Les cellules souches somatiques présentent habituellement un comportement très différent des cellules souches pluripotentes. Les bases moléculaires de l’auto-renouvellement des cellules souches embryonnaires ont été récemment déchiffrées grâce à la facilité avec laquelle nous pouvons maintenant les purifier et les maintenir en culture durant de longues périodes de temps. Par contre, il en va tout autrement pour les cellules souches hématopoïétiques. Dans le but d’en apprendre davantage sur le fonctionnement moléculaire de l’auto-renouvellement des cellules souches hématopoïétiques, j’ai d’abord conçu une nouvelle méthode de criblage gain-de-fonction qui répond aux caprices particuliers de ces cellules. Partant d’une liste de plus de 700 facteurs nucléaires et facteurs de division asymétrique candidats, j’ai identifié 24 nouveaux facteurs qui augmentent l’activité des cellules souches hématopoïétiques lorsqu’ils sont surexprimés. J’ai par la suite démontré que neuf de ces facteurs agissent de manière extrinsèque aux cellules souches hématopoïétiques, c’est-à-dire que l’effet provient des cellules nourricières modifiées en co-culture. J’ai également mis à jour un nouveau réseau de régulation de transcription qui implique cinq des facteurs identifiés, c’est-à-dire PRDM16, SPI1, KLF10, FOS et TFEC. Ce réseau ressemble étrangement à celui soutenant l’ostéoclastogénèse. Ces résultats soulèvent l’hypothèse selon laquelle les ostéoclastes pourraient aussi faire partie de la niche fonctionnelle des cellules souches hématopoïétiques dans la moelle osseuse. De plus, j’ai identifié un second réseau de régulation impliquant SOX4, SMARCC1 et plusieurs facteurs identifiés précédemment dans le laboratoire, c’est-à-dire BMI1, MSI2 et KDM5B. D’autre part, plusieurs indices accumulés tendent à démontrer qu’il existe des différences fondamentales entre le fonctionnement des cellules souches hématopoïétiques murines et humaines. / Somatic stem cells usually exhibit a very different behavior compared to pluripotent stem cells. The molecular basis of embryonic stem cell self-renewal was recently decrypted by the relative straightforwardness with which we can now purify and maintain these cells in culture for long periods of time. However, this is not the case with hematopoietic stem cells. In order to elucidate the molecular mechanisms of hematopoietic stem cell self-renewal, I developed a novel gain-of-function screening strategy, which bypasses some constraints found with these cells. Starting from a list of more than 700 candidate nuclear factors and asymmetric division factors, I have identified 24 new factors that increase hematopoietic stem cell activity when overexpressed. I have also found that nine of these factors act extrinsically to hematopoietic stem cells, i.e., the effect comes from the engineered feeder cells in co-culture. Moreover, I have revealed a new transcriptional regulatory network including five of the factors identified, i.e., PRDM16, SPI1, KLF10, FOS and TFEC. This network is particularly similar to that involved in osteoclastogenesis. These results raise the hypothesis that osteoclasts might also be part of the functional hematopoietic stem cell niche in the bone marrow. Furthermore, I have identified a second regulatory network involving SOX4, SMARCC1 and several factors previously identified in the laboratory, i.e., BMI1, MSI2 and KDM5B. Besides, several lines of evidence tend to show that there are fundamental differences between mouse and human hematopoietic stem cells.

As doenças falciformes (DF) constituem um grupo de doenças hereditárias monogênicas. São doenças extremamente relevantes no contexto de saúde pública no Brasil, portanto diferentes estratégias terapêuticas devem ser avaliadas. As oclusões vasculares afetam praticamente todos os órgãos, inclusive o baço e a medula óssea, porém não existem dados na literatura se estas comprometem também o tecido tímico. Os pacientes apresentam maior suscetibilidade às infecções cujas causas não são ainda totalmente esclarecidas Embora as infecções observadas nos pacientes sejam atribuídas à disfunção esplênica, o quadro inflamatório crônico e possíveis alterações no timo e na medula óssea, também poderiam causar uma disfunção imunológica. O objetivo deste trabalho foi avaliar a neogênese de células T e B e a diversidade do repertório de células T periféricas em pacientes com anemia falciforme (AF) sem tratamento (N = 15), tratados com hidroxiuréia (N = 20) ou transfusão crônica (N = 21) e em pacientes com DF tratados com transplante de células-tronco hematopoéticas (TCTH) alogênico (N = 29). Pacientes sem tratamento apresentaram menores níveis de sjTREC e ?-TREC, e menor taxa de divisão celular intratímica, demonstrando alterações importantes na neogênese das células T. A produção tímica de novas células T naïve foi reestabelecida em um ano pós-transplante, com normalização dos níveis de sjTREC e ?-TREC. O desenvolvimento de doença do enxerto contra o hospedeiro (DECHa) e reativação de citomegalovírus comprometeu a timopoiese nos primeiros seis meses pós-transplante, com diminuição significativa dos níveis de sjTRECs e ?-TRECs. Análises do repertório da cadeia V? dos receptores de células T (TCRs), pelo método TCRBV CDR3 spectratyping, indicaram que os pacientes com AF apresentaram um repertório menos diverso, composto predominantemente de famílias V? com padrão skewed e picos de CDR3 monoclonais, sendo a família V?3 mais frequente. A composição do repertório de células T foi alterada após o transplante, adquirindo um perfil mais policlonal dos picos de CDR3 ao longo do tempo. A família V?22 foi a mais expressa no período pré-transplante e em todos os seguimentos pós-transplante. Os pacientes com DF apresentaram aumento de linfócitos B naive, demonstrado pelos altos níveis de sjKRECs e pela taxa de proliferação homeostática. As análises multivariadas demonstraram que as alterações esplênicas influenciam diretamente os níveis de sjKREC, indicando que a baixa função esplênica leva ao aumento da produção de células B naive pela medula óssea, sugerindo um mecanismo compensatório. Os resultados desse trabalho demonstraram a existência de um desequilíbrio na neogênese de células T e B e consequentemente nesses compartimentos celulares periféricos, que pode conferir aos pacientes com DF uma maior susceptibilidade a infecções. Entre as diferentes modalidades terapêuticas, o TCTH alogênico sobressaiu-se em relação aos tratamentos convencionais, melhorando a neogênese de células T e B a longo prazo. / Sickle Cell Disease (SCD) are a group of monogenic hereditary diseases. These are extremely relevant diseases in the context of public health in Brazil, thus, different therapeutic strategies must be studied. Vascular occlusions affect practically all organs, including the spleen and bone marrow. However, there are no literature data about the impact of vaso-occlusions on the thymic tissue. Patients are more susceptible to infections whose causes are not fully elucidated. Although the infections observed in these patients are assigned to splenic dysfunction, the chronic inflammatory state and possible alterations of the thymus and bone marrow could also lead to immune dysfunction. The goal of this work was to evaluate the neogenesis of T and B cells and the diversity of peripheral T cell repertoire in patients with sickle cell anemia (SCA) without treatment (N = 15), treated with hydroxyurea (N = 20) or chronic transfusions (N = 21) and in patients with SCD treated with hematopoietic stem cell transplantation (N = 29) allogeneic. Patients without treatment had lower levels of sjTREC and ?-TREC, and lower rate of intrathymic cell division, demonstrating important alterations in the neogenesis of T cells. The thymic production of new naïve T cells was reestablished at one-year post transplantation, with normalization of sjTREC and ?-TREC levels. The development of graft-versus-host disease (aGVHD) and cytomegalovirus activation compromised thymopoiesis in the first six months post transplantation, with a significant decrease of sjTRECs and ?-TRECs levels. Analysis of the TCR V? chain repertoire by TCRBV CDR3 spectratyping indicate that patients with SCA showed a less diverse repertoire, mainly composed by V? families with a skewed pattern and monoclonal CDR3 peaks, being the V?3 family the most frequent one. The composition of the T-cell repertoire was altered after transplantation, changing over time to more polyclonal profile of the CDR3 peaks. The V?22 family was the more expressed at pre-transplantation and at all follow-up periods. Patients with SCD presented increased numbers of naive B cells, demonstrated by higher levels of sjKRECs and homeostatic proliferation. Multivariate analysis demonstrated that splenic function directly influenced sjKREC levels, indicating that compromised splenic function leads to increase of naive B cell output by the bone marrow, suggesting a compensatory mechanism. The results of this study showed the existence of an imbalanced T and B cell neogenesis and, consequently on these peripheral cell compartments, which may confer to patients with SCD an increased susceptibility to infections. Among different therapeutic modalities, allogeneic HSCT stood out in relation to the conventional treatments, improving long-term T and B cell neogenesis.

O diabetes mellitus tipo 1 (DM-1) é uma doença autoimune órgão-específica caracterizada pela destruição seletiva das células pancreáticas produtoras de insulina. A imunossupressão em altas doses seguida do transplante autólogo de células-tronco hematopoéticas (TACTH) constitui uma alternativa terapêutica recente e promissora para o DM-1 recém-diagnosticado, impedindo a progressão da destruição das células pancreáticas produtoras de insulina e induzindo independência insulínica por um período prolongado na maioria dos pacientes. O princípio dessa terapia baseia-se na eliminação das células autorreativas pela imunossupressão intensa e na reconstituição de um sistema imunológico novo e tolerante após o transplante. Com o objetivo de avaliar a função do timo e sua contribuição na geração do repertório de células T nos pacientes com DM-1 após o TACTH, nesse trabalho foram avaliados os níveis de T cell receptor excision circles (TRECs) em células T do sangue periférico e a diversidade do repertório de células T dos pacientes com DM-1 (n=23) antes e em diversos períodos após o transplante. A quantificação absoluta dos níveis de TRECs (número de moléculas de TRECs/100g de DNA) foi realizada pela técnica de PCR em tempo real e a avaliação do repertório de células T foi realizada pela técnica de TCRBV CDR3 Spectratyping. Dentre os vinte e três pacientes, vinte alcançaram a independência insulínica por períodos variáveis de tempo e três não responderam ao tratamento. Não foi observada a restrição do repertório de células T nos pacientes com DM-1 no período pré-transplante, ou seja, quando recém-diagnosticados. Foram identificadas cinco famílias V (7, 18, 19, 20 e 22) em expansão clonal nos pacientes com DM-1. As famílias V 7, 18, 19, 20 apresentaram-se em expansão clonal antes do transplante e se mantiveram com frequência elevada após o transplante, enquanto a família V 22 apresentou aumento da frequência somente nos períodos mais tardios após o transplante. Nos primeiros meses após o transplante, houve redução do número de moléculas de TRECs e restrição do repertório de células T. Contudo, um ano após o transplante, o número de moléculas de TRECs atingiram valores normais e o repertório de células T apresentou-se com ampla diversidade. Nossos resultados mostraram que o TACTH foi capaz de induzir mudanças na composição do repertório de células T dos pacientes com DM-1 após a terapia de IAD/TACTH, evidenciadas por alterações qualitativas e quantitativas dos picos de CDR3 do TCR, sugerindo a reconstituição de um repertório de células T diverso até dois anos pós-transplante. Embora tenha ocorrido reativação da função tímica após o transplante, evidenciada pelo aumento dos níveis de TRECs de um ano e meio a cinco anos pós-transplante, a diversidade do repertório das células T diminuiu a partir de dois anos e meio pós-transplante, sugerindo uma reconstituição tímica de novo de células T naive que expressam preferencialmente algumas cadeias V. Estas evidências imunológicas poderiam explicar a melhora clínica (independência insulínica) temporária observada na maioria dos pacientes após a terapia de IAD/TACTH. / Type 1 diabetes mellitus (T1D) is an organ-specific autoimmune disease characterized by insulin-producing pancreatic cell destruction. High-dose immunosuppression followed by autologous hematopoietic stem cell transplantation (AHSCT) is a recent and promising therapeutic approach for treatment of T1D, preventing the progress of destruction of pancreatic cells and inducing insulin independence for a prolonged period in most patients. The rationale of the AHSCT is based on the elimination of autoreactive cells by the intense immunosuppression and on the reconstitution of a new and tolerant immune system after transplantation. Aiming at assessing the thymic role in the production of new T cell repertoire in T1D patients after AHSCT, in this study was evaluated the levels of T cell receptor excision circles (TRECs) in T cells of peripheral blood as well as the clonality and diversity of T cell repertoire in T1D patients (n=23) before and several periods after transplantation. The absolute quantification of TRECs levels (number of molecules of TRECs/100ng of DNA) was performed by real-time PCR and the analysis of T cell repertoire was performed by TCRBV CDR3 Spectratyping. Among the twenty-three patients, twenty achieved insulin independence for variable periods and three did not respond to the treatment. The T cell repertoire in T1D patients was not restricted in pre-transplantation, i.e., when newly diagnosed. It was identified five V families (7, 18, 19, 20 e 22) in the clonal expansion in T1D patients. The V families 7, 18, 19, 20 were in clonal expansion before transplantation and maintained with high frequency after transplantation, whereas the V 22 family increased its frequency only in the later periods after transplantation. It was observed that the numbers of molecules of TRECs decreased and the T cell repertoire was restricted in the early months after transplantation. However, the levels of TRECs were normalized and the T cell repertoire showed diversity one year after transplantation. Our results indicate that AHSCT was able to induce changes in the composition of the T cell repertoire of patients after AHSCT, evidenced by qualitative and quantitative changes in the composition of T-cell receptor -chain CDR3 peaks, suggesting the reconstitution of diverse T cell repertoire up to two years after transplantation. Although there was reactivation of thymic function after transplantation, as evidenced by increased levels of TRECs from one and a half year to five years after transplantation, the diversity of the T cells repertoire decreased from two and a half years after transplantation, suggesting a reconstruction of new naive T cells that preferentially express some V chains. These immunological evidences could explain the temporary clinical improvement (insulin independence) observed in most patients after IAD / AHSCT therapy.

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.

A boca é local de frequentes complicações relacionadas ao transplante de células tronco hematopoiéticas (TCTH) tais como xerostomia, disgeusia, disfagia, mucosite, infecções oportunistas e doença do enxerto contra hospedeiro (DECH). Sabe-se que estas complicações podem comprometer a qualidade de vida do paciente e interferir na morbidade pós-TCTH. O dentista é o profissional da saúde que deverá intervir no momento correto para tratar e minimizar esses efeitos secundários do TCTH. Para tanto é importante conhecermos o momento em que cada complicação ocorre para que a intervenção seja pronta e eficiente. O objetivo principal deste estudo foi identificar e quantificar as alterações bucais em indivíduos submetidos ao TCTH em cinco momentos consecutivos desde antes do início do condicionamento pré-TCTH até o dia 100 pós-TCTH. Como objetivos secundários buscamos investigar possíveis relações entre a severidade da mucosite oral e a manifestação da DECH com dados demograficos (sexo, idade), com o status de saude bucal (por meio dos índices IHO-S, CPOD, número de dentes cariados) e com a realização de adequação bucal pré-TCTH, e ainda, somente para a DECH, também foi investigada a possível relação entre esta doença com infecção sistêmica por citomegalovírus e com a manifestação de mucosite oral severa. Foram incluídos no estudo 27 indivíduos com doenças hematológicas do Serviço de Transplante de Medula Óssea do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP), com idade 12 anos que receberam TCTH alogênico. Os indivíduos foram examinados em cinco momentos consecutivos. No primeiro momento, pré-TCTH, foi realizada a coleta de índices de saúde bucal e aplicação de questionário sobre o histórico de tratamentos odontológico prévios. Nos momentos de 10, 20, 60 e 100 dias pós-TCTH foram avaliadas as manifestações bucais presentes. A incidência de mucosite oral foi de 82,6% considerando todos os momentos avaliados. Mucosite oral severa, ou seja, graus 3 e 4 (OMS) foi observada em 57,9% dos pacientes avaliados nos momentos 2 e 3. Dez (37%) pacientes apresentaram GVHD em algum órgão, e destes, 8 (80%) apresentaram GVHD de boca. Infecção sistêmica por CMV foi diagnosticada em 6 (22,2%) pacientes. Concluímos que entre as queixas levantadas, dor bucal e disfagia foram as mais referidas. O período de maior incidência das complicações bucais foi nos segundo e terceiro momentos, ou seja, D+10 e D+20, representando deste forma, o período de maior morbidade do tratamento. Não houve associação entre a severidade de mucosite oral e idade, sexo, fonte de células, regime de condicionamento, número de dentes cariados, IHO-S, CPOD e preparo bucal pré-TCTH. Para a DECH a única relação encontrada foi para fonte de células, tendo sido observada menor chance de ocorrer DECH quando a fonte de células foi o sangue periférico. / The mouth is a well-known site of complications of the hematopoietic stem cell transplantation (HSCT) such as dry mouth, dysgeusia, dysphagia, mucositis, opportunistic infections and graft versus host disease (GVDH). It is known that these complications can compromise the patients quality of life and morbidity post-HSCT. The dentist is the health professional who should interfere at the right time to treat and minimize these side effects of HSCT. Thus, it is important to know the time at which each complication occurs to be dynamic and efficient. The main objective of this study was to identify and quantify the oral complications in patients treated with HSCT in five consecutive moments starting before conditioning chemotherapy until day 100 post-HSTC. As secondary objectives we seek to investigate possible relationships between the severity of oral mucositis and the manifestation of GVHD with demographic data (gender, age), with the oral health status (IHO-S, CPOD, number of decayed teeth) and dental treatment previously HSCT, and, only for GVHD, was also investigated the possible relationship between this disease with systemic cytomegalovirus infection and the manifestation of severe oral mucositis. It was included in the study 27 patients with hematologic diseases who were admitted in the Unit of Bone Marrow Transplantation, Hospital of Clinics, Faculty of Medicine, University of Sao Paulo (HC-FMUSP), 12 years old whom received allogeneic HSCT. The subjects were examined in five consecutive moments. At the first moment, before HSCT, the oral health índex evaluation and a questionnaire about history of previous dental treatments were performed. Besides that, 10, 20, 60 and 100 days after HSCT they were evaluated for oral manifestations. Oral mucositis incidence was 82,6% and 57,9% of these patients presented severe mucositis. Ten (37%) patients had GVHD in any organ, and of these, 8 (80%) had oral GVHD. Infection by CMV was diagnosed in 6 (22.2%) patients. We conclude that among the complaints raised, mouth pain and dysphagia were the most mentioned. The period of increased incidence of oral complications was the second and third times (D +10 and +20), representing the increased morbidity period. There was no association between the severity of oral mucositis and age, sex, cell source, conditioning regimen, number of decayed teeth, IHO-S, CPOD and dental treatment pre-HSCT. For GVHD the only relation found was with source of cells, in which, GVHD was less likely to occur when the source of cells was peripheral blood.

O HPgV (Pegivirus Humano), conhecido anteriormente por GBV-C, causa infecção assintomática, persistente e com alta carga viral. Sendo o vírus de RNA sem patologia associada mais prevalente no mundo até os dias de hoje, os estudos in vitro ainda não foram capazes de mimetizar a replicação in vivo, permanecendo pouco esclarecidos vários aspectos de sua biologia. Estudos em macacos mostraram que os órgãos responsáveis pela maior replicação viral são o baço e a medula óssea, no entanto as células específicas permissíveis à infecção ainda não foram determinadas. Na década de 90, o HPgV ganhou notoriedade por diminuir os efeitos patológicos do HIV e aumentar a sobrevida de pacientes coinfectados HIV/HPgV, através da diminuição da ativação do sistema imune. Devido a essa característica, diversos estudos tentaram associar a presença de viremia a doenças hematológicas e um deles mostrou que a viremia de HPgV parece estar associada com o desenvolvimento de linfoma não- Hodgkin, porém muitas variáveis de confusão parecem influenciar esse resultado. Em conjunto, os dados mostram que o HPgV pode ter influência sobre a maturação e liberação de células progenitoras da medula óssea e, portanto, tornou-se primordial avaliar o impacto da viremia em pacientes com doenças hematológicas e definir o tropismo viral. Para tanto, o presente trabalho analisou a dinâmica de exposição à viremia de HPgV em pacientes submetidos ao transplante de células-tronco hematopoiéticas (HSCT), em cadáveres autopsiados pelo Sistema de Verificação de Óbitos da Capital de São Paulo (SVOC-SP) e em pacientes submetidos à cirurgia de artroplastia pelo Instituto de Ortopedia e Traumatologia (IOT) do HCSP. A carga viral de HPgV foi mensurada por qRT-PCR em Tempo Real levando em consideração valores adquiridos com um curva padrão desenvolvida para este trabalho. Para os pacientes HSCT, o efeito da presença de viremia antes e após o procedimento foi avaliada quanto aos principais desfechos do transplante: doença enxerto-hospedeiro aguda (aGVHD), recaída da doença hematológica primária e mortalidade. Para amostras SVOC e IOT, células específicas dos tecidos (cérebro, fígado, baço, linfonodo, medula óssea e sangue) foram avaliadas quanto à quantidade de RNA viral de polaridade positiva e negativa, a fim de quantificar a replicação e definir o tropismo do vírus. Os resultados mostraram que a prevalência de HPgV encontrada nos pacientes HSCT foi maior do que em estudos similares: 42% para as amostra pré- HSCT e 31% para as amostras pós-HSCT; porém, para as amostras SVOC, a prevalência foi menor do que o esperado: 1,2%. A presença de alta carga viral de HPgV em pacientes HSCT foi associada com aumento da taxa de incidência de aGVHD (95% CI 1,05-5,37, p=0,038). Dada a alta prevalência de HPgV na população brasileira, é essencial confirmar esse achado em outras coortes, de modo a determinar se o monitoramento do HPgV pode beneficiar o cuidado a esses pacientes. Nas amostras SVOC e IOT, a medula óssea apresentou maior número de populações celulares com replicação viral, com destaque para as células B progenitoras e para as células dendríticas. Porém, devido às condições da coleta e do processamento das amostras, o tipo celular permissível à infecção não pôde ser identificado com clareza, portanto fazendo-se necessária a coleta de um número maior de amostras SVOC / HPgV (Human Pegivirus), previously known as GBV-C, causes asymptomatic, persistent and high viral load infection. To date, it is the most prevalent RNA virus without associated pathologies in the world, yet in vitro studies have not yet been able to mimic the in vivo replication, consequently many aspects of its biology remaining unclear. Studies in non-humam primatas have shown that the organs responsible for the greatest viral replication are spleen and bone marrow, however the specific cells permissible for infection have not yet been determined. In the 1990s, HPgV gained notoriety by decreasing the pathological effects of HIV and increasing the survival of coinfected HIV/HPgV patients, by decreasing the activation of the immune system. Because of this characteristic, several studies have attempted to associate the presence of viremia with haematological diseases, and one has shown that HPgV appears to be associated with the development of non-Hodgkin\'s lymphoma, but many confounding variables still seems to influence this outcome. Together, the data show that HPgV may influence the maturation and release of bone marrow progenitor cells and therefore it has become crucial to evaluate the impact of HPgV viremia in patients with haematological diseases and to define viral tropism. Therefore, the present study analyzed the dynamics of HPgV viremia exposure in patients submitted to hematopoietic stem cell transplantation (HSCT), in cadavers autopsied by the São Paulo City Death Verification System (SVOC-SP) and in patients submitted to arthroplasty surgery by the Orthopedics and Traumatology Institute (IOT) of the Clinics Hospital of Sao Paulo. The HPgV was identified by real-time qRT-PCR and the viral load quantification was estimated by a standard curve developed for this work. For HSCT patients, the effect of viremia before and after the procedure was evaluated for the main transplant outcomes: acute graft vs host disease (aGVHD), relapse of primary haematological disease and mortality. For SVOC and IOT samples, tissue-specific cells (brain, liver, spleen, lymph node, bone marrow and blood) were evaluated for the amount of viral positive and negative RNA strains, in order to quantify replication and define virus tropism. The results showed that the prevalence of HPgV found in HSCT patients was higher than in similar studies: 42% for pre-HSCT samples and 31% for post-HSCT samples; however, for SVOC samples, the prevalence was lower than expected: 1.2%. The presence of high viral load of HPgV in HSCT patients was associated with an increase in the incidence rate of aGVHD (95% CI 1.05-5.37, p = 0.038). Given the high prevalence of HPgV in the Brazilian population, it is essential to confirm this finding in other cohorts, in order to determine if HPgV monitoring can improve the care of these patients. In the SVOC and IOT samples, the bone marrow presented a higher number of cell populations with viral replication, especially the progenitor B cells and the dendritic cells. However, due to the conditions of specimen collection and processing, the major permissible cell could not be clearly identified, thus making it necessary to collect a larger number of SVOC samples

L’allogreffe de cellules souches hématopoïétiques (CSH) est un traitement efficace des Hémopathies malignes. Cependant, les complications des allogreffes parmi lesquelles les infections virales sont associées parfois à une morbidité et une mortalité importantes. Ces infections surviennent en l’absence de reconstitution immunitaire. Un monitoring régulier de la charge virale des principaux agents infectieux impliqués est réalisé mais amène parfois à la mise en oeuvre abusive de traitements anti-viraux qui ne sont pas dénués de toxicité.Dans ce travail, nous proposons d’associer à ce monitoring un suivi régulier de la reconstitution immunitaire spécifique afin de cibler parmi les patients présentant une réactivation ceux qui nécessitent un traitement curatif de ceux qui pourront maîtriser l’infection par leur système immunitaire. Nous illustrons ce propos avec le virus d’Epstein Barr (EBV) et avons en cours une étude sur l’Adénovirus (ADV).Dans certains cas parfaitement ciblés, les traitements anti-viraux s’avèrent inefficaces. C’est pourquoi dans ce travail, nous présentons la mise au point d’une technique de grade clinique de production de lymphocytes T cytotoxiques anti-ADV (CTL anti-ADV) en condition GMP (Good Manufacturing Practice), grâce au système CliniMACS et au Cytokine Capture System de Miltenyi, afin de proposer une immunothérapie adoptive.Nous décrivons par la suite trois expériences cliniques de traitement compassionnel d’une infection ADV post-allogreffe de CSH. Enfin, nous présentons les résultats préliminaires de la production de CTL bispécifique anti-ADV et CMV / Hematopoietic stem cells Transplantation (HSCT) is a well recognized strategy for treatment of haematological malignancies. However, HSCT complications among which the viral infections a reassociated with high morbidity and mortality. These infections arise in the absence of immune reconstitution. Monitoring of viral reactivations after allogeneic HSCT is necessary, to identify patients at risk of viral infections, but not sufficient, as patients may be abusively treated. In this work we propose to combine viral DNA load assessment with specific immune monitoring to target patients who need to be treated. We report a retrospective study investigating EBV infection and EBV-specific immune recovery using the functional IFN Elispot assay in 40 allogeneic HSCT patients. We initiated a similar study with ADV which is pending. However, although patients are correctly targeted, anti-viral treatment is sometimes not effective. We present a study on the development of a complete clinical grade generation of Human anti-Adenovirus cytotoxic T cells in GMP (Good Manufacturing Practice) conditions, thanks to the system CliniMACS and the Cytokine Capture System, to propose an adoptive immunotherapy to the recipient.We describe afterwards three clinical experiments of treatment of an ADV infection after HSCT.Finally, we present the preliminary results of the anti-ADV and -CMV bi-specific CTL production.

L’allogreffe de cellules souches hématopoïétiques (CSH) est un des seuls traitements curatifs des hémopathies bénignes ou malignes et des déficits immunitaires primitifs. Cependant, les infections notamment virales ainsi que la réaction du greffon contre l’hôte comptent parmi les complications les plus fréquentes des allogreffes associées à une morbidité et une mortalité élevées. Les infections virales surviennent souvent en l’absence de reconstitution immunitaire spécifique dans un contexte d’immunosuppression liée à la GVHD elle-même ou à la prophylaxie ou au traitement de la GVHD. Les traitements médicamenteux anti-viraux préconisés présentent une efficacité inconstante dans ce contexte d’immunodéficience et ne sont pas dénués de toxicité. L’alternative thérapeutique prometteuse est l’immunothérapie adoptive cellulaire notamment celle qui consiste en l’injection de lymphocytes T spécifiques anti-viraux isolés par technique immunomagnétique (VSTs). Cependant, ces lymphocytes T peuvent être la cible des traitements immunosuppresseurs administrés pour la GVHD mais également par eux-mêmes être potentiellement la cause de la survenue ou de la réactivation d’une GVHD. Nous avons montré dans ce travail que l’efficacité des VSTs, qui repose sur leur expansion in vivo lors de la rencontre avec le virus circulant, est principalement permise par les sous-populations lymphocytaires les plus immatures, même si elles ne sont présentes qu’en faible proportion. Nous défendons dans ce travail le fait que l’efficacité des VST ainsi que leur persistance repose prioritairement sur la présence des sous-populations lymphocytaires T les plus immatures et ce quel que soit le degré de compatibilité HLA entre les VSTs et le receveur. De plus, leur sensibilité modérée aux corticoïdes, que nous avons étudiée in vitro, ne justifie pas la modulation de l’immunosuppression lors de l’injection des ADV-VSTs, comme observé in vivo dans le protocole clinique multicentrique de phase I/II que nous avons mené entre 2012 et 2015. En effet, ce protocole clinique ne rapporte aucune GVHD de novo après injection d’ADV-VSTs ; en revanche, la modulation de l’immunosuppression peut potentiellement être incriminée dans la réactivation de GVHD dans les semaines suivant l’injection des ADV-VSTs. La réalisation d’un essai comparatif de phase II permettra de prouver très clairement le rôle des VSTs dans la réactivation de GVHD. / Hematopoietic stem cell transplantation (HSCT) is one of the only curative treatments for benign or malignant hematological diseases and primary immune deficiencies. However, viral infections and graft-versus-host disease (GVHD) are among the most frequent complications after HSCT associated with high morbidity and mortality. Viral infections often occur in the absence of specific immune reconstitution in the context of immunosuppression related to GVHD itself or to the prophylaxis or treatment of GVHD. The recommended anti-viral drug treatments have an inconsistent efficacy in this context of immunodeficiency and are not devoid of toxicity. The promising therapeutic alternative is adoptive immunotherapy, in particular the infusion of specific anti-viral T lymphocytes isolated by immunomagnetic technique (VSTs). However, these T lymphocytes may be targeted by immunosuppressive treatments administered for GVHD, but also may be the cause of the onset or reactivation of GVHD. We have shown in this work that the efficacy of VSTs, which is based on their in vivo expansion when they encounter the circulating virus, is mainly allowed by the most immature lymphocyte subpopulations, even in a small proportion. We argue in this work that the efficacy of VSTs and their persistence is mainly based on the presence of the most immature T lymphocyte subpopulations and this regardless of the degree of HLA compatibility between the VSTs and the recipient. Moreover, their moderate sensitivity to corticosteroids, which we have studied in vitro, does not justify the modulation of immunosuppression at the time of infusion of ADV-VSTs, as observed in vivo in the multicenter phase I / II clinical trial we conducted between 2012 and 2015. Indeed, this clinical trial does not report any de novo GVHD after ADV-VSTs infusion. On the other hand, modulation of immunosuppression may potentially be incriminated in the reactivation of GVHD within weeks of ADV-VST infusion. A Phase II comparative trial will bring the evidence of efficacy and will clearly determine the role of VSTs in the reactivation of GVHD

De par leurs propriétés d’autorenouvellement et de multipotentialité, les cellules souches hématopoïétiques (CSH) présentent un intérêt médical majeur et sont au cœur de nombreuses thérapies génique et cellulaire. Cependant, leur utilisation en clinique est encore limitée par leur rareté, leur hétérogénéité fonctionnelle, et leur perte durant l’étape de manipulation in vitro qui précède la transplantation.Dans ce contexte, nous avons mis au point une stratégie de marquage cellulaire par des codes-barres moléculaires afin d’évaluer simultanément et quantitativement les capacités de repopulation des CSH in vivo. Cette stratégie nous a permis de cribler 1200 composés chimiques afin d’identifier des molécules capables (1) d’améliorer l’efficacité de transduction des CSH par un vecteur lentiviral, (2) de maintenir voire d’expandre, lors de l’étape de culture in vitro, les CSH capables de repopulation à long terme. Plusieurs molécules candidates sont en cours de validation.Par ailleurs, grâce au marquage par des codes-barres, nous avons révélé l’hétérogénéité des CSH en comparant leur capacité d’autorenouvellement, la taille de la descendance qu’elles génèrent et leur capacité de différenciation. Notre technique nous a permis non seulement de confirmer l’existence de CSH biaisées vers le lignage myéloïde ou vers le lignage lymphoïde, mais aussi de quantifier leur fréquence.L’application de cette stratégie à un autre système cellulaire nous a permis d’étudier l’influence de facteurs environnementaux et génétiques sur la croissance cellulaire de populations exprimant différentiellement l’-synucléine, une protéine impliquée dans la maladie de Parkinson et certains cancers. / Considerable hope is placed on the use of hematopoietic stem cells (HSC) in engineered gene- and cell- based therapy protocols. Their clinical utilization is, however to some extent, limited by their scarce representation, their heterogeneity and their loss during the ex vivo manipulation procedure. Within this context, we developed a barcode tagging strategy to simultaneously evaluate in vivo the repopulating capacity of HSC cultured in vitro. Barcode deconvolution demonstrated that our strategy constitute a powerful tool for tracking HSC quantitatively even using several dozens of different conditions. Using this strategy, we screened 1200 chemical compounds in order to identify molecules acting (1) on HSC transduction efficiency using a lentiviral vector, (2) on maintenance or even amplification during the in vitro culture step of long-term repopulating HSC. Several candidate molecules are currently under validation. In addition, using this barcoding strategy, we reveal heterogeneous behaviour of HSC examining their proliferation capacity, self-renewal ability and their differentiation lineage option. Our technique allowed us not only to confirm the occurrence of myeloid- and lymphoid-biased HSC, but also to quantify their frequency.We apply this strategy to another cell system to address the effect of different genetic and environmental factors on proliferation of cells expressing -synuclein, a protein involved in Parkinson disease and some cancers.

L’hématopoïèse est un processus finement régulé pour permettre sa pérennité et son adaptation aux contraintes physiologiques et pathologiques. Ce potentiel repose en grande partie sur les capacités de quiescence, auto-renouvellement, division asymétrique et multipotence des cellules souches hématopoïétiques (CSH). Les CSH et progéniteurs hématopoïétiques (CSPH) sont principalement régulés de façon extrinsèque au sein des niches hématopoïétiques médullaires et cette régulation fait intervenir, des contacts intercellulaires et des facteurs diffusibles. Le phénotype « side-population » (SP), secondaire à l’efflux actif d’un colorant fluorescent (Hoechst 33342) par des pompes de type multidrugresistance, est une caractéristique des cellules souches de la plupart des tissus. Au sein de l’hématopoïèse, le phénotype SP est un excellent moyen pour identifier les CSH murines et est associé à leur quiescence et à leur adhésion à la niche endostéale, mais sa valeur comme marqueur des CSH est plus discutée chez l’homme. Les cellules SP, de par leur nature, sont également étudiées en oncologie, et sont associées aux cellules tumorales les plus résistantes et les plus tumorogènes. La compréhension des mécanismes régulant la fonctionnalité SP devrait permettre d’ouvrir des pistes en physiologie quand à la compréhension de la régulation des CSPH par les niches mésenchymateuses et en pathologie pour cibler les mécanismes de chimiorésistance.Dans ce travail nous montrons pour la première fois chez l’homme que l’acquisition du phénotype SP est un phénomène dynamique et versatile sous le contrôle du stroma médullaire. Le stroma médullaire est en effet capable de maintenir le phénotype SP de CSPH médullaires et d’induire le phénotype SP de CSPH circulants. L’acquisition du phénotype SP par les cellules circulantes nécessite à la fois un « nichage » au sein du stroma et des facteurs diffusibles. Les cellules circulantes capables d’acquérir le phénotype SP contiennent des CSPH au regard de (i) leur expression du CD34, (ii) leur richesse en cellules quiescentes, (iii) leur capacité clonogénique et proliférative en cultures secondaires, (iv) leur expression des gènes de « nichage » et de « souchitude », (v) leur capacité de migration en réponse à un gradient de CXCL12, (vi) leur activité LT-SRC in vivo. De plus nous avons mis en évidence, au sein de ces CSPH SP+CD34+ révélés par le stroma médullaire, une sous-population CD44-/faible qui pourrait contenir les cellules plus immatures en raison de sa quiescence et de l’intensité de son efflux du Hoechst 33342. Les études mécanistiques montrent que l’acquisition du phénotype SP par les cellules circulantes est sous la dépendance de l’intégrine VLA-4 et du CD44. La transduction du signal implique des protéines G et la famille des Src-kinases. Nous montrons également que le stroma médullaire peut induire/maintenir/amplifier la fonctionnalité SP de blastes circulants de leucémie aigüe myéloblastique de façon ß1-intégrine dépendante et que cette fonctionnalité est associée à une capacité d’efflux de Mitoxantrone. Ce mécanisme de modulation de l’activité d’ABC-transporteurs par l’adhésion au stroma correspond à un mécanisme encore jamais décrit de CAM-DR. / Hematopoiesis is a finely tuned process to allow its long-term efficiency and its adaptation to various physiological and pathological stresses. Hematopoietic stem cell (HSC) is the keystone of hematopoiesis through its multipotency, quiescence, asymmetrical division and self-renewing properties. HSC bone marrow (BM) niches mainly regulate hematopoietic stem and progenitor cells (HSPC) through intercellular contacts and diffusible factors. Side-population (SP) cells are characterized by their capability to actively efflux Hoechst 33342 dye through multidrug resistance-like pumps. SP phenotype is a characteristic of stem cells in many tissues and especially, it is a stringent criterion to purify murine HSCs. In mice, this phenotype has been demonstrated to be related to quiescence and resistance to drugs/environmental stresses and to be controlled by endosteal niche adhesion. SP cells are also studied in oncology and are associated to chemo-resistance and tumor initiating capacity. At steady state, SP cells are mainly present in the BM and are mostly absent from the circulation except in stress conditions, raising the hypothesis of the versatility of the SP functionality. Therefore, studying SP phenotype regulation is of importance to understand how BM niches regulate HSPC and how to interfere with cancer cells chemo-resistance.In this work, we demonstrate for the first time and in human that SP phenotype acquisition is a dynamic phenomenon under control of stromal BM cells. Stromal cells from healthy donors maintain SP phenotype of BM HSPC and promote SP phenotype acquisition in circulating ones. SP phenotype promotion depends of stroma nesting and of diffusible factors secretion. This stroma-induced circulating SP cell fraction contains HSPC, as ascertained by (i) CD34 expression, (ii) proportion of cells in G0, (iii) clonogenic and proliferative potential, (iv) nesting and “stemness” gene expression, (v) CXCL12-related migration capability and (vi) LT-SRC activity. Moreover, we describe an SP+CD34+CD44-/low sub-population that could contain most immature HSPCs with regards to their quiescence and Hoechst efflux intensity. Mechanistic studies show that the stoma-mediated SP promoting effect is VLA-4/4ß1-integrin and CD44 dependent, and implicate G-protein and Src-kinase pathways. We also demonstrate that BM stroma from healthy donors can induce/maintain/amplify in a ß1-integrin dependent manner an SP sub-population with mitoxantrone efflux capability in blast cells from acute myeloid leukemia. The existence of a similar mechanism in circulating leukemic blasts suggests the possibility to interfere with the chemo-resistant phenotype of blast cells through integrin/CD44 axis blockade.