A esclerose múltipla (EM) é uma doença desmielinizante inflamatória crônica recorrente, restrita ao sistema nervoso central (SNC), cuja característica histológica é a ocorrência de desmielinização relacionada com infiltrado inflamatório perivenular com relativa preservação axonal. A forma clássica da doença se caracteriza pela recorrência de ataques (surtos), seguidos de remissão dos sintomas (RRMS), com a presença de múltiplas lesões focais dispersas pelo SNC (dissociação espacial) com processo inflamatório exuberante na fase aguda, coexistindo com lesões crônicas (dissociação temporal) sem atividade inflamatória evidenciavel pela quebra de barreira hematoencefálica e realce na fase contrastada na imagem de ressonância magnética (MRI). Alguns pacientes tem uma evolução benigna, permanecendo livre de sequelas significativas por mais de 20 anos da doença. Outros pacientes têm uma forma agressiva da doença, ficando restritos à cadeira de rodas em 8 a 10 anos de evolução. Um desafio é modificar o curso desta forma agressiva, o que pode ser feito com o uso de imunomoduladores, quimioterápicos e, eventualmente, transplante autólogo de células tronco hematopoiéticas (aHSCT). O objetivo da utilização do aHSCT é a restauração da atividade imunológica livre dos ataques à mielina (\"autoimune reset\"). Uma das maneiras de se monitorar a eficácia do tratamento é a identificação da ocorrência de novas lesões e de lesões com reforço visíveis em exames de RM seriados. Objetivo: Testar a hipótese de que o tratamento de pacientes com EM, utilizando AHSCT, foi eficaz em evitar a recorrência de inflamação e o aparecimento de novas lesões visíveis na SB ao exame de MRI, a longo prazo. Resultados: Na nossa Instituição, cerca de 66 pacientes portadores de EM foram tratados com aHSCT no período de 2004 a 2011, sendo seguidos desde então pelas disciplinas de hematologia, neurologia/neuroimunologia e pela seção de RM do CCIFM-HCRP. Método: Foram revisados os exames de RM de encéfalo adquiridos de maneira prospectiva e protocolada, de 76 pacientes submetidos ao aHSCT, com seguimento por MRI há mais de cinco anos. As imagens de RM foram arquivadas nos servidores do CCIFM, foram recuperadas, anonimizadas e revistas por pelo menos dois radiologistas experientes, de maneira independente e por confrontação. Foi considerada falha terapêutica a identificação de lesões novas e/ou lesões com reativação inflamatória. Resultados: Dez pacientes foram excluídos. Doze pacientes (18,18%) apresentaram novas lesões ou recorrência do processo inflamatório, com reforço. Conclusão: Na nossa amostra o aHSCT foi capaz de controlar a recorrência de lesões e da atividade inflamatória perceptível na RM em mais de 87% dos casos, caracterizando uma importante opção terapêutica de segunda linha nos casos de maior agressividade da doença / Multiple sclerosis (MS) is a recurrent chronic inflammatory demyelinating disease, restricted to the central nervous system (CNS), whose histological feature is the occurrence of perivenular inflammatory infiltrate, leading to demyelination with relative axonal preservation. The classic form of the disease is characterized by the recurrence of attacks (outbreaks), followed by remission of symptoms (RRMS), with the presence of multiple focal lesions dispersed by the CNS (spatial dissociation) with exuberant inflammatory process in the acute phase, coexisting with chronic lesions (Temporal dissociation) without inflammatory activity evidenced by the breakdown of blood-brain barrier and contrast-enhanced contrast-enhanced magnetic resonance imaging (MRI). Some patients have a benign course, remaining free of significant sequelae for more than 20 years of the disease. Other patients have an aggressive form of the disease, being restricted to the wheelchair in 8 to 10 years of evolution. One challenge is to modify the course in this aggressive way, which can be done with the use of immunomodulators, chemotherapeutics and, eventually, autologous hematopoietic stem cell transplantation (aHSCT). The goal of using aHSCT is to restore immune activity free of myelin attacks (\"autoimmune reset\"). One of the ways to monitor treatment efficacy is to identify the occurrence of new lesions and visible reinforcing lesions on serial MRI scans. Objective: To test the hypothesis that the treatment of patients with MS using AHSCT was effective in avoiding the recurrence of inflammation and the appearance of new visible lesions in SB at the long-term examination of MRI. Results: At our institution, approximately 66 patients with MS were treated with aHSCT from 2004 to 2011, followed by the hematology, neurology / neuroimmunology and MRI sections of the CCIFM-HCRP. Methods: Brain and MRI scans acquired in a prospective and protocolized way from 76 patients who underwent aHSCT were followed up with MRI for more than five years. The MR images were archived on the CCIFM servers, retrieved, anonymised and reviewed by at least two experienced radiologists, independently and by confrontation. The identification of new lesions and / or lesions with inflammatory reactivation was considered therapeutic failure. Results: Ten patients were excluded. Twelve patients (18.18%) presented new lesions or recurrence of the inflammatory process, with reinforcement. Conclusion: In our sample, aHSCT was able to control the recurrence of lesions and the inflammatory activity detected in MRI in more than 87% of the cases, characterizing an important second line therapeutic option in the cases of greater aggressiveness of the disease.

Submitted by Fabiana Gonçalves Pinto (benf@ndc.uff.br) on 2015-12-03T17:07:24Z No. of bitstreams: 1 Dulcinea Luzia de Oliveira Lima Marques.pdf: 1782106 bytes, checksum: 004f6a4a517b9e777f74bc04168ea7c5 (MD5) / Made available in DSpace on 2015-12-03T17:07:24Z (GMT). No. of bitstreams: 1 Dulcinea Luzia de Oliveira Lima Marques.pdf: 1782106 bytes, checksum: 004f6a4a517b9e777f74bc04168ea7c5 (MD5) Previous issue date: 2011 / Mestrado Acadêmico em Ciências do Cuidado em Saúde / As atividades desenvolvidas pelo enfermeiro nos Bancos de Sangue de Cordão Umbilical Placentário demanda alta complexidade assistencial e funcional, fato que imputam a responsabilidade de identificar situações que devam ser avaliadas, pesquisadas e reformuladas, como por exemplo os critérios de doação de sangue de cordão umbilical e placentário (SCUP), critérios de inclusão e exclusão de doadoras, padronização de técnicas e demais situações que envolvem a atuação do enfermeiro. A celularidade tem sido alvo de atenção por parte dos enfermeiros, devido a necessidade de obtenção de um número adequado células-tronco hematopoiéticas. Este estudo objetivou discutir a associação entre a celularidade do sangue do cordão umbilical e placentário e a hipertensão arterial e; traçar perfil das gestantes com hipertensão arterial e sem hipertensão arterial. A metodologia correspondeu a um estudo observacional do tipo caso-controle. O local da pesquisa foi o Hospital Maternidade Oswaldo de Nazareth e o Laboratório de Processamento e Criopreservação do Instituto Nacional de Câncer e os sujeitos do estudo foram setenta de três gestantes internadas, e que tiveram o sangue de cordão umbilical e placentário coletados após a dequitação. A análise estatística foi processada pelo software estatístico SAS® System, versão 6.11 e o critério de determinação de significância adotado foi o nível de 5%. A análise estatística apresentou resultados satisfatórios, constatamos que a celularidade inicial do sangue do cordão umbilical de gestantes hipertensas não apresentou diferença significativa comparado com a celularidade de gestantes não-hipertensas. Das bolsas de SCUP coletas de gestantes hipertensas, 80% apresentou celularidade ≥ 5 x 108 e 20% celularidade < 5 x 108 (de acordo com padrão estabelecido pela RDC 56). A partir deste estudo, podemos concluir que gestantes com hipertensão arterial apresentaram uma quantidade adequada no total de células nucleadas, atendendo aos critérios estabelecidos pela RDC 56. Com base nestes resultados e com a intensificação de pesquisas neste campo, sugerimos a reavaliação dos critérios de doação do sangue do cordão umbilical e placentário das gestantes com hipertensão arterial. A celularidade do SCUP apresentou uma relação positiva com alguns fatores neonatais e características da placenta/cordão, o conhecimento destes fatores permitirá as equipes dos BSCUP, a identificação de características importantes relacionadas à mãe, ao recém-nascido e as características da placenta e do cordão que possam otimizar o quantitativo de células obtidas do SCUP. / The activities performed by nurses in the Blood Banks Umbilical Cord Placental high demand and functional complexity of care, a fact that indirectly responsible to identify situations that should be evaluated, researched and reworked, such as the criteria for donation of umbilical cord blood and placental (SCUP), inclusion and exclusion criteria for donors, standardization of techniques and other situations that involve the performance of nurses. WBC has been the subject of attention from nurses, due to the need to obtain an adequate number of hematopoietic stem cells. This study aimed to discuss the relation between cellularity and cord blood and placental and hypertension, a profile of pregnant women with hypertension and no hypertension. The methodology corresponded to an observational case-control. The research site was the Maternity Hospital and Oswaldo de Nazareth Processing and Cryopreservation Laboratory of the National Cancer Institute and the study subjects were pregnant women admitted seventy three, and had the blood of the umbilical cord and placenta collected after dequitação. Statistical analysis was performed with SAS ® System software, version 6.11 and the criterion for determining significance was set at 5%. Statistical analysis showed satisfactory results, we found that the initial cellular cord blood of pregnant women with hypertension showed no significant difference compared with the cellularity of pregnant women not hypertensive. SCUP collections of the stock of pregnant women with hypertension, 80% had WBC ≥ 5 x 108 and 20% cellularity <5 x 108 (according to standards established by the DRC 56). From this study, we conclude that pregnant women with hypertension had an adequate amount of total nucleated cells, meeting the criteria established by the RDC 56. Based on these results and the intensification of research in this field, we suggest a reassessment of the criteria for donation of umbilical cord blood and placenta of pregnant women with hypertension. The cellularity of the SCUP presented a positive relationship with some factors and characteristics of neonatal placental / cord, the knowledge of these factors will allow teams of BSCUP, the identification of important features related to the mother, the newborn and the characteristics of the placenta and umbilical that can optimize the quantity of cells obtained from SCUP.

As graft source for lymphoma or leukemia treatment, hematopoietic stem and progenitor cells (HSPCs) have been the focus of translational medicine for decades. HSPCs are defined by their self-renewing capacity and their ability to give rise to all mature blood cells. They are found anchored to a specialized microenvironment in the bone marrow (BM) called the hematopoietic niche. HSPCs can be enriched by sorting them based on the presence of the surface antigen CD34 before clinical or tissue engineering use. As these cells represent a minority in most graft sources and the amount of applicable cells is limited, ex vivo expansion-cultures were established using cytokine cocktails or small molecules. However, in vitro culture of HSPCs as suspension-cultures result in heterogeneous cell populations with undefined cellular identities. In the BM niche, HSPCs are not exclusively maintained by cytokines but also by cell-matrix adhesions mediated by integrins (ITGs). Thus, β1 and β2 ITGs were found to promote initial contact of HSPCs with mesenchymal stromal cells (MSCs) and ITGβ3 expression was shown to be a marker for long-term repopulating HSPCs in vivo. Consequently, ex vivo remodeling of the BM niche using co-cultures of HSPCs and niche cells like MSCs came into spotlight and was proven to be a promising tool for stem cell expansion. However, in clinical and research applications, direct contact of two cell populations necessitates HSPC post-culture purification. To address these problems, we established a novel culture method for remodeling the BM extra cellular stroma in vitro wherein we used decellularized extracellular matrix (ECM) scaffolds derived from immortalized mesenchymal stromal cells (SCP-1). Such scaffolds were found to be highly reproducible and served as in vitro niche for HSPCs by being more effective for the expansion of CD34+ cells, compared to classical suspension cultures. ECMs were shown to consist of multiple proteins including fibronectins, collagens, and a major niche chemokine responsible for BM homing and retention of HSPCs in vivo, namely, stromal derived factor 1 (SDF-1). SDF-1 is known to be secreted by MSCs and is anchored to matrix proteins. This reveals that ECM scaffolds produced by SCP-1 cells are a naïve reconstructed microenvironment. When CD34+ cells were seeded, only around 20% of the cells adhered to the provided ECM scaffold. These cells recognized SDF-1 via C-X-C chemokine receptor type 4 (CXCR-4), as shown by laser scanning confocal microscopy. Thus, adhesive sides as they are present in the BM niche are provided. However, CD34+ cells isolated from G-CSF mobilized peripheral blood of healthy donors were found to be heterogenous with respect to adhesion capacity. Nonetheless, it was similar to HSPC co-cultures with SCP-1 cells as feeder layer. Therefore, we separated and analyzed two cell fractions, the adherent (AT-cells) and the non- adherent supernatant (SN-cells) cells. Other signals provided by the BM extracellular stroma to HSPCs are physical cues that control HSPC fate. HSPCs sense these physical features through focal contacts and accordingly remodel their morphological and biomechanical properties. Using real-time deformability cytometry (RT-DC) to uncover biomechanical phenotypes of freshly isolated HSPCs, SN-cells, AT-cells, and classical suspension cultured HSPCs in plastic culture dishes (PCD) were analyzed. We found freshly isolated cells to be less deformable and small. AT-cells displayed actin polymerization to stress fibers, and exhibited a stiffer mechanical phenotype compared to PCD-cultured or SN-cells. This might constitute the first hint of functional adaptation. Integrins are known to establish mechanosensing focal contacts. Thus, we analyzed ITG surface expression and identified ITGαIIb, ITGαV, and ITGβ3 to be enriched on AT-cells compared to freshly isolated cells or SN-cells. Active integrins need to form heterodimers consisting of one α- and one β subunit. Interestingly, the identified ITGs exclusively interact with each other to form RGD peptide receptors. RGD is a tripeptide consisting of the amino acids arginine, glycine, and aspartic acid and was identified as an adhesion sequence within fibronectin and other extracellular proteins. Consequently, we could confirm an important role for ITGαVβ3 in HSPC- ECM interaction with respect to adhesion and migration. However, we also identified ITGβ3 expression on a subset of CD34+ cells either freshly isolated or ECM cultured cells, as a marker for erythrocyte differentiation. These findings demonstrate that, in vitro, the ECM compartment acts as a regulator of HSPC fate and portray mechanical recognition as a potent driver of differentiation. In this context, targeted modulation of ECM scaffolds could enhance cell-ECM interactions and accelerate stem cell expansion or differentiation. These modulations could also provide further insights into HSPC-niche regulation. We demonstrate that ECMs derived from osteogenic differentiated SCP-1 cells increase HSPC expansion but do not lead to increased cell adhesion. As ECM adhesion preliminary alters HSPC function, we aimed at developing ECM scaffolds with increased adhesion capacity. Using lentiviral transduction, we generated a stable knock down of fibulin-1 in SCP-1 cells. Fibulin-1 is an ECM protein known to form anti-adhesion sites with fibronectin. However, we failed to increase adherent cell numbers or enhance HSPC expansion in the fibulin-1 knock down ECMs. Taken together, SCP-1 cell-derived ECM protein scaffolds provide an in vitro niche for HSPCs capable of stem cell expansion. Integrin mediated signaling altered the biomechanical and functional properties of HSPCs and hints at suspension cultures as being inappropriate to study the physiological aspects of HSPCs. Targeted modulation of ECM scaffolds could theoretically generate suitable ex vivo environments with the capacity to gain detailed insight into HSPC regulation within their niche. This will enhance the functionality of new biomaterials and will lead to improved regenerative therapies like BM transplantation.

Les anticorps à réactivité croisée sont caractérisés par leur capacité à reconnaitre des épitopes différents de ceux qui ont causé leur induction. Cela se produit lorsque des similitudes structurales entre les deux déterminants antigéniques deviennent suffisantes pour permettre une liaison spécifique. Nous rapportons ici pour la première fois la présence, à une haute fréquence, d'anticorps dans des échantillons de sang provenant de sujets vivant en France avec une protéine de Trypanosoma cruzi. Nous avons appelé ces anticorps ''Trypanosoma cruzi Cross-reactive antibodies'' ou TcCRA. Nos résultats montrent une forte séroprévalence des anticorps à réaction croisée, suggérant qu'ils sont induits par un immunogène largement répandu, acquis dès l’enfance et qui ne semble pas être associé à des agents pathogènes communs en clinique humaine. Les recherches effectuées in silico orientent vers un virus de la famille des Herpesviridae. Cette hypothèse est renforcée par la documentation d’un profil sérologique de séroconversion chez un patient qui a subi une transplantation de cellules souches allogéniques. Ce premier travail va servir de base à la mise en oeuvre d’investigations cliniques rétrospectives et prospectives destinées à élucider l’étiologie et l’importance clinique du biomarqueur TcCRA / Cross-reactive antibodies are characterized by their recognition of antigens that are different from the trigger immunogen. This happens when the similarity between two different antigenic determinants becomes adequate enough to enable a specific binding. Here, we report for the first time the presence, at an ‘‘abnormal’’ high frequency in blood samples from French human subjects, of antibodies that cross-react with a protein of Trypanosoma cruzi. We called these antibodies ‘‘Trypanosoma cruzi Cross-Reactive Antibodies’’ or TcCRA. Our findings show a large seroprevalence of cross-reactive antibodies and suggest that they are induced by a widely spread immunogen, acquired during childhood. Furthermore TcCRA serology does not seem to be associated with commonly known pathogens in clinical routine. Our hypothesis of the implication of a viral agent in the induction of TcCRA was further put forward when we documented a seroconversion pattern in a patient after allogenic stem cell transplantation. This initial exploratory work will serve as the basis for organizing prospective and retrospective clinical investigations, where we will pursue the analysis of TcCRA in order to elucidate its etiology and clinical importance

O transplante de células-tronco hematopoéticas (TCTH) consiste em um procedimento complexo e relacionado à ocorrência de diversas complicações, dentre elas os processos infecciosos decorrentes do longo período de imunossupressão vivenciado após a instituição do regime de condicionamento. Inúmeras medidas têm sido empregadas visando à prevenção e controle de infecções, porém, observam-se divergências em relação à utilização das mesmas; sendo que o emprego da prática baseada em evidências possibilita ao profissional tomar decisões em relação à sua prática fundamentadas em resultados de pesquisas científicas atuais. Esta revisão integrativa da literatura teve como objetivo identificar e avaliar as evidências disponíveis na literatura e publicadas nos últimos 20 anos em relação ao uso de três medidas de prevenção de infecção em pacientes submetidos ao TCTH durante o período de internação: uso de filtros de ar de alta eficiência, isolamento protetor e máscaras. Para a seleção dos artigos foram utilizadas as bases de dados LILACS, PUBMED, CINAHL, EMBASE e a Biblioteca Cochrane. A amostra foi composta por 15 estudos, sendo que apenas um apresentou nível de evidência forte (nível I), dois apresentaram nível de evidência moderado (nível IV e V) e doze consistiram em estudos com evidências fracas (nível VI e VII). Dez estudos abordaram a utilização dos filtros HEPA, sendo recomendado seu emprego para pacientes submetidos ao transplante alogênico durante o período de neutropenia. A necessidade de seu uso para pacientes submetidos ao transplante autólogo ainda é controversa. Nove trabalhos abordaram o uso do isolamento protetor e, embora alguns autores relatem que o emprego do mesmo parece apresentar benefícios quando não se dispõe de filtros HEPA, a utilização desta medida já não é mais indicada tanto pelos Centers for Disease Control and Prevention (CDC) quanto pela maioria dos estudos analisados. Em relação à utilização de máscaras por pacientes, profissionais de saúde ou visitantes dentro das unidades de internação para TCTH, não foram encontrados estudos com evidências fortes que justifiquem o seu uso. No entanto, recomenda-se que sejam seguidas as diretrizes dos CDC quanto ao uso de respiradores especiais (como as máscaras N95) pelos pacientes imunocomprometidos submetidos ao TCTH ao deixar a unidade de transplante provida de filtro HEPA quando próximo a ela houver áreas de construção/reforma ou atividades geradoras de poeira. Embora os dados evidenciados auxiliem na tomada de decisão para a implementação da assistência de enfermagem a estes pacientes, verificou-se a necessidade de realização de estudos com nível de evidência forte que comprovem ou refutem a efetividade destas medidas. / Hematopoietic stem cell transplantation (HSCT) is a complex procedure related to the occurrence of different complications, including infectious processes deriving from the long period of immunosuppression experienced after the establishment of the conditioning regimen. Countless measures have been used for infection prevention and control, but divergences are observed with regard to their use; evidence-based practice allows professionals to make decisions for practice based on current scientific research results. This integrative literature review aimed to identify and assess evidence available in literature and published in the last 20 years about the use of three infection prevention measures in patients submitted to HSCT during hospitalization: use of high-efficiency air filters, protective isolation and masks. LILACS, PUBMED, CINAHL, EMBASE and the Cochrane Library were used to select the articles. The sample comprised 15 studies, only one of which presented strong evidence (level I), while two presented moderate evidence (levels IV and V) and twelve were studies with weak evidence (levels VI and VII). Ten studies discussed the use of HEPA filters, recommended for patients submitted to allogeneic transplantation during the neutropenia period. It remains controversial whether these filters need to be used for patients submitted to autologous transplant. Nine studies addressed the use of protective isolation and, although some authors report that using this measure can be beneficial when HEPA filters are unavailable, neither the Centers for Disease Control and Prevention (CDC) nor by most of the studies under analysis indicate it any longer. With regard to the use of masks by patients, health professionals or visitors inside HSCT hospitalization units, no studies with strong evidence were found that justify its use. However, it is recommended that CDC recommendations be followed regarding the use of special respirators (like N95 masks) by immunocompromised patients submitted to HSCT when they leave the transplantation unit with a HEPA filter in case of nearby construction/reform areas or activities that generate dust. Although the evidenced data support decision making with a view to nursing care delivery to these patients, research with strong evidence is needed to prove or reject the efficacy of these measures.

Aujourd'hui, l'un des modèles de souris humanisées le plus robuste est obtenu en injectant des cellules souches hématopoïétiques humaines (HSC) issues de foie fœtal humain et en implantant du thymus fœtal autologue. Ce modèle, appelé BLT (Bone marrow/Liver/Thymus), s'est révélé capable de supporter une reconstitution, une maturation et une sélection optimales des cellules T. Les souris BLT sont utilisées pour de nombreuses études telles que la compréhension de la biologie du VIH ou plus récemment en médecine régénérative. Grâce à ce modèle, nous avons pu d’une part étudier le rôle des cellules dendritiques plasmacytoïdes (pDC) lors de l’infection par le VIH mais aussi mieux comprendre la formation in vivo de tératomes lors de l’utilisation d’iPSC. Cependant, l'une des principales limites de cette technique réside dans l'obtention du tissu fœtal. Ici, nous avons décrit un nouveau protocole de souris humanisées greffées avec du thymus humain en utilisant des matériaux plus accessibles: du thymus humain retiré lors d’une chirurgie cardiaque chez des nouveaux-nés ou des enfants, et des HSC de sang de cordon. Des morceaux de ces thymus ont été implantés dans les quadriceps de souris immunodéficientes, après avoir été mis en culture. Ces souris CCST (Cord blood and Cardiac Surgery Thymus) ont permis une prise de greffe importante et un meilleur développement des lymphocytes T humains que les souris humanisées sans thymus. Les lymphocytes T des souris CCST et BLT ont montré une fonction similaire, évaluée par des tests de prolifération ex vivo et par rejet de lignées de cellules leucémiques allogéniques in vivo. Nous avons testé l’intérêt de cette nouvelle stratégie dans le modèle de l’infection au VIH-1, qui représente le modèle type de l’utilité des BLT. Nous avons montré que les souris CCST sont sensibles à l'infection par le VIH-1 par voie muqueuse ou intrapéritonéale, comme l'indique la détection de l'ADN du VIH et des cellules p24 +, similairement aux souris BLT. Les souris CCST ont présenté des réponses de lymphocytes T spécifiques du VIH-1 ex vivo plus efficaces que les BLT. Lors du traitement antirétroviral, les souris CCST, comme les BLT, ont vu leur charge virale diminuer. Ces résultats démontrent que les souris CCST représentent une alternative au modèle de souris BLT classique. Ces thymus, éthiquement plus facile à obtenir, peuvent être utilisés pour générer un grand nombre de souris par rapport aux thymus fœtaux. / Immunodeficient mice engrafted with human immune system provide an exciting in vivo model for a better understanding of its functioning and for development of new therapies. Today, one of the most robust humanized mouse model is achieved by injecting human hematopoietic stem cells (HSC) from fetal liver along with an implantation of autologous fetal thymic tissue. This model, called BLT, was shown to be able to support an optimal T cell reconstitution, maturation and selection. BLT mice are extensively used for many studies such as understanding HIV biology or in regenerative medicine. Indeed, our work used BLT mice on one hand to study the role of plasmacytoid dendritic cells (pDC) during the HIV infection and on the other hand to better understand the formation of teratomes from iPSCs in vivo. However, one of the biggest limitations of this technique is the procurement of the fetal tissue. Here we describe a new protocol to do humanized mice engrafted with human thymus pieces by using more accessible materials: human thymus obtained during cardiac surgery and cord blood HSC. Indeed, thymus is spontaneously removed during cardiac surgery in neonates and young children, thus it is an easy and ethical way to obtain this tissue. Those thymuses pieces were implanted in the quadriceps of a immunodeficient mice, after being put in culture. CCST mice (Cord blood and Cardiac Surgery Thymus) exhibited a significant engraftment of T-cells, compared to humanized mice without thymus. T-cells from both CCST and BLT mice showed a similar function as evaluated by proliferation assays upon PHA stimulation ex vivo and rejection of allogeneic leukemic cells lines in vivo. CCST mice were susceptible to HIV-1 infection via mucosal or intraperitoneal route, as shown by detectable viral load, HIV DNA and p24+ cells, at similar levels to those of BLT mice. Importantly, CCST mice displayed more effective ex vivo HIV-1-specific T-cell responses compared to BLT. Upon antiretroviral treatment, CCST mice, like BLT, were able to diminish the viral load. Our data suggest that CCST mice represent an alternative to the regular BLT mouse model. Those easy-to-access thymuses can be used to generate a large number of mice compared to fetal thymuses.

Les lymphocytes T régulateurs (Treg) jouent un rôle majeur dans la modulation de l’alloréactivité après allogreffe de cellules souches hématopoïétiques et permettent notamment de contrôler la réaction du greffon contre l’hôte (GVH) dans des modèles expérimentaux. Le potentiel thérapeutique des Treg est donc très important dans ce domaine, mais aussi dans l’auto-immunité ou en cancérologie. Cependant, de nombreuses barrières rendent difficile l’élaboration de stratégies thérapeutiques reposant sur le transfert adoptif de Treg chez l’homme et une meilleure compréhension des facteurs et mécanismes contrôlant la prolifération et les capacités suppressives de ces cellules permettrait de les cibler directement et si possible spécifiquement in vivo.Dans ce travail, après avoir élaboré un nouveau système d’évaluation clinique de la GVH chez la souris et démontré sa simplicité, sa reproductibilité et sa performance, nous avons pu montrer que l’action suppressive des Treg dans la GVH dépendait de l’interaction entre le TNFα produit par les lymphocytes T conventionnels (Tconv) du donneur et le récepteur TNFR2 exprimé par les Treg. En effet, en bloquant cette interaction de 3 façons différentes, à savoir par un anticorps monoclonal bloquant anti-TNFR2, ou en utilisant soit des Treg n’exprimant pas TNFR2 soit des Tconv ne produisant pas de TNFα, nous avons à chaque fois montré que l’effet protecteur des Treg était aboli en l’absence du signal TNF. Le récepteur TNFR2 étant exprimé préférentiellement par les Treg par rapport aux Tconv, nos résultats ouvrent la voie au ciblage des Treg in vivo via TNFR2, soit pour activer ce récepteur par un agoniste et donc stimuler les Treg afin de contrôler la GVH, soit à l’inverse pour bloquer l’axe TNFα/TNFR2 par un antagoniste et ainsi inhiber les Treg, ce qui permettrait alors de lever un frein à l’alloréactivité dans les situations où l’on cherche à la stimuler pour renforcer l’effet anti-tumoral, comme par exemple dans le cas des rechutes post-allogreffe. / Regulatory T cells (Tregs) are key players involved in the modulation of alloreactivity after hematopoietic stem cell transplantation. Indeed, Tregs can prevent graft-versus-host disease (GVHD) in experimental models. Therefore, the therapeutic potential of these cells in GVHD is substantial, as it is in other fields like auto-immunity or oncology. However, many obstacles still make the application of cellular therapy strategies based on the adoptive transfer of Tregs in humans quite complicated. A better understanding of factors and mechanisms that control the proliferation and suppressive capacities of Tregs could allow for a direct and specific targeting of these cells in vivo.In this work, after designing a new clinical grading system for murine GVHD and demonstrating its ease of use, reproducibility and performance, we have shown that the suppressive action of Tregs in GVHD depends on the interaction between TNFα produced by donor conventional T cells (Tconvs) and TNFR2 expressed by Tregs. Using 3 different ways to block this interaction, i.e. with an anti-TNFR2 blocking monoclonal antibody, or Tregs that do not express TNFR2 or donor Tconvs that cannot produce TNFα, we were able to show in each situation that blocking TNF signaling resulted in a loss of protection by Tregs. TNFR2 being highly expressed by Tregs as compared with Tconvs, our results pave the way for in vivo targeting of Tregs through TNFR2, either to activate this receptor with an agonist and therefore stimulate Tregs to control GVHD, or to block the TNFα/TNFR2 axis with an antagonist and in this case inhibit Tregs, which could boost alloreactivity, as expected in some particular settings like post-transplant relapse.

Clinical application of natural killer (NK) cells against leukemia is an area of intense investigation. In human leukocyte antigen-mismatched allogeneic hematopoietic stem cell transplantations (HSCT), alloreactive NK cells exert powerful anti-leukemic activity in preventing relapse in the absence of graft-versus-host disease, particularly in acute myeloid leukemia patients. Adoptive transfer of donor NK cells post-HSCT or in non-transplant scenarios may be superior to the currently widely used unmanipulated donor lymphocyte infusion. This concept could be further improved through transfusion of activated NK cells. Significant progress has been made in good manufacturing practice (GMP)-compliant large-scale production of stimulated effectors. However, inherent limitations remain. These include differing yields and compositions of the end-product due to donor variability and inefficient means for cryopreservation. Moreover, the impact of the various novel activation strategies on NK cell biology and in vivo behavior are barely understood. In contrast, reproduction of the thirdparty NK-92 drug from a cryostored GMP-compliant master cell bank is straightforward and efficient. Safety for the application of this highly cytotoxic cell line was demonstrated in first clinical trials. This novel ‘off-theshelf’ product could become a treatment option for a broad patient population. For specific tumor targeting chimeric-antigen-receptor-engineered NK-92 cells have been designed.

No description available.

As graft source for lymphoma or leukemia treatment, hematopoietic stem and progenitor cells (HSPCs) have been the focus of translational medicine for decades. HSPCs are defined by their self-renewing capacity and their ability to give rise to all mature blood cells. They are found anchored to a specialized microenvironment in the bone marrow (BM) called the hematopoietic niche. HSPCs can be enriched by sorting them based on the presence of the surface antigen CD34 before clinical or tissue engineering use. As these cells represent a minority in most graft sources and the amount of applicable cells is limited, ex vivo expansion-cultures were established using cytokine cocktails or small molecules. However, in vitro culture of HSPCs as suspension-cultures result in heterogeneous cell populations with undefined cellular identities. In the BM niche, HSPCs are not exclusively maintained by cytokines but also by cell-matrix adhesions mediated by integrins (ITGs). Thus, β1 and β2 ITGs were found to promote initial contact of HSPCs with mesenchymal stromal cells (MSCs) and ITGβ3 expression was shown to be a marker for long-term repopulating HSPCs in vivo. Consequently, ex vivo remodeling of the BM niche using co-cultures of HSPCs and niche cells like MSCs came into spotlight and was proven to be a promising tool for stem cell expansion. However, in clinical and research applications, direct contact of two cell populations necessitates HSPC post-culture purification. To address these problems, we established a novel culture method for remodeling the BM extra cellular stroma in vitro wherein we used decellularized extracellular matrix (ECM) scaffolds derived from immortalized mesenchymal stromal cells (SCP-1). Such scaffolds were found to be highly reproducible and served as in vitro niche for HSPCs by being more effective for the expansion of CD34+ cells, compared to classical suspension cultures. ECMs were shown to consist of multiple proteins including fibronectins, collagens, and a major niche chemokine responsible for BM homing and retention of HSPCs in vivo, namely, stromal derived factor 1 (SDF-1). SDF-1 is known to be secreted by MSCs and is anchored to matrix proteins. This reveals that ECM scaffolds produced by SCP-1 cells are a naïve reconstructed microenvironment. When CD34+ cells were seeded, only around 20% of the cells adhered to the provided ECM scaffold. These cells recognized SDF-1 via C-X-C chemokine receptor type 4 (CXCR-4), as shown by laser scanning confocal microscopy. Thus, adhesive sides as they are present in the BM niche are provided. However, CD34+ cells isolated from G-CSF mobilized peripheral blood of healthy donors were found to be heterogenous with respect to adhesion capacity. Nonetheless, it was similar to HSPC co-cultures with SCP-1 cells as feeder layer. Therefore, we separated and analyzed two cell fractions, the adherent (AT-cells) and the non- adherent supernatant (SN-cells) cells. Other signals provided by the BM extracellular stroma to HSPCs are physical cues that control HSPC fate. HSPCs sense these physical features through focal contacts and accordingly remodel their morphological and biomechanical properties. Using real-time deformability cytometry (RT-DC) to uncover biomechanical phenotypes of freshly isolated HSPCs, SN-cells, AT-cells, and classical suspension cultured HSPCs in plastic culture dishes (PCD) were analyzed. We found freshly isolated cells to be less deformable and small. AT-cells displayed actin polymerization to stress fibers, and exhibited a stiffer mechanical phenotype compared to PCD-cultured or SN-cells. This might constitute the first hint of functional adaptation. Integrins are known to establish mechanosensing focal contacts. Thus, we analyzed ITG surface expression and identified ITGαIIb, ITGαV, and ITGβ3 to be enriched on AT-cells compared to freshly isolated cells or SN-cells. Active integrins need to form heterodimers consisting of one α- and one β subunit. Interestingly, the identified ITGs exclusively interact with each other to form RGD peptide receptors. RGD is a tripeptide consisting of the amino acids arginine, glycine, and aspartic acid and was identified as an adhesion sequence within fibronectin and other extracellular proteins. Consequently, we could confirm an important role for ITGαVβ3 in HSPC- ECM interaction with respect to adhesion and migration. However, we also identified ITGβ3 expression on a subset of CD34+ cells either freshly isolated or ECM cultured cells, as a marker for erythrocyte differentiation. These findings demonstrate that, in vitro, the ECM compartment acts as a regulator of HSPC fate and portray mechanical recognition as a potent driver of differentiation. In this context, targeted modulation of ECM scaffolds could enhance cell-ECM interactions and accelerate stem cell expansion or differentiation. These modulations could also provide further insights into HSPC-niche regulation. We demonstrate that ECMs derived from osteogenic differentiated SCP-1 cells increase HSPC expansion but do not lead to increased cell adhesion. As ECM adhesion preliminary alters HSPC function, we aimed at developing ECM scaffolds with increased adhesion capacity. Using lentiviral transduction, we generated a stable knock down of fibulin-1 in SCP-1 cells. Fibulin-1 is an ECM protein known to form anti-adhesion sites with fibronectin. However, we failed to increase adherent cell numbers or enhance HSPC expansion in the fibulin-1 knock down ECMs. Taken together, SCP-1 cell-derived ECM protein scaffolds provide an in vitro niche for HSPCs capable of stem cell expansion. Integrin mediated signaling altered the biomechanical and functional properties of HSPCs and hints at suspension cultures as being inappropriate to study the physiological aspects of HSPCs. Targeted modulation of ECM scaffolds could theoretically generate suitable ex vivo environments with the capacity to gain detailed insight into HSPC regulation within their niche. This will enhance the functionality of new biomaterials and will lead to improved regenerative therapies like BM transplantation.:List of contents I List of figures IV List of tables VI Abbreviations VII 1 Introduction 1 1.1 The stem cell microenvironment 3 1.1.1 The cellular endosteal bone marrow microenvironment 6 1.1.1.1 Mesenchymal stem/stromal cells 7 1.1.1.2 Hematopoietic stem and progenitor cells 8 1.1.2 Extracellular bone marrow microenvironment 10 1.1.2.1 Extracellular matrix 11 Chemokines and Cytokines 12 Cell adhesion to ECM 13 1.2 Native ex vivo ECM scaffolds 16 2 Aim of the study 19 3 Materials and methods 21 3.1 Materials 21 3.1.1 Chemicals and reagents 21 3.1.2 Kits 23 3.1.3 Media 24 3.1.4 Antibodies 24 3.1.5 Primers, sh-RNA sequences, and vectors 25 3.1.6 Equipment 26 3.1.7 Software 27 3.2 Methods 27 3.2.1 Cell preparation and culture 27 3.2.1.1 Mesenchymal stromal cells 27 3.2.1.2 Hematopoietic stem cells 28 3.2.1.3 Single cell picked clone 1 (SCP-1) cells 28 3.2.2 Generation of surface immobilized ECM preparations 29 3.2.2.1 Surface functionalization 29 3.2.2.2 ECM preparation 29 3.2.3 Flow cytometry and fluorescent activated cell sorting 30 3.2.4 Cell cycle analyses 30 3.2.5 Proliferation analyses 31 3.2.6 Colony forming unit cell assay (CFU-GEMM) 31 3.2.7 Migration assays 31 3.2.7.1 Transwell migration 31 3.2.7.2 Live cell migration 32 3.2.8 Confocal laser scanning microscopy 32 3.2.9 Real-time deformability cytometry (RT-DC) 32 3.2.10 Molecular biological methods 33 3.2.10.1 RNA isolation, reverse transcription, and PCR 33 3.2.10.2 Lentiviral shRNA transduction 34 3.2.10.3 Western blot 35 3.2.10.4 ELISA 36 3.2.11 Statistical analysis 37 4 Results 38 4.1 Extracellular matrix scaffolds for HSPCs 38 4.1.1 ECM properties 39 4.1.2 HSPC survival in ECM and PCD cultures 40 4.1.3 HSPC expansion in ECM and PCD cultures 41 4.2 HSPC morphological and mechanical adaptation to ECM 44 4.2.1 Actin polymerization and polarization 45 4.2.2 Biomechanical phenotype 46 4.3 Bioactive SDF-1 is incorporated in ECM scaffolds 49 4.3.1 CXCR4 polarization towards ECM 50 4.4 HSPC integrin expression and migration 52 4.4.1 Integrin surface expression on HSPC subsets 52 4.4.2 Focal contact formation 53 4.4.3 Integrin activation via ECM adhesion 55 4.4.4 Clonogenicity of ECM cultured HSPCs 57 4.4.5 HSPC migration when attached to ECM scaffolds 60 4.4.5.1 Reduced migratory behavior via ITGαVβ3 inhibition 61 4.4.5.2 SDF-1 induces migration but not adhesion 64 4.5 Targeted modulation of ECM scaffolds 65 4.5.1 Fibulin-1 knock down in SCP-1 cells 66 4.5.2 HSPC support of fibulin-1 reduced ECM scaffolds 70 5 Discussion 73 5.1 SCP-1 cells as a source for ECM scaffold production 74 5.2 Cell adhesion and focal contact formation 75 5.3 HSPC multilineage potential 78 5.4 ECM scaffold modulation 79 6 Summary 83 7 Zusammenfassung 86 Bibliography 89 Danksagung 108 Anlagen 110 Erklärung zur Eröffnung des Promotionsverfahrens [Formblatt 1.2.1] 110 Erklärung zur Einhaltung rechtlicher Vorschriften [Formblatt 1.1] 110