Indiana University-Purdue University Indianapolis (IUPUI) / Hematopoietic stem (HSC) and progenitor (HSPC) cells reside in low oxygen (~1- 4%, low O2) bone marrow niches which provide critical signals for maintenance, selfrenewal, and differentiation. Exposure of HSC/HSPCs to air (~21%) for less than 10 minutes irreversibly diminishes numbers of phenotypic and functional stem cells, a phenomenon termed extra physiologic oxygen stress/shock. Yet, most studies harvest and analyze HSC/HSPCs in air and often in fixed cells, leaving endogenous signaling mechanisms unidentified. To better understand the endogenous mechanisms regulating HSCs and HSPCs, we generated the first low O2 landscape of phenotypic/functional/signaling alterations in live, low O2 harvested/sorted HSC/HSPCs utilizing novel technology. HSC (LSKCD150+) and HSC/HSPC (LSK) expression, frequency, and stem cell maintenance retention were enhanced in low O2 relative to historic data and our air data. Transcriptomics uncovered low O2 differential pathway regulation of HSC/HSPCs and HSCs with analysis identifying low O2 enrichment of genes/pathways including Ca2+ ion binding, altered sodium hydrogen (Na+/H+) activity, viral entry, and transmembrane receptor activity in both HSCs and HSPCs. In exploring the low O2 landscape, we investigated differential low O2 regulation of Ca2+ and SARS-CoV-2 related pathways/mechanisms in HSCs and HSPCs. Differential Ca2+ regulation was observed in our transcriptional/proteomic analysis corroborated by phenotypic/functional data demonstrating increases in low O2 of cytosolic and mitochondrial Ca2+ flux, ABC Transporter (ABCG2) and Na+/H+ (NHE1) expression, discovery of a novel low O2 Ca2+ high HSPC population that enhances HSC maintenance compared to Ca2+ low populations and blunting of this population and subsequent enhanced stem cell maintenance upon NHE1 inhibition (Cariporide). Multi-omics analyses also identified enhancements in COVID19-related pathways in low O2 that corresponded with enhanced expression of SARS-CoV-2 receptors/co-receptors, SARS-CoV-2 spike protein (SP) binding, and expansion of SP-bound HSC/HSPCs in low O2 compared to air, as well as enhanced stem cell maintenance of SP-bound, versus unbound, cells in low O2. Together, these data presented show low O2 harvest/retention of HSC/HSPCs enhances stem cell maintenance, which could be utilized to improve HSC expansion, and leads to differential pathway/signaling regulation of various biological pathways in HSC/HSPCs including Ca2+ and SARS-CoV-2/viral infection that results in phenotypic and functional consequences. / 2024-11-01

Embryonic stem (ES) cells possess the remarkable ability to form cells and tissues from all three germ layers, a characteristic known as pluripotency. In particular, the generation of ES cell-derived hematopoietic cells could serve as an alternate source of hematopoietic stem cells for transplantation in place of bone marrow cells, which are limited by donor availability and high immunogenicity. The advantages of ES cell-derived hematopoietic cells over bone marrow cells include a greater proliferative capacity, which alleviates the problems of donor shortage, and low level expression of MHC antigens, which suggests immune privilege. However, it is unclear whether the immune system is capable of recognizing and rejecting ES cell-derived hematopoietic cells following transplantation. The observation that ES cell-derivatives express low levels of MHC class I, the predominant inhibitory ligand for NK cells, led us to hypothesize that ES cell-derived hematopoietic progenitor cells (HPC) are susceptible to NK cell-mediated killing. To test this hypothesis, we first generated HPCs from murine ES cells ectopically expressing HOXB4, a homeobox transcription factor that confers hematopoietic self-renewal, and confirmed that HPCs expressed low levels of MHC class I antigens. To specifically investigate the role of NK cells in regulating the in vivo engraftment of HPCs, we transplanted NK-replete Rag2-/- or NK-deficient Rag2-/-γc-/- mice with HPCs. We observed permanent HPC engraftment in Rag2-/-γc-/- mice; however, HPC engraftment was significantly reduced in Rag2-/- mice and was eventually eliminated over time. Bone marrow harvested from these animals showed that HPC-derived Lin-c-kit+ and Lin-Sca-1+ progenitor cells, critical progenitor cells for long-term hematopoietic engraftment, were deleted in Rag2-/- but not in Rag2-/-γc-/- mice. Next, we focused on the mechanism of NK cell activation by HPCs. Increased expression of the cytotoxic proteins Granzyme B and Perforin in the NK cells of HPC-transplanted Rag2-/- mice confirmed in vivo NK cell activation. Phenotypic analysis of HPCs revealed high level expression of H60, a ligand of the NK activating receptor NKG2D, and neutralization of H60 rescued HPCs from NK cell-mediated killing. Altogether, our results demonstrate that NK cells are a major barrier to the successful engraftment of ES cell-derived hematopoietic cells, underlining an important role of the innate immune system in regulating the long-term engraftment of ES cell derivatives.

Indiana University-Purdue University Indianapolis (IUPUI) / Cell cycle checkpoints guarantee movement through the cell cycle in an appropriate manner. The spindle assembly checkpoint (SAC) ensures the proper segregation of chromosomes into daughter cells during mitosis. Mitotic arrest deficiency 2 (Mad2), a member of the mitotic checkpoint proteins, appears to be crucial for generating the wait anaphase signal to prevent onset of anaphase. We first studied the SAC in hematopoietic stem cells (HSC) to ensure that it was functional. Our previous studies found that prolonged SAC activation was uncoupled from apoptosis initiation in mouse and human embryonic stem cells (ESC). We found that upon treatment with a microtubule-destabilizing agent, HSC arrested in M-phase and subsequently initiated apoptosis. Thus unlike ESC, HSC exhibit coupling of prolonged SAC activation with apoptosis. We studied the effects of Mad2+/- on in vivo recovery of bone marrow HPC from cytotoxic effects and also effects of cytostatic agents on HPC growth in vitro using Mad2-haploinsufficient (Mad2+/-) mice. We found that Mad2+/- HPCs were protected from the cytotoxic effects of cytarabine (Ara-C), a cycle specific agent, consistent with Mad2+/- HPCs being in a slow or non-cycling state. Mad2 haploinsufficiency did not affect recovery of functional HPC after treatment with cyclophosphamide or high sub-lethal dose irradiation, both non-cycle specific agents. There were no differences in immunophenotype defined HSCs in Mad2+/- and Mad2+/+ mice, data confirmed by functional HSC competitive repopulation assays. To better understand the role of Mad2 in HPC, E3330, a cytostatic agent, was used to assess the redox function of Ape1/Ref-1, and colony formation in vitro was examined under normoxic and lowered O2 tension. Mad2+/- HPCs were less responsive to E3330 than Mad2+/+ HPCs, and E3330 was more effective under lowered O2 tension. Mad2+/- HPCs did not exhibit enhanced growth in lowered oxygen tension, in contrast to Mad2+/+ HPCs. Our studies have unexpectedly found that Mad2 haploinsufficiency is protective from the cytotoxic effects of a cycle specific DNA synthesis agent in vivo, and Ape1/Ref-1 inhibitor in vitro.

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

Orientador: Márjorie de Assis Golim / Resumo: O sistema de qualidade é de suma importância em laboratórios clínicos para avaliação de processos analíticos de maneira que os resultados liberados sejam verdadeiros. Para a metodologia de imunofenotipagem celular por citometria de fluxo as amostras devem ser frescas e os exames realizados preferencialmente dentro de 48 horas. É relevante utilizar amostras de controle de qualidade internos (CQI) padronizadas, de modo que possam ser repetidas rotineiramente, como referencial de qualidade. No Brasil, poucos serviços comercializam amostras preservadas para uso como CQI. Deste modo, a padronização in house com validação de processo para obtenção de amostras que possam ser utilizadas para esta finalidade é relevante. O objetivo deste trabalho foi desenvolver controle de qualidade interno para as rotinas de quantificação de células progenitoras hematopoéticas (CPH), utilizando solução preservante e avaliar a reprodutibilidade e estabilidade ao longo do tempo. Foram preparadas soluções preservantes contendo diferentes concentrações de anticoagulantes e fixadores, e destas, foi selecionada uma composição, originalmente padronizada neste estudo. Foram utilizados 5mL de sangue periférico, sendo este acrescido da solução a ser testada. Imediatamente, realizou-se a quantificação das populações de CPH em tubo Trucount®, usando anti-CD45, anti-CD34 e 7-AAD, conforme indicado pelo fabricante. A leitura foi realizada em citômetro de fluxo modelo FACSCalibur®-BD, para obtenção dos valores abs... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The quality system is of paramount importance in clinical laboratories for evaluating analytical processes in order to consider true the released results. The samples must be performed fresh preferably within 48 hours for the cell immunophenotyping methodology by flow cytometry. It is relevant to use standardized internal quality control (IQC) samples, thus they could be repeated routinely, as a quality benchmark. In Brazil, only a few services commercialize preserved samples for use as IQC. Therefore, it is relevant to use in-house standardization with process validation to obtain samples that can be used for this purpose. The objective of this work was to develop an IQC for a daily routine quantification of hematopoietic stem cells (HSCs) by using a preservative solution and to evaluate the reproducibility and stability over time. Preservative solutions containing different concentrations of anticoagulants and fixatives were prepared, and from these, a composition was selected, which was previously originally standardized in this study. 5mL of peripheral blood were used, which was added to the solution to be tested. The HSCs populations were immediately quantified in a Trucount® tube, using anti-CD45, anti-CD34 and 7-AAD, as indicated by the manufacturer. The reading was performed in a flow cytometer model FACSCalibur®-BD in order to obtain the absolute values of HSCs on day zero, 7, 21, 35 and 49. During this period, the samples were kept refrigerated (2 to 8ºC). The value... (Complete abstract click electronic access below) / Mestre

Introdução. As células progenitoras hematopoéticas (CPH) têm sido utilizadas no tratamento de algumas doenças malignas hematológicas e de desordens hematopoéticas. Dentre estas células estão as CPHs provenientes de cordão umbilical e placentário (SCUP). Para coleta e armazenamento destas células foram criados os Bancos de Sangue de Cordão Umbilical e Placentário (BSCUP). No Brasil existe a rede BrasilCord e o BSCUP do Hemocentro de Ribeirão Preto é um dos que compõem esta rede. Objetivo. Avaliar a viabilidade financeira de um banco de sangue de cordão umbilical e placentário público comparando os custos de seus procedimentos com os valores ressarcidos definidos pela tabela SUS. Metodologia: Este estudo utilizou a metodologia de Custeio Baseado em Atividades (Activity-Based Costing - ABC), que procura reduzir as distorções provocadas pelo rateio arbitrário dos custos indiretos utilizando direcionadores de custos. Foram avaliados os custos diretos e indiretos da coleta, transporte, processamento, testagem e criopreservação de CPH proveniente de SCUP no primeiro semestre de 2015. Para os custos indiretos foram definidos os direcionadores de custo. Resultados Os resultados mostram que o BSCUP do Hemocentro de Ribeirão Preto foi deficitário no primeiro semestre de 2015. O déficit apurado por unidade foi de R$1.155,69 para o processamento semiautomatizado e R$1.703,78 para o processamento automatizado. O déficit total no período foi de R$ 100.376,50 quando 50 unidades foram processadas utilizando o método semiautomatizado e 25 pelo método automatizado. Conclusão. O valor ressarcido pelo SUS não cobre os gastos do BSCUP do Hemocentro de Ribeirão Preto. Isso pode ser atribuído a várias causas como: sistemática de pagamento pelo SUS apenas pelo produto criopreservado, elevados índices de rejeição das doadoras na maternidade e de descarte das unidades coletadas, embora este último seja menor que o descrito na literatura, e o custo do armazenamento em longo prazo. Os custos do BSCUP são menores que os descritos na literatura e poderiam ser reduzidos com melhorias de processos de gestão e aumento do número de unidades criopreservadas, bem como, por meio de descentralização de coletas para processamento centralizado. / Introduction. Hematopoietic progenitor cells (HPC) are being used in some hematologic malignancies and hematopoietic disorders treatment. Among these cells are the HPCs from umbilical blood cord (UCB). Umbilical cord blood banks were created to collect and store these cells. In Brazil there is a net called Brasilcord and the umbilical cord blood bank (UCBB)of the Hemocentro de Ribeirão Preto belongs to this net. Objective. To evaluate the financial viability of a public umbilical cord blood bank and compare the costs from its procedure with the values reimbursed defined by the SUS table. Methodology.This study used the ActivityBased Costing, which tries to reduce distortions caused by arbitrary apportionment of indirect costs using cost drivers. Direct and indirect costs of collection, transport, processing, testing and cryopreservation of HPCs from umbilical cord blood were evaluated. Cost drivers were defined for indirect costs. Results. The results showed that there was a deficit in the UCBB of the Hemocentro de Ribeirão Preto in the first semester of 2015. The deficit was R$1.155,69 when the unit was processed by the semi-automated method and R$1.703,78 when it was processed by an automated method. The total deficit in the period was R$100.376,50 as 50 units were processed by a semi-automated method and 25 by an automated method. Conclusion. The amount reimbursed by SUS does not cover the UCBB of Hemocentro de Ribeirão Preto expenses. This can be attributed to several causes such as: systematic of reimbursement used by SUS that only cryopreserved units are payed, high percentage of deferral in the maternity and of discard of collected units, although this latter is smaller than that described in the literature and the cost of storage of the units for long periods. The costs of UCBB are lower than that described in the literature and could be reduced with improvements in managing process and increase the number of cryopreserved units as well as decentralization of collections for centralizes processing.

Introdução. As células progenitoras hematopoéticas (CPH) têm sido utilizadas no tratamento de algumas doenças malignas hematológicas e de desordens hematopoéticas. Dentre estas células estão as CPHs provenientes de cordão umbilical e placentário (SCUP). Para coleta e armazenamento destas células foram criados os Bancos de Sangue de Cordão Umbilical e Placentário (BSCUP). No Brasil existe a rede BrasilCord e o BSCUP do Hemocentro de Ribeirão Preto é um dos que compõem esta rede. Objetivo. Avaliar a viabilidade financeira de um banco de sangue de cordão umbilical e placentário público comparando os custos de seus procedimentos com os valores ressarcidos definidos pela tabela SUS. Metodologia: Este estudo utilizou a metodologia de Custeio Baseado em Atividades (Activity-Based Costing - ABC), que procura reduzir as distorções provocadas pelo rateio arbitrário dos custos indiretos utilizando direcionadores de custos. Foram avaliados os custos diretos e indiretos da coleta, transporte, processamento, testagem e criopreservação de CPH proveniente de SCUP no primeiro semestre de 2015. Para os custos indiretos foram definidos os direcionadores de custo. Resultados Os resultados mostram que o BSCUP do Hemocentro de Ribeirão Preto foi deficitário no primeiro semestre de 2015. O déficit apurado por unidade foi de R$1.155,69 para o processamento semiautomatizado e R$1.703,78 para o processamento automatizado. O déficit total no período foi de R$ 100.376,50 quando 50 unidades foram processadas utilizando o método semiautomatizado e 25 pelo método automatizado. Conclusão. O valor ressarcido pelo SUS não cobre os gastos do BSCUP do Hemocentro de Ribeirão Preto. Isso pode ser atribuído a várias causas como: sistemática de pagamento pelo SUS apenas pelo produto criopreservado, elevados índices de rejeição das doadoras na maternidade e de descarte das unidades coletadas, embora este último seja menor que o descrito na literatura, e o custo do armazenamento em longo prazo. Os custos do BSCUP são menores que os descritos na literatura e poderiam ser reduzidos com melhorias de processos de gestão e aumento do número de unidades criopreservadas, bem como, por meio de descentralização de coletas para processamento centralizado. / Introduction. Hematopoietic progenitor cells (HPC) are being used in some hematologic malignancies and hematopoietic disorders treatment. Among these cells are the HPCs from umbilical blood cord (UCB). Umbilical cord blood banks were created to collect and store these cells. In Brazil there is a net called Brasilcord and the umbilical cord blood bank (UCBB)of the Hemocentro de Ribeirão Preto belongs to this net. Objective. To evaluate the financial viability of a public umbilical cord blood bank and compare the costs from its procedure with the values reimbursed defined by the SUS table. Methodology.This study used the ActivityBased Costing, which tries to reduce distortions caused by arbitrary apportionment of indirect costs using cost drivers. Direct and indirect costs of collection, transport, processing, testing and cryopreservation of HPCs from umbilical cord blood were evaluated. Cost drivers were defined for indirect costs. Results. The results showed that there was a deficit in the UCBB of the Hemocentro de Ribeirão Preto in the first semester of 2015. The deficit was R$1.155,69 when the unit was processed by the semi-automated method and R$1.703,78 when it was processed by an automated method. The total deficit in the period was R$100.376,50 as 50 units were processed by a semi-automated method and 25 by an automated method. Conclusion. The amount reimbursed by SUS does not cover the UCBB of Hemocentro de Ribeirão Preto expenses. This can be attributed to several causes such as: systematic of reimbursement used by SUS that only cryopreserved units are payed, high percentage of deferral in the maternity and of discard of collected units, although this latter is smaller than that described in the literature and the cost of storage of the units for long periods. The costs of UCBB are lower than that described in the literature and could be reduced with improvements in managing process and increase the number of cryopreserved units as well as decentralization of collections for centralizes processing.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13159号 / 論医博第2146号 / 新制||医||1029(附属図書館) / (主査)教授 河本 宏, 教授 前川 平, 教授 髙折 晃史 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

L'atteinte vasculaire cérébrale est une complication grave et fréquente chez les enfants drépanocytaires, car elle impacte leur pronostic, en termes de morbidité (handicap) et de mortalité. L’accélération des vitesses mesurées par le doppler transcrânien (DTC) est prédictive du risque d'infarctus cérébral et implique une modification de la prise en charge thérapeutique. Chez l’enfant drépanocytaire, l'infarctus cérébral est d'origine multifactorielle, lié à la vasculopathie cérébrale sténotique ainsi qu'à une hypercoagulabilité et une activation cellulaire. Nous avons étudié de manière prospective l'association de marqueurs biologiques au DTC chez 108 enfants porteurs de syndrome drépanocytaire majeur et recherché des éléments prédictifs d'événement vasculaire périphérique ou cérébral. Nous avons ainsi réalisé une analyse approfondie de la fonction endothéliale, de l’activation de l’hémostase primaire et de la coagulation, de l'activation cellulaire et de la mécanique artérielle. L’atteinte vasculaire cérébrale a été estimée en considérant les données du DTC comme une variable continue plutôt que catégorielle. Le principal résultat est le rôle prédictif du nombre des cellules souches hématopoïétiques CD34+ pour la survenue d'événements cliniques vasculaires. Nous avons également mis en évidence un profil particulier de coagulation chez les enfants drépanocytaires présentant des céphalées récurrentes ou des accès migraineux. Ceci supporte l'hypothèse que les céphalées chez l'enfant drépanocytaire, et notamment celles répondant aux critères de la migraine, peuvent être le reflet d'événements ischémiques cérébraux ultra-transitoires. Elles représentent donc peut-être un indicateur indirect de risque ischémique cérébral. Nous avons par ailleurs montré que le risque hémorragique cérébral chez les enfants drépanocytaires restait proportionnellement stable par rapport au risque ischémique, malgré l'utilisation en routine de stratégies de prévention du risque ischémique. L'observation de lésions sténotiques et d'anévrismes permet de supposer que ces atteintes vasculaires cérébrales procèdent de mécanismes physiopathologiques communs. L'amélioration de la compréhension des mécanismes physiopathologiques des complications neurovasculaires et la mise en évidence de facteurs prédictifs d'événements cliniques est un pas supplémentaire vers l'amélioration de la sensibilité diagnostique de la vasculopathie cérébrale drépanocytaire, de la compréhension des mécanismes des accidents vasculaires cérébraux de ces enfants et probablement de leur pronostic neurologique en permettant une prise en charge thérapeutique adaptée plus précoce. / Cerbrovascular involvement is frequent in children with sickle-cell disease (SCD). It is severe in terms of morbidity (handicap) and mortality. Accelerated intracranial arterial blood flow velocity measured by transcranial doppler (TCD) is predictive for stroke occurrence and leads to therapeutic modifications. In SCD children, ischemic stroke results from stenotic cerebral vasculopathy associated with hypercoagulability, and cell activation. We prospectively addressed associations between biological markers and TCD velocity in 108 children with sickle-cell anemia (HbSS or HbSβ°) and looked for predictive factors for vascular peripheral or cerebral events. We performed extensive work-up of endothelial function, coagulation activation, cell activation, and arterial wall mechanics. Cerebral vasculopathy was defined using TCD velocity (continuous data) rather than the classical category classification. The main result is the demonstration of the role of hematopoietic stem cell CD34+ for the prediction of clinical vascular event occurrence. We also demonstrated an imbalanced coagulation profile in SCD children with recurrent cephalalgia or migraine. This finding supports the hypothesis that recurrent cephalalgia, especially migraine, could be a symptom of ultra-transient ischemic cerebrovascular events in SCD children. Therefore, this symptom may also indicate increased cerebrovascular ischemic risk. We demonstrated that the ratio cerebral hemorrhagic risk / cerebral ischemic risk in SCD children remains stable, despite the routine use of strategies aiming at reducing ischemic stroke risk. The concurrent observation of intracranial arterial stenotic lesions and aneurysm suggests common pathophyiological mechanisms. Improving pathophysiological understanding of cerebrovascular complications and demonstrating predictive risk factors for clinical events may help clinicians to improve early diagnosis of SCD-associated cerebral vasculopathy, to better understand stroke mechanism in this population, and probably to improve neurological outcome with earlier and more adapted management

Indiana University-Purdue University Indianapolis (IUPUI) / Adult hematopoietic stem cells (HSC) are routinely used to reconstitute hematopoiesis after myeloablation; however, transplantation efficacy and multilineage reconstitution can be limited by inadequate HSC number, or poor homing, engraftment or self-renewal. We have demonstrated that mouse and human HSC express prostaglandin E2 (PGE2) receptors, and that short-term ex vivo exposure of HSC to PGE2 enhances their homing, survival and proliferation, resulting in increased long-term repopulating cell and competitive repopulating unit (CRU) frequency. HSC pulsed with PGE2 are more competitive, as determined by head-to-head comparison in a competitive transplantation model. Enhanced HSC frequency and competitive advantage is stable and maintained upon multiple serial transplantations, with full multi-lineage reconstitution. PGE2 increases HSC CXCR4 mRNA and surface expression and enhances their migration to SDF-1α in vitro and homing to bone marrow in vivo and stimulates HSC entry into and progression through cell cycle. In addition, PGE2 enhances HSC survival, associated with an increase in Survivin mRNA and protein expression and reduction in intracellular active caspase-3. While PGE2 pulse of HSC promotes HSC self-renewal, blockade of PGE2 biosynthesis with non-steroidal anti-inflammatory drugs (NSAIDs) results in expansion of bone marrow hematopoietic progenitor cells (HPC). We co-administered NSAIDs along with the mobilizing agent granulocyte-colony stimulating factor (G-CSF) and evaluations of limiting dilution transplants, assays monitoring neutrophil and platelet recoveries, and secondary transplantations, clearly indicate that NSAIDs facilitate mobilization of a hematopoietic graft with superior functional activity compared to the graft mobilized by G-CSF alone. Enhanced mobilization has also been confirmed in baboons mobilized with G-CSF and a NSAID. Increases in mobilization are the result of a reduction of signaling through the PGE2 receptor EP4, which results in marrow expansion and reduction in the osteoblastic HSC niche. We also identify a new role for cannabinoids, an eicosanoid with opposing functions to PGE2, in hematopoietic mobilization. Additionally, we demonstrate increased survival in lethally irradiated mice treated with PGE2, NSAIDs, or the hypoxia mimetic cobalt chloride. Our results define novel mechanisms of action whereby eicosanoids regulate HSC and HPC function, and characterize novel translational strategies for hematopoietic therapies.