Das Ubiquitin-Proteasom-System sorgt in eukaryontischen Zellen für einen kontrollierten Abbau von Proteinen. Das 20S Proteasom ist als Multikatalytischer Protease Komplex der zentrale Bestandteil dieses Systems. In der vorliegenden Arbeit konnte gezeigt werden, dass sich gereinigtes 20S Proteasom aus HeLa-Zellen chromatographisch in Subtypen auftrennen lässt, die sich strukturell und in ihrer proteolytischen Aktivität unterscheiden. Nach Induktion der Zellen mit gamma-Interferon (gamma-IFN) werden Immuno-Proteasomen gebildet und es kommt zu einer Veränderung des Subtypen-Musters und der Aktivitäten. Unter dem Einfluss von gamma-IFN bilden sich hauptsächlich Mischkomplexe mit sowohl konstitutiven als auch Immuno-Untereinheiten. Weiterhin konnte gezeigt werden, dass in den Zellkompartimenten Cytoplasma, Zellkern und Microsomen von HeLaS3-Zellen unterschiedliche 20S Proteasom-Subtypen vorkommen. Dies war unter anderem auf eine unterschiedliche Glykosylierung einzelner proteasomaler Untereinheiten zurückzuführen. Die genaue Kenntnis von Struktur und Funktion der 20S Proteasom-Subtypen ist im Hinblick auf neue diagnostische und therapeutische Ansätze in der Humanmedizin von großem Interesse. / The Ubiquitin-proteasome system is responsible for the regulated protein degradation in eucaryotic cells. The 20S proteasome is as a multicatalytic protease the central complex of these system. This study has shown that it is possible to separate 20S proteasome subtypes from HeLa cells by chromatography. 20s proteasome subtypes differ in structure and proteolytic activity. The subtype-pattern and the activity are significantly changed after an induction of the cells with gamma-Interferon (gamma-IFN) under formation of immuno proteasomes. After gamma-IFN induction mainly mixed complexes have been formed with both constitutive and immuno subunits. Further it has been shown that in cell compartements cytoplasm, microsomes and nucleus of HeLaS3 cells different 20S proteasome subtypes are located. Among other things glycosylation of some subunits is responsible for that phenomenon. With regard to new strategies in diagnostic and therapy of human diseases the exactly knowledge of structure and function of the proteasome subtypes is a case of interest.

A terapia endodôntica visa o selamento biológico do complexo sistema apical, contribuindo para isso, as substâncias usadas no tratamento e a resposta imune do paciente. A LILT tem mostrado atividade antiinflamatória, favorecendo o processo reparacional. Sendo assim, este trabalho objetivou analisar o efeito da LILT na atividade secretória de macrófagos, previamente ativados por IFN-? e LPS de E.coli, e estimulados por substâncias liberadas de três tipos de cimentos endodônticos, um a base de óxido de zinco e eugenol, outro a base de hidróxido de cálcio e um terceiro resinoso. A citotoxicidade dessas substâncias foi avaliada usando a técnica de análise do MTT. Macrófagos ativados foram estimulados por essas substâncias ou não (controle) e então, irradiados ou não (controle) e a secreção de proteínas próinflamatórias (interleucina-1 b, fator de necrose tumoral-a e metaloproteinase da matriz-1) foram analisadas pelo teste ELISA. As irradiações foram realizadas com um laser GaAlAs (780 nm, 70 mW, ponta da fibra de 4 mm2, 1.67 seg, 3 J/cm2). Foram usadas duas aplicações de irradiação com intervalo de 6 h. Os dados obtidos foram tratados por Análise de Variância, quando de distribuição normal, ou teste de Friedman, quando de distribuição não normal, com nível de significância de 5 % (p = 0,05). A viabilidade dos controles e células tratados pelos cimentos endodônticos foi similar. Produção de IL-1 b e TNF-a foram observadas. Houve alta produção de MMP-1. Entretanto, sem diferenças estatísticas entre os grupos experimentais. Os grupos irradiados apresentaram resultados similares aos não irradiados. Substâncias liberadas pelos cimentos endodônticos testados não se mostraram citotóxicas nas condições deste experimento. Essas substâncias, bem como a LILT, no parâmetro utilizado, não causam alteração na atividade de secreção de MMP-1, IL-1 b e TNF-a por macrófagos ativados. / The endodontic therapy seeks the dental root canal biological sealing, depending on substances used in this process and patient’s defense immune factors. LILT has shown an anti-inflammatory activity, improving the periapical repair process. This in vitro study aimed to analyze the effect of LILT at the secretory activity of macrophages previously activated by interferon-gamma and lypopolisaccharide from E.coli, and stimulated by substances leached from three endodontic sealers (zinc oxide-eugenol based, resinous and calcium hydroxide-based). Cytotoxicity of these substances was assessed by the MTT test. Activated macrophages were stimulated by the substances or not (control) and then, irradiated or not (control) and the secretion of pro-inflammatory proteins (interleukin-1 b, tumor necrosis factor-a and matrix metalloproteinase-1) was analyzed by ELISA test. The LILT was performed using a GaAlAs laser (780 nm, 70 mW, focal spot of 4.0 mm2, 1.67 sec, 3 J/cm2). Two irradiations with 6 h-intervals were done. The data was compared by either ANOVA test or Friedman’s test. The cell viabilities of controls and cells treated by the sealers were similar. Production of IL -1 b and TNF-a were observed. There was a high production of MMP-1. However, statistical differences were not observed amongst the groups. The irradiated groups presented results similar to those of non irradiated groups. Substances leached from the endodontic sealers are non cytotoxic at these experiments conditions . These substances, as well as the LILT, at the parameter used, were not able to change the secretion of MMP-1, IL-1 b e TNF-a by activated macrophages.

A introdução da terapia anti-retroviral de alta potência no tratamento da infecção pelo HIV reduziu significativamente as taxas de morbi-mortalidades relacionadas à imunodeficiência. Entretanto, o tratamento medicamentoso é acompanhado de vários efeitos colaterais, dentre eles, a síndrome da lipodistrofia (SL), caracterizada por alterações morfológicas e metabólicas. Apesar de sua patogenia não estar totalmente esclarecida, é sabido que aumento dos níveis de algumas citocinas inflamatórias estão relacionados com o desenvolvimento da SL. Diversos sítios polimórficos têm sido descritos por influenciarem a transcrição de genes, levando a variações nos níveis de produção de citocinas, como os da região promotora da interleucina-18 (IL-18 -607 C/A e IL-18 -137 C/G) e do gene do interferon gama (IFN- +874 T/A). Diante disso, esse estudo tipificou os polimorfismos da IL-18 e do IFN- em 88 pacientes portadores do HIV com a SL, em 79 portadores do HIV sem a SL, todos sob terapia anti-retroviral e em 133 indivíduos saudáveis, por meio da técnica de reação em cadeia da polimerase com iniciadores de seqüência específica. Este estudo foi aprovado pelo Comitê de Ética em Pesquisa. A presença do alelo -607A e do genótipo -607AA na IL-18 estava significativamente aumentada nos pacientes portadores do HIV com SL quando comparados aos sem a SL, conferindo susceptibilidade ao desenvolvimento da síndrome. De maneira oposta, o alelo -607C e o genótipo -607CC estavam significativamente aumentados em pacientes portadores do HIV sem SL quando comparados aos com a SL, conferindo proteção ao desenvolvimento da síndrome. Os haplótipos -137G/-607A and -137C/-607A, que comportam o alelo -607A, também estavam associados com a susceptibilidade à SL e o haplótipo -137G/-607C estava fortemente associado com proteção contra a SL. Nenhuma diferença significativa na distribuição alélica e genotípica da IL-18 -137 e do IFN- +874 foram observadas entre os grupos de pacientes e o grupo controle. Este é o primeiro estudo que avaliou o polimorfismo da IL-18 e do IFN- na SL e os resultados sugerem que a região promotora da IL-18 está associada com o desenvolvimento da SL em pacientes portadores do HIV. / The introduction of highly active antiretroviral therapy in the treatment of HIV infection significantly reduced the rates of morbidity and mortality related to immunodeficiency. However, the drug treatment is accompanied by various side effects, including the lipodystrophy syndrome (LD), characterized by morphological and metabolic changes. Although its pathogenesis is not totally clear, it is known that increased levels of some inflammatory cytokines are related to the development of LD. Several polymorphic sites have been described by influencing transcription of genes, leading the variations in the levels of cytokine production, such as the promoter region of interleukin-18 (IL-18 -607 C/A and IL-18 -137 C/G) and the interferon gamma gene (IFN- +874 T/A). Thus, this study typifies the polymorphism of the IL-18 and IFN- in 88 HIV-infected patients with LD, in 79 HIV-infected patients without LD, all under antiretroviral therapy and in 133 healthy controls, using the sequence-specific primers-polymerase chain reaction. This study was approved by the Ethics Committee at the place of study. The presence of -607A allele and -607AA genotype in IL-18 gene were significantly increased in HIV patients presenting LD as compared with HIV patients without LD, resulting in susceptibility to the development of LD. Conversely, the -607C allele and -607CC genotype were significantly increased in HIV patients without LD as compared with the HIV patients with LD, offering protection against LD. Haplotypes -137G/-607A and -137C/-607A, carrying the -607A allele, were also associated with susceptibility to LD. The haplotype -137G/-607C was strongly associated with protection against LD. No significant differences in IL-18 -137 and IFN- +874 genotype and allele distribution were observed in patients when compared to a control group. This is the first study evaluating the IL-18 and IFN- polymorphisms in LD and the results suggest that the promoter region of the IL-18 gene is associated with LD development in HIV-infected patients.

Les traitements antirétroviraux combinés limitent la morbidité et la mortalité liées au SIDA après l'infection par le VIH, mais une hyperactivation du système immunitaire persiste, notamment au niveau des cellules myéloïdes, en corrélation avec une morbidité et une mortalité métabolique et cardio-vasculaire plus précoces que celles de la population générale. Il existe deux types de VIH ; l'infection par le VIH-2, prévalente en Afrique de l'Ouest et dans des communautés émigrées de cette région, conduit moins rapidement et moins fréquemment au SIDA que l'infection par le VIH-1, pour des raisons de relation hôte-virus encore à élucider. Lors de l'infection aigüe par le VIH-1, un pic sérique d'interféron (IFN) de type I et d'autres cytokines est observé, puis régulé négativement. Il est accompagné d'une forte réponse des gènes stimulés par l'IFN, qui persiste lors du passage à l'infection chronique, avec hyperactivation du système immunitaire. Les IFN de type I sont produits par toutes les cellules, mais en particulier par les cellules dendritiques plasmacytoïdes (pDC). Les IFN de type III (lambda) sont liés à la résolution de l'infection par le VHC, mais leur production en réponse au VIH reste à explorer. Durant mon doctorat, j'ai montré pour la première fois qu'in vitro, le VIH-1 et le VIH-2 stimulent la production d'IFN-lambda par les PBMC de donneurs sains de façon comparable. Chez des donneurs sains, les pDC produisent ces IFN de façon intrinsèque, mais pas les autres DC myéloïdes. Les pDC ont également un rôle dans la réponse immunitaire adaptative contre le VIH. L’équipe a en effet montré qu’elles peuvent effectuer la présentation croisée d’antigènes de cellules mortes infectées par le VIH, comme le font les autres DC. Elles peuvent ainsi activer des réponses T cytotoxiques spécifiques qui vont éliminer les cellules infectées. J’ai étudié ce mécanisme et montré que l’activation spécifique des lymphocytes T par les pDC est potentialisée par une pré-activation non-spécifique. En effet, les pDC s’activent en présence du virus et sécrètent des cytokines qui pré-activent une production intracellulaire d'IFN-gamma par les lymphocytes T CD8. Ces lymphocytes ne relarguent l'IFN-gamma qu'après reconnaissance spécifique par leur récepteur T d'un complexe peptide-CMH. Les résultats de cette thèse pourraient donner une place aux IFN-lambda ou à leur inhibition dans l'arsenal thérapeutique contre le VIH, ainsi qu'à l'activation des pDC pour conduire à une meilleure détection et élimination des réservoirs viraux par présentation croisée. / Combined antiretroviral treatments limit AIDS-related morbidity and mortality after HIV infection. But hyperactivation of the immune system persists, notably within the myeloid cell compartment, in correlation with metabolic and cardiovascular morbidity, which occurs earlier than in the general population. Two types of HIV have been described: HIV-2 infection, prevalent in West Africa and in emigrated communities originating from this area, leads less frequently and less rapidly to AIDS compared to HIV-1 infection, because of host-virus, which still need to be characterized. During acute HIV-1 infection, in the plasma, peak levels of type I Interferon (IFN) and other cytokines are observed, then they are down-modulated. A strong IFN stimulated gene (ISG) response is also observed. During chronic infection, the ISG response persists, with hyperactivation of the immune system. Type I IFN are produced by all cell types, but more specifically by plasmacytoid Dendritic Cells (pDC). Certain type III IFN (lambda) gene variants correlate with clearance of HCV infection, but IFN-lambda production in response to HIV remained to be studied. During my PhD, I showed for the first time that, in vitro, HIV-1 and HIV-2 induce IFN-lambda production by healthy donors PBMC at comparable levels. Plasmacytoid DC from healthy donors produce these IFN intrinsically, but not conventional DC. Plasmacytoid DC also have a role in the induction of adaptive immune responses against HIV. Our team demonstrated that they can crosspresent antigens from HIV infected apoptotic cells, like other DC. They can therefore activate specific cytotoxic T cell responses which eliminate infected cells. I studied this mechanism and showed that the activation of specific T cell by pDC is potentiated by non-specific pre-activation. Indeed, pDC become activated in the presence of virus and secrete cytokines which pre-activate intracellular IFN-gamma production by CD8 T cells. IFN-gamma is then secreted only after cognate MHC-peptide-T cell receptor interaction. The results of this thesis potentially give a role to IFN-lambda or their blockade in HIV treatment, and to the activation of pDC to induce better detection and elimination of HIV reservoirs through crosspresentation.

<p>The mechanisms of β-cell destruction leading to type 1 diabetes are complex and not yet fully understood, but infiltration of the islets of Langerhans by autoreactive immune cells is believed to be important. Activated macrophages and T-cells may then secrete cytokines and free radicals, which could selectively damage the β-cells. Among the cytokines, IL-1β, IFN-γ and TNF-α can induce expression of inducible nitric synthase (iNOS) and cyclooxygenase-2. Subsequent nitric oxide (NO) and prostaglandin E₂ (PGE₂) formation may impair islet function.</p><p>In the present study, the ability of melatonin (an antioxidative and immunoregulatory hormone) to protect against β-cell damage induced by streptozotocin (STZ; a diabetogenic and free radical generating substance) or IL-1β exposure was examined. <i>In vitro</i>, melatonin counteracted STZ- but not IL-1β-induced islet suppression, indicating that the protective effect of melatonin is related to interference with free radical generation and DNA damage, rather than NO synthesis. <i>In vivo</i>, non-immune mediated diabetes induced by a single dose of STZ was prevented by melatonin.</p><p>Furthermore, the effects of proinflammatory cytokines were examined in islets obtained from mice with a targeted deletion of the iNOS gene (iNOS -/- mice) and wild-type controls. The <i>in vitro</i> data obtained show that exposure to IL-1β or (IL-1β + IFN-γ) induce disturbances in the insulin secretory pathway, which were independent of NO or PGE₂ production and cell death. Initially after addition, in particular IL-1β seems to be stimulatory for the insulin secretory machinery of iNOS –/- islets, whereas IL-1β acts inhibitory after a prolonged period. Separate experiments suggest that the stimulatory effect of IL-1β involves an increased gene expression of phospholipase D1a/b. In addition, the formation of new insulin molecules appears to be affected, since IL-1β and (IL-1β + IFN-γ) suppressed mRNA expression of both insulin convertase enzymes and insulin itself.</p>

Allergic diseases are influenced by genetics and the environment. Maternal allergy appears to confer a higher risk for allergic sensitization than paternal allergy, suggesting an in utero influence. A decrease in particular infections or a lower exposure to microbial components during infancy is suggested to contribute to the high allergy prevalence in affluent societies. Toll-like receptors (TLR) 2 and 4 recognize peptidoglycan (PGN) and LPS respectively, are expressed on e.g. monocytes, and have been implicated in modulating the risk of IgE-sensitization. This thesis aimed to study the influence of maternal allergy and early microbial exposure on monocyte function and allergic sensitization during childhood. Blood samples from children participating in a prospective allergy cohort were used. Two-year old infants with allergic mothers had lower IL-6 production and reduced activation of the TLR-signalling intermediate p38-MAPK in response to PGN than children with non-allergic mothers. In 5-year old children, allergic disease and not maternal allergy influenced monocytic TLR2-regulation. Five-year olds who were seropositive for Epstein-Barr virus (EBV) at 2-years of age had a lower risk of persistent IgE-sensitization while EBV contraction after 2-years of age related to a higher risk of IgE-sensitization. Upon in vitro stimulation, NK cells from EBV+ 2-year olds produced lower IFN-g levels. EBV+ 2-year olds had also lower systemic IFN-g. In comparison to CD14++CD16- monocytes, CD14+CD16+ cells induced NK-cell IFN-g more potently in vitro, and EBV+ infants tended to have lower proportions of these CD14+CD16+ monocytes. This thesis highlights the importance of early-life microbial (EBV) exposure for a proper allergy-protective immunity. Also, maternal allergic heredity appears to influence monocytic microbial responses in early infancy. All these aspects relate to altered monocyte functionality, which suggest that they could have a role in allergic sensitization.

<p>This study shows that herbs can be effectively screened for potiential bio-activity using in vitro methods. Further studies will be needed to better explore Artemisia afra&rsquo / s effect on immunoregulation, particularly long term effects of the herb on the immune system and its effect on other disease states.</p>

The mechanisms of β-cell destruction leading to type 1 diabetes are complex and not yet fully understood, but infiltration of the islets of Langerhans by autoreactive immune cells is believed to be important. Activated macrophages and T-cells may then secrete cytokines and free radicals, which could selectively damage the β-cells. Among the cytokines, IL-1β, IFN-γ and TNF-α can induce expression of inducible nitric synthase (iNOS) and cyclooxygenase-2. Subsequent nitric oxide (NO) and prostaglandin E2 (PGE2) formation may impair islet function. In the present study, the ability of melatonin (an antioxidative and immunoregulatory hormone) to protect against β-cell damage induced by streptozotocin (STZ; a diabetogenic and free radical generating substance) or IL-1β exposure was examined. In vitro, melatonin counteracted STZ- but not IL-1β-induced islet suppression, indicating that the protective effect of melatonin is related to interference with free radical generation and DNA damage, rather than NO synthesis. In vivo, non-immune mediated diabetes induced by a single dose of STZ was prevented by melatonin. Furthermore, the effects of proinflammatory cytokines were examined in islets obtained from mice with a targeted deletion of the iNOS gene (iNOS -/- mice) and wild-type controls. The in vitro data obtained show that exposure to IL-1β or (IL-1β + IFN-γ) induce disturbances in the insulin secretory pathway, which were independent of NO or PGE2 production and cell death. Initially after addition, in particular IL-1β seems to be stimulatory for the insulin secretory machinery of iNOS –/- islets, whereas IL-1β acts inhibitory after a prolonged period. Separate experiments suggest that the stimulatory effect of IL-1β involves an increased gene expression of phospholipase D1a/b. In addition, the formation of new insulin molecules appears to be affected, since IL-1β and (IL-1β + IFN-γ) suppressed mRNA expression of both insulin convertase enzymes and insulin itself.

B virus (Macacine herpesvirus 1, Cercopithecine herpesvirus 1, herpes B virus) is an Old World monkey simplex virus endemic in macaques. B virus infection in its natural host, macaque, is very similar to HSV-­‐1 infection in humans causing mild or asymptomatic infection. On the other hand, zoonotic infection in humans results in death in the absence of early initiation of antiviral drugs. Viruses evade host immune responses in order to survive and propagate. Most herpes viruses including HSV-­‐1 down-­‐regulate major histocompatibility complex class I (MHC class I) surface expression on infected cells in order to prevent CD8+ T-­‐cell recognition and subsequent cell lysis. MHC class I molecules bind to the inhibitory receptors of NK cells and prevent NK cell activity. Thus, this mechanism protects HSV-­‐1 infected cells from CD8+ T-­‐cell lysis, making them sensitive to natural killer (NK) cell cytotoxicity. To investigate if B virus pathogenicity is a result of novel immune evasion mechanisms employed by B virus, we determined NK cell regulation during B virus infection. To this end, our experiments demonstrate that B virus does not down-­‐ regulate MHC I expression as effectively as HSV-­‐1, leading us to hypothesize that B virus in-­‐ fected cells are resistant to NK cell activity. We examined the expression of MHC I chain related genes (MICA/ MICB), which are activation ligands to NKG2D receptors on NK cells. Our results show that there is no significant difference in MICA and MICB expression between HSV-­‐1 and B virus infected cells. Furthermore, we tested for the up-­‐regulation of cytokines and chemokines responsible for NK cell activation and migration. Our results indicate a significant up-­‐regulation of IFN-­‐α from PBMCs co-­‐cultured with HSV-­‐1 infected cells, which plays an important role in activating NK cells. NK cells within these PBMCs up-­‐regulate perforin release indicative of NK cell activity. PBMCs co-­‐cultured with B virus infected cells do not up-­‐regulate any cytokines or chemokines responsible for NK cell activity. As a result the NK cells within these PBMCs do not significantly up-­‐regulate perforin release. These results demonstrate that B virus employs a novel immune evasion mechanism to subvert NK cell activity.

Les cellules dendritiques plasmacytoïdes (PDC) représentent une nouvelle entité distincte de cellules dendritiques. Elles peuvent sécréter de grandes quantités d'interféron de type I après stimulation par un virus ou des produits bactériens tels que les CpG ODN grâce à leur expression sélective des Toll-like récepteurs (TLR)7 et 9.<br />Notre laboratoire a récemment développé une lignée de PDC (GEN2.2) à partir de leucémies à PDC (LPDC), qui résistent aux thérapies conventionnelles. Les GEN2.2 partagent la plupart des caractéristiques phénotypiques et fonctionnelles des PDC normales. Nous avons d'abord utilisé cette lignée comme modèle de LPDC et nous montrons qu'elles sont sensibles à l'apoptose induite par TRAIL (TNF-Related Apoptosis-Inducing Ligand) via l'expression du récepteur DR5, comme la plupart des LPDC, alors que les PDC normales ne le sont pas, ce qui permettrait la mise en place de thérapies des leucémies à PDC utilisant des agonistes de TRAIL.<br />Les PDC normales sont difficiles à isoler ou générer. Nous avons donc ensuite utilisé la lignée GEN2.2 comme modèle de PDC normales. Nous avons ainsi découvert que ces cellules, une fois activées par des ligands des TLR7 et 9, acquièrent une fonction cytotoxique via l'expression de TRAIL et peuvent tuer des cellules tumorales. Les PDC pourraient donc jouer un rôle crucial dans l'éradication des cancers après activation.<br />Enfin, nous avons cherché à préciser les mécanismes moléculaires d'induction de TRAIL dans les PDC après activation par des ligands des TLR7 et 9.<br />L'ensemble des travaux suggère que les PDC pourraient représenter une cible de choix dans le développement de nouvelles approches thérapeutiques anti-tumorales.