Die rheumatoide Arthritis ist eine chronisch entzündliche Gelenkentzündung, deren Ursachen bis dato ungeklärt bleibt. Der Verlauf der Erkrankung führt zu irreversiblem Zerstörung von Knorpel, Knochen und umgebenden Geweben in den betroffenen Gelenken. Es scheint jedoch klar, dass Synovialzellen, Knorpelzellen und Zytokine eine entscheidende Rolle im Fortschreiten der Erkrankung spielen. IL17 ist ein kürzlich entdecktes Zytokine, welches im besonderen Maßen mit der rheumatoiden Arthritis in Verbindung gebracht wird. Es liegt in der Synovialflüssigkeit und im Serum von Patienten mit rheumatoider Arthritis in deutlich höherer Konzentration vor als in anderen Gelenks-zerstörenden Erkrankungen wie z.B. der Osteoarthritis. Eine kürzlich entdeckte Zellpopulation konnte als chondrogene Vorläuferzellen charakterisiert werden. Da sie einen IL17-Rezeptorkomplex exprimieren, ist anzunehmen, dass eine IL17 Stimulation der chondrogenen Vorläuferzellen zu Veränderungen in deren Synthesemustern führt. Methode: Chondrogenen Vorläuferzellen konnten aus menschlichem Knorpel von Patientin mit rheumatoider Arthritis gewonnen und in Kultur gehalten werden. Die Zellen wurden mit IL17 inkubiert und deren Reaktion durch Zellkulturstudien, real-time-PCR, Immunfluoreszenz und western blot untersucht. Ergebnisse: Die IL17 Stimulation der chondrogenen Vorläuferzellen führte zu einer Suppression des Zellwachstums in den ersten Tagen der Zellkultur. Folgende mRNA Moleküle konnten erstmals in den chondrogenen Vorläuferzellen nachgewiesen werden: IL6, IL1,MMP3, MMP14,MMP15, MMP17 NFkB, , MAPK1, IL1, Aggrecan, ADAMTS-5 und TIMP3. Die IL17 Stimulation führte zu einer Erhöhung der Expression von MMP3, MMP17, IL6 und NFkB in den chondrogenen Vorläuferzellen. MMP14 und MMP15 zeigten sich nicht beeinflusst in ihrem Expressionsmuster. Anschließend konnten die Proteine MMP3 und IL6 stellvertretend in den chondrogenen Vorläuferzellen nachgewiesen werden. Ein IL17 Antikörper konnte den zuvor gezeigten Effekt von IL17 auf die IL6 Expression erfolgreich blocken. Schlussvolgerung: chondrogenen Vorläuferzellen scheinen in einem bestimmten Expressionsmuster auf die IL17 Stimulation zu reagieren. Gerade diese Reaktion scheint zum weiteren Fortschreiten der rheumatoiden Arthritis zu führen. Aufgrund der vielen bereits gezeigten Effekte von IL17 auf Gewebe in der rheumatoiden Arthritis scheint IL17 ein vielversprechendes Ziel in der zukünftigen Therapie der Erkrankung darzustellen.

Regenerative medicine is a rapidly developing field, merging engineering and biological life sciences to create biological replacements for damaged tissue and organ function. Development of cellular based therapies has the potential of curing present untreatable diseases and conditions, such as diabetes. The identification of protein expression patterns, that guide undifferentiated cells to different lineages, can provide important information about the progression of cellular differentiation at various stages. This research project utilizes proteomics and in vitro live-cell microscopy to investigate two distinct cellular systems: (1) the signaling pathways of calmodulin (CaM) in the differentiation of a human glioblastoma cell line; and (2) the effect of islet neogenesis associated protein (INGAP) on human islet-derived progenitor cells (hIPCs). Using a proteomic readout with a long term live-cell imagining approach, it was hypothesized that highly specific binding proteins of a CaM-mutant, and proteins in hIPCs perturbed by INGAP, could be identified and studied in vitro, characterizing specific signaling pathways which control the function of CaM in brain tumour cells and the mechanism(s) of INGAP in islet-derived progenitor cells. This thesis presents the utility of a proteomics and an in vitro cell microscopy approach to investigate therapeutic proteins, such as INGAP, on cell culture systems. The results have established the limitations and the utility of DIGE, differential binding of a CaM-mutant versus calcium-CaM, and the cell specific uptake feasibility of using the TAT-binding domain. In the hIPC system, proteomic, phenotypic, motility, proliferation and nuclear effects of INGAP were determined. Specifically, hIPCs exposed to INGAP had 50% decrease in average nuclear speed, the translocation of two identified proteins caldesmon and tropomyosin and INGAP was found to bind specifically to hIPCs. However, hIPCs had no changes in insulin specific hormone expression.

Les données cliniques supportent le concept et offrent l’espoir que la thérapie cellulaire trouvera sa place parmi les stratégies thérapeutiques des pathologies hépatiques. Cependant deux obstacles majeurs limitent l'étendue de son application clinique: la faible disponibilité d’hépatocytes humains de qualité et en quantité importante, et une faible efficacité de greffe conduisant à une survie et une fonctionnalité seulement à court terme. L’objectif de ce travail était de développer des modèles animaux d’insuffisance hépatique après hépatectomie et d’analyser la réponse régénérative après transplantation de progéniteurs hépatiques humains isolés et caractérisés dans 2 laboratoires de recherche (INSERM U1040 Montpellier et laboratoire PEDI UCL Bruxelles), en comparaison à des hépatocytes fraichement isolés.Le premier modèle consistait à réaliser une hépatectomie de 30% chez des souris NOD SCID, associée à l’injection préalable de rétrorsine (blocage de la prolifération cellulaire endogène) et d’injecter dans le même temps directement dans le parenchyme 1 million de cellules progénitrices exprimant constitutivement le gène rapporteur Luciférase. Les résultats ont montré la bonne implantation des cellules jusqu’à 1 mois après l’injection avec une différenciation fonctionnelle des cellules mise en évidence par la sécrétion d’albumine humaine dans le sang circulant des animaux.Le deuxième modèle consistait à réaliser une hépatectomie large de 70% chez des souris immunodéprimées RAG 2-/- γ-/- pour augmenter la souffrance hépatocellulaire et à comparer deux timing d’injection (voie intrasplénique) des cellules progénitrices génétiquement marquées avec la Green Fluorescent Protein. Les résultats ont montré une meilleure tolérance clinique (moins de mortalité) et une plus grande quantité de cellules implantées lorsque l’injection était réalisée 48h après l’hépatectomie. La régénération hépatique endogène était plus importante et plus rapide chez les souris injectées avec les progéniteurs qu’avec les hépatocytes primaires, suggérant un effet paracrine bénéfique de ces cellules.Ces travaux ont mis en évidence la possibilité d’utiliser ces cellules progénitrices comme alternative aux hépatocytes avec des propriétés régénératrices certaines mais soulèvent les problèmes d’implantation de ces cellules qui reste faible dans des foies hépatectomisés remaniés. Définir le meilleur environnement pour favoriser la survie, la fonctionnalité et éventuellement l’intégration effective des cellules transplantées reste une question clé pour avancer dans cette voie.En parallèle de ces travaux de recherche, un projet de recherche clinique de biothérapie a été développé et accepté pour transplanter des hépatocytes frais humains en intrahépatique chez des patients ayant une insuffisance hépatocellulaire terminale (hépatite alcoolique aigue, cirrhose grave, après résection hépatique large). Il devrait débuter fin 2013. / Clinical data support the concept and offer the hope that cell therapy will find its place among the therapeutic strategies in liver diseases. However, two major obstacles limit the scope of its clinical application: the limited availability of human hepatocytes quality and in large quantities, and low efficiency leading to graft survival and only a short-term functionality. The objective of this work was to develop animal models of liver failure after hepatectomy and analyze the regenerative response after transplantation of human hepatic progenitors isolated and characterized in two research laboratories (INSERM U1040 Montpellier laboratory PEDI UCL Brussels) compared to freshly isolated hepatocytes.The first model was to achieve a 30% hepatectomy in mice NOD SCID associated with prior injection retrorsine (blocking of endogenous cellular proliferation) and injected at the same time directly into the parenchyma 1 million progenitor cells constitutively expressing the luciferase reporter gene. The results showed good cell implantation until 1 month after injection with a functional differentiation as evidenced by secretion of human albumin in the circulating blood cells of animals.The second model was to achieve a wide 70% hepatectomy in mice immunocompromised RAG 2 - / - γ-/ - to increase the suffering hepatocellular comparing two injection timing (channel intrasplenically) progenitor cells genetically marked with the Green Fluorescent Protein. The results showed better clinical tolerance (less mortality) and a greater amount of implanted when the injection was performed 48 hours after hepatectomy cells. Endogenous hepatic regeneration was greater and faster in mice injected with the progenitors with primary hepatocytes, suggesting a beneficial paracrine effect of these cells.These studies have highlighted the possibility of using these progenitor cells as an alternative to hepatocytes with regenerative properties but raise some problems implementing these cells remains low in hepatectomized livers reworked. Define the Define the best environment to promote the survival, function and possibly the effective integration of transplanted cells remains a key issue for progress in this direction.In parallel with this research, a clinical research project biotherapy was developed and agreed to transplant human hepatocytes in intrahepatic costs in patients with terminal liver failure (acute alcoholic hepatitis, severe cirrhosis, after extensive liver resection). It should begin in late 2013.

A hematopoese tem sido bem descrita em modelos murinos nas últimas décadas, contudo, trabalhos demonstrando os mecanismos da hematopoese em humanos ainda são escassos. A derivação da primeira linhagem de células-tronco embrionárias humanas (CTEhs) em 1998, gerou novas perspectivas tanto para o estudo da hematopoese na tentativa de mimetizar o que ocorre naturalmente durante o desenvolvimento embrionário, quanto para a aplicação clínica das células hematopoéticas obtidas a partir da diferenciação dessas células. Contudo, apesar de inúmeros trabalhos terem demonstradoa obtenção de células hematopoéticas a partir de CTEhs, os protocolos têm gerado quantidades variáveis de células, com baixa eficiência e com propriedades funcionais de células primitivas. Desse modo, este trabalho procurou estabelecer um modelo próprio de diferenciação de CTEhs-H1 em células progenitoras hematopoéticas para que estas pudessem ser melhor caracterizadas e obtidas de forma mais eficiente. Para isto, foi desenvolvido um sistema de diferenciação baseado no co-cultivo da linhagem de CTEh-H1 com fibroblastos de embrião de camundongo (MEFs), em meio de diferenciação suplementado soro fetal bovino (SFB) e citocinas e fatores de crescimento hematopoéticos em baixas concentrações. Como resultado, o desenvolvimento do presente trabalho permitiu o estabelecimento de um método para geração de populações mistas de células enriquecidas em CPHs positivas para o marcador CD45, o qual mostrou ser coexpresso com outros marcadores hematopoéticos (CD31, CD43, CD71 e CD38), e células hematopoéticas maduras positivas para marcadores mielóide-específicos (235a, CD14, CD15, CD16) e com características morfológicas típicas. Foi demonstrado que as células obtidas expressavam genes relativos ao sistema hematopoético (CD45, CD31, runx1, tal1, lmo2, prom1, CD34 e notch1), e possuíam potencial clonogênico in vitro da ordem de 1/574 células plaqueadas. Em adição, corroboramos os achados de que as células hematopoéticas apresentam duas origens distintas: a partir do endotelio hemogênico e a partir de células com propriedades hemangioblásticas independentes do endotélio hemogênico. / Hematopoiesis has been well described in murine models in recent decades, however, studies demonstrating the mechanisms of hematopoiesis in humans are still scarce. The first human embryonic stem cells line (hESCs) derived in 1998, has generated new perspectives about the study of hematopoiesis as in attempting to mimic what naturally occurs during embryonic development, as for clinical application of hematopoietic cells obtained from the differentiation of these cells. However, although numerous studies have shown the production of hematopoietic cells derived from hESCs, the protocols have generated varying quantities of cells with low efficiency and functional properties of primitive stem cells. Thus, this study sought to establish our own model for hESC-H1 differentiation in hematopoietic progenitor cells so that they could be better characterized and obtained more efficiently. For this way, we developed a differentiation system based on co-culture of hESC-H1 line with inactivated mouse embryonic fibroblasts (MEFs) in differentiation medium supplemented with fetal calf serum (FCS) and cytokines and hematopoietic growth factors in low concentrations. As a result, the development of this study allowed the establishment of a method for generation of mixed population of cells enriched in hematopoietic progenitor cells positive for the marker CD45, which proved to be co-expressed with other hematopoietic markers (CD31, CD43, CD71 and CD38), and mature hematopoietic cells positive for myeloid-specific markers (235a, CD14, CD15, CD16) and morphological characteristics typical. It was shown that these cells expressed genes related to the hematopoietic system (CD45, CD31, runx1, TAL1, LMO2, prom1, CD34 and NOTCH1), and had clonogenic potential in vitro of 1/574 plated cells. In addition, we corroborate the findings that hematopoietic cells have two distinct origins: they can arise as from an hemogenic endothelium as from cells with hemangioblastic properties by an hemogenic endothelium-independent way.

O saco vitelino é o único anexo embrionário presente em todas as espécies dos embriões vertebrados, répteis, aves e mamíferos. Em mamíferos domésticos o saco vitelino é inicialmente grande, pois nestas espécies ele é transitório. Após a implantação, surge no mesênquima lateral à notocorda agrupamentos de células, denominados ilhotas sanguíneas, que representam os progenitores dos sistemas vascular e hematopoético: os hemangioblastos. Os hemangioblastos centrais das ilhas sanguíneas formam as primeiras células-tronco hematopoéticas, enquanto os hemangioblastos periféricos se diferenciam em angioblastos, os precursores dos vasos sanguíneos. O desenvolvimento inicial da atividade hematopoética no saco vitelino conduz a hipótese de que esse tecido é o local primário de desenvolvimento hematopoético e que as células-tronco derivadas dele semeiam os outros sítios intraembriônicos. Foi possível observar nas análises microscópicas que realmente existe uma relação entre ambas linhagens. Nas análises de expressão gênica, alguns genes expressos pelo hemangioblasto apresentaram alta expressão nas análises D+0 e outros genes também específicos do hemangioblasto, porém em estágios secundários de diferenciação como os encontrados na região aórtica, a nível de endotélio hemogênico apresentaram altos níveis de expressão após 3 dias em cultivo. Concluímos portanto, que o saco vitelino por ser o local primário de formação das células sanguíneas e endoteliais nos estágios iniciais da embriogênese, por serem primitivas e, portanto não expressarem marcadores de células maduras na sua superfície, tornam estas células uma importante fonte de células-tronco relevante para a Terapia Celular para hemofilia e muitas outras doenças humanas. / The yolk sac is the single attachment embryo present in all species of vertebrate embryos, reptiles, birds and mammals. In domestic mammals the yolk sac is initially large, since these species it is transient. After implantation, appears in the lateral mesenchyme to the notochord cell clusters, called \"blood islands\" that represent the progenitors of vascular and hematopoietic systems: the hemangioblasts. The central islands hemangioblasts form the first blood hematopoietic stem cells, while peripheral hemangioblasts, the angioblastic differentiate into the precursors of blood vessels. The initial development of the yolk sac hematopoietic activity leads to the hypothesis that this tissue is the primary site of development and that hematopoietic stem cells derived from them sow other intraembryos sites. It was observed in the microscopic analysis that there is indeed a relationship between the two lineages. In the analysis of gene expression, some genes expressed by hemangioblasts showed high expression in D+0 and other specific genes also hemangioblasts, but in secondary stages of differentiation as found in the aortic region, the level of hemogenic endothelium showed high levels of expression after 3 days in culture. We therefore conclude that the yolk sac to be the primary site of formation of blood and endothelial cells in the early stages of embryogenesis, for its cells be primitive and therefore do not express markers of mature cells on the surface, these cells become an important source of cells relevant to stem cell therapy for hemophilia and many other human diseases.

Skin is daily exposed to sun radiation. Among them, UVA reach the basal layer of epidermis composed of keratinocyte stem cells (KSC) and progenitors commonly called transitory amplifying cells (TA). KSC and TA, responsible of the epidermal renewal, are sensitive to genotoxic agents and more particularly to UV. Indeed, KSC, usually quiescent but composing the stem cell pool all lifelong, as well as TA, are specific targets for photocarcinogenesis and photoageing. In this context, the aim of the project was to develop a method able to isolate KSC and TA for characterizing their response to UVA, and in a more industrial objective, to value a photoprotective and/or genoprotective ingredient by investigating their mechanisms of action. A parallel aim was to define culture conditions suitable for maintaining the stemness of KSC in culture, which is quickly lost from their enter into a proliferative state. Thus we showed that adjunction of adipose derived stem cells (ASC) to fibroblasts in dermis of a skin equivalent model (SE) in order to reproduce the physiological environment of KSC, significantly increases the thickness of the epidermis and preserves the keratinocytes from their senescence. ASC act partially via the increase of fibroblasts proliferation in dermis and potentially via a synergic effect of factors secreted by the combination of ASC and fibroblasts. To compare the behavior of KSC to the one of TA, we firstly optimized the rapid adhesion method ; then compared it to the cell sorting by flow cytometry following the alpha6high/CD71low phenotype, which appeared more efficient. Thus, KSC (alpha6high/CD71low) and TA (alpha6high/CD71high) were then irradiated to UVA. KSC showed a photoresistance compared to TA with a better cell viability and a clonogenic potential superior as well as a better ability to reconstruct a pluristratified epidermis in vitro. We also investigated mechanisms of resistance. Our results demonstrate that the induction of the three types of DNA damage immediately induced by UVA is similar in both populations, but that the repair of single strand breaks (SSB) and of thymin dimers (CPDs) is faster for KSC. Finally, PE1, ingredient preselected by Gattefossé was characterized for its photoprotective and genoprotective effect. We showed that PE1 is able to i) preserve capacity of keratinocytes to form holoclones after UVA radiation, ii) decrease DNA damage, notably 8oxoGuanin and CPDs, and iii) improve several repair genes expression and activities. To conclude, this thesis project showed for the first time that KSC are more resistant to UVA radiation than their direct progeny, TA notably via improved DNA repair systems. Moreover, it allowed to identify a plant extract (PE1) able to protect genome of proliferative keratinocytes to UVA radiation / La peau est quotidiennement exposée aux rayons UV du soleil dont les UVA qui atteignent la couche basale de l'épiderme, composée de cellules souches kératinocytaires (KSC) et de cellules progénitrices appelées communément les cellules d'amplification transitoire (TA). Les KSC et TA, responsables du renouvellement de l'épiderme, sont vulnérables à l'action des agents génotoxiques et plus particulièrement aux rayonnements UV. En effet, Les KSC normalement quiescentes mais constituant la réserve de cellules souches tout au long de la vie de l'organisme, comme les TA, sont des cibles préférentielles pour la photocarcingénèse et le photovieillissement cutanés. Dans ce contexte, le but du projet était de développer une méthode capable d'isoler les KSC et les TA afin de caractériser leur réponse biologique vis-à-vis des UVA et dans un objectif plus industriel, de valoriser un actif photoprotecteur et/ou génoprotecteur par l'investigation de ses mécanismes d'action. Un objectif parallèle était de définir des conditions de culture optimales pour garder le phénotype souche des KSC en culture, qui est rapidement perdu dès leur entrée en prolifération. Ainsi, nous avons montré que l'ajout des cellules souches adipeuses (ASC) aux fibroblastes d'une peau reconstruite (PR), pour reproduire l'environnement des KSC, augmente significativement l'épaisseur de l'épiderme et surtout préserve les kératinocytes de leur entrée en sénescence, en partie par l'augmentation de la prolifération des fibroblastes au niveau du derme et potentiellement par un effet synergique des facteurs solubles sécrétés par la combinaison des ASC/fibroblastes. Afin de comparer le comportement des KSC à celui des TA nous avons donc tout d'abord optimisé la méthode d'adhésion rapide, puis l'avons comparée au tri par cytométrie de flux selon le phénotype alpha6high/CD71low, qui s'est avérée plus efficace. Les KSC (alpha6high/CD71low) et les TA (alpha6high/CD71high) ont ensuite été irradiés aux UVA. Les KSC ont montré une photorésistante plus importante que les TA avec une viabilité cellulaire et un potentiel clonogénique supérieurs ainsi qu'une meilleure capacité à reconstruire un épiderme pluristratifié in vitro. Nous avons aussi recherché les mécanismes de résistance. Nos résultats démontrent que l'induction des 3 types de dommages à l'ADN immédiatement après irradiation est identique pour les deux populations mais que la réparation des cassures simple brin (SSB) et des dimères de pyrimidine (CPDs) est plus rapide pour les KSC. Enfin, PE1, actif préselectionné par Gattefossé, a été caractérisé pour son effet photoprotecteur et génoprotecteur. Nous avons montré que PE1 est capable de (i) préserver la capacité des kératinocytes à former des holoclones après irradiation, (ii) diminuer les lésions à l'ADN, notamment la 8oxoGuanine et les CPDs (iii) améliorer l'expression de plusieurs gènes et les activités de réparation de l'ADN. Pour conclure, ce travail de thèse a montré pour la première fois que les KSC (alpha6high/CD71low), sont plus résistantes vis-à-vis des UVA que les TA (alpha6high/CD71high) notamment grâce à des systèmes de réparation de l'ADN plus actifs dans les KSC, et a permis d'identifier un extrait végétal (PE1) capable de protéger le génome des kératinocytes proliférants contre les UVA

La peau est un organe très exposé à l’environnement et fournit la première ligne de défense contre de nombreux pathogènes. Cette fonction est remplie dans l’épiderme murin par les cellules de Langerhans (LCs) et les cellules T dendritiques de l’épiderme (DETCs). Alors que le développement de ces cellules a bien été étudié, peu d’expériences ont été effectuées sur leur renouvellement en condition homéostatique chez des animaux adultes sans manipulations. Nous avons alors développé un système de traçage cellulaire par fluorescence multicolore pour étudier l’homéostasie des LCs et des DETCs. Cette approche de «fate mapping» m’a permis de mettre en évidence un modèle dans lequel le réseau adulte des LCs est formé d’unités prolifératives adjacentes composées de LCs en division et leurs cellules filles. Nous avons identifié que les cellules en division étaient majoritairement représentées par la fraction la plus immature des LCs, suggérant que ces LCs peuvent régénérer leur réseau grâce à une capacité de prolifération limitée. Lors d’une inflammation importante, les LCs sont renouvelées par des progéniteurs issus de la moelle osseuse et s’organisent également en unités de prolifération. Je me suis ensuite intéressé à l’homéostasie des DETCs. Ce réseau est formé de la même manière par des unités prolifératives de DETCs. Un modèle de greffe de peau nous a permis de montrer que les DETCs semblent renouveler les cellules disparues dans une zone restreinte. En conclusion, mes travaux de thèse ont permis de révéler les dynamiques cellulaires qui régissent l’homéostasie des cellules immunitaires de l’épiderme. / The skin is an organ very much exposed to the environment and supplies the primary line of defence against several pathogens. In the mouse model epidermis, this function is fulfilled by Langerhans’ cells (LCs) and dendritic T cells (DETCs). While LCs and DETCs development have thoroughly been studied, few experiences have been carried out concerning the renewal of these cells through homeostatic conditions in adult “nonmanipulated” animals. Then we have designed a new system of fate mapping, by way of multi-coloured fluorescence to study the LCs and DETCs homeostasis. This method of fate mapping allowed me to highlight a model in which the adult network of LCs is made up of adjacent proliferating units, made of dividing LCs and of their daughter cells. We have identified that the dividing cells were mainly represented by the most immature fraction of LCs, suggesting that these LCs can renew their network thanks to a limited ability to proliferate. During significant inflammation, LCs are renewed by progenitors coming from the bone marrow and organize themselves in proliferation units as well. I also took an interest in the homeostasis of DETCs. In the same way as for the LCs, this network seems to be made up of DETCs proliferating units. A model of skin graft led us to show that the DETCs seem to renew the missing cells in a restricted area. As a conclusion, my research work allowed me to reveal the cellular dynamism which governs the homeostasis of the epidermis’ immune cells.

Les cellules progénitrices endothéliales formant des colonies (EFCFs) sont issues decellules CD34+ de la moelle osseuse humaine. Peu de données concernent l’expression dufacteur tissulaire (FT) lors de cette différenciation endothéliale. Outre son rôle dansl’initiation de la génération de thrombine, le FT est impliqué dans l’angiogenèse.Nous montrons que les cellules CD34+ expriment le FT mais non ses isoformes. LesECFCs expriment peu de FT à l’état basal. En revanche, leur stimulation par le TNF-α induitune augmentation de l’expression de FT, et la génération de microparticules pro-coagulantes.Nous avons analysé les modifications fonctionnelles induites par cette stimulation. Nosrésultats montrent que l’expression de FT par les ECFCs est responsable d’une activité procoagulante majeure, alors que les propriétés angiogéniques ne semblent pas affectées.L’expression du tissue factor pathway inhibitor (TFPI) a été évaluée, ainsi que la capacité desmicroparticules issues de ECFCs à générer des métalloprotéinases (MMP2-, MMP-9).Une évaluation de la stabilité chromosomique des cb-ECFCs durant leur expansion aété réalisée, mettant en évidence des anomalies de nombre, mais pas d’anomalies destructures. Les conséquences de ces résultats en termes de thérapie cellulaire appliquées auxpathologies cardio-vasculaires sont discutées. Enfin, nous évoquons la possibilité deconsidérer l’expression de FT comme un marqueur de différenciation cellulaire. / Endothelial colony-forming cells (ECFCs) can be obtained from human bone marrowCD34+ cells. In spite of the essential role of the tissue factor (TF) in coagulation triggeringand angiogenesis, its expression during endothelial differentiation is not established. We showthat CD34+ cells express TF, but not TF splicing forms. ECFCs express a small amount of TFat baseline level. In contrast, ECFCs express TF high levels of TF on response to TNF-α andcan generated highly pro-coagulant microparticles. We have examined the functionalproperties induced by TNF-α stimulation. TF expression confers to ECFCs a strong thrombingeneration capacity without influencing their non-coagulant properties. We have examinedthe co-expression of the tissue factor pathway inhibitor (TFPI) and the ability of ECFCs togenerate microparticles producing metalloproteins (MMP-2, MMP-9).We have performed an evaluation of cb-ECFCs chromosomal stability during theirexpansion. We found quantitative but no structural chromosomal abnormalities. Theconsequences of our observations in the use of cell therapy in cardiovascular diseases arediscussed. We conclude that TF expression may be considered as cell differentiation marker

A malária é uma infecção causada por plasmódios, cujo controle depende do baço, o responsável pelo clareamento dos eritrócitos parasitos. O aumento da parasitemia induz uma ampliação do baço para resolver a infecção, onde participam células precursoras que apresentam CCD34+ na sua superfície. Estudamos a distribuição e a quantidade de células CD34+ em baços de roedores durante malárias de roedores, para compreender sua participação na ampliação do baço e no controle da infecção. Camundongos C57Bl/6j infectados com as cepas AJ e CR de Plasmodium chabaudi, e com a cepa ANKA de Plasmodium berghei, tiveram seus baços removidos e encaminhados para histologia e citometria de fluxo. A distribuição das células CD34+ mostrou-se mais intensa no 4º dia p.i. e menos intensa no 8º dia p.i.. As células CD34+ livres, por citometria de fluxo, surgem com uma onda no 4º dia p.i.. Sua quantidade é similar entre os modelos de P. chabaudi, mas diferente no P. berghei. Neste trabalho, o influxo de células CD34+ no baço não se relaciona com o controle da infecção. / Malaria is caused by Plasmodium sp., which control depends on the spleen, responsible for parasite clearing. The increase of parasitemia implies in spleen amplification to control the infection, with participation of CD34+ cells. We studied the distribution and amount of CD34+ cells in spleen during rodent malaria, to define the role of those cells in spleen amplification and infection control. C57Bl/6j mice were infected with strains CR and AJ of Plasmodium chabaudi, and ANKA strain of Plasmodium berghei. The spleen was removed and processed for histology and flow cytometry. Spleen CD34+ cells was increased in 4th day, p.i., and decreases in 8th day p.i. in all models. By flow cytometry, free CD34+ cells appears as a wave in the 4th day p.i.. P. chabaudi models presented the same level of those cells, which was larger in the P. berghei mice. In this work, increase of spleen CD34+ cells do not correlate with infection control.

O transplante autólogo de células progenitoras hematopoéticas (CPH) requer a mobilização dessas células da medula óssea para o sangue periférico, de onde são coletadas. Essa mobilização pode ser realizada com a administração de filgrastima (G-CSF do inglês, granulocyte-colony stimulating factor) de forma isolada ou associada à quimioterapia (G-CSF / QT). Os produtos de CPH obtidos por esses dois métodos de mobilização apresentam diferenças no conteúdo celular, o que poderia resultar em diferentes desfechos clínicos, como recuperação hematológica e reações adversas (RA) à infusão do produto. Este estudo retrospectivo teve como objetivo avaliar as taxas de RA da infusão do produto de acordo com o tipo de mobilização celular, ou seja, G-CSF isolado ou associado à quimioterapia. Desenho do estudo / Método: Um total de 611 pacientes com linfoma ou mieloma múltiplo (MM) foram submetidos a mobilização e coleta de CPH para transplante autólogo nos últimos 15 anos, destes 267 utilizaram G-CSF e 344 G-CSF / QT (285 dos quais foram submetidos ao transplante em nossa instituição). O procedimento de aférese resultou em 2 bolsas (100 mL cada), conforme padronização local, que foram criopreservadas com DMSO a 10% mantidas em recipiente de nitrogênio líquido até serem descongeladas e infundidas. As RA avaliadas foram náusea / vômito, diarreia, arritmia, dispneia e anormalidades neurológicas (cefaleia e encefalopatia) (5 possibilidades de RA para cada paciente) durante a infusão celular ou logo após o seu término. Resultados: A mediana (faixa) de idade foi de 54 (46-60) e 41 (29-55) anos para os grupos G-CSF e G-CSF / QT, respectivamente (p <0,0001). O pico de células CD34 + / µL foi de 16,6 (8,88 - 29,18) e 31,1 (16,15 - 71,9) para os grupos GCSF e G-CSF / QT, respectivamente (p <0,0001). Os produtos obtidos no grupo GCSF continham um número maior de granulócitos (x 108/mL): 155,2 (113,2-205,1) vs 114,4 (68,31-178,2) (p <0,0001) e plaquetas (x 108 / mL): 1.590 (1010-2190) vs 392 (209,5-800) (p <0,001). O grupo G-CSF recebeu infusão de uma dose maior de DMSO (g/kg): 0,21 (0,14-0,57) vs 0,17 (0,11-0,71) (p = 0,012) e uma dose inferior de células CD34 (x 106 / kg): 3,28 (2,46 -3,99) vs 3,72 (2,58-5,48) (p <0,0001). A recuperação hematológica (neutrófilos >= 500 / µL) ocorreu nos dias 12 (11-14) e 11 (10-12) nos grupos G-CSF e G-CSF +QT, respectivamente (p <0,0001). As RA ocorreram em 58,27% e 50,94% dos pacientes dos grupos G-CSF e G-CSF / QT, respectivamente (p = 0,234), entretanto, o número de reações foi de 132 (em 635 possibilidades) e 126 (em 795 possibilidades) nos grupos G-CSF e G-CSF / QT, respectivamente (p = 0,016). Nos pacientes que receberam >= 2 bolsas de CPH (e dose semelhante de DMSO), observou-se maior número de RA no grupo G-CSF (122 vs 75, p = 0,02). O sexo feminino foi associado a uma maior taxa de náusea/vômito (23,84% vs 46,49%, p = 0,0001). Conclusão: a mobilização de CPH com G-CSF isoladamente, apesar de apresentar muitas vantagens, resulta em maior número de células indesejáveis, como granulócitos e plaquetas no produto final, o que poderia explicar, pelo menos em parte, a maior taxa de reações adversas observada durante a infusão celular, além de resultar em menor número de células CD34, com consequente recuperação hematológica ligeiramente mais tardia / Autologous hematopoietic progenitor cell (HCP) transplantation requires the mobilization of these cells from the bone marrow into the peripheral blood from which they are collected. Such mobilization may be performed with the administration of filgrastim (granulocyte-colony stimulating factor) alone or in combination with chemotherapy (G-CSF / CT). The HPC products obtained by these two methods of mobilization present differences in cellular content, which could result in different clinical outcomes, such as hematological recovery and adverse reactions (RA) to infusion of the product. This retrospective study aimed to evaluate the RA rates of infusion of the product according to the type of cellular mobilization, in other words, GCSF isolated or associated with chemotherapy. A total of 611 patients with lymphoma or multiple myeloma (MM) underwent mobilization and collection of MCH for autologous transplantation in the last 15 years, of which 267 used G-CSF and 344 G-CSF / CT (285 of which were transplanted at our institution). The apheresis procedure resulted in 2 pockets (100 mL each), according to local standardization, which were cryopreserved with 10% DMSO kept in a liquid nitrogen container until thawed and infused. The RAs evaluated were nausea / vomiting, diarrhea, arrhythmia, dyspnea and neurological abnormalities (headache and encephalopathy) (5 possibilities of RA for each patient) during the cellular infusion or soon after its completion. Results: The median age range was 54 (46-60) and 41 (29-55) years for the G-CSF and G-CSF / CT groups, respectively (p <0.0001). The CD34 + / ?L peak was 16.6 (8.88 - 29.18) and 31.1 (16.15 - 71.9) for the G-CSF and G-CSF / CT groups, respectively (p <0.0001). The products obtained in the G-CSF group contained a greater number of granulocytes (x 108 / ml): 155.2 (113.2-205.1) vs 114.4 (68.31-178.2) (p <0, 0001) and platelets (x 108 / ml): 1590 (1010-2190) vs 392 (209.5-800) (p <0.001). The G-CSF group received infusion of a higher dose of DMSO (g / kg): 0.21 (0.14-0.57) vs 0.17 (0.11-0.71) (p = 0.012) and a lower dose of CD34 cells (x 106 / kg): 3.28 (2.46 -3.99) vs 3.72 (2.58-5.48) (p <0.0001). Haematological recovery (neutrophils >= 500 / ?L) occurred on days 12 (11-14) and 11 (10-12) in the G-CSF and G-CSF / QT groups, respectively (p <0.0001). The RAs occurred in 58.27% and 50.94% of patients in the G-CSF and G-CSF / CT groups, respectively (p = 0.234), however, the number of reactions was 132 (in 635 possibilities) and 126 (in 795 possibilities) in the G-CSF and G-CSF / CT groups, respectively (p = 0.016). In patients receiving >= 2 pockets of MHC (and similar dose of DMSO), there was a greater number of RAs in the G-CSF group (122 vs 75, p = 0.02). The female sex was associated with a higher rate of nausea / vomiting (23.84% vs 46.49%, p = 0.0001). Conclusion: mobilization of CPH with G-CSF alone, despite having many advantages, results in a higher number of undesirable cells, such as granulocytes and platelets in the final product, which could explain, at least in part, the higher rate of adverse reactions observed during the cellular infusion, in addition to resulting in a smaller number of CD34 cells, with consequent slightly later hematological recovery