Regensburg, Univ., Diss., 2009.

Regensburg, Univ., Diss., 2008.

Techn. Hochsch., Diss., 2000--Aachen.

Universiẗat, Diss., 2005--Kiel.

Les cytokines sont d’importants médiateurs dans la physiopathologie des lymphomes hodgkiniens (LH). A partir d’une cohorte de 464 patients, nous avons évalué l’impact pronostique de onze SNPs parmi les gènes de cytokines : IL10 (rs1800890, rs1800896, rs1800871, rs1800872), TNFA (rs1800629) ; IL6 (rs1800795) ; IL1B (rs16944) ; ILRN (rs419598) ; INFG (rs2430561) ; IL12 (rs3212227) ; CCL17 (rs223828). Le génotypage du SNP de l’IL12 montre une distribution différente de celle attendue dans la population générale selon le test de Hardy-Weinberg. Ce résultat suggère que les variations génétiques de l’IL12 pourraient être impliquées dans la susceptibilité au LH. Les patients porteurs du génotype IL10-1082AA présentent un taux de rémission complète au traitement initial supérieur aux patients présentant un autre génotype (95% vs. 88% P = .02). Pour les patients de stade avancé III-IV, le taux de survie globale à 6 ans est statistiquement différent entre les génotypes IL10-592AA/CC/AC et IL10-819TT/CC/CT (100%, 94%, 78%, P = .03). Ce résultat est retrouvé pour les patients porteurs de LH n’exprimant pas l’EBV. Pour les LH EBV négatif, le taux de survie sans progression à 6 ans est différent en fonction du génotype du TNFA-308AA/GG/AG (100%, 84%, 68%, P = .03). Il n’a été pas retrouvé de corrélation entre les génotypes et les dosages plasmatiques de l’IL-10, TNFA, IL-1RA, IL-6. Cette étude montre que le ‘‘fond génétique immun’’ est important à prendre en considération pour définir le pronostic des patients. Le rôle des SNPs de l’IL10 et du TNFA dans les LH EBV négatif devra être confirmé ainsi que l’influence des variations génétiques de l’IL12 dans la susceptibilité au LH. / Cytokines are important immune mediators implicated in Hodgkin lymphoma (HL) pathogenesis but little is known on the role of immune gene variations. We assessed prospectively the prognostic role of cytokine gene single nucleotide polymorphisms (SNPs) in HL patients (pts) : IL10 (rs1800890, 448 pts; rs1800896, 459 pts ; rs1800871, 446 pts ; rs1800872, 447 pts), TNFA (rs1800629, 464 pts) ; IL6 (rs1800795, 201 pts) ; IL1B (rs16944, 198 pts) ; ILRN (rs419598, 199 pts) ; INFG (rs2430561, 200 pts) ; IL12 (rs3212227, 259 pts) ; CCL17 (rs223828, 198 pts). IL12 genotype distribution appears significantly different from what observed in general population according to Hardy-Weinberg test which was already observed in another published study. IL10-1082AA genotype was associated with better complete response than other IL10 genotypes (95% vs. 88% P = .02). For patients with stage III-IV HL, the 6-year overall survival was statistically different between IL10-592AA/CC/AC and IL10-819TT/CC/CT genotypes (100%, 94%, 78%, P = .03). This prognostic effect was observed in EBV-negative but not in EBV-positive HL. In EBV-negative HL, TNFA-308AA/GG/AG genotypes had a different 6-year progression-free survival (100%, 84%, 68%, P = .03). No correlation was observed between genotypes and IL-10, TNFA, IL-1RA, IL-6 cytokine levels. This exploratory study suggests an effect of IL10 and TNFA SNPs in predicting HL outcome but other studies are needed to decipher the role of the host immunogenetic background, in particular the relation with EBV. Regarding the IL12 genotyping results, whether IL12 polymorphism is implicated in HL susceptibility needs also to be clarify.

Les cellules souches de la pulpe dentaire humaine (DPSCs) sont des cellules souches mésenchymateuses (MSCs) isolées de la pulpe dentaire. Les DPSCs sont capables de s’auto-renouveler et se différencier en plusieurs types cellulaires tel que les odontoblastes, les ostéoblastes, les chondrocytes, les neuroblastes et les adipocytes. Les propriétés immunitaires des DPSCs sont de plus en plus étudiées, elles hébergent des récepteurs de types Toll à la surface, possedent une activité immuno-modulatrice.Cependant, les propriétés immunitaires comme celles décrites dans les cellules immunitaires professionnelles telles que la phagocytose, la production de composés anti-microbiens et le nouveau concept « Trained immunity » pourraient être étudiées. une brève revue a été élaborée pour mettre en évidence l'ensemble des propriétés immunitaires des DPSCs décrites dans la littérature. Ensuite, expérimentalement, nous avons montré que les DPSCs pouvaient internaliser le pathogène bactérien Bartonella quintana. En outre, nous avons décrit la capacité des DPSCs à développer une immunité entrainée “trained immunity”. Il s’agit d’une mémoire inflammatoire concernant deux cytokines IL-6 et MCP-1. La stimulation des DPSCs avec le ligand bactérien LPS ou PGN induit une augmentation de l’expression et de la production de l'IL-6 et du PGN après un second stimulus. Dans l'ensemble, l'étude des propriétés immunitaires des DPSCs montre que ces dernières peuvent agir comme des cellules immunitaires. / Dental pulp Stem cells (DPSCs) are mesenchymal stem cells (MSCs) isolated from the dental pulp. DPSCs are able to self-renew and differentiate into several cell types such as odontoblasts, osteoblasts, chondrocytes, neuroblasts and adipocytes.The immune properties of DPSCs are being studied more and more, they harbor Toll-like receptor on the surface and have an immunomodulatory activity.However, immune properties such as those described in professional immune cells such as phagocytosis, production of antimicrobial compounds and the new concept "Trained immunity" could be studied.A brief review has been developed to highlight the set of immune properties of DPSCs described in the literature. Then, experimentally, we showed that DPSCs could internalize the bacterial pathogen Bartonella quintana.In addition, we have described the ability of DPSCs to develop trained immunity. It is an inflammatory memory concerning two cytokines IL-6 and MCP-1. Priming DPSCs with the bacterial ligand LPS or PGN induces an increase in the expression and production of IL-6 and PGN after a second stimulus.Overall, the study of the immune properties of DPSCs shows that DPSCs can act as immune cells.

Escherichia coli enteropatogênica (EPEC) é um dos principais agentes etiológicos da diarreia infantil tanto em países desenvolvidos como em países em desenvolvimento. Esta bactéria possui dois fatores de virulência comprovadamente envolvidos na patogênese, intimina e bundle-forming pilus (BFP). Este patotipo está dividido em EPEC típica e EPEC atípica, ambos apresentam uma ilha de patogenicidade cromossomal denominada locus of enterocyte effacement (região LEE) onde está localizado o gene eae (E. coli attachment effacement), que codifica a intimina, uma proteína de membrana externa que medeia a adesão íntima da bactéria ao enterócito. Diferente da EPEC típica, as cepas de EPEC atípica não possuem o plasmídeo EAF (EPEC adherence factor) no qual encontra-se o operon bfp (bundle forming pilus) constituído por 14 genes incluindo bfpA o qual codifica a bundlina (BfpA), principal subunidade da fímbria Bfp, que possibilita a agregação bacteriana. Na infecção por EPEC ocorre grave disfunção da barreira epitelial, e uma das conseqüências é a inflamação. Na literatura, é bem descrito a interação entre as proteínas efetoras de EPEC com as células epiteliais e os processos iniciais da interação bactéria à célula hospedeira. Entretanto, poucos são os estudos que analisam a produção de citocinas em infecções por EPEC ou suas moléculas efetoras com relação a ativação de macrófagos, fundamentais para o controle do processo inflamatório e geração da resposta imune durante esta infecção. A análise das citocinas produzidas constitui uma parte importante da resposta imune e representa a tentativa do hospedeiro em lidar com um determinado microrganismo. Em função disto analisou-se o papel da intimina e do BfpA na capacidade de ativar a resposta inata mediada por macrófagos in vitro, onde avaliou-se a produção de citocinas pró-inflamatórias (TNF-α, IL-1, IL-6 e IL-12), citocina antiinflamatória (IL-10) e quimiocina (MCP-1). Os resultados demonstraram que as proteínas recombinantes intimina e BfpA são potentes ativadores de macrófagos, de forma dose dependente, produzindo TNF-α, IL-12 e IL-6, IL-10 e MCP-1, mas não IL-1β. Neste estudo não foi observado efeito sinérgico na produção de citocinas pró-inflamatórias ao associar intimina e BfpA, entretanto em dose mais elevada potenciou a produção de IL-10, um mediador antiinflamatório. O efeito imune obtido foi atribuído majoritariamente a estas proteínas uma vez que o tratamento destas com polimixina B não alterou a produção de TNF-α. Conclui-se que intimina e BfpA são potentes ativadores de macrófagos durante a resposta inata podendo colaborar para o controle do processo inflamatório durante a infecção por EPEC. / Enteropathogenic Escherichia coli (EPEC) is a common cause of childhood diarrhea in developed countries as well as developing countries. This bacterium has two proven virulence factors involved in pathogenesis, intimin and bundle-forming pilus (BFP). This pathotype EPEC is divided into typical and atypical EPEC, both having a chromosomal pathogenicity island called locus of enterocyte effacernent (LEE region) which contains the gene eae (E. coli attachment effacement). eae encodes intimin, an outer membrane protein that mediates the intimate adherence of bacteria to the enterocyte. Unlike typical EPEC, atypical EPEC strains do not possess the plasmid EAF (EPEC adherence factor) which is in the operon bfp (bundle forming pilus) consisting of 14 genes including bfpA, which encodes bundlin (BfpA), the main subunit of BFP allowing bacterial aggregation. EPEC infection occurs in severe dysfunction of the epithelial barrier, and one consequence is inflammation. In the literature, the interaction between effector proteins of EPEC and epithelial cells and the initial processes of bacterial interaction with the host cell are well described. However, there are few studies that have examined cytokine production in EPEC infections or their effector molecules with respect to macrophage activation, essential for controlling inflammation and immune response during this infection. The production of cytokines is an important part of the immune response and represents the host\'s attempt to deal with a particular microorganism. Therefore, we examined in vitro the role of intimin and BfpA in the ability to activate the innate response mediated by macrophages, where we analyzed the production of the proinflammatory cytokines TNF-α, IL-1, IL-6 and IL-12, and the antiinflammatory cytokine IL-10 and chemokine MCP-1. The results show that recombinant intimin and BfpA are potent activators of macrophages in a dose-dependent manner, where the stimulated cells produce TNF-α, IL-12 and IL-6, IL-10 and MCP-1, but not IL-1β. In this study, no synergistic effect was observed in the production of proinflammatory cytokines by combining BfpA and intimin, although production of IL-10, an antiinflammatory mediator, was potentiated at a higher dose. The effect obtained was largely attributed to these proteins, as the treatment of proteins with polymyxin B did not alter the production of TNF-α. We conclude that intimin and BfpA are potent activators of macrophages during the innate response and may contribute to the control of inflammation during infection with EPEC.

Escherichia coli enteropatogênica (EPEC) é um dos principais agentes etiológicos da diarreia infantil tanto em países desenvolvidos como em países em desenvolvimento. Esta bactéria possui dois fatores de virulência comprovadamente envolvidos na patogênese, intimina e bundle-forming pilus (BFP). Este patotipo está dividido em EPEC típica e EPEC atípica, ambos apresentam uma ilha de patogenicidade cromossomal denominada locus of enterocyte effacement (região LEE) onde está localizado o gene eae (E. coli attachment effacement), que codifica a intimina, uma proteína de membrana externa que medeia a adesão íntima da bactéria ao enterócito. Diferente da EPEC típica, as cepas de EPEC atípica não possuem o plasmídeo EAF (EPEC adherence factor) no qual encontra-se o operon bfp (bundle forming pilus) constituído por 14 genes incluindo bfpA o qual codifica a bundlina (BfpA), principal subunidade da fímbria Bfp, que possibilita a agregação bacteriana. Na infecção por EPEC ocorre grave disfunção da barreira epitelial, e uma das conseqüências é a inflamação. Na literatura, é bem descrito a interação entre as proteínas efetoras de EPEC com as células epiteliais e os processos iniciais da interação bactéria à célula hospedeira. Entretanto, poucos são os estudos que analisam a produção de citocinas em infecções por EPEC ou suas moléculas efetoras com relação a ativação de macrófagos, fundamentais para o controle do processo inflamatório e geração da resposta imune durante esta infecção. A análise das citocinas produzidas constitui uma parte importante da resposta imune e representa a tentativa do hospedeiro em lidar com um determinado microrganismo. Em função disto analisou-se o papel da intimina e do BfpA na capacidade de ativar a resposta inata mediada por macrófagos in vitro, onde avaliou-se a produção de citocinas pró-inflamatórias (TNF-α, IL-1, IL-6 e IL-12), citocina antiinflamatória (IL-10) e quimiocina (MCP-1). Os resultados demonstraram que as proteínas recombinantes intimina e BfpA são potentes ativadores de macrófagos, de forma dose dependente, produzindo TNF-α, IL-12 e IL-6, IL-10 e MCP-1, mas não IL-1β. Neste estudo não foi observado efeito sinérgico na produção de citocinas pró-inflamatórias ao associar intimina e BfpA, entretanto em dose mais elevada potenciou a produção de IL-10, um mediador antiinflamatório. O efeito imune obtido foi atribuído majoritariamente a estas proteínas uma vez que o tratamento destas com polimixina B não alterou a produção de TNF-α. Conclui-se que intimina e BfpA são potentes ativadores de macrófagos durante a resposta inata podendo colaborar para o controle do processo inflamatório durante a infecção por EPEC. / Enteropathogenic Escherichia coli (EPEC) is a common cause of childhood diarrhea in developed countries as well as developing countries. This bacterium has two proven virulence factors involved in pathogenesis, intimin and bundle-forming pilus (BFP). This pathotype EPEC is divided into typical and atypical EPEC, both having a chromosomal pathogenicity island called locus of enterocyte effacernent (LEE region) which contains the gene eae (E. coli attachment effacement). eae encodes intimin, an outer membrane protein that mediates the intimate adherence of bacteria to the enterocyte. Unlike typical EPEC, atypical EPEC strains do not possess the plasmid EAF (EPEC adherence factor) which is in the operon bfp (bundle forming pilus) consisting of 14 genes including bfpA, which encodes bundlin (BfpA), the main subunit of BFP allowing bacterial aggregation. EPEC infection occurs in severe dysfunction of the epithelial barrier, and one consequence is inflammation. In the literature, the interaction between effector proteins of EPEC and epithelial cells and the initial processes of bacterial interaction with the host cell are well described. However, there are few studies that have examined cytokine production in EPEC infections or their effector molecules with respect to macrophage activation, essential for controlling inflammation and immune response during this infection. The production of cytokines is an important part of the immune response and represents the host\'s attempt to deal with a particular microorganism. Therefore, we examined in vitro the role of intimin and BfpA in the ability to activate the innate response mediated by macrophages, where we analyzed the production of the proinflammatory cytokines TNF-α, IL-1, IL-6 and IL-12, and the antiinflammatory cytokine IL-10 and chemokine MCP-1. The results show that recombinant intimin and BfpA are potent activators of macrophages in a dose-dependent manner, where the stimulated cells produce TNF-α, IL-12 and IL-6, IL-10 and MCP-1, but not IL-1β. In this study, no synergistic effect was observed in the production of proinflammatory cytokines by combining BfpA and intimin, although production of IL-10, an antiinflammatory mediator, was potentiated at a higher dose. The effect obtained was largely attributed to these proteins, as the treatment of proteins with polymyxin B did not alter the production of TNF-α. We conclude that intimin and BfpA are potent activators of macrophages during the innate response and may contribute to the control of inflammation during infection with EPEC.

Les hypothèses retraçant les mécanismes physiopathologiques de la maladie de Behçet (MB), une vascularite inflammatoire non liée aux anticorps anti-cytoplasme des polynucléaires neutrophiles (ANCA), sont multiples. Cette étude propose une compilation exhaustive des mécanismes immunopathologiques décrits dans la littérature contemporaine, et fournit un résumé détaillé des aspects cliniques de la maladie et de ses différents traitements. Cette étude inclut également une analyse statistique de 20 signatures de protéines proposées comme biomarqueurs potentiels de la MB. Vingt-deux patients avec une MB active (MBA) et 46 patients avec une MB inactive (MBI), répondant aux critères de l’International Criteria for Behçet Disease (2013), ainsi que 47 donneurs sains (DR) et 98 patients subissant une angiographie coronaire (AC) ont fourni des échantillons de sérums pour une étude de dosage multiplex. Les résultats indiquent que les protéines sériques ICAM-1, SAA, THBD, et VCAM-1 jouent un rôle essentiel dans la différenciation entre les patients MB et les DR. De même, Caldesmon, Clusterin, CRP, IL-8, SELP et SICMA3 permettent un tri entre les patients MB et AC. Les modèles de signatures des biomarqueurs proposés dans cette étude et qui séparent entre les patients atteints par la MB, les DR et / ou les AC, représentent une nouvelle piste pour le développement de tests sériques pour la MB, avec une sensibilité et une spécificité élevées. Ceci peut éventuellement compléter les outils de diagnostic clinique établis. Ces résultats apportent une contribution significative à l’interprétation actuelle de la pathogénie de la MB en tant que vascularite auto-immune non-ANCA. Cette enquête fournit un bilan à la fois qualitatif et quantitatif aux cliniciens et aux chercheurs dans ce domaine. / Hypotheses concerning the specific pathophysiological mechanisms of Behçet’s Disease (BD), a non-antineutrophil cytoplasmic antibodies (ANCA) inflammatory vasculitis, are numerous. This study offers an exhaustive review of the disease in an attempt to recap the immunopathological pathways described by extant literature, and provides a detailed summary of the clinical aspects of, and treatment options for the disease. In addition, this investigation completed a statistical analysis of 20 protein signatures that were proposed as potential biomarkers for BD. Twenty-two patients with active BD (BDA) and 46 patients with inactive BD (BDI) fulfilling the International Criteria for Behçet's Disease, 47 healthy donors (HD), and 98 coronary angiography patients (CA) provided serum samples for a multiplex assay study. Findings indicate that serum proteins ICAM-1, SAA, THBD, and VCAM-1 play a significant role in differentiating BD patients from HD. Likewise, Caldesmon, Clusterin, CRP, IL-8, SELP, and sICAM-3 segregate between BD and CA. The biomarker predictive models proposed in this study that segregate between BD, HD, and / or CA represent a significant avenue for the development of sera testing specific to BD with a high level of sensitivity and specificity. This may serve as a supplement to established clinical diagnostic tools. These results represent a noteworthy complement to the current interpretation of the pathogenesis of BD as an autoimmune non-ANCA vasculitis. This investigation provides expert clinicians and researchers with both qualitative and quantitative outcomes.

L’endocytose permet l’internalisation d’éléments présents dans le milieu extracellulaire tels que les nutriments. Ce processus prend place dans la membrane plasmique. Les courbures de la membrane jouent un rôle essentiel dans l’endocytose pour générer une invagination initiale, un puits, puis une vésicule qui se sépare ensuite de la membrane plasmique pour fusionner avec les compartiments intracellulaires. Il existe plusieurs voies d’endocytose qui peuvent être classées selon des critères tels que la taille des vésicules produites, la médiation par un récepteur ou la présence d’un manteau recouvrant les vésicules. La voie d’endocytose la mieux caractérisée est celle dépendante de la clathrine. Mon laboratoire d’accueil travaille sur l’entrée du récepteur de l’interleukine 2 (IL-2R). Ce récepteur peut entrer de façon constitutive ou induite en présence de son ligand l’IL-2. Les deux voies sont indépendantes de la clathrine. Lors de mon arrivée dans le laboratoire, ces voies étaient encore peu caractérisées, notamment les facteurs induisant les courbures membranaires restaient à identifier. Ces facteurs doivent être particulièrement impliqués car les vésicules contenant l’IL-2R sont dépourvues de manteaux. Un crible par interférence à ARN, réalisé avant mon arrivée avait permis de proposer des protéines candidates pouvant courber les membranes. La première partie de ma thèse a consisté à confirmer l’importance de certaines protéines issues de ce crible puis à étudier leurs rôles dans la voie constitutive de l’IL-2R. Parmi ces protéines confirmées, deux familles de facteurs étaient particulièrement intéressantes pour leur capacité à courber les membranes, les phospholipases D et les endophilines. Ces dernières ont permis d’identifier une nouvelle voie d’entrée nommée « Fast Endophilin Mediated Endocytosis » FEME dans laquelle l’endophiline joue un rôle essentiel et qui est empruntée par de nombreux récepteurs transmettant le signal. La voie FEME partage plusieurs facteurs communs avec la voie d’endocytose induite de l’IL-2R. Pour finir, mes travaux de thèse ont porté sur l’orchestration de l’endophiline et de la dynamine dans la voie d’endocytose constitutive de l’IL-2R. Ces deux facteurs sont impliqués en fin d’endocytose, pour scinder les vésicules de la membrane plasmique. Cependant, ces deux protéines n’ont pas la même orchestration. Nos travaux montrent une action distincte de l’endophiline et de la dynamine dans les voies d’endocytose dépendante et indépendante de la clathrine. / Endocytosis allows the uptake of elements from the extracellular fluid such as nutriments. This process takes place at the plasma membrane. The membrane curvatures play an important role in endocytosis for the production of initial invagination to form a pit that will be then separate from the plasma membrane and will go to the intracellular compartments. Several routes of endocytosis exist and can be classified depending on vesicles size formed, receptor mediated endocytosis or coat on vesicles. The well-known characterized endocytosis pathway is the clathrin mediated one. My lab is working on interleukin 2 receptor (IL-2R) entry. This receptor can enter either constitutively or upon induction by the ligand IL-2. Both uptake pathways are independent of clathrin. When I arrived in the lab, those pathways were still under characterization, in particular the factors inducing the membrane curvature. Their role should be important since IL-2R containing vesicles are coated free. A small interfering RNA screen performed before my phD, allowed to identify new candidates. The first part of my thesis was to verify the involvement of some of them in the IL-2R constitutive pathway and then to study their role in this pathway. Among them, 2 families of proteins were particularly interesting as they can curve membranes, phospholipases D and endophilins. The endophilin allowed the discovery of a new route called “Fast Endophilin Mediated Endocytosis” (FEME) in which it plays an essential role and which is used by numerous receptors that transmit signal. The FEME pathway shares several factors that are common with the IL-2 induced endocytosis pathway. Then, I conducted a work on the orchestration of endophilin and dynamin during the constitutive IL-2R endocytosis. Both factors are recruited at the end of the mechanism, to separate the vesicles from the plasma membrane. However, both proteins do not have the same orchestration. Our works show a distinct action of endophilin and dynamin in clathrin dependent and independent endocytosis.