Parasitische Nematoden beeinflussen gezielt die Abwehrreaktionen ihres Wirtes. Dies wird besonders während der chronischen Infektionsphase durch eine herabregulierte T-Zell-Antwort auf Parasitenantigene und andere Stimuli ersichtlich. In dieser Arbeit wurde die T-Zell-Antwort gegen einen intestinalen Nematoden untersucht. Mäuse wurden mit dem Trichostrongyliden Heligmosomoides polygyrus infiziert und in der Folge Effektor- sowie regulatorische T-Zellen (Tregs) untersucht. Subpopulationen von CD4+ T-Zellen wurden aus chronisch infizierten Mäusen isoliert und in naive Empfänger transferiert, welche nachfolgend infiziert wurden. Dabei zeigte sich, dass der Transfer von CD4+ Effektor-T-Zellen zu einer verminderten Wurmlast in den Empfängertieren führte, diese Zellen also einen partiellen Schutz gegen die Primärinfektion vermitteln. Der gleichzeitige Transfer von Tregs beeinflusste diesen Effekt nicht. Tregs allein zeigten keinerlei Einfluss auf die Wurmlast der Empfänger. Die Protektion durch Transfer von Effektor-T-Zellen kann vermutlich auf eine kleine Antigen-spezifische Population von CD4+ Zellen zurückgeführt werden. Diese Zellen wurden durch die Expression von CD40-L (CD154) nach Restimulation mit Parasitenantigen in vitro charakterisiert und enthielten einen Großteil der Zytokinproduzenten unter den CD4+ Zellen. Während diese Effektorzellen ein deutliches Th2-Zytokinprofil durch Produktion von Interleukin-4 (IL-4) und IL-13 zeigten, reagierte eine Treg-Subpopulation mit der Sekretion hoher Mengen von IL-10 auf Antigenstimulation. Diese Tregs waren durch Expression des Integrins AlphaE (CD103)Beta7 sowie CD25 und Foxp3 charakterisiert und vermittelten in vitro die stärkste Suppression anderer T-Zellen, wenn sie aus chronisch infizierten Mäusen isoliert wurden. Durch Untersuchung der zellulären Zusammensetzung von mesenterialen Lymphknoten und Milz konnte gezeigt werden, dass die Frequenz solcher regulatorischer Zellen im Verlauf der Infektion dauerhaft und überproportional zunimmt. Im Gegensatz dazu wurde am Infektionsort nur eine vorübergehende Akkumulation von Tregs (Foxp3+) während der akuten Phase der Infektion nachgewiesen. Diese Ergebnisse zeigen den Einfluss einer intestinalen Nematodeninfektion auf die Aktivität von Tregs und das Potential parasitenspezifischer CD4+ Effektor-Zellen zur Vermittlung von Schutz gegen die Infektion. Ein weiteres Projekt dieser Arbeit wahr die Verabreichung eines immunmodulatorischen Parasitenproteins, des Filariencystatins Av17, in einem Mausmodell entzündlicher Darmerkrankungen. In Mäusen wurde eine kolitisartige Entzündung durch eine Chemikalie im Trinkwasser induziert. Die regelmäßige Verabreichung von rekombinant exprimiertem Cystatin verminderte die Entzündungsreaktion signifikant. Damit konnte in dieser Arbeit gezeigt werden. dass Entzündungsreaktionen, die nicht durch den Parasiten selbst hervorgerufen werden, durch die Applikation einer einzelnen Parasitenkomponente unterdrückt werden können. / Parasitic nematodes specifically modulate the immune response of their hosts. A cellular hyperreactivity, especially during the chronic phase of infection, is a distinct finding of such infections. The T cell response against an intestinal nematode was analyzed in this work. Mice were infected with the trichostrongylid Heligmosomoides polygyrus and surveyed for changes concerning effector and regulatory T cells (Tregs). Subpopulations of CD4+ T cells were isolated from chronically infected mice and adoptively transferred to naive recipients, which were subsequently infected. The Transfer of CD4+ effector cells conferred partial protection, seen as decreased worm burdens in recipients. This effect was unimpaired by simultaneous transfer of Tregs. The transfer of purified Tregs alone showed no effect on worm burdens. The protection by transfer of effector T cells was probably due to a small parasite-specific population, which was characterized by the expression of CD40-L (CD154) after antigen-restimulation. The CD154+ population contained high frequencies of cells reacting with production of the Th2 key cytokines interleukin-4 (IL-4) and IL-13. On the other hand, a subpopulation of Tregs secreted high amounts of IL-10 in response to the antigen. These Tregs were characterized by the expression of the integrin AlphaE (CD103)Beta7, as well as CD25 and Foxp3. They showed a peculiar strong suppressive efficacy on the proliferation of other T cells, especially when derived from chronically infected donors. Analyzing the cellular composition of mesenteric lymph nodes and spleens in response revealed a lasting and over-proportional increase in frequencies of these Tregs. In clear contrast, only a transient increase of Foxp3-expressing Tregs was detected at the site of infection during the acute phase. These results point out the changes Treg activity during an intestinal nematode infection and show the potential of CD4+ effector cells in mediating protection against infection. A second project of this work was the application of an immunomodulatory parasite protein, the filarial cystatin Av17, in a mouse model of inflammatory bowel disease. Mice developed an inflammatory response to a chemical applied in the drinking water. The repeated application of recombinantly expressed cystatin significantly diminished the inflammatory response. Hence, this work showed the potential of a single parasite component in suppressing inflammatory processes not caused by the parasite itself.

Infektionen mit dem intestinalen Parasiten Giardia duodenalis, verursachen weltweit eine der häufigsten humanen Parasitosen. Bislang konnten keine eindeutigen Virulenz- oder Pathogenitätsmarker des Erregers beschrieben werden. Es wird allerdings vermutet, dass potentielle G. duodenalis Virulenzfaktoren Enzyme sind, die während des Kontaktes des Erregers mit den Dünndarmepithelzellen sezerniert werden. Eines dieser Enzyme ist die Arginin Deiminase (ADI), die Arginin zu Citrullin umwandelt. Ziel dieser Arbeit war es Merkmale zu identifizieren, die für die ADI als Virulenzfaktor sprechen. Dazu wurde das Enzym zunächst hinsichtlich seiner Bedeutung für die Wirt-Pathogen-Interaktion untersucht. Die mit rekombinanter, katalytisch aktiver ADI (Assemblage A) behandelten LPS-stimulierten humanen moDC zeigten eine Veränderung in ihrem Phänotyp als auch in ihrer Cytokinsekretion. Diese ließ sich auf die durch das Enzym hervorgerufene Arginindepletion und/oder auf die Bildung der Metabolite, Citrullin und NH4+, zurückführen. Weiterhin konnte gezeigt werden, dass Parasitenisolate verschiedener G. duodenalis Assemblage A-Subtypen, vermutlich durch die katalytische Aktivität der ADI, die Stickstoffmonoxid-Bildung einer intestinalen Epithelzelllinie inhibiert. Neben dem Einfluss auf die Wirtsimmunantwort wurde auch die Variabilität in der kodierenden Sequenz des Enzyms in verschiedenen Parasitenisolaten analysiert. Anschließend erfolgte die funktionelle Charakterisierung des nativen (verschiedene Assemblage A-Subtypen) als auch des rekombinant aufgereinigten Enzyms (Assemblage A, B und E). Dabei zeigten sich Unterschiede in der Substrataffinität der ADI für Arginin, sowohl zwischen unterschiedlichen Assemblage A-Subtypen als auch unterschiedlichen Assemblage-Klassen. Zusammenfassend wurde gezeigt, dass die G. duodenalis ADI immunmodulatorische Effekte hat und das vermutlich eine Korrelation zwischen der Variation in der Primärstruktur und der Funktion des Enzyms besteht. / Giardia duodenalis (G. duodenalis) is an intestinal protozoan parasite that causes giardiasis, one of the most prevalent parasitic diseases worldwide. So far, little is known concerning host-parasite interaction, in particular what determines the parasite’s pathogenicity. Several potential virulence factors, among them the arginine deiminase (ADI) that hydrolyzes arginine into citrulline and NH4+, are discussed. The ADI was identified to be released upon contact with intestinal epithelium by Giardia trophozoites and was recognized as an immunoreactive protein during acute human giardiasis. Aim of the study was to identify hints for G. duodenalis ADI to be a virulence factor. First, to analyze the enzyme’s impact on host-parasite interplay, its influence on human monocyte-derived dendritic cells (moDC) was investigated. Treatment of LPS-stimulated cells with recombinant ADI of assemblage A changed DC phenotype (CD83, CD86) and cytokine secretion (TNF-α, IL-10, IL-12p40). These immunomodulatory changes in DC response were due to arginine depletion and the formation of reaction products, in particular, ammonium ions. Furthermore, trophozoites of different assemblage subtypes were shown, probably due to consumption of arginine by ADI, to reduce nitric oxide formation by intestinal epithelial cells in vitro. Second, variation in the ADI coding sequence of different G. duodenalis isolates being collected in a Giardia biobank was analyzed by sequencing. Subsequently, functional genetics were performed with native ADI of different assemblage A subtypes expressed by these strains as well as with purified, recombinant ADI of assemblage A, B and E. It was recognized that enzymes of the same subtype as well as of different assemblages types had different substrate affinities for arginine. To sum up, this report identified G. duodenalis ADI to be immunomodulatory and gives first indications of a correlation between enzyme function and variation of the protein primary structure.

A epidemia HIV/AIDS é um grave problema de saúde enfrentado no Brasil e no mundo. Desde o surgimento do vírus, na década de 80, muitos esforços foram realizados para esclarecer o curso da infecção que resulta no comprometimento do sistema imune em indivíduos sem tratamento. A ativação imune crônica pode levar a um status de imunossenescência exacerbada, morte celular, alteração da resposta imune e uma imunodeficiência generalizada. Percebe-se que diversos fatores do hospedeiro interferem na progressão para Aids, como deleção de 32 pares de base do gene CCR5 (CCR5delta32), perfis de HLA desfavoráveis (*B35) e coinfecções, principalmente citomegalovírus, tuberculose e hepatites B e C. Estudos recentes com o GBV-C, pertencente à família Flaviviridae, gênero Pegivirus, possibilitaram uma nova perspectiva no entendimento do curso da infecção causada pelo HIV, uma vez que nenhuma doença foi relacionada à presença do vírus GB tipo C, além de promover um atraso na progressão para a Aids e aumento da sobrevida dos pacientes portadores do vírus. Assim, o objetivo desse estudo foi avaliar o perfil de ativação, senescência e exaustão celular em indivíduos recém-infectados pelo HIV-1 e coinfectados pelo GBVC. Foram investigados a contagem de linfócitos T CD4+ e CD8+, razão CD4/CD8, presença do GBV-C e HIV-1, além da análise da expressão de marcadores de ativação (CCR5, CD38 e HLA-DR), senescência e exaustão celular (PD-1, CD95, CD57 e CD28). Diante dos critérios de inclusão do estudo, foram selecionados nove pacientes com infecção persistente com o vírus GBV-C para o grupo 1 (HIV-1+/GBV-C+), e oito pacientes sem viremia para GBV-C foram incluídos no grupo 2 (HIV-1+/GBV-C-), sendo a média de idade dos pacientes selecionados de 31,6 e 31,7 anos, respectivamente, sexo masculino e homens que fazem sexo com homens (HSH). Na visita de inclusão no estudo (V1) nenhum dos dados analisados (células T CD4+ e CD8+, carga viral e razão CD4/CD8) apresentou diferença estatística, assim como os marcadores de ativação, senescência e exaustão celular. Na análise longitudinal da diferença (deltaVn-V1), percebeu-se uma diminuição dos marcadores de ativação e senescência no grupo HIV-1+/GBV-C +, sem significância estatística entre esses dados. Foi observado, contudo, que houve uma diminuição de células T CD4+ e CD8+ naïve no grupo HIV-1+/GBV-C+, também notou-se redução na subpopulação de células T CD8+ naïve e memória central expressando CD28, houve uma diminuição das subpopulações de memória intermediária e efetora terminal, assim como na subpopulação efetora terminal expressando HLA-DR+, no grupo HIV-1+/GBV-C+. Os resultados demonstraram que a infecção pelo GBV-C reflete na diminuição da estimulação imune, ativação celular e também na redução de marcadores de senescência e exaustão celular nas subpopulações de células T, sugerindo um envolvimento na modulação da progressão do HIV / The HIV/AIDS epidemic is a serious health problem in Brazil and in the world. Since its emergence in the 1980s, many efforts have been made to understand this infection, resulting in a compromised immune system if left untreated. Chronic immune activation may lead to exacerbated immunosenescence, cell death, altered immune response, and a generalized immunodeficiency. Several host factors play an important role in the progression to AIDS, such as the 32 base pairs deletion in the CCR5 gene (CCR5delta32), unfavorable HLA molecules (*B35), and coinfections, mainly cytomegalovirus, tuberculosis, and hepatitis B and C. Recent studies with the GBV-C (Flaviviridae family, genus Pegivirus) have provided a new perspective in the understanding of the HIV infection natural history. GBV-C coinfection delays progression to Aids and increases patient survival. In addition, no symptoms have been associated to its occurrence. The aim of this study was to evaluate the profile of cellular activation, senescence, and exhaustion in recently HIV-infected individuals coinfected with GBVC. Patients were selected from a prospective cohort diagnosed with recent HIV-1 infection with known results for levels of CD4+ and CD8+ T lymphocytes, CD4/CD8 ratio, GBV-C plasma levels, HIV-1 plasma viremia, and markers for cellular activation (CCR5, CD38, and HLA-DR) and senescence and exhaustion (PD-1, CD95, CD28, and CD57). Nine presented persistent GBV-C infection and were selected for group 1 (HIV- 1/GBV-C+), mean age of 31.6 years. Another set of eight patients without GBV-C viremia were selected as controls and included in group 2 (HIV-1/GBV-C-), mean age of 31.7 years. All participants were male, in most cases men who have sex with men (MSM). At baseline visit (V1), no variable (levels of CD4+ and CD8+ lymphocytes, viral load, CD4/CD8 ratio, and cellular activation, senescence, and exhaustion markers) presented no statistical significant differences, suggesting that all selected patients shared similar characteristics. Longitudinal analysis (delta, Vn-V1) revealed a nonsignificant decrease in activation and senescence markers for both groups. However, it was observed a decrease in naïve CD4+ and CD8+ T cells in group 1, and also a reduction in the subpopulations of naïve and central memory (CD28+) CD8+ T cells. The HIV+/GBV-C+ group also presented diminished intermediate memory and terminal effector subpopulations, as well a decrease in HLA-DR+ terminal effector cells. The data demonstrate that GBV-C infection results in reduced immune stimulation, cellular senescence, and cell exhaustion, suggesting an involvement in the modulation of HIV progression

A natureza sempre constituiu uma fonte extremamente rica de compostos e substâncias utilizados pelo homem para diferentes fins, principalmente na área médica, pois os produtos naturais abrangem uma variedade de agentes terapêuticos para diversas doenças, como moléstias infecciosas, câncer, desordens lipídicas e imunológicas. Com a necessidade de se encontrar novos fármacos, tanto para doenças bem definidas, quanto para novas doenças, a pesquisa de substâncias com atividades biológicas torna-se fundamental. Fontes exóticas como o ambiente marinho e, recentemente o universo dos micro-organismos, ganharam grande interesse no meio científico devido ao fato de terem sido pouco conhecidas e exploradas. Nesse contexto, o objetivo desse estudo foi avaliar se diferentes frações da macroalga marinha Bostrychia tenella e extratos de micro-organismos endofíticos isolados dessa mesma espécie possuíam atividade imunomoduladora sobre o sistema complemento (SC) humano, que é reconhecido como um dos maiores efetores do processo inflamatório. Iniciado por três vias distintas (clássica, alternativa e das lectinas), ele leva à formação de produtos de clivagem e complexos de ativação que exercem diferentes funções biológicas importantes, como quimiotaxia, ativação de fagócitos, desgranulação de mastócitos, citólise, ativação celular e apoptose. Sendo assim, este sistema desempenha papel importante em várias reações inflamatórias, bem como em diferentes mecanismos patogênicos de dano tecidual. Através do ensaio de atividade hemolítica, 10 das 20 amostras iniciais foram selecionadas por modularem esta atividade do SC no pool de soro humano normal (SHN). As 10 amostras (dentre frações, subfrações e extratos de B. tenella e seus micro-organismos endofíticos) foram capazes de induzir a geração de fragmentos de C3, componente central do SC, observados tanto em técnicas imunoeletroforéticas, quanto em Western blotting. Também, observou-se a formação do complexo de ataque à membrana (MAC) em ensaio imunoenzimático, indicando que houve ativação total do SC. Através dos resultados obtidos no Western blotting para fragmentos de C4 e Fator B foi possível identificar quais vias do SC são ativadas, quando correlacionados com os resultados dos ensaios hemolíticos para via clássica/das lectinas (VC/VL) e via alternativa (VA). Desse modo, foi possível concluir que as 10 amostras selecionadas possuem efeito imunomodulador sobre o SC humano por serem capazes de ativá-lo, seja pela VC/VL ou pela VA. Conclui-se, também, que o ensaio hemolítico cinético constitui uma importante ferramenta de triagem, assim como o Western blotting do SHN para detecção de fragmentos do SC. / Nature has always provided an extremely rich source of compounds and substances used by man for various purposes, mainly in the medical field because natural products include a variety of therapeutic agents for various diseases such as infectious diseases, cancer, lipid and immune disorders. With the need to find new drugs for either well defined diseases as for new ones, the search for substances with biological activities becomes essential. Exotic sources such as marine environment, and recently the world of micro-organisms have gained great interest in the scientific community due to the fact that they were little known and explored. In this context, the objective of this study was to assess whether different fractions of marine macroalgae Bostrychia tenella and extracts of endophytic micro-organisms isolated from this species possess immunomodulatory activity on the human complement system (CS), which is recognized as one of the major effectors of the inflammatory process. Started by three distinct pathways (classical, alternative and lectin), it leads to the formation of cleavage products and activation complexes which have various important biological functions such as chemotaxis, activation of phagocytes, mast cell degranulation, cytolysis, cellular activation and apoptosis. Thereby, this system plays an important role in various inflammatory reactions, as well as various pathogenic mechanisms of tissue damage. Through the hemolytic activity assays, 10 of the initial 20 samples were selected because they were able to modulate this activity of the CS in the pool of normal human serum (NHS). The 10 samples (among fractions, subfractions and extracts of B. tenella and its endophytic micro-organisms) were able to induce the generation of fragments from C3, a central component of the CS, which was observed both in immuno electrophoresis technique and Western blotting. Also, we observed the formation of the membrane attack complex (MAC) in enzyme immunoassay, indicating that there was total activation of the CS. By the results obtained in Western blotting for fragments of C4 and Factor B was possible to identify which pathways are activated in CS, when correlated with the results of hemolytic assays for the classical/lectin (VC/VL) and alternative pathway (VA). Thus, we conclude that the 10 selected samples have immunomodulatory effects in human CS because they were able to activate it either by VC/VL or VA. It follows also that the hemolytic assay by the kinetic method is an important tool for screening, as well as Western blotting of NHS to detect fragments of the CS.

O presente estudo avaliou a ocorrência de imunoparalisia e sua associação com pior prognóstico em pacientes pediátricos internados em uma UTI de hospital terciário. Para determinar a presença de imunoparalisia procedeu-se a dosagem da expressão de mHLA-DR usando o QuantiBRITE TM Anti HLA-DR/ Anti- Monocyte, um novo reagente que padroniza os valores da citometria de fluxo para o mHLA-DR. Determinamos a expressão de mHLA-DR em 30 pacientes com sepse grave ou choque sépticos admitidos na UTI Pediátrica no período do estudo, mHLA-DR foi quantificado por duas vezes: entre os dias 3 a 5 (mHLA-DR1) e 5 a 7 (mHLA-DR2) após o inicio do quadro séptico. Também foi calculado o deltamHLA-DR (mHLA-DR2 - mHLA-DR1). Dosamos, ainda, o mHLA-DR em vinte e um controles hígidos. O objetivo do estudo foi determinar se a expressão de mHLA-DR correlaciona-se com a mortalidade em pacientes sépticos pediátricos. Os resultados mostram que o mHLA-DR foi significativamente menor nos pacientes sépticos do que nos controles (p = 0.0001). A mortalidade foi de 46% nos pacientes com valores negativos ou < 1000 mAb/cell de deltaHLA-DR, e 7% em pacientes com valores positivos ou > 1000 mAb/cell de deltaHLADR. O deltamHLA-DR médio foi significativamente diferente entre sobreviventes e pacientes que foram a óbito (p = 0.023). Dessa forma, após a análise estatística dos resultados concluímos que o deltaHLA-DR correlaciona-se com a mortalidade em pacientes pediátricos com sepse grave e choque séptico / This study analysis the presence of Immunoparalysis and its association with prognosis in pediatric septic patients of a Tertiary Intensive Care Unit. To determine the presence of immunoparalysis we performed the mHLA-DR dosage using the QuantiBRITE TM Anti HLA-DR/ Anti- Monocyte, a novel reagent that standardizes flow cytometry values. We determined mHLA-DR expression in 30 patients with severe sepsis or septic shock admitted to PICU, mHLA-DR expression was quantified between days 3-5 and 5-7 after the onset of sepsis and calculated the deltamHLA-DR (mHLA-DR2 - mHLADR1). We also measured mHLA-DR levels in twenty-one healthy patients. The objective of this study was to determine if mHLA-DR values correlate with mortality in pediatric septic patients. The results showed that the mean mHLA-DR expression was significantly lower in septic patients compared with controls (p = 0.0001). Mortality was 46% in patients with negative deltaHLA-DR or < 1000 mAb/cell and 7% in patients with positive deltaHLA-DR or > 1000 mAb/cell. Mean deltamHLA-DR levels were significantly different between survivors and non-survivors (p = 0.023). After statistical analysis we concluded that deltaHLA-DR correlates with mortality in pediatric patients with septic shock or severe sepsis

O presente estudo avaliou a ocorrência de imunoparalisia e sua associação com pior prognóstico em pacientes pediátricos internados em uma UTI de hospital terciário. Para determinar a presença de imunoparalisia procedeu-se a dosagem da expressão de mHLA-DR usando o QuantiBRITE TM Anti HLA-DR/ Anti- Monocyte, um novo reagente que padroniza os valores da citometria de fluxo para o mHLA-DR. Determinamos a expressão de mHLA-DR em 30 pacientes com sepse grave ou choque sépticos admitidos na UTI Pediátrica no período do estudo, mHLA-DR foi quantificado por duas vezes: entre os dias 3 a 5 (mHLA-DR1) e 5 a 7 (mHLA-DR2) após o inicio do quadro séptico. Também foi calculado o deltamHLA-DR (mHLA-DR2 - mHLA-DR1). Dosamos, ainda, o mHLA-DR em vinte e um controles hígidos. O objetivo do estudo foi determinar se a expressão de mHLA-DR correlaciona-se com a mortalidade em pacientes sépticos pediátricos. Os resultados mostram que o mHLA-DR foi significativamente menor nos pacientes sépticos do que nos controles (p = 0.0001). A mortalidade foi de 46% nos pacientes com valores negativos ou < 1000 mAb/cell de deltaHLA-DR, e 7% em pacientes com valores positivos ou > 1000 mAb/cell de deltaHLADR. O deltamHLA-DR médio foi significativamente diferente entre sobreviventes e pacientes que foram a óbito (p = 0.023). Dessa forma, após a análise estatística dos resultados concluímos que o deltaHLA-DR correlaciona-se com a mortalidade em pacientes pediátricos com sepse grave e choque séptico / This study analysis the presence of Immunoparalysis and its association with prognosis in pediatric septic patients of a Tertiary Intensive Care Unit. To determine the presence of immunoparalysis we performed the mHLA-DR dosage using the QuantiBRITE TM Anti HLA-DR/ Anti- Monocyte, a novel reagent that standardizes flow cytometry values. We determined mHLA-DR expression in 30 patients with severe sepsis or septic shock admitted to PICU, mHLA-DR expression was quantified between days 3-5 and 5-7 after the onset of sepsis and calculated the deltamHLA-DR (mHLA-DR2 - mHLADR1). We also measured mHLA-DR levels in twenty-one healthy patients. The objective of this study was to determine if mHLA-DR values correlate with mortality in pediatric septic patients. The results showed that the mean mHLA-DR expression was significantly lower in septic patients compared with controls (p = 0.0001). Mortality was 46% in patients with negative deltaHLA-DR or < 1000 mAb/cell and 7% in patients with positive deltaHLA-DR or > 1000 mAb/cell. Mean deltamHLA-DR levels were significantly different between survivors and non-survivors (p = 0.023). After statistical analysis we concluded that deltaHLA-DR correlates with mortality in pediatric patients with septic shock or severe sepsis

Μέχρι σήμερα δεν υπάρχει εμβόλιο για καμιά μορφή των λεϊσμανιάσεων και τα φάρμακα που χρησιμοποιούνται δεν είναι ασφαλή, αποτελεσματικά και οικονομικά προσιτά. Υπάρχει άφθονη επιστημονική πληροφορία για τα αντιλεϊσμανιακά φάρμακα που χρησιμοποιούνται με αναγνωρισμένα μειονεκτήματα όπως η τοξικότητα, η ανάπτυξη αντοχής, η υποχρεωτική παραμονή στο νοσοκομείο και το υψηλό κόστος. Επιπλέον η συλλοίμωξη με τον ιό του AIDS (HIV/VL) έχει σοβαρές επιπτώσεις στην επιδημιολογία, τη διάγνωση και την πρόγνωση της νόσου. Είναι προφανές ότι η εκδήλωση της σπλαγχνικής λεϊσμανίασης είναι “ευκαιριακή” όπως αποδεικνύεται από τα στοιχεία του Π.Ο.Υ., σύμφωνα με τα οποία οι ασθενείς με AIDS έχουν 3.000 φορές υψηλότερη συχνότητα VL σε σύγκριση με τον γενικό πληθυσμό. Για τα διάφορα είδη του γένους Leishmania υπάρχουν αξιόλογα ευρήματα από παρασιτολογικές, βιοχημικές, μοριακές και ανοσολογικές μελέτες, όμως τα θεραπευτικά πρωτόκολλα ενάντια των διαφορετικών κλινικών μορφών δεν είναι ικανοποιητικά. Γεγονός αποτελεί η αδήριτη ανάγκη για την ανάπτυξη νέων θεραπευτικών προσεγγίσεων κατά των λεϊσμανιάσεων που μαστίζουν έως και 1,7 εκατομμύρια ανθρώπους σε όλη την γη. Σήμερα το ενδιαφέρον για φυσικά προϊόντα βρίσκεται σε επικαιρότητα κυρίως με την αναζήτηση νέων χημικών ενώσεων αλλά και την επανεξέταση παλαιοτέρων σε σύγχρονα πλέον πειραματικά πρωτόκολλα. Μεταξύ αυτών των προϊόντων η ελαιοευρωπεΐνη, που προέρχεται από το δένδρο της ελιάς (Olea europaea), χαρακτηρίζεται από αντιοξειδωτική, αντιμικροβιακή και αντιφλεγμονώδη δράση. Η βιοφαινόλη αυτή φαίνεται να συμβάλλει επίσης στην μακροζωία αλλά και όταν χορηγείται σε πειραματόζωα με καρκινικούς όγκους είναι ικανή στο να τους συρρικνώνει ή ακόμη και να τους εξαφανίζει. Ο σκοπός αυτής της διατριβής είναι η διερεύνηση φυσικών προϊόντων προερχόμενων από τα φύλλα και τους καρπούς του δέντρου της ελιάς αλλά και από πάρα-προϊόντα του όπως τα υγρά απόβλητα ελαιοτριβείου. Επίσης εξετάσθηκαν δύο αδρά εκχυλίσματα από τον ερυθρό οίνο ποικιλίας ξινόμαυρο. Αμφότερες οι πηγές των εκχυλισμάτων αποτελούν θεμελιώδεις λίθους της Μεσογειακής διατροφής και οι βιβλιογραφικές αναφορές τα καθιστούν ως υποσχόμενους αντιλεϊσμανιακούς παράγοντες. Τα αποτελέσματά έδειξαν ότι τα δύο καθαρά φυσικά προϊόντα που εξετάσθηκαν, η υδροξυτυροσόλη και η ελαιοευρωπεΐνη επέδειξαν επιλεκτική και σημαντική αντιλεϊσμανιακή δραστικότητα εναντίον τριών ειδών πρωτοζώων του γένους Leishmania και συγκεκριμένα των L. infantum, L. donovani και L. major. Η διατριβή εστιάστηκε στη ελαιοευρωπεΐνη και αναδείχθηκε η ικανότητά της να προκαλεί ρυθμιζόμενο κυτταρικό θάνατο αποδεικνυόμενο από μορφολογικές αλλαγές σε προμαστιγότες L. donovani, λογαριθμικής φάσης ανάπτυξης, με τη χρήση συνεστιακής μικροσκοπίας. Η στρογγυλοποίηση των προμαστιγοτών, η συμπύκνωση της χρωματίνης καθώς και η έκθεση της φωσφατιδυλοσερίνης στην εξωτερική επιφάνεια της κυτταροπλασματικής μεμβράνης με τη χρήση κυτταρομετρίας ροής είναι φαινοτυπικά χαρακτηριστικά της επαγωγής ρυθμιζόμενου παρασιτικού θανάτου που αποδείχθηκε ότι ο μηχανισμός πρόκλησής του είναι ανεξάρτητος της παραγωγής ROS. Επιπλέον η ελαιοευρωπεΐνη προκάλεσε μια καθυστέρηση στο φυσιολογικό κυτταρικό κύκλο του παρασίτου οδηγώντας στον κατακερματισμό του DNA. Η αντιλεϊσμανιακή δράση της ελαιοευρωπεΐνης αποδείχτηκε και εναντίον των αμαστιγωτικών μορφών του είδους L. donovani που είχαν in vitro παρασιτήσει σε μακροφάγα της κυτταρικής σειράς J774Α.1. Αυτή η δράση συνοδεύτηκε από μία ενδομακροφαγική αύξηση ROS ενώ η in vitro χορήγηση ελαιοευρωπεΐνης σε μυελοειδή DCs αύξησε τον πληθυσμό που παράγει IL-12 δίχως να προκαλεί παράλληλη ωρίμανσή τους. Τα ανωτέρω οδήγησαν στη μελέτη της δράσης της ελαιοευρωπεΐνης σε in vivo πειραματικό μοντέλο σπλαχνικής λεϊσμανίασης. Σε L. donovani μολυσμένα BALB/c ποντίκια χορηγήθηκε ενδοπεριτοναϊκώς ελαιοευρωπεΐνη κάθε δεύτερη ημέρα για 28 συνεχόμενες ημέρες σε τρεις διαφορετικές δόσεις (45, 15 και 5mg/kg σωματικού βάρους). Βρέθηκε ότι και οι τρεις δόσεις περιόρισαν το παρασιτικό φορτίο του σπλήνα και του ήπατος στις 3 ημέρες και στις 6 εβδομάδες μετά το πέρας της χορήγησης της ελαιοευρωπεΐνης. Διαπιστώθηκε ότι τα πειραματόζωα που έλαβαν ελαιοευρωπεΐνη και περιόρισαν την ενδοκυτταρική εξάπλωση του παρασίτου στο σπλήνα και στο ήπαρ ανέπτυξαν μια ενισχυμένη ΤΗ1 τύπου ανοσολογική απόκριση με χαρακτηριστικό ισοτυπικό προφίλ αντισωμάτων IgG2α/IgG1 και μεταγραφικών παραγόντων Tbx21/GATA-3. Η υπερ-έκφραση των IL-12p40, IFNγ, TNFα και iNOS επιβεβαιώνει την πόλωση προς τον ΤΗ1 υποτύπο ανοσολογικής απόκρισης, ενώ η έκφραση των κυτταροκινών ρυθμιστές της TΗ2 ανοσολογικής απάντησης δεν είχαν καμία αυξητική τάση. Αυτό το φαινόμενο που προέρχεται αρχικώς από την ικανότητα της ελαιοευρωπεΐνης να διατηρεί χαμηλά τα επίπεδα της προφλεγμονώδους κυτταροκίνης IL-1β, επιτρέπει τον ανοσολογικό μηχανισμό του ξενιστή να ενεργοποιηθεί (μεταγραφή του Tbx21) αποτρέποντας την είσοδο του ξενιστή σε κατάσταση χρόνιας φλεγμονικής διεργασίας που ευνοεί τον παρασιτικό πολλαπλασιασμό. Απεναντίας ενεργοποιούνται μηχανισμοί παραγωγής των μικροβιοκτόνων μορίων ROS και NO• στο σπλήνα και στο ήπαρ. Η παραγωγή των ROS σε σπληνοκύτταρα ποντικών μολυσμένων με L. donovani ήταν δοσοεξαρτώμενη ενώ αντιθέτως η παραγωγή των ΝΟ• ήταν μη-δοσοεξαρτώμενη τόσο σε κύτταρα του σπλήνα όσο και του ήπατος. Αυτή η παραγωγή των ΝΟ• φαίνεται να επιδρά στην επιβίωση των παρασίτων χωρίς να αλλοιώνει τη φυσιολογική λειτουργία των σπληνοκυττάρων και των ηπατοκυττάρων εφ’ όσον τα μόρια γλουταθειόνης στα κύτταρα του ξενιστή ήταν επαρκή για να συντηρούν τη συνεχή επιβίωση του κυττάρου ξενιστή όπως παρατηρήθηκε με την μη-ενεργοποίηση του NF-kB. Επίσης η ελαιοευρωπεΐνη επιλεκτικά ρυθμίζει την γονιδιακή έκφραση των ενζύμων που σχηματίζουν τη γλουταθειόνη και την τρυπανοθειόνη εις βάρος των αντιοξειδωτικών αμυντικών μηχανισμών του παρασίτου. Συμπερασματικά, το σύνολο των αποτελεσμάτων που περιεγράφηκαν στη διδακτορική αυτή διατριβή, προτείνουν ότι η ελαιοευρωπεΐνη δρα ως ένα αντιλεϊσμανιακό φάρμακο με ανοσοτροποποιητικές ιδιότητες. Η διερεύνηση της συνδυαστικής δράσης κλασσικών αντιλεϊσμανιακών φαρμάκων με ασφαλή, μη-τοξικά και “φθηνά” φυσικά προϊόντα όπως η ελαιοευρωπεΐνη, παραμένει μια υποσχόμενη μελλοντική προοπτική. Επιπροσθέτως, ενδιαφέρουσα θα ήταν η διερεύνηση της δράσης της ελαιοευρωπεΐνης στην έκφραση έτερων γονιδίων που κωδικοποιούν αντιοξειδωτικά ένζυμα καθώς και στους κυτταρικούς πληθυσμούς που εμπλέκονται επίσης σε in vivo πειραματικό μοντέλο σπλαχνικής λεϊσμανίασης όπως τα δενδριτικά κύτταρα, τα κύτταρα φυσικοί φονιάδες και τα CD8+. Τέλος η ελαιοευρωπεΐνη μπορεί να δοκιμασθεί σε HIV/L. donovani πειραματικό πρωτόκολλο αφού η ελαιοευρωπεΐνη είναι ικανή στο να σταματάει τον πολλαπλασιασμό του ιού HIV. / Up to now there has been neither a vaccine against any form of leishmaniasis found nor drugs that are safe, effective and inexpensive. Plethora of recent data has showed that the existing antileishmanial drugs have numerous disadvantages such as toxicity, development of resistance, long hospitalization and high cost. In addition, HIV/VL coinfection has important epidemiological, clinical, diagnostic and prognostic implications. The two diseases are mutually reinforcing. Clearly the opportunistic manifestation of VL is demonstrated by the WHO records showing that 50-75% new VL cases in South Europe concern HIV patients that bring the incidences of VL 3,000 times more frequent than in the general population. Despite the advances in the parasitological, biochemical, molecular and immunological research using various Leishmania species, the treatment protocols applied against the different forms of the disease are not satisfactory. There is an unconquerable need for the development of new therapeutic treatments to combat leishmaniasis. Nowadays, the interest concerning plant natural products is certainly undergoing a renaissance. The remarkable chemical diversity present in natural products and the accumulated research data produced so far, demonstrate their significant effectiveness against parasitic diseases suggesting the hypothesis to be tested also against leishmaniasis. This was particularly evident in the case of natural products of the olive tree (Olea europaea). Among them oleuropein has most of olive oil’s antioxidant, anti-inflammatory, and disease-fighting characteristics and it is the biophenol that provides life extending benefits. In addition to its antimicrobial and antioxidant activity, when oleuropein was given to animals with tumors, the tumors completely regressed or in some cases even disappeared. The aim of this thesis is to investigate natural products obtained from the olive tree leaves, products and their by-products (olive mill waste waters) and red wine (xinomavro variety), major cornerstones of the Mediterranean diet, as natural sources for promising antileishmanial drugs in accordance with the considerations outlined above. Our results showed that both pure constituents, hydroxytyrosol and oleuropein, exhibited the highest and the most selectively significant leishmanicidal activity against parasites of three Leishmania species, L. infantum, L. donovani and L. major, among the natural products tested. We focused on oleuropein and we discovered that it was capable of causing substantial morphological alterations upon L. donovani logarithmic promastigotes depicted with confocal microscopy namely, cell rounding up, cellular volume reduction and chromatin condensation that were accompanied with phosphatidylserine exposure to the outer leaflet of the plasma membrane. Moreover, oleuropein was found responsible for a delay on normal L. donovani cell cycle progression which was extended up to DNA fragmentation. This regulated parasitic cell death induced by oleuropein appeared to be ROS-independent. Oleuropein’s leishmanicidal capacity was also evident against the amastigote form of the parasite using an in vitro experimental model based on J774.A1 macrophages infected with L. donovani. This activity is accompanied by an increase of inracellular ROS production and the ability of oleuropein to increase the percentage of IL-12 producing myeloid DCs when is administrated in vitro. This data suggested testing the leishmanicidal activity of oleuropein upon in vivo murine visceral model. Intraperitoneal administration of oleuropein every other day for 28 days in L. donovani infected BALB/c mice, led to the dramatic reduction of the parasite load in spleen and liver. Interestingly, oleuropein treated L. donovani infected BALB/c mice, switch towards a TH1 type of immune response characterized by relevant IgG2a/IgG1 isotype and Tbx21/GATA-3 transcription factor ratio. Analysis of the whole spleen tissue revealed small but significant fold changes in gene expression of TH1 versus TH2 type of cytokines between treated and non-treated L. donovani infected BALB/c mice. Particularly, the dominance of TH1 type of response was confirmed by the up regulation of IL-12p40, IFN-γ and TNF-α which triggers elevated expression of iNOS in splenocytes in oleuropein treated L. donovani infected BALB/c mice. Despite the presence of TH2 type of response, the abiding down regulation of IL-1β permits Tbx21 expression and prevents prolonged inflammatory process and thus to disease exacerbation. We also found that oleuropein promoted a dose-dependent ROS production on spleen cells from BALB/c mice infected with L. donovani while it was able to evoke a dose-independent NO production on spleen and liver cells. NO production seems to affect parasitic viability but did not alter physiological function of spleen and liver cells since glutathione cell reservoir is capable of maintaining the continuity of host cell survival confirmed by the absence of NF-kB2 expression increase. Furthermore, oleuropein selectively regulates the transcription of glutathione and trypanothione gene which encode their synthesis enzymes at expense of parasitic antioxidant defense mechanism. The overall conclusion of the research described in this thesis suggests that oleuropein acts as an effective antileishmanial drug with demonstrable immunostimulating properties and selectively induces parasitic death by microbicidal molecules. The option of testing the combining effects of classical antileishmanial drugs with safe, non-toxic and cost effective natural products like oleuropein remains a promising future perspective. Moreover, interesting remains the effect of oleuropein upon other antioxidant genes and cell populations that play crucial roles in the in vivo experimental visceral leishmaniasis such as mDCs, NK and CD8+ cells. Finally, oleuropein could be used upon HIV/L. donovani experimental protocol since oleuropein is also capable to cease viral multiplication.

La réaction du greffon contre l’hôte (GvH) est responsable d’un grand taux de morbidité et de mortalité chez les patients recevant des greffes de cellules souches (GCSH) allogéniques. Dans ce contexte, les cellules T régulatrices sont largement étudiées et semblent avoir un grand potentiel d’utilisation dans le domaine de la thérapie cellulaire de la GvH. Parmi les populations cellulaires T régulatrices, les lymphocytes T CD4-CD8- TCRαβ+ « Doubles-Négatifs » (DN), qui ne représentent que 1-3% des lymphocytes T, ont été décrits. Ces cellules ont des propriétés inhibitrices de la réponse immunitaire qui s’avèrent spécifiques aux antigènes auxquels elles ont préalablement été exposées. La répression de la réponse immunitaire par les cellules T DN régulatrices semble être un mécanisme important impliqué dans l’induction de la tolérance aux allo-antigènes. De plus, ces cellules confèrent une tolérance immunitaire dans des modèles de greffes allogéniques et xénogéniques. En effet, ces cellules ont la capacité d’inhiber la réaction contre un allo-antigène auquel elles ont été exposées, sans inhiber la réaction contre un allo-antigène inconnu. Les cellules T DN ont été isolées et caractérisées chez l’homme où elles ont la capacité d’interagir avec des cellules présentatrices d’antigènes (APCs) par un contact cellulaire, comme chez la souris. Cependant, leur capacité immunomodulatrice reste inconnue chez l’humain. Notre objectif consistait donc principalement à étudier le rôle et le mécanisme d’action des cellules T DN régulatrices humaines in vitro, en étudiant leur capacité à inhiber une réaction lymphocytaire mixte (MLR). Nous avons montré que les cellules T DN stimulées par un allo-antigène donné inhibent des cellules syngéniques effectrices dirigées contre ce même alloantigène mais n’inhibent pas des cellules syngéniques effectrices dirigées contre un autre alloantigène, démontrant ainsi la spécificité aux antigènes de ces cellules. De plus, les T DN non stimulées par un allo-antigène n’ont pas de rôle inhibiteur. Cependant, durant cette inhibition, nous n’observons pas de modulation de l’expression des marqueurs d’activation et d’induction de l’apoptose. Afin d’étudier le mécanisme d’action des cellules T DN, nous avons mesuré l’expression intracellulaire de la granzyme B. Les résultats démontrent que les cellules T DN stimulées expriment un niveau significativement plus élevé de granzyme B que les cellules T DN non-stimulées par l’allo-antigène. Ceci suggère que l’immunosuppression induite par les cellules T DN stimulées pourrait passer par la voie granzyme B. Le mécanisme utilisé par ces cellules reste à être confirmé par nos futures expériences. / Graft-versus-Host Disease (GvHD) is a major cause of morbidity and mortality in patients receiving an allogeneic Hematopoietic Stem Cell Transplantation (HSCT). Many regulatory T cell populations have been studied and shown to have immunosuppressive properties in GvHD. Among these populations, Double Negative CD4-CD8-TCRαβ+ regulatory T cells (DN T) have been described. These cells represent 1-3% of all T cell lymphocytes and are known to have antigen-specific inhibitory functions of the immune response. The suppression of an immune response by DN T cells seems to be an important mechanism involved in the induction of tolerance to allo-antigens. Moreover, these cells also confer immune tolerance in models of allogeneic and xenogenic grafts. DN T cells have the ability to suppress syngeneic T CD4+ and T CD8+ cells in an antigen-specific manner. Therefore, these DN T cells can inhibit the reaction caused by syngeneic effector cells against a specific alloantigen to which they have been previously exposed. However, they cannot inhibit a reaction directed against an unknown alloantigen. Human DN T cells have been isolated and characterized as cells that have the capacity to interact with APCs by cell-to-cell contact, just like in mice. However, their immunomodulatory properties are still unknown in humans. The goal of our project was to study the role and immunomodulatory functions of human DN T cells in Mixed Lymphocyte Reactions (MLR). The MLRs have allowed us to demonstrate that DN T cells, after having been stimulated by an allo-antigen, have an antigen-specific inhibitory function towards the syngeneic effector cells reacting against the same alloantigen that they have been exposed to. Interestingly, they do not inhibit the reaction of these effector cells against an unknown alloantigen. However, stimulated DN T cells did not modulate the expression of the activation markers expressed by the effector cells and did not give a death signal to these cells either. Moreover, we also wanted to study how DN T cells have an immunosuppressive activity. Therefore, we compared the expression of Granzyme B in stimulated versus non-stimulated cells. Our results suggest that DN T cells may use the Granzyme B pathway to immunosuppress the effector cells. In conclusion, our results demonstrate that DN T cells have an antigen-specific inhibitory function. The mechanism used by these DN T cells remains to be confirmed with our future experiments.

A natureza sempre constituiu uma fonte extremamente rica de compostos e substâncias utilizados pelo homem para diferentes fins, principalmente na área médica, pois os produtos naturais abrangem uma variedade de agentes terapêuticos para diversas doenças, como moléstias infecciosas, câncer, desordens lipídicas e imunológicas. Com a necessidade de se encontrar novos fármacos, tanto para doenças bem definidas, quanto para novas doenças, a pesquisa de substâncias com atividades biológicas torna-se fundamental. Fontes exóticas como o ambiente marinho e, recentemente o universo dos micro-organismos, ganharam grande interesse no meio científico devido ao fato de terem sido pouco conhecidas e exploradas. Nesse contexto, o objetivo desse estudo foi avaliar se diferentes frações da macroalga marinha Bostrychia tenella e extratos de micro-organismos endofíticos isolados dessa mesma espécie possuíam atividade imunomoduladora sobre o sistema complemento (SC) humano, que é reconhecido como um dos maiores efetores do processo inflamatório. Iniciado por três vias distintas (clássica, alternativa e das lectinas), ele leva à formação de produtos de clivagem e complexos de ativação que exercem diferentes funções biológicas importantes, como quimiotaxia, ativação de fagócitos, desgranulação de mastócitos, citólise, ativação celular e apoptose. Sendo assim, este sistema desempenha papel importante em várias reações inflamatórias, bem como em diferentes mecanismos patogênicos de dano tecidual. Através do ensaio de atividade hemolítica, 10 das 20 amostras iniciais foram selecionadas por modularem esta atividade do SC no pool de soro humano normal (SHN). As 10 amostras (dentre frações, subfrações e extratos de B. tenella e seus micro-organismos endofíticos) foram capazes de induzir a geração de fragmentos de C3, componente central do SC, observados tanto em técnicas imunoeletroforéticas, quanto em Western blotting. Também, observou-se a formação do complexo de ataque à membrana (MAC) em ensaio imunoenzimático, indicando que houve ativação total do SC. Através dos resultados obtidos no Western blotting para fragmentos de C4 e Fator B foi possível identificar quais vias do SC são ativadas, quando correlacionados com os resultados dos ensaios hemolíticos para via clássica/das lectinas (VC/VL) e via alternativa (VA). Desse modo, foi possível concluir que as 10 amostras selecionadas possuem efeito imunomodulador sobre o SC humano por serem capazes de ativá-lo, seja pela VC/VL ou pela VA. Conclui-se, também, que o ensaio hemolítico cinético constitui uma importante ferramenta de triagem, assim como o Western blotting do SHN para detecção de fragmentos do SC. / Nature has always provided an extremely rich source of compounds and substances used by man for various purposes, mainly in the medical field because natural products include a variety of therapeutic agents for various diseases such as infectious diseases, cancer, lipid and immune disorders. With the need to find new drugs for either well defined diseases as for new ones, the search for substances with biological activities becomes essential. Exotic sources such as marine environment, and recently the world of micro-organisms have gained great interest in the scientific community due to the fact that they were little known and explored. In this context, the objective of this study was to assess whether different fractions of marine macroalgae Bostrychia tenella and extracts of endophytic micro-organisms isolated from this species possess immunomodulatory activity on the human complement system (CS), which is recognized as one of the major effectors of the inflammatory process. Started by three distinct pathways (classical, alternative and lectin), it leads to the formation of cleavage products and activation complexes which have various important biological functions such as chemotaxis, activation of phagocytes, mast cell degranulation, cytolysis, cellular activation and apoptosis. Thereby, this system plays an important role in various inflammatory reactions, as well as various pathogenic mechanisms of tissue damage. Through the hemolytic activity assays, 10 of the initial 20 samples were selected because they were able to modulate this activity of the CS in the pool of normal human serum (NHS). The 10 samples (among fractions, subfractions and extracts of B. tenella and its endophytic micro-organisms) were able to induce the generation of fragments from C3, a central component of the CS, which was observed both in immuno electrophoresis technique and Western blotting. Also, we observed the formation of the membrane attack complex (MAC) in enzyme immunoassay, indicating that there was total activation of the CS. By the results obtained in Western blotting for fragments of C4 and Factor B was possible to identify which pathways are activated in CS, when correlated with the results of hemolytic assays for the classical/lectin (VC/VL) and alternative pathway (VA). Thus, we conclude that the 10 selected samples have immunomodulatory effects in human CS because they were able to activate it either by VC/VL or VA. It follows also that the hemolytic assay by the kinetic method is an important tool for screening, as well as Western blotting of NHS to detect fragments of the CS.

L’activité anti-tumorale des cellules NK est régulée par des récepteurs de surface activateurs et inhibiteurs qui reconnaissent des ligands exprimés par les cellules cibles. Afin d’augmenter le potentiel cytotoxique des cellules NK face à des cellules cancéreuses autologues, Innate Pharma a développé des Ac bloquant les interactions entre certains récepteurs inhibiteurs de la cellule NK, les KIRs, et leurs ligands, des molécules du CMH de classe I.Dans un premier temps, nous avons mis en place un modèle tumoral d’efficacité des Ac anti-KIR dans des souris transgéniques pour l’expression d’un KIR. Ce modèle nous a permis de démontrer l’efficacité thérapeutique anti-tumorale du blocage des interactions entre les KIRs exprimés par les cellules NK et les molécules HLA exprimées par la tumeur. Ce modèle nous a également permis de tester la combinaison de l’Ac anti-KIR avec un Ac thérapeutique utilisé dans le traitement des lymphomes B non-hodgkiniens, rituximab. Utilisé en combinaison, ces deux Ac augmentent significativement la survie de souris greffées avec une lignée tumorale d’origine B humaine par rapport à chacun des deux traitements seuls.Ces données précliniques nous amènent à proposer une stratégie thérapeutique pour le traitement de tumeurs hématologiques, basée sur la combinaison d’un Ac anti-KIR avec un Ac induisant de l’ADCC afin de potentialiser la réponse anti-tumorale des cellules NK. / NK cell anti-tumoral activity is regulated by inhibitory and activating receptors interacting with target cell ligands. Innate Pharma has developed anti-KIR Abs (IPH2101 and lirilumab), directed against inhibitory NK cell receptors interacting with HLA molecules, in order to increase NK cell activity against autologous tumor cells.We have set up a preclinical model to assess anti-KIR anti-tumoral efficacy in transgenic mice expressing a KIR receptor. With this model, we have also shown the therapeutic benefit of combining lirilumab with rituximab, a therapeutic Ab mediating ADCC.These results support the rationale of combining anti-KIR Ab with Ab mediating ADCC as a therapeutic strategy for hematological malignancies.