves et centrales mémoires. Nous avons montré qu’un cycle d’injections d’IL-7 induisait une augmentation significative de la quantité de cellules infectées circulantes 28 jours et 3 mois post-injection. Cependant, malgré l’accroissement de la fréquence de LT-CD4 infectés 28 jours post-injection, nous avons observé une diminution significative de la charge virale ADN par million de LT-CD4 chez la majorité des patients 3 mois après l’initiation de la thérapie, suggérant une élimination partielle de cellules infectées. Suite au second cycle d’injections, nous n’avons pas observé d’évolution de la quantité de cellules infectées circulantes ni de la fréquence de LT-CD4 infectés, suggérant un impact différent des 2 cycles d’injections sur la dynamique du réservoir viral périphérique. Enfin, certains patients ayant développé des anticorps neutralisants anti-IL-7 (Nab) suite au second cycle d’injections d’IL-7, nous avons cherché à identifier des facteurs prédictifs de l’apparition de ces anticorps ainsi que leurs conséquences physiologiques in vivo. Le seul paramètre caractérisant ces patients est l’amplitude de la reconstitution T-CD4 au cours du premier cycle d’injections d’IL-7. Il semble donc qu’une meilleure réponse à l’IL-7 ait pour conséquence de faciliter le développement de la réponse immune contre cette cytokine. Cependant, ces anticorps ne sont détectables que de façon transitoire chez les patients. De plus, nous avons observé une diminution significative, mais transitoire, de la prolifération des thymocytes chez les patients présentant des Nab, démontrant un impact fonctionnel de ces anticorps sur l’activité biologique de l’IL-7 endogène. L’injection systémique d’IL-7 induit la migration des cellules circulantes vers différents compartiments tissulaires lymphoïdes et non lymphoïdes. Dans une seconde partie de mon travail de thèse, j’ai étudié le pouvoir adjuvant de cette cytokine administrée localement par pulvérisation à la surface de la muqueuse vaginale. Dans le modèle macaque rhésus, nous avons mis en évidence une augmentation de la production d’un large spectre de chimiokines dans le chorion et l’épithélium vaginal des animaux 48 heures après l’administration vaginale d’IL-7. Cette surexpression de chimiokines s’accompagne d’une migration massive de LT-CD4, CD8, macrophages, cellules dendritiques et cellules NK dans cette muqueuse, suggérant l’augmentation de la vigilance immunologique. L’effet adjuvant de cette cytokine a été confirmé par l’analyse de la réponse humorale muqueuse de macaques vaccinés par pulvérisation vaginale d’antigènes 48h après l’administration du spray d’IL-7. Dans les lavages cervicovaginaux (CVL) des animaux traités à l’IL-7, nous avons mis en évidence des réponses spécifiques de type IgA et IgG plus rapides, plus fortes et plus durables que chez les animaux contrôles, démontrant la capacité de l’IL-7 à préparer la muqueuse vaginale à répondre à une stimulation antigénique locale. / Highly Active Antiretroviral Therapy (HAART) has led to significant reduction of HIV-associated mortality by maintaining an undetectable viral load and inducing progressive CD4-T cell restoration. However, some patients, defined as poor immunological responders (PIR), fail to restore their CD4 counts to 500cells/µL during treatment, a threshold considered as the protective against AIDS related or non AIDS related malignancies, opportunistic infections and cardiovascular events. Interleukin-7 (IL-7), an essential cytokine for thymopoïesis and T cell homeostasis has been used in clinical trials aimed at restoring and maintaining CD4 counts in PIR patients. The first part of my thesis project aimed at assessing the impact of IL-7 therapy on circulating HIV reservoir. In the clinical study we worked on (INSPIRE 3, Cytheris), cycles of IL-7 injections led to a significant increase of the number of circulating CD4 and CD8 T-cells, with a predominance of naïve and central memory T cell expansion. We have shown that one cycle of IL-7 injections induced a significant increase in the number of circulating infected cells 28 days and 3 months post-injections. However, despite a significant increase in the frequency of infected CD4 T-cells 28 days post-injections, we observed a significant decrease of HIV-DNA load in CD4 T-cells in the majority of patients 3 months after the therapy initiation. These data suggest a partial elimination of HIV infected cells. After the second cycle of IL-7 injections, we did not observed any change in the number or frequency of circulating infected cells, suggesting a differential impact of the two IL-7 injection cycles on the dynamics of circulating HIV-reservoir. Finally, considering that some patients developed anti-IL-7 neutralizing antibodies (Nab) after the second cycle of IL-7 injections, we looked for predictive factors of this immunogenicity and analyzed its physiological consequences in vivo. The only parameter that distinguished Nab and non-Nab patients was the extent of CD4 T-cell reconstitution during the first cycle of therapy. This suggests that a better response to IL-7 also facilitates the development of auto-antibodies to the cytokine. However, these antibodies were only transiently detectable after the second cycle of therapy. Moreover, the appearance of Nab was associated with a significant but transient decrease of thymocyte proliferation, suggesting a functional impact of these antibodies on the endogenous IL-7 function. Systemic injection of IL-7 induces circulating T cells homing from the blood into lymphoid and non-lymphoid tissues. In the second part of my thesis project, I evaluated whether this cytokine could be used as an adjuvant when sprayed on the vaginal mucosa. Ten micrograms of IL-7 directly sprayed in the vaginal tract of rhesus monkeys induced, 48h after administration, the production of a large pattern of chemokines in the vaginal chorion and epithelium. This chemokine expression was accompanied by massive homing of CD4 and CD8-T cells, macrophages, dendritic cells and NK cells in the vaginal mucosa, suggesting an increased immunological vigilance. Finally, the adjuvant potential of this cytokine was confirmed by analyzing local humoral immune response after vaginal administration of antigens 48h following IL-7 spray. In cervicovaginal washes (CVL) of treated animals, we observed a faster, stronger and longer-lasting specific IgA and IgG response than in control animals, highlighting the capacity of IL-7 to prepare the vaginal mucosa response to local antigen stimulation.

A sinalização da resposta Imune Inata exerce um papel fundamental na eliminação de patógenos bem como no recrutamento de progenitoras para recuperação de tecidos nervosos lesionados. Nossos estudos iniciais que buscavam a compreensão desta participação da atividade inflamatória na neurorregeneração e degenerção do OE constataram que a administração tópica de lipopolissacarídeo (LPS) gera perda de seus neurônios olfatórios (OSNs), enquanto que camundongos deficientes do gene Myd88 apresentam uma taxa de reposição neuronal pós-lesão mais elevada que a de selvagens e o uso consecutivo de DEX retarda a neurorregeneração olfatória em curto prazo. Prosseguimos agora no esclarecimento destes resultados em três vertentes: A primeira busca descrever as respostas do OE de animais selvagens e trangênicos Tlr4-/-, Myd88-/-, Ticam1-/-, Il1r1-/-, Casp1-/-/Casp4-/- e Casp1-/-/Casp4-/-/Casp4tg a estímulos inflamatórios provenientes de infusões intranasais de ligantes e citocinas recombinantes envolvidas na sinalização dos TLRs. Isto através de análises histológicas por marcação nuclear e análise da espessura e alterações anatômicas do OE, e da expressão de IL1b e Nf&#954bia por hibridização in situ (ISH), ou ainda pela contagem de suas células TUNEL-positivas pós-tratamento. Neste estudo propomos ainda um modelo de lesão para estudos de neurorregenereração através da análise por imunofluorescência (IF) de OSNs em função do tempo após uma infusão intranasal (IN) de LPS. A segunda analisa por microarranjos de oligonucleotídeos (microarray) as alterações de expressão gênicas associadas à ausência do gene Myd88 no OE pós-lesão seguida de análises histológicas de sua regeneração em camundongos com deleções em genes selecionados. Por fim o terceiro estudo investiga a degeneração e a neurorregeneração do OE sob o efeito do uso tópico consecutivo de DEX. O corticoide é co-aplicado topicamente com um insulto inflamatório para avaliar o seu efeito preventivo e consecutivamente por três dias em dois modelos de lesão para avaliar sua interferência na neurorregeneração 14 dias após o tratamento. Foram realizadas IF para quantificação dos volumes neuronais, espessura do OE, proliferação celular, síntese proteica e morte celular por TUNEL. O uso tópico de LPS promove a degeneração do OE via TLR4 a partir de regiões com expressão de Il1b e Nf&#954bia e número de células TUNEL elevada. Este efeito é via MyD88-dependente sem a participação da TRIF-dependente. O gene Myd88 é tão crucial nesta degeneração do epitélio quanto na gerada por rmIL-1β e rmTNFα. Sua ausência não promove citoproteção contra o gás NO. Possivelmente, CASP1 e IL-1R estejam também envolvidos. O modelo de lesão imunológica para estudos de neurorregeneração é rápido e eficaz. A ausência do gene Myd88 acompanha uma redução na expressão da enzima degradadora de insulina (IDE) no OE pós-lesão. Camundongos que não a expressam apresentam uma reposição celular do epitélio mais rápida. A co-IN de DEX com LPS impede a degeneração do OE. 10µl deste corticoide a 40ng/µl administrada por IN não é tóxica a este epitélio, porém seu uso consecutivo em curto prazo promove aberrações anatômicas nos modelos de lesão imunológica, além de interferir na sua dinâmica de reposição neural, elevação da taxa de síntese proteica e proliferação celular, sem alterar a diferenciação, após lesão induzida por metimazol. / The innate immune response signaling plays a key role in the elimination of pathogens and in the recruitment of new cells to recover the injured nervous tissues. Our preliminary studies on the roles of the inflammatory activity in OE degeneration and neuroregeneration process showed that the topical administration of lipopolysaccharide (LPS) generates the loss of olfactory sensorial neurons (OSNs), Myd88 gene-deficient mice exhibit a higher neuronal replacement rate after injury than wild-type mice, and that the consecutive use of DEX provokes retarding olfactory neuroregeneration over the short term. We seek now to clarify these results in three ways: The first describes the OE response in wild-type animals Tlr4-/-, Myd88-/-, Ticam1-/-, Il1r1-/-, Casp1-/-/Casp4-/- and Casp1-/-/Casp4-/-/Casp4tg animals to inflammation through the intranasal infusion (IN) of ligands and cytokines associated with Toll-Like Receptors (TLR) signaling. This was acomplished through histological analysis of the thickness and anatomical changes in DAPI stained OE, Il1b and Nfκbia expression analysis by in situ hybridization (ISH), and TUNEL-positive cells counting after treatment. In this study we propose an inflammatory lesion for neuroregeneration studies using immunofluorescence (IF) analysis of the OE in function of time after the intranasal infusion of LPS. The second part analyzes the Myd88 gene loss in the OE gene expression of after a lesion using microarray. This is followed by the histological analysis of regeneration using IF in transgenic mice with the deletion of specific genes (microarray versus literature review). Finally, the third study evaluates the OE degeneration and neuroregeneration under the influence of consecutive topical use of the DEX. The corticosteroid is co-administered with the inflammatory stimulus in order to evaluate its protective effect and consecutively for three days in two models of lesion to assess their influence on neuroregeneration 14 days after treatment. The IF analysis was performed in order to quantify the neuronal volumes, the thickness of the OE, cell proliferation, protein synthesis (by incorporation the identification of puromycin) and cell death by TUNEL. The topical use of LPS promotes the degeneration of OE by TLR4 from sites that feature an overexpression of Il1b and Nfκbia and a high number of TUNEL-positive cells. This effect is MyD88-dependent. The Myd88 gene is as crucial in this degeneration of the epithelium as in those generated by rmIL-1β and rmTNFα. Their absence does not promote cytoprotection against the cytotoxic gas nitric oxide (NO). It is possiby that CASP1 and IL-1R are also involved. The immunologic lesion model for neuroregeneration studies is fast and effective. The absence of Myd88 gene reduces the expression of insulin-degrading enzyme (IDE) in the post-lesion OE. Mice without this enzyme show a rapid cellular restoration in the epithelium. The Co-IN of DEX with LPS prevents the degeneration EO. The doses adopted by us are nontoxic; however its short-term consecutive use promotes anatomical aberrations in the immunological lesion model, and interferes with the dynamics of neural replacement by impairing both the rate of protein synthesis and proliferation of the epithelium without halting their differentiation.

Aggregatibacter actinomycetemcomitans está associado à periodontite agressiva e produz CDT. Visando contribuir no entendimento do papel da CDT na regulação da resposta imune, foi determinada sua atividade sobre macrófagos e a resposta humoral contra a toxina. CDT inibiu a proliferação de células epiteliais OBA-9 e macrófagos Raw 264.7 e também a produção de NO por células Raw 264.7 e macrófagos peritoneais de camundongos C3H/HePas e C3H/HeJ, mas estimulou a produção de IL-12. Na imunidade humoral, 75% dos soros de indivíduos com PAgL e 81,8% dos PAgG foram reativos para A. actinomycetemcomitans. Não houve diferença na resposta contra CDTA e CDTB entre o soro de pacientes com diferentes condições periodontais. Todos os pacientes PAgG foram soropositivos para a CDTC, porém apenas 8,3% dos indivíduos com PAgL, nenhum dos PC e 25% dos saudáveis foram positivos. CDT tem atividade imunomodulatória e a resposta humoral difere entre indivíduos infectados pela bactéria. / Aggregatibacter actinomycetemcomitans is associated with aggressive periodontitis and produces CDT. Aiming to contribute in the understanding the CDT activity in the immune response regulation, its action on macrophages was determined and the response against the toxin analyzed. CDT inhibited the proliferation of OBA-9 epithelial cells and Raw 264.7 macrophages and also inhibited the NO production by Raw 264.7 cells and peritoneal macrophages of C3H/HePas and C3H/HeJ mice, however, stimulated the IL-12 production. In the humoral immunity, 75% of sera from LAgP subjects and 81.8% were reactive to A. actinomycetemcomitans. There was not difference in the response against CDTA and CDTB among sera of patients with different periodontal conditions. All GAgP subjects were sera-reactivity to CDTC, however only 8.3% LAgP subjects, none in CP and 25% of healthy subjects were positive. CDT has immunomodulatory activity and the humoral response differ among bacteria infected subjects.

A passagem do século XIX para o século XX acarretou muitas dúvidas e incertezas no pensamento e na cultura ocidentais. A crise do positivismo, que impulsionou os literatos e os intelectuais a se questionarem sobre o conceito de real, gerou um nítido conflito entre tudo aquilo que remetia ao racional e à ciência em contraposição a um renovado tipo de subjetivismo e de relativismo, definindo, assim, uma nova abordagem da literatura e das artes na primeira metade do século XX. Neste trabalho é feita uma análise do papel da imprensa e do intelectual no final do século XIX, para depois retomá-la no período no qual se insere a obra de Pirandello. Estuda-se, ainda, a presença estrangeira nos periódicos italianos e o conceito de literatura popular e cultura de massa que estão intrinsecamente ligados à função do intelectual e sua relação com o povo/nação. Pirandello é um escritor que tem a maior parte de sua produção literária publicada no início do século XX, a qual vai desde obras em dialeto siciliano, nas quais são trabalhados os aspectos mais regionais - como tradições e costumes rurais e folclóricos, crenças populares e místicas e, por isso, de certa forma mais populares, até obras que atingem uma complexidade narrativa comparadas a grandes clássicos literários nacionais e europeus. Nesta pesquisa será apresentada de que forma se dá o comprometimento do intelectual e do artista Pirandello com a sociedade daquele período e de que modo algumas questões subjetivas do autor também influem em sua obra. Como base e modelo que guiará toda a discussão deste trabalho, apresentamos, ainda, uma análise de Il fu Mattia Pascal (1904), uma das obras mais representativas da modernidade europeia e na qual se encontram os elementos principais da poética e da filosofia do autor que, além de resgatar algumas técnicas do romance-folhetim francês, as emprega na publicação seriada desta obra, traduzindo toda a crise da modernidade e resgatando, mesmo que de maneira atenuada, alguns aspectos do romance popular. / The passage between the XIX and the XX centuries brought a lot of doubts and uncertainties in the occidental thought and culture. The positivism crisis, which forced the men of letter and the intellectuals to debate the concept of real, generated a sharp conflict between everything which concerned the logical and the science and a renewed kind of subjectivism and relativism, defining in this manner a new approach to arts and literature at the first half of XX century. This work presents an analysis about the press and the literate man at the end of the XX century, to be introduced again during the period in which Pirandello is set in. Moreover is studied the foreign presence in the italian magazines and the concepts of popular literature and mass culture which are strictly connected to the intellectuals function and his relationship with the population/nation. Pirandello is a writer whose literary productions was published at the beginning of the XX century and it goes from sicilian dialect works, in which more characteristic and regional aspects can be found such as popular and folkloristic customs and traditions, popular and mystic lores and therefore more popular, to works where a complex narrative is involved until reaching a national and european level. The aim of this research is to show the compromise between the artist Pirandello and the society of that period and in what way a few personal issues of the author influence his opus. To enlighten the whole discussion is introduced an analysis of Il fu Mattia Pascal (1904) - one of the most representative works of european modernity - in which can be found characteristic elements of Pirandellos poetry and philosophy. Pirandello revives some tecniques from the french roman-feuilleton and employs them in a serial publication of this play that, beyond traslating the whole crisis of modernity, partially rescues some aspects of the popular novel.

Durante a ativação policlonal induzida pelo P. chabaudi a maior fonte de IL-2 são as células CD4+ ativadas, além disso, acorre a expansão de células Treg. No dia 7 após a infecção, a ausência de células Treg leva a uma exacerbação da ativação de células CD4+, além de altos níveis de anticorpos anti-P.chabaudi e auto-anticorpos. A neutralização da IL-2, com Mab anti-IL-2 JES6-1, na fase aguda da infecção leva a uma redução no número de células Treg. No dia 20 de infecção, a freqüência de células CD4+ ativadas esteve elevada e as células Treg voltaram aos níveis basais. Experimentos in vitro mostraram que a neutralização da IL-2 não altera a proliferação antígeno-específica de células CD4+ da fase aguda da infecção, porém, em tempos tardios da infecção houve um drástico aumento na freqüência de células CD4+ que proliferam em resposta a eritrócitos parasitados. Podemos concluir que a IL-2 e as células Treg são capazes de limitar a ativação policlonal induzida pelo P. chabaudi ainda que com cinéticas distintas. / Polyclonal activation during P. chabaudi infection results on a huge IL-2 production by activated CD4+ T cells, besides a considerable expansion of Treg cells. At day 7 after infection in the absence of Treg cells there is an enhanced response of activated CD4+ T cells, an increase of Abs anti-P.chabaudi and autoantibody production. Neutralization of IL-2 with Mab anti-IL2 JES6-1 during acute infection reveals a markedly reduction in Treg-cells number. At day 20 of infection we can observe an increase on activated CD4+ T cells frequency. Moreover, Treg cells return to values similar to controls. IL-2 in vitro assays during acute infection results on Ag-specific CD4+ T cells proliferation, on the other hand, at the late infection, we observed a huge increase of CD4+ T cells frequency that strongly response to PRBC. Our findings suggest that IL-2 and Treg cells are capable of restricting PLA during P.chabaudi infection, although with different kinetics.