IntroduÃÃo â AlteraÃÃes na expressÃo ou funÃÃo das molÃculas de adesÃo (MA) nas cÃlulas leucÃmicas podem contribuir para a evoluÃÃo e no comportamento biolÃgico das leucemias agudas. A expressÃo aumentada nas LLAs parece relacionar-se aos mecanismos de disseminaÃÃo extramedular dos linfoblastos, infiltraÃÃo do SNC e formaÃÃo de massas tumorais. Objetivos â Analisar a expressÃo da L-selectina e do CD44 nas LLAs em crianÃas e adolescentes. Avaliar os fatores prognÃsticos (idade, sexo, leucometria ao diagnÃstico, imunofenÃtipo, classificaÃÃo FAB, EGIL, Ãndice de DNA e resposta ao tratamento de induÃÃo) e as apresentaÃÃes extramedulares das LLAs e correlacionÃ-los com essas MA. Pacientes e MÃtodos â Foram avaliados 76 pacientes com LLA, tratados com o Protocolo GBTLI-LLA. O diagnÃstico foi baseado em critÃrios FAB, imunofenotÃpicos (EGIL) e citogenÃticos. A expressÃo das MA foi avaliada por citometria de fluxo, utilizando tripla marcaÃÃo. O anticorpo monoclonal CD45-PerCP (ImmunotechÂ) foi utlizado como marcador dos linfoblastos. O CD44-PE (Clone HP2/9 - ImmunostepÂ) e o CD62L-FITC (Clone HI62L - ImmunostepÂ) foram utilizados para a marcaÃÃo das MA. Para a anÃlise das amostras e o cÃlculo da intensidade mÃdia de fluorescÃncia foi utilizado o programa Cell Quest. Na anÃlise estatÃstica utilizou-se o software SPSS 16.0. A associaÃÃo entre as variÃveis, os fatores prognÃsticos e resposta foi realizada com os testes de Qui-quadrado, exato de Fisher e Mann-Whitney. Sobrevida global foi determinada por curvas Kaplan-Meier e teste log-rank. AnÃlise multivariada por modelo proporcional de Cox foi utilizada para assegurar a independÃncia dos fatores prognÃsticos. Resultados â A mÃdia de idade foi 6,3Â0,5 anos (5m -17a) e predominou o sexo masculino (65%). Ao diagnÃstico os achados clÃnicos foram: hepatomegalia (63%), esplenomegalia (58%) e linfadenomegalia (44%). A infiltraÃÃo SNC ocorreu em 6,6% dos casos e o alargamento de mediastino em 11,8%. Quanto ao risco, 54% eram baixo risco e 46% alto risco. A classificaÃÃo FAB determinou 83% como L1 e 17% L2. DiagnÃstico de LLA-B foi mais frequente (89,5%) e o da LLA-T em 10,5% dos pacientes. O subtipo EGIL mais prevalente foi B II e B III, 51,5% e 45% respectivamente. O IDNA &#8805; 1.16 foi encontrado em 19% dos pacientes e associou-se a bom prognÃstico. Na avaliaÃÃo do D8, 95% dos pacientes apresentaram contagem de blastos <1000/mm3 e leucÃcitos < 5.000/mm3. A taxa de remissÃo de induÃÃo foi de 95% e ocorreram 2,6% de Ãbitos na induÃÃo. Observou-se uma maior expressÃo do CD44 na LLA-T (87,5%/ IMF=150,44Â20,29), porÃm sem significÃncia estatÃstica. LLAs com massa tumoral apresentaram 84% de expressÃo do CD44, quando comparada a 52% das LLAs sem massa tumoral (p=0.01; OR=4,8). ExpressÃo aumentada da L-selectina na LLA-T (87,5%/IMF=272,33Â52,72) foi estatisticamente significante (p=0,004), comparado a LLA-B (54,5%/ IMF= 115,90Â12,75). NÃo houve correlaÃÃo entre os outros fatores prognÃsticos e essas MA. Na anÃlise multivariada as variÃveis de maior impacto para a sobrevida foram: a leucometria ao diagnÃstico, sexo, imunofenÃtipo T e a L-selectina. ConclusÃo â A expressÃo da L-selectina e do CD44 estÃo aumentadas nas LLAs estudadas, principalmente na LLA-T. O CD44 correlacionou-se com LLAs com massas tumorais e parece estar relacionado aos mecanismos de disseminaÃÃo extramedular dos linfoblastos / Introduction â Altered expression or function of adhesion molecules on leukemic blasts may contribute to the evolution of acute leukemia and its biological behavior. The elevated expression of adhesion molecules in ALL might be correlated with the extramedullary dissemination of blast cells, CNS involvement and leukemia tumor burden. Purpose â To analyze the expression of L-selectin and CD44 in ALL in children and adolescents. As well as to evaluate the prognostic factors (age, gender, initial leukocyte count, immunophenotype, FAB and EGIL classification, DNA index and early response to treatment) and the extramedullary presentation of ALL, to finally correlate the prognostic factors with these adhesion molecules. Patients and Methods â From November 2007 to November 2008, 76 patients with newly diagnosed ALL started on Brazilian GBTLI-ALL Protocol. The diagnosis was based on cytological, immunophenotypic, and cytogenetic methods. The mean fluorescence intensity (MFI) and the percentage of the adhesion molecules blasts cells was measured by flow cytometry using triple staining with McAb directly conjugated. CD45-PerCP positive cells were gated for blasts analysis. Anti-CD44-PE (Clone HP2/9 - ImmunostepÂ) and CD62L-FITC (Clone HI62L - ImmunostepÂ) were used to mark the adhesion molecules. The Cell Quest program was used for data acquisition and analysis. Statistical analysis was done by SPSS 16.0 Software. The association of features, prognosis and response to treatment was assessed by Chi-square, Fisher exact and Mann-Whitney tests. Overall survival curves were constructed by the Kaplan-Meier method and the log-rank test. Multivariate Cox regression analysis showed independent prognostic factors. Results â The mean age at diagnosis was 6.3Â0.5 years (range 9mo to 17yr) and 65% of them were boys. Clinical findings were hepatomegaly (63%), splenomegaly (58%), lymphadenopathy (44%). CNS involvement was detected in 6.6% of cases and mediastinal mass appeared in 11.8% of them. Patients were classified into low risk (54%) and high risk (46%). FAB classification identified 83% as L1 and 17% as L2. Immunophenotypically, 89.5% of patients were classified as B-lineage ALL and 10.5% as T-lineage ALL. The most frequent EGIL subtype was B common and pre-B-ALL (51.5% and 45.5%, respectively). DNA index greater than 1.16 was found in 19% of the patients and was associated with favorable prognosis. On the D8 evaluation, 95% of the patients had blast count lower than 1.000/mm3 and leukocyte count lower than 5.000/mm3. The remission induction rate was 95% and there was a rate of 2.6% of death during induction therapy. CD44 had greater expression to the rate of 87.5% in T-cell ALL (MFI=150.44Â20.29) with no statistical correlation. A significant positive correlation was demonstrated between 84% of CD44 expression and Leukemia burden tumor cases (p=0.01; OR=4.8). There was statistical correlation between L-selectin expression (87.5%/MFI=272.33Â52.72) and T-cell ALL (p=0,004). No significant correlation was detected between L-selectin and CD44 expression and other prognostic factors. Multivariate statistical analysis (adjusted for overall survival) indicated that initial leukocyte count, gender, T immunophenotype and L-selectin were independent factors. Conclusion â L-selectin and CD44 expressions were elevated in ALL studied, mainly in T-cell ALL. The research demonstrated that there is an association between CD44 expression and leukemia tumor burden, which might be involved in the dissemination of leukemic cells and the progression of the disease.

Hematopoietic cells exploit the multistep paradigm of cell migration to ultimately enable them to perform their function. This process is dictated by the ability of adhesion molecules on the circulating hematopoietic cells to find their counter-receptors on endothelial cells. Of those molecules, the selectin family and their respective ligands induce the initial transient interactions between circulating cells and the opposing endothelium. In this thesis, I focused on studying E-selectin mediated cellular migration in two hematopoietic cell types, namely human hematopoietic stem and progenitor cells (HSPCs) and human T-lymphocytes. HSPCs derived from pluripotent sources theoretically offers a novel, unlimited source for hematopoietic stem cell transplantation therapy. In vitro pluripotent stem cell derived- hematopoietic stem/progenitor cells (ES/iPS-HSPCs) behave much like somatic HSPCs in that they exhibit clonal expansion and multilineage hematopoietic capacity. However, unlike somatic sources, ES/iPS-HSPCs do not give rise to effective hematopoietic repopulation, which may be due to insufficient HSPCs homing to the bone marrow. HSPCs exploit E- and P-selectin to home and engraft into bone marrow niches. Thus, one of my objectives in this thesis was to study the expression of E-selectin ligands associated with ES/iPS-HSPCs. I showed that ES/iPS-HSPCs lack functional E-selectin ligand(s). In an effort to enhance the interaction between Eselectin and ES/iPS-HSPCs, we decorated the cell surface with sialyl-Lewis x (sLex) using the ex-vivo glycan engineering technology. However, this decoration did not improve the engraftment capacity of ES/iPS-HSPCs, in vivo. Induction of E-selectin expression during inflammation is key to recruitment of immune cells and therefore I also focused on analyzing the expression of E-selectin ligands on activated human T-cells. I identified several novel glycoproteins that may function as E-selectin ligands. Specifically, I compared the role of the known E-selectin ligands, namely PSGL-1 and CD43, to CD44. I showed that CD44 purified from in vitro human activated T-cells or from psoriasis patients acts as a functional E-selectin ligand. Furthermore, our knock-down studies demonstrated that CD44, and not CD43, cooperates with P-selectin glycoprotein ligand-1 (PSGL-1) as a major E-selectin ligand.

Selectins are key adhesion molecules responsible for initiating a multistep process that leads a cell out of the blood circulation and into a tissue or organ. The adhesion of cells (expressing ligands) to the endothelium (expressing the selectin i.e.,E-selectin) occurs through spatio-temporally regulated interactions that are mediated by multiple intra- and inter-cellular components. The mechanism of cell adhesion is investigated primarily using ensemble-based experiments, which provides indirect information about how individual molecules work in such a complex system. Recent developments in single-molecule (SM) fluorescence detection allow for the visualization of individual molecules with a good spatio-temporal resolution nanometer spatial resolution and millisecond time resolution). Furthermore, advanced SM fluorescence techniques such as Förster Resonance Energy Transfer (FRET) and super-resolution microscopy provide unique opportunities to obtain information about nanometer-scale conformational dynamics of proteins as well as nano-scale architectures of biological samples. Therefore, the state-of-the-art SM techniques are powerful tools for investigating complex biological system such as the mechanism of cell adhesion. In this project, several constructs of fluorescently labeled E-selectin will be used to study the conformational dynamics of E-selectin binding to its ligand(s) using SM-FRET and combination of SM-FRET and force microscopy. These studies will be beneficial to fully understand the mechanistic details of cell adhesion and migration of cells using the established model system of hematopoietic stem cells (HSCs) adhesion to the selectin expressing endothelial cells (such as the E-selectin expressing endothelial cells in the bone marrow).

L'élaboration de nouvelles stratégies pour la détection de l'activité de la sclérose en plaques (SEP) est importante pour améliorer le diagnostic et le suivi de cette pathologie. Pour cela, nous avons utilisé des microparticules d'oxyde de fer (MPIO) couplées à un anticorps spécifique de la protéine P-sélectine ou de MAdCAM-1. Durant cette thèse, nous avons démontré que l’IRM moléculaire spécifique de la P-sélectine est capable de détecter les événements pathologiques qui se déroulent dans la moelle épinière de modèles murins de SEP chronique et récurrente-rémittente. De façon intéressante, nous montrons ici que cette technique d'IRM peut prédire l'apparition des poussées et des récupérations dans l’EAE. De plus, nous avons démontré que l’IRM moléculaire de MAdCAM-1 est capable de détecter l’inflammation intestinale dans des modèles de pathologies intestinales et de SEP. Les techniques novatrices d'IRM développées dans cette étude pourraient apporter de nouvelles avancées dans le diagnostic et le pronostic des rechutes de la SEP en ciblant l'activation vasculaire. Enfin, nous rapportons dans la dernière partie de cette thèse que le système glymphatique existe également dans le parenchyme de la moelle épinière de la souris. Dans l’EAE, l’activité de ce système est réduite dans la moelle épinière mais pas dans le cerveau ou le cervelet. Cette altération est associée à l'accumulation de cellules inflammatoires dans l'espace péri-vasculaire, à la désorganisation de l'AQP4 et entraine une forte augmentation du volume ventriculaire. Ces perturbations pourraient contribuer à la physiopathologie de la SEP. Nos résultats sont très prometteurs pour l'élaboration de nouvelles stratégies thérapeutiques. / Developing new strategies to detect disease activity in multiple sclerosis (MS) is essential to improve the diagnosis and follow-up of this pathology. To this aim, we used microparticles of iron oxide (MPIO) coupled to an antibody specific to the P-selectin or MAdCAM-1 protein. In this thesis, we establish that molecular MRI specific to P-selectin protein is able to detect the pathological events that take place in the spinal cord of chronic and relapsing-remitting models of MS in mice. Interestingly, we show here that this MRI technique can predict the apparition of relapses and recoveries in EAE. Moreover, we demonstrate that MRI specific to MAdCAM-1 protein is able to detect the gut inflammation that takes place in models of bowel diseases or MS. The innovative MRI techniques developed in this study could bring new advances in the diagnosis and prognosis of MS relapses by targeting gut inflammation. In the last part of this work, we report that the glymphatic system also exists in the spinal cord parenchyma of the mouse. In EAE, the activity of this system is reduced in the spinal cord but not in the brain or cerebellum. This alteration is associated to inflammatory cell accumulation within the perivascular space, AQP4 disorganization and leads to a large increase of ventricular volume. These disruptions could contribute to the MS pathophysiology. Our results hold significant promise for the development of new therapeutic strategies.

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京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第21715号 / 薬科博第106号 / 新制||薬科||11(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 山下 富義, 教授 髙倉 喜信, 講師 樋口 ゆり子 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

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