<p>Megakaryocyte cultures are a strong tool for the in vitro investigation of platelet production in platelet disorders. Peripheral blood derived hematopoietic progenitor cells (PB-HPCs) are the most accessible source of HPCs with high potential to produce mature megakaryocytes in vitro; however, they are present in low numbers making peripheral blood an inefficient source. Additionally, a megakaryocyte culture with an optimized thrombopoietin (TPO) concentration is required which can reliably allow the investigation of suppressive effects of antibodies/plasma from immune thrombocytopenia (ITP) patients. In this study, we developed a megakaryocyte culture with the utilization of human PB-HPCs in an efficient fashion resulting in the production of high purity megakaryocytes in a TPO-dependent manner.</p> <p>The mononuclear fraction was collected from 180 mL of peripheral whole blood and CD34+ cells were isolated by a positive selection yielding the average of 5.5 x 105 ± 2.5 x 105 CD34+ cells (n = 18). Using 96-well tissue-culture plates and seeding 10,000 CD34+ cells/well, the average of 13 experiments in triplicate can be set up utilizing isolated CD34+ in an efficient manner. Capitalizing on a TPO dose-dependent megakaryocyte production experiment, 20 ng/mL was established as the TPO concentration which resulted in the production of mature megakaryocytes without reaching the plateau in megakaryopoiesis response. On day 11 of culture, the expression of megakaryocytic lineage (CD41/61+) and maturation (CD41/61+CD42+) markers peaked at 90.65% and 76.10%. In conclusion, this culture system has broad application for investigation of platelet disorders and drug discovery which can be accessible to all researchers.</p> / Master of Science (MSc)

Il est désormais admis que le caractère invasif d'une tumeur est lié, non seulement, au génotype des cellules cancéreuses, mais aussi à leurs interactions avec le microenvironnement tumoral (MT). Au sein du MT, une déstabilisation de la matrice stromale favorise la progression tumorale et la dissémination métastatique. Le remaniement de la matrice extracellulaire est souvent piloté par des enzymes protéolytiques. En revanche, les effets de l'inhibition de la formation de cette matrice sont peu étudiés. C’est dans cette optique que nous nous sommes intéressés à la protéine C (PC) et son récepteur endothélial (EPCR) et à leur rôle dans la tumorigenèse des leucémies et des cancers solides.L’EPCR est exprimé par un grand nombre de lignées cellulaires cancéreuses. Il est aussi détecté dans le compartiment tumoral chez des patients atteints de pathologie tumorale. Son gène est hautement conservé. Il possède cependant plusieurs polymorphismes. Un de ces SNPs (single nucleotide polymorphism) - 6936A/G - se traduit par la libération d'une forme soluble circulante de l'EPCR (EPCRs) résultant de la protéolyse de la forme membranaire. Chez des patients leucémiques, une fréquence élevée du SNP 6936A/G est observée et associée à la survenue de thrombose. D'autre part, l’EPCR est détecté in situ dans la majorité des biopsies tumorales testées et sécrété en grande quantité dans les ascites. La fixation de la PC sur l’EPCR et son activation augmentent la survie et le potentiel migratoire des cellules cancéreuses. Aussi, la PCA est capable de moduler, par communication paracrine, la sécrétion de plusieurs interleukines et cytokines. Ainsi, la stimulation de cellules du cancer de l'ovaire par la PCA induit la synthèse d'une thrombopoéïtine ovarienne fonctionnelle. Cette cytokine étant régulatrice de la production de plaquettes, la PCA semble être de nouveau à l'interface entre troubles de l'hémostase et pathologie cancéreuse. L’élucidation du rôle complexe de la PCA et de son récepteur endothélial dans la carcinogenèse permettrait non seulement de dégager de nouvelles approches thérapeutiques, mais aussi de prévenir le risque de thrombose associée au cancer et d’en réduire la morbidité. / It is now recognized that the invasiveness of tumor cells is not only related to the genotype of these cells but also to their interaction with tumor microenvironment (TM). Within the TM, stromal matrix destabilization promotes tumor progression and metastatic dissemination. The extracellular matrix remodeling is often driven by proteolytic enzymes. However, few studies have investigated the effects of an impairment of the matrix formation. Given these facts and circumstances, we were interested in protein C (PC) and its endothelial receptor (EPCR), as well as in their role in tumorigenesis in leukemia and solid cancers. EPCR is expressed by a wide range of cancer cell lines. It is also detected within the tumor compartment in patients with malignant diseases. EPCR gene is highly conserved but nevertheless contains polymorphisms. One of these SNPs (single nucleotide polymorphism) - 6936A/G – reflects – in the release of a soluble circulating form (EPCRs) resulting from the proteolysis of membrane-associated form. In leukemic patients a high incidence of 6936A/G SNP is observed and associated with thrombosis events. Moreover, EPCR is detected in the majority of tumor biopsies and is abundantly secreted in ascitic fluid. The PC attachment to EPCR and its activation promotes cell survival and migratory potential of tumor cells. Also, APC is able to modulate, by a paracrine manner, interleukins and cytokines secretion. Thus, ovarian cancer cells stimulation by APC induces the synthesis of a functional ovarian thrombopoietin. As this cytokine has a regulatory effect on platelet production, APC may be once again at the interface between hemostasis disorders and coagulation. The elucidation of the intricate role of APC and its endothelial receptor could permit not only to identify new therapeutic approaches but also to prevent cancer-associated thrombosis risk and to decrease morbidity in cancer patients.

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

The overall aim of this thesis is to improve knowledge about the prevention of infectious and bleeding complications in patients with hematological malignancies, primarily in those with chronic lymphocytic leukemia (CLL) and myelodysplatic syndrome (MDS). Hypogammaglobulinemia, impaired production of immunoglobulins (Ig), is an established risk factor for infection, but the impact of IgG pure subclass deficiency (IgG subclass deficiency with adequate production of IgG, IgA, and IgM) has been debated. In a retrospective single institution study, we concluded that pure IgG subclass deficiency in CLL patients is rare and is not associated with an increased risk of infection. Hence, routine analysis of IgG subclasses in patients with CLL is not warranted. There is no consensus on recommending vaccination against Streptococcus pneumoniae to CLL patients mainly because comparative studies are lacking. In our randomized trial, the efficacy of a conjugated pneumococcal vaccine on immune response was superior or equal to a polysaccharide vaccine for all pneumococcal serotypes common for the two vaccines. A conjugate pneumococcal vaccine should therefore be included in vaccination programs for patients with CLL. Bronchoalveolar lavage (BAL) is a well-established invasive method to identify the cause of pulmonary infiltrates in immunocompromised patients. In a retrospective trial, we have studied the diagnostic yield of BAL in patients with hematological malignancies. We concluded that BAL is highly useful in either verifying or excluding some of the important respiratory tract infections affecting these patients, particularly invasive pulmonary aspergillosis (IPA) and Pneumocystis jirovecii pneumonia (PJP). However, standardized procedures for BAL sampling should be continually revised to avoid unnecessary microbiological tests. Thrombocytopenia, an adverse prognostic factor in patients with MDS, can be aggravated by azacitidine, first-line treatment for high-risk MDS. Eltrombopag, a thrombopoietin-receptor agonist (TPO-R), alleviates thrombocytopenia in patients with immune thrombocytopenic purpura (ITP). In a phase I clinical trial, we concluded that the combination of eltrombopag and azacitidine in high-risk MDS patients with thrombocytopenia is feasible and well tolerated in doses up to 200 mg eltrombopag daily.

Leukemia is a hematopoietic cancer that is characterized by the abnormal differentiation and proliferation of hematopoietic cells. It is ranked 7th by death rate among cancer types in USA, even though it is not one of the top 10 cancers by incidence (USCS, 2010). This indicates an urgent need for more effective treatment strategies. In order to design the new ways of prevention and treatment of leukemia, it is important to understand the molecular mechanisms involved in development of the disease. In this study, we investigated mechanisms involved in the development of acute myeloid leukemia (AML) that is associated with CBF fusion genes. The RUNX1 and CBFB genes that encode subunits of a transcriptional regulator complex CBF, are mutated in a subset (20 – 25%) of AML cases. As a result of these mutations, fusion genes called CBFB-MYH11 and RUNX1-ETO arise. The chimeric proteins encoded by the fusion genes provide block in proliferation for myeloid progenitors, but are not sufficient for AML development. Genetic studies have indicated that activation of cytokine receptor signaling is a major oncogenic pathway that cooperates in leukemia development. The main goal of my work was to determine a role of two factors that regulate cytokine signaling activity, the microRNA cluster miR-17-92 and the thrombopoietin receptor MPL, in their potential cooperation with the CBF fusions in AML development. We determined that the miR-17-92 miRNA cluster cooperates with Cbfb-MYH11 in AML development in a mouse model of human CBFB-MYH11 AML. We found that the miR-17-92 cluster downregulates Pten and activates the PI3K/Akt pathway in the leukemic blasts. We also demonstrated that miR-17-92 provides an anti-apoptotic effect in the leukemic cells, but does not seem to affect proliferation. The anti-apoptotic effect was mainly due to activity of miR-17 and miR-20a, but not miR-19a and miR-19b. Our second study demonstrated that wild type Mpl cooperated with RUNX1-ETO fusion in development of AML in mice. Mpl induced PI3K/Akt, Ras/Raf/Erk and Jak2/Stat5 signaling pathways in the AML cells. We showed that PIK3/Akt pathway plays a role in AML development both in vitro and in vivo by increasing survival of leukemic cells. The levels of MPL transcript in the AML samples correlated with their response to thrombopoietin (THPO). Moreover, we demonstrated that MPL provides pro-proliferative effect for the leukemic cells, and that the effect can be abrogated with inhibitors of PI3K/AKT and MEK/ERK pathways. Taken together, these data confirm important roles for the PI3K/AKT and RAS/RAF/MEK pathways in the pathogenesis of AML, identifies two novel genes that can serve as secondary mutations in CBF fusions-associated AML, and in general expands our knowledge of mechanisms of leukemogenesis.