T-cells are lymphocytes that make up part of the adaptive arm of the immune system, and are essential for efficient protection from and eradication of viruses and pathogens. T-cells not only play an important role in protection from external agents, but also regulate and prevent activation towards self-peptides and detect and remove erratically growing cells. Alterations in T-cell activation and suppression contribute to auto-immunity, immunocompromised disorders, and cancer progression. The immune system, and T-cells in particular, provides daily surveillance, recognition and destruction of aberrant cells. Although the immune system is proficient at suppressing malignant progression, tumor cells acquire various methods of immune evasion. Myelodysplastic Syndrome (MDS) is a pre-malignant dysplastic disorder of the bone marrow characterized by ineffective hematopoiesis and clonality in the myeloid lineage, where lack of immune response has been implicated in the propensity for progression to acute myeloid leukemia (AML). Leukemia progression is associated with the acquisition of complex genetic abnormalities. Alterations in immune system regulation have been implicated in various stages of the disease process, although the role of the immune system in response to several therapies in MDS has not been fully discovered. Lenalidomide is a small molecule therapeutic preferentially effective in MDS patients with an interstitial chromosome 5q deletion (del(5q)). Improved erythropoiesis has also been reported to occur in 20-30% of low-risk, non-del(5q) patients. Although lenalidomide is a potent immunomodulatory drug that potentiates T-cell and NK-cell responses, the T-cell compartment in MDS is highly deregulated by aberrant repertoire skewing, decreased function and abnormal naïve and memory cell homeostasis. The presence of lymphoid infiltrates in the bone marrow of lenalidomide-responsive patients suggests that T-cells may participate in the hematopoietic response, but it is unclear if lenalidomide is capable of reversing these functional T-cell defects. We therefore assessed immunological changes in low-risk MDS patients before and after 16-weeks of lenalidomide therapy, and assessed the relationship to erythroid response. Although MDS T-cells were anergic prior to treatment, we have shown that T-cells in responders have a significant increase in antigen-induced proliferative response and T helper type-1 (Th1) cytokine production (IL-2, IFN-γ, TNF-α) compared to non-responders. The change in function positively correlated with an increase in naïve T-cells and a decrease in memory cells, indicating that lenalidomide has immunomodulatory activity to reverse anergy in MDS. Although it is known that lenalidomide may increase T-cell activation and proliferation in the absence of co-stimulatory signals, a direct mechanism of action has yet to be elucidated. Since CD28 is one of the most important co-stimulatory molecules deregulated in cancer, we therefore set out to determine if the expression of CD28 was essential for lenalidomide's mechanism in T-cells. We knocked out CD28 expression in healthy donor T-cells, and sorted on inherently deficient, CD28null, T-cells that accumulate in older healthy donors and found that lenalidomide-induced proliferation and function were completely ablated within the CD28null subset. These data indicate the immunotyrosine-based activation motifs (ITAMs) on the intracellular domain of the CD28 receptor are necessary for lenalidomide action. Interestingly, during the natural aging process, repeated exposure to antigens results in the accumulation of CD28null T-cells that are phenotypically distinct and functionally deficient due to excessive proliferative history in vivo. We therefore examined whether CD28 expression on MDS patient T-cells affected responses to lenalidomide, and if this could be used as a predictive biomarker of responsiveness. We found that patients who fail lenalidomide therapy had higher CD8+ Terminal Effector Memory (TEM), which are inherently CD28null, and that non-responders had an overall increase in CD4+ and CD8+CD28null T-cells, as well as an increase in CD28null cells within the TEM compartment compared to hematologic responders. We then sought to determine the particular protein target of lenalidomide responsible for increased CD28 receptor signaling in T-cells. Several targets in a variety of cell types have been postulated, although the direct mechanism in T-cells is unclear. Our group has previously shown that lenalidomide inhibits the activity of two haplodeficient phosphatases located within the commonly deleted region (CDR) on chromosome 5q in the MDS myeloid clone, Protein Phosphatase 2A (PP2A) and Cdc25c. PP2Ac is known to bind CD28 and is hypothesized to inhibit T-cell co-stimulation. Therefore, it is plausible that lenalidomide and other IMiDs inhibit the phosphatase activity of PP2A which leads to increased activation of T-cell proximal signals dependent on CD28 expression. We examined this hypothesis using molecular modeling and virtual screening and found that all of the IMiDs (lenalidomide, pomalidomide, and thalidomide) can theoretically interact with the catalytic pocket of the PP2A heterotrimer, potentially inhibiting PP2Ac activity. In vitro phosphatase activity assays supported these findings as lenalidomide-inhibition of PP2Ac activity was seen in both ad293 and Jurkat cell lines, and in primary T-cells. Mutations of theorized lenalidomide hydrogen-bond sites within the catalytic pocket of PP2A rendered the enzyme catalytically dead, indicating that these are important residues for enzymatic activity, but unfortunately could not be used to determine if lenalidomide activity was disrupted by mutation of those sites. These data together suggest that the ability of lenalidomide to augment immune activation in vivo in MDS patients, and potentially other diseases, is extremely important to patient response. Also, that CD28 expression on T-cells is essential for lenalidomide immune-mediated tumor eradication through CD28 downstream signaling, and potentially through inhibition of PP2A. These results are useful in designing future lenalidomide-combination therapy trials in other hematologic and solid malignancies, and could be used to help stratify patients for future therapeutic decisions in MDS and other malignancies.

Η αιμοποιητική φωλαιά (hematopoietic stem cell niche) περιέχει οστεοβλάστες, οι οποίοι ρυθμίζουν τη φυσιολογική αιμοποίηση. Ωστόσο, λίγα στοιχεία είναι γνωστά, έως τώρα, για το ρόλο των οστεοβλαστών στη διαδικασία της αιμοποίησης σε ασθενείς με Μυελοδυσπλαστικό Σύνδρομο (ΜΔΣ). Το ΜΔΣ, αποτελεί μια ετερογενή ομάδα κλωνικών αιματολογικών διαταραχών, με αυξημένο κίνδυνο εκτροπής προς Οξεία Μυελογενή Λευχαιμία (ΟΜΛ). Μελέτες σε ex-vivo συστήματα καλλιεργειών (co-cultures) περιγράφουν την επίδραση των μεσεγχυματικών κυττάρων (“feeder cells”) στο δυναμικό πολλαπλασιασμού, στη μεταναστευτική ικανότητα, καθώς και στη διατήρηση (stemness) των αρχέγονων αιμοποιητικών κυττάρων (HSCs) φυσιολογικών δοτών. Η μελέτη αυτή στοχεύει στη διερεύνηση των βιολογικών χαρακτηριστικών των οστεοβλαστών από ασθενείς με ΜΔΣ καθώς και τις αλληλεπιδράσεις φυσιολογικών HSCs και οστεοβλαστών ασθενών με ΜΔΣ. Για το σκοπό αυτό δημιουργήθηκε ένα σύστημα δισδιάστατης καλλιέργειας (2-D culture system) χρησιμοποιώντας οστεοβλάστες που παρήχθησαν από μεσεγχυματικά κύτταρα μυελού των οστών (human marrow mesenchymal stem cells-MSCs). Τα MSCs απομονώθηκαν από το μυελό των οστών ασθενών με ΜΔΣ και υγιών δοτών και καλλιεργήθηκαν σε κατάλληλο θρεπτικό μέσο. Ακολούθησε επαγωγή της διαφοροποίησης των MSCs, μετά από συνεχόμενες καλλιέργειες σε οστεοβλάστες. Στη μελέτη χρησιμοποιήθηκαν 13 δείγματα μυελού των οστών από ασθενείς με ΜΔΣ (6 RA, 3 RAEBI, 2 RAEBII, 1 5q- και 1 υποπλαστικό MDS) και 8 δείγματα μυελού φυσιολογικών μαρτύρων όμοιας ηλικίας. Για τη μελέτη της επίδρασης των οστεοβλαστών από ασθενείς με ΜΔΣ στην αιμοποίηση χρησιμοποιήθηκαν φυσιολογικά HSCs από κινητοποιημένο περιφερικό αίμα υγιών δοτών (mPB, n=4), τα οποία τοποθετήθηκαν πάνω στους ήδη εγκατεστημένους οστεοβλάστες (osteoblast confluent monolayer cultures). Τα MSCs και οι οστεοβλάστες που αναπτύχθηκαν ελέγχθηκαν μορφολογικά και ανοσοφαινοτυπικά, με τη χρήση μικροσκοπίας και κυτταρομετρίας ροής αντίστοιχα. Μονοπύρηνα κύτταρα από δείγματα κινητοποιημένου περιφερικού αίματος υγιών δοτών τοποθετήθηκαν στο δισδιάστατο καλλιεργητικό σύστημα, σε καλλιεργητικό υλικό αιμοποιητικών κυττάρων, χωρίς την εξωγενή προσθήκη κυτταροκινών. Με τη χρήση κυτταρομετρίας ροής ελέγχθηκε η έκφραση των μορίων που σχετίζονται με την προσκόλληση των αιμοποιητικών κυττάρων στην αιμοποιητική φωλαιά καθώς και την εγκατάσταση και διατήρησή τους σε αυτή. Ο έλεγχος έγινε στις 36 ώρες και τις 7 ημέρες συγκαλλιέργειας και αφορούσε τα μόρια CXCR4, το οποίο ρυθμίζει την άμεση πρόσδεση των HSCs στην φωλαιά κατά τη διαδικασία του “homing”, CD49d (Very Late Antigen-4- VLA4) και CD49e (Very Late Antigen-5- VLA5), τα οποία παρέχουν σήματα επιβίωσης ή προάγουν την ενεργοποίηση μιας φάσης ηρεμίας (quiescence) στα HSCs μετά την είσοδο τους στη φωλαιά (localization). Η έκφραση των μορίων αυτών μελετήθηκε στους υποπληθυσμούς των CD34+, CD34+/CD38+ και CD34+/CD38- κυττάρων. Παράλληλα εκτιμήθηκε το ποσοστό (συχνότητα) των CD34+ αιμοποιητικών κυττάρων καθώς επίσης και η προσκόλλησή τους στους οστεοβλάστες. Μετά τη συγκαλλιέργεια, οι οστεοβλάστες που προήλθαν από υγιείς δότες προκάλεσαν τον πολλαπλασιασμό των CD34+ κυττάρων των φυσιολογικών αιμοποιητικών κυττάρων που τοποθετήθηκαν πάνω στο εγκατεστημένο στρώμα των οστεοβλαστών (3-fold και 9-fold αύξηση στις 36ώρες και τις 7ημ., αντίστοιχα). Αύξηση επίσης, παρατηρήθηκε (2 fold αύξηση) στα CD34+ κύτταρα στις συγκαλλιέργειες των 36h, φυσιολογικών HSCs με οστεοβλάστες που παρήχθησαν από ασθενείς με ΜΔΣ, ενώ καμία διαφορά δεν παρατηρήθηκε μεταξύ των διαφορετικών υποτύπων ΜΔΣ. Στις 7 ημέρες συγκαλλιέργειας από την άλλη, δεν 12 παρατηρήθηκε καμία διαφορά στη συχνότητα εμφάνισης ενός πιο άωρου φαινοτύπου των φυσιολογικών HSCs που αναπτύχθηκαν σε οστεοβλάστες από ασθενείς με χαμηλού κινδύνου ΜΔΣ (low risk MDS). Αντιθέτως, τα CD34+ κύτταρα αυξήθηκαν κατά πολύ (16- fold αύξηση), όταν φυσιολογικά HSCs, τοποθετήθηκαν σε οστεοβλάστες ασθενών με υψηλού κινδύνου ΜΔΣ (high risk MDS). Επιπλέον, παρατηρήθηκε αύξηση της έκφρασης των μορίων CXCR4, CD49d και CD49e στα CD34+ κύτταρα μετά από συγκαλλιέργεια φυσιολογικών HSCs και οστεοβλαστών από υγιείς δότες, συγκριτικά με τα επίπεδα έκφρασης των μορίων αυτών πριν την τοποθέτηση τους στο σύστημα συγκαλλιέργειας. Η αύξηση της έκφρασης του μορίου CXCR4 ήταν λιγότερο εμφανής στην περίπτωση συγκαλλιέργειας των φυσιολογικών HSCs με οστεοβλάστες από ασθενείς με ΜΔΣ, όπου η μεγαλύτερη διαφορά παρατηρήθηκε στο σύστημα που περιείχε τους οστεοβλάστες από ασθενείς χαμηλού κινδύνου ΜΔΣ (3- και 1,7- fold αύξηση στις 7ημέρες καλλιέργειας με οστεοβλάστες από υγιείς δότες και χαμηλού κινδύνου ΜΔΣ ασθενείς, αντίστοιχα). Το πρότυπο έκφρασης των μορίων CD49d και CD49e ήταν όμοιο στα κύτταρα που τοποθετήθηκαν τόσο σε οστεοβλάστες προερχόμενους από υγιείς δότες, όσο και οστεοβλάστες από ΜΔΣ ασθενείς. Ο φαινότυπος, τόσο όσον αφορά τα μορφολογικά όσο και τα ανοσοφαινοτυπικά χαρακτηριστικά, των MSCs ήταν ίδιος και στις δυο ομάδες μελέτης, ενώ η διαφοροποίηση των MSCs προς οστεοβλάστες ήταν όμοια τόσο στα MSCs που προήλθαν από φυσιολογικούς δότες όσο και σε αυτά που προήλθαν από ασθενείς με ΜΔΣ, δείχνοντας παρόμοια έκφραση των ειδικών οστεοβλαστικών πρωτεϊνών αλλά και της διαδικασίας της ενασβεστοποίησης. Σύμφωνα με τα αποτελέσματα που λάβαμε, οι οστεοβλάστες από υγιείς δότες προώθησαν την αύξηση του ποσοστού των προγονικών αιμοποιητικών κυττάρων και οδήγησαν στην επαγωγή της έκφρασης του μορίου CXCR4, ενός πολύ σημαντικού μορίου για τη μετανάστευση, την εγκατάσταση αλλά και την ανάπτυξη. Ωστόσο, η διαφορετική δραστηριότητα, τόσο όσον αφορά το ποσοστό των CD34+ όσο και την έκφραση του μορίου CXCR4, όταν τα φυσιολογικά αιμοποιητικά κύτταρα συγκαλλιεργήθαν με οστεοβλάστες που προήλθαν από ασθενείς με ΜΔΣ, οδηγεί στην υπόθεση ότι υπάρχει μεταβολή στη λειτουργία των οστεοβλαστών, οπότε προβλέπεται και μια επακόλουθη αλλαγή στη ρύθμιση της εγκατάστασης των HSC στην αιμοποιητική φωλαιά, στους ασθενείς με ΜΔΣ. / The hematopoietic stem cell niche contains osteoblasts that regulate normal hematopoiesis. However, little is known about the role of osteoblasts in MDS hematopoiesis so far. Myelodysplastic syndrome comprises a heterogeneous group of clonal stem cell disorders with dismal prognosis and difficulty in their therapeutic approach, which is characterized by ineffective hematopoiesis. It appears with dysplastic hematopoietic cells, peripheral blood cytopenias and high risk of evolution to acute myeloid leukemia (AML). Data derived from ex vivo co-culture systems using mesenchymal stromal cells as a feeder cell layer suggest that cell-cell contact has a significant impact on the expansion, migratory potential and “stemness” of hematopoietic stem cells. In this study, we investigated the biological characteristics of osteoblasts from MDS patients and the interactions between these cells and normal hematopoietic stem cells (HSCs). Osteoblasts were differentiated from marrow MSCs from 13 MDS patients (6 RA, 3 RAEBI, 2 RAEBII, 1 5q- and 1 hypoplastic MDS) and 8 age-matched healthy individuals. To study the effect of MDS osteoblasts on hematopoiesis, normal HSCs from mobilized peripheral blood from healthy individuals (n=4) were seeded onto osteoblast confluent monolayer cultures using a culture medium appropriate for the culture of HSCs, without the exogenous addition of cytokines. We studied the morphology and immunophenotype of MSCs and osteoblasts by microscopy and flow cytometric analysis, respectively. Cytometric analyses of homing associated molecules were performed 36h and 7d later. These molecules are CXCR4, which regulates the direct adhesion of HSCs to the bone marrow niche during “homing”, CD49d (Very Late Antigen-4- VLA4) and CD49e (Very Late Antigen-5- VLA5), which produce survival signals or promote the maintenance of a quiescent state for HSCs after entering the stem cell niche (localization). We investigated the expression of these molecules in CD34+, CD34+/CD38+ and CD34+/CD38- cell populations. Furthermore we studied the frequency of CD34+ hematopoietic cells and also their ability to adhere osteoblasts.Osteoblasts from healthy individuals increased the frequency of CD34+ cells by 3- and 9-fold increase in normal hematopoietic cells after 36h and 7d co-cultures respectively. A 2-fold increase was also seen in CD34+ cells when normal HSCs grown on MDS-osteoblasts for 36h and no difference was seen between the MDS subtypes. When the culture period was extended to 7d, there was no change in the frequency of immature phenotype of normal HSCs in osteoblast cultures from low-risk MDS patients. In contrast, CD34+ cells increased several fold (16-fold increase) when normal HSCs were cultured on high-risk MDS 14 osteoblasts, twice the values obtained in osteoblast co-cultures from healthy individuals and low risk patients. The expression of adhesion molecules CXCR4, CD49d and CD49e on CD34+ cells from normal HSCs was increased in co-cultures with osteoblasts from healthy individuals compared to the values obtained before culture (3-fold increase at 7d). The increase in CXCR4 expression was less pronounced in the presence of osteoblasts from MDS patients with the largest difference being found in low-risk MDS patients (1,7-fold increase at 7d). The expression pattern of CD49d and CD49e was identical between cells grown on MDS- and normal- osteoblast co-cultures.The morphological and immunophenotypical analysis of MSCs show the same results for the two study groups, while the differentiation of MSCs to osteoblasts was similar for both healthy individuals and MDS patients, after having similar expression of bone specific proteins and mineralization activity. According to our data, osteoblasts from healthy individuals promoted the expansion of immature hematopoietic progenitors and induced the cell surface expression of CXCR4, an important molecule in HSCs homing, retention and development. However, the different expression of CXCR4 and the change in frequency of CD34+ cells that were detected when normal HSCs co-operated with MDS-osteoblasts, suggests alteration in osteoblast function and the subsequent regulation of the HSC residency in the niche in MDS patients compared with healthy individuals.

Made available in DSpace on 2014-06-11T19:26:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-17Bitstream added on 2014-06-13T20:54:04Z : No. of bitstreams: 1 freitas_pc_me_sjrp.pdf: 528091 bytes, checksum: d630cd8e1a7a4fdc34c7e9200a36b8b5 (MD5) / Síndromes Mielodisplásicas (SMD) compreendem um conjunto heterogêneo de doenças hematopoéticas caracterizadas por hematopoese ineficaz, que geralmente apresentam citopenias no sangue periférico, medula óssea hipercelular, diferenciação celular displásica e propensão ao desenvolvimento de leucemia mielóide aguda. São classificadas em oito tipos e a incidência anual é estimada entre dois e 12 casos por 100.000 pessoas da população em geral e em até 50 casos por 100.000 indivíduos com idades superiores a 60 anos. A análise cromossômica das células da medula óssea dos doentes ao diagnóstico detecta alterações diretamente relacionadas com o prognóstico em aproximadamente 50% dos casos. Alguns genes também foram relacionados à etiologia e prognóstico das mielodisplasias. O gene FOXO3, um supressor de tumor, embora não estudado anteriormente em SMD, é um dos genes que mais se expressam no tecido hematopoético normal. Alterações neste gene poderiam resultar em hematopoese anormal, pois já foram relacionadas a outros tipos de câncer, com mutações descritas no éxon 1. O objetivo deste trabalho foi estudar células da medula óssea de doentes com SMD de qualquer tipo, ao diagnóstico, para investigar a presença de alterações cromossômicas e de mutações no éxon 1 do FOXO3. A análise citogenética foi realizada em metáfases submetidas ao bandamento GTG, obtidas de culturas de curta duração de células da medula, sem estimulação mitogênica. Para a análise molecular foi extraído o DNA, realizada a amplificação gênica pela Reação em Cadeia da Polimerase e realizado o sequenciamento direto do éxon 1. Entre os 25 casos analisados, três (12%) apresentaram alterações cromossômicas clonais isoladas: deleção intersticial do braço longo do cromossomo 5; monossomia do cromossomo 21 e monossomia do cromossomo 22. Todas puderam ser relacionadas... / Myelodysplastic syndrome (MDS) constitute a heterogeneous group of hematopoietic diseases characterized by ineffective hematopoiesis usually with peripheral blood cytopenia, hypercellular bone marrow, dysplastic differentiation and a tendency to evolve to acute myeloid leukemia. They are classified in eight categories by the World Health Organization. The annual incidence is estimated at between two and 12 cases per 100,000 individuals in the general population and up to 50 cases per 100,000 of over 60-year olds. A chromosomal analysis of bone marrow cells at diagnosis identifies changes directly related to prognosis in approximately 50% of cases. Additionally, some genes are also associated to the etiology and prognosis of myelodysplasia. Although not previously studied in respect to MDS, a tumor suppressor, FOXO3, is one of the most commonly expressed genes in normal hematopoietic tissue. Changes in this gene could therefore result in abnormal hematopoiesis, as mutations described in exon 1 have already been associated with other types of cancer. The aim of this study was to investigate chromosomal alterations and mutations in exon 1 of FOXO3 in bone marrow cells from patients diagnosed with any type of MDS. Cytogenetic analysis was performed on metaphases submitted to GTG banding, obtained from short-term cultures of bone marrow cells without mitogenic stimulation. To evaluate mutations in the FOXO3 gene, DNA was extracted from the bone marrow, gene amplification was achieved by polymerase chain reaction and direct sequencing was performed. Of the 25 cases analyzed, three (12%) showed clonal chromosomal abnormalities in isolation characterized as the interstitial deletion of the long arm of chromosome 5, monosomy 21 and monosomy 22. All were correlated to the diagnosis and/or prognosis of patients. No mutations were detected in exon 1, but the 159C>T polymorphism was detected... (Complete abstract click electronic access below)

Orientador: Irene Lorand-Metze / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T20:45:18Z (GMT). No. of bitstreams: 1 Reis-Alves_SuiellenCarvalho_M.pdf: 2640206 bytes, checksum: 0fe8b3a89ccee8a9cd543d9d74f965c3 (MD5) Previous issue date: 2009 / Resumo: A análise multiparamétrica por citometria de fluxo é útil para o diagnóstico diferencial de Síndromes Mielodisplásicas (SMD) nos casos com poucos elementos displásicos na medula óssea (MO) e cariótipo normal. Vários estudos têm relatado hipogranularidade dos granulócitos, anormalidades no padrão de expressão de antígenos, alterações nas quantidades e expressões anômalas nas células CD34+, assim como uma diminuição das células precursoras-B, além do aumento dos monócitos. No entanto, não há um consenso sobre qual o melhor painel a ser aplicado, pois, os painéis geralmente utilizados incluem um grande número de anticorpos monoclonais (AcMo). Neste estudo, a utilidade de um painel racionalizado (10 AcMo) foi avaliada, em combinações de quatro cores, na rotina laboratorial, permitindo o estudo de diversos parâmetros para o diagnóstico de SMD, podendo sugerir fatores prognósticos. Foi examinado MO de pacientes com diagnóstico recente de SMD. O diagnóstico foi baseado na contagem de sangue periférico, citologia de medula óssea e cariótipo. O critério OMS foi utilizado. Foram analisados: dispersão lateral da luz (side scatter -SSC) e padrão de maturação dos granulócitos e dos monócitos, além dos subtipos presentes na população de células CD34+, sendo estas características comparadas com a MO normal. As combinações de AcMo utilizadas foram: HLA-DR/CD14/CD45/CD33; CD16/CD34/CD45/CD13; CD19/CD34/CD45/CD117 e HLA-R/CD123/CD45/CD33. No mínimo 50.000 eventos/caso foram adquiridos. Este estudo incluiu 24 casos de MO normal e 54 casos de SMD com idade mediana de 62 anos (23-93). Os tipos OMS foram 2 casos de AR, 2 SMD del 5q, 25 CRDM, 8 CRDMSA, 7 AREB-I, 10 AREB-II. Os casos foram grupados em SMD com <5% de blastos (baixo risco) e SMD >5% de blastos (alto risco) para análise e comparados com MO normal. Foram detectadas 16 alterações: 4 nos precursores granulocíticos, 4 nos monócitos, 6 na população de blastos como o aumento das células CD34+, mieloblastos e células imaturas não definidas, diminuição dos precursores de células B, e 2 nas populações minoritárias. O total de número de alterações em casos com < 5% de blastos na MO foram de 6 (2-15) porcentagem de blastos na citologia (r= 0.38; p= 0.001), porcentagem de células CD34+ (r= 0.40; p< 0.001), células CD34+/CD13+ (r= 0.61; p< 0.0001), e com as células imaturas não linfóides CD34+/CD13- e CD34+/CD117- (r= 0.30 p=0.02 e r=0.55 p=0.0003, respectivamente). Os precursores de células-B (r=-0.39; p= 0.001) e a hemoglobina (r= -0.30; p= 0.001) diminuíram de acordo com a extensão do número de alterações. Houve correlação entre o número de alterações com o tipo OMS (r= 0.38; p=0.002) e o IPSS (r=0.27 p=0.02). Mesmo com o uso de um painel restrito de 10 AcMo, concluiu-se que este painel foi suficiente para fazer o diagnóstico em 91% dos casos, e permitiu detectar características associadas com a agressividade dos casos. / Abstract: Flow cytometric analysis is useful for the diagnosis of myelodysplastic syndromes (MDS) in cases of few dysplastic elements in bone marrow (BM) and a normal karyotype. Several studies have reported decreased side scatter (SSC) in the granulocytic gate, abnormalities in the maturation pattern antigens, number and abnormal co-expressions in CD34+ cells besides decreased of B-cell precursors and increased of monocytes. There is no uniformly accepted panel for these analyses that usually comprise a large number of monoclonal antibodies (MoAbs). We examined the utility of a small panel of MoAbs in four-color combinations, used routinely in our laboratory that allows the evaluation of several parameters for the diagnosis of MDS and may suggest prognostic factors. Bone marrow aspiration was performed in the diagnostic routine. The diagnosis was based on peripheral blood counts, BM morphology and karyotype. The WHO criteria were used. We examined: SSC and maturation pattern of granulocytes and monocytes; subsets present in the CD34+ population. These features were compared to the values found in normal BM. Combinations of MoAbs:HLA-DR/CD14/CD45/CD33; CD16/CD34/CD45/CD13; CD19/CD34/CD45/CD117; HLA-R/CD123/CD45/CD33. At least 50 000 events/case were acquired. Data were acquired in a FACS CaliburTM flow cytometer and the Paint-A-Gate software was used for data analysis. Normal BMs: 24 cases; MDS 54 cases. Median age: 62 years (23-93). WHO types: 2 RA, 2 MDS del 5q, 25 RCMD, 8 RCMD-RS, 7 RAEB-I, 10 RAEB-II. We could detect 16 alterations: 4 in granulocytic precursors, 4 in monocytes, increase in CD34+ cells, myeloblasts and not defined immature cells, decrease in B-cell precursors, changes in precursor lymphoid dendritic cells and basophilic precursors. The total number of changes in cases with <5% BM blasts was 6 (2-15) and in RAEB 8 (4-12). The number of alterations had a positive correlation with the WHO type, IPSS, %blasts (cytology) (r=0.38; p=0.001), %CD34+ cells (r=0.40; p=0.001), CD34+/CD13+ cells (r=0.61; p<0.0001), but also non-lymphoid immature cells (CD34+/CD13- r=0.30; p=0.02 e CD34+/CD117- r=0.55; p=0.0003). The B-precursors cells and hemoglobin decreased with the increase of number alterations (r=-0.39; p=0.001 and r=-0.30; p=0.01 respectively). We conclude that our panel, although small, was sufficient to make the diagnosis of MDS in 91% of our cases, and permitted to detect features associated with aggressive cases. / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

As sÃndromes mielodisplÃsicas (SMD) representam um grupo heterogÃneo de doenÃas clonais que acometem a cÃlula precursora hematopoÃtica pluripotente, caracterizando-se por baixa contagem de cÃlulas no sangue perifÃrico, displasia em uma ou mais linhagens celulares, hematopoese ineficiente, alÃm do risco aumentado de progressÃo para leucemia mielÃide aguda. Embora a doenÃa possa acometer pacientes de outras faixas etÃrias, Ã mais frequente naqueles com idade avanÃada, com mÃdia ao diagnÃstico de 60 a 75 anos. As anormalidades cromossÃmicas sÃo observadas em aproximadamente 50% de todos os casos de SMD, estando, em alguns casos, relacionadas com achados clÃnicos e morfolÃgicos. O objetivo deste trabalho foi determinar, atravÃs da tÃcnica de FISH (hibridizaÃÃo in situ por fluorescÃncia), a frequÃncia de alteraÃÃes envolvendo os genes MLL, RB e TP53 em pacientes com SMD e associar estas alteraÃÃes com os achados citogenÃticos. Os casos inseridos no estudo foram oriundos do ambulatÃrio de SMD do Hospital UniversitÃrio Walter CantÃdio. Dos 33 pacientes selecionados, 17 pertenciam ao grupo com idade acima de 60 anos. 52% dos pacientes foram classificados, segundo a OMS, como citopenia refratÃria com displasia em mÃltiplas linhagens (CRDM) e 61% estratificados, segundo o IPSS, como de risco intermediÃrio 1 (INT-1). Um total de 78% dos pacientes apresentaram alteraÃÃes citogenÃticas, dentre eles 31% possuÃam cariÃtipos complexos (mais de 3 alteraÃÃes por metÃfase). A tÃcnica de FISH permitiu identificar em 18% dos pacientes alteraÃÃes envolvendo um dos trÃs genes avaliados. TrÃs pacientes apresentaram alteraÃÃo do gene TP53, sendo detectada em dois deles (registros 31 e 6) a deleÃÃo de um Ãnico alelo ou de ambos os alelos do gene, respectivamente, e no terceiro (registro 2), detectou-se a amplificaÃÃo do gene TP53, sendo estas alteraÃÃes nÃo visualizadas atravÃs da citogenÃtica clÃssica, por se tratar de um tÃcnica menos sensÃvel. Detectou-se em 6% dos pacientes (registros 7 e 22) rearranjo do gene MLL, no primeiro a FISH descartou a suposta deleÃÃo do gene alegada pela citogenÃtica, provando que o mesmo estava presente no genoma do paciente, porÃm de forma rearranjada e no segundo a citogenÃtica nÃo conseguiu demonstrar o rearranjo do gene. Quanto ao gene RB, a FISH permitiu identificar apenas um paciente (3%) com deleÃÃo de um dos alelos do gene, sendo esta alteraÃÃo tambÃm nÃo detectada pela citogenÃtica clÃssica. A FISH possibilitou identificar, durante a avaliaÃÃo do gene TP53, dois pacientes (registros 5 e 10) apresentando pelo menos 6 cÃpias extras do cromossomo 17, devendo essa alteraÃÃo se tratar de um pequeno clone hiperdiplÃide detectado parcialmente no primeiro paciente e nÃo detectado no segundo. Nos seis pacientes que apresentaram alteraÃÃo dos genes avaliados, a FISH proveu informaÃÃes que adicionaram, confirmaram ou alteraram o resultado previamente emitido pela citogenÃtica clÃssica, sendo estas uma das principais aplicaÃÃes desta tÃcnica devido sua alta sensibilidade quando comparada ao mÃtodo clÃssico. / Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal disorders affecting the hematopoietic pluripotent cell, characterized by low cell counts in peripheral blood, dysplasia in one or more cell lines, inefficient hematopoiesis and increased risk of progression to acute myeloid leukemia. Although the disease can affect patients of other age groups, they are more frequent in those with advanced age with an average 60 to 75 years at diagnosis. Chromosomal abnormalities are observed in approximately 50% of all cases of MDS and are related with clinical and morphological findings. The aim of this study was to determine, through the technique of FISH (fluorescence in situ hybridization), the frequency of changes involving the MLL, RB, and TP53 genes in patients with MDS and associate these changes with cytogenetic findings. The cases included in the study were selected in the ambulatory of SMD from University Hospital Walter CantÃdio. Thirty three patients were selected, 17 had aged over 60 years. 52% of patients were classified, according to WHO criteria, as refractory cytopenia with dysplasia in multiple lineages (RCDM) and 61% stratified, according to IPSS, as intermediate risk 1 (INT-1). 78% of patients had abnormalities detected by cytogenetics, among them 31% had complex karyotypes (more than 3 changes per metaphase). 18% of patients had changes at least in one of the three genes valued in this study by FISH. Three patients showed alterations of TP53 gene, being detected in two patients (records 31 and 6) the deletion of a single allele or both alleles of the gene, respectively, and in the third (record 2), we detected amplification of TP53 gene, all this changes were not detected by classical cytogenetics, because it is a less sensitive technique. 6% of patients (records 7 and 22) had rearrangement of MLL gene. In the first case, FISH discarded the gene deletion alleged by cytogenetic, proving that it was present in the genome of the patient, but in a rearranged form, and in the second case cytogenetics failed to demonstrate rearrangement of the gene. For the RB gene, FISH identified only one patient (3%) with deletion of one allele of the gene, and this change was also not detected by classical cytogenetics. During evaluating the TP53 gene, FISH allowed identification of two patients (records 5 and 10) presenting at least six extra copies of chromosome 17, probably representing a small hyperdiploid clone partially detected in the first patient and not detected in the second . In the six patients who showed abnormalities of the genes analyzed, FISH has provided information that added, changed or confirmed the result previously given by classical cytogenetics, which are a major application of this technique due to its high sensitivity compared to the traditional method.

A síndrome mielodisplásica (SMD) é uma doença hematológica heterogênea, caracterizada por hematopoese anormal, displasia e instabilidade genômica, portanto, a análise citogenética é determinante no diagnóstico, prognóstico e acompanhamento evolutivo da doença. Considerando que as células hematopoéticas (CHs) e as estromais mesenquimais multipotentes (CTMs) estão em estreita associação, estudos que visem à caracterização destas poderão contribuir para elucidar os mecanismos que governam a progressão tumoral e identificar novos alvos terapêuticos. Objetivo: Caracterizar e comparar as CHs e CTMs derivadas de pacientes através da citogenética convencional e molecular para os genes aurora cinase A e B. Avaliar as propriedades biológicas das CTMs derivadas de SMD e controles saudáveis. Métodos: o estudo iniciou-se com a avaliação clinica de 25 pacientes e 8 controles saudáveis doo HCFMRP-USP e HAC-Jaú. Em seguida, foi realizada a análise cariótipica das CHs e CTMs da medula óssea pelo bandamento G e por FISH para os genes aurora A e B e o perfil imunofenotípico, bem como potencial de diferenciação em adipócito e osteócito das CTMs de pacientes portadores de SMD e controles saudáveis. Resultados: A avaliação clínica mostrou plaquetopenia (76%), neutropenia (100%), hemoglobina baixa (16%). A análise citogenética das CHs revelou cariótipo alterado em 13 pacientes (52%), com cariótipo complexo resultando em alterações numéricas e estruturais. Ao contrário, nas CTMs, o cariótipo se mostrou alterado em sete pacientes (28%) e um padrão de menor complexidade, apenas quatro pacientes apresentaram alterações nas duas populações celulares, porém, diferentes. Foram encontradas apenas alterações numéricas (sendo 86% monossomia e 14% ganho de cromossomo). As CHs e CTMs dos controles apresentaram cariótipos 100% normais. Na análise de FISH não foi evidenciada amplificação dos genes AURKA e AURKB. As CTMs dos pacientes e controles apresentaram-se semelhantes quanto à morfologia e potencial de diferenciação. Entretanto, as CTMs de pacientes mostraram-se alteradas para dois antígenos de superfície, CD90 e CD146, os quais mostraram níveis de expressão mais elevados nas amostras dos pacientes (p= 0,04, p = 0,001 respectivamente). Conclusão: Observou-se que as CTMs se encontram alteradas embora em menor frequência e diferindo das alterações encontradas nas CHs. Esses dados sugerem que as CTMs devem exercer importante papel na progressão tumoral e devem ser consideradas como alvos na busca de novas terapias e melhor esclarecimento dos mecanismos que governam a progressão tumoral. Apesar de não ter evidenciado amplificação dos genes AURKA e AURKB em SMD, estudos futuros que visem avaliar o nível de expressão dessas enzimas em pacientes portadores ou não de alterações citogenéticas poderão contribuir para a compreensão do envolvimento ou não desse gene com a evolução da doença. Além disso, não foi evidenciada associação de anemia profunda e citogenética alterada. / The myelodysplastic syndrome (MDS) is a heterogeneous hematologic disease characterized by abnormal hematopoiesis, dysplasia and genomic instability, therefore, cytogenetic analysis is crucial in the diagnosis, prognosis and monitoring of disease evolution. Whereas hematopoietic cells (CHs) and stromal multipotent mesenchymal (MSCs) are in close association studies aimed at the characterization of these may help to elucidate the mechanisms that govern tumor progression and identify novel therapeutic targets. Objective: To characterize and compare the CHs and MSCs derived from patients by conventional cytogenetics and molecular genes aurora kinase A and B. To evaluate the biological properties of MSCs derived from MDS and healthy controls. Methods: The study began with the clinical evaluation of 25 patients and eight healthy controls HCFMRP dooUSP and CH-Jau. Next, we performed a karyotypic analysis of CHs and MSCs from bone marrow by G-banding and FISH for aurora A and B genes and immunophenotypic profile and potential to differentiate into adipocytes and osteocytes of MSCs in patients with MDS and controls healthy. Results: The clinical evaluation showed thrombocytopenia (76%), neutropenia (100%), low hemoglobin (16%). The cytogenetic analysis revealed karyotype of CHs changed in 13 patients (52%), resulting in complex karyotype with numerical and structural changes. In contrast, in MSC, the karyotype was abnormal in seven patients (28%) and a pattern of lower complexity, only four patients had changes in both cell populations, however, different. Were found only numerical changes (monosomy being 86% and 14% gain in chromosome). The CHs and MSCs controls showed 100% normal karyotypes. In FISH analysis there was no evidence of gene amplification and AURKA AURKB. The MSCs of patients and controls were similar regarding the morphology and differentiation potential. However, the CTMs of patients proved to be changed to two surface antigens, CD90 and CD146, which showed higher expression levels in samples of patients (p = 0.04, p = 0.001 respectively). Conclusion: Furthermore, it was observed that the MSCs are changed although less frequently and differing from changes found in CHs. These data suggest that MSCs should play an important role in tumor progression and should be considered as targets in the search for new therapies and better explain the mechanisms that govern tumor progression. Although not shown AURKA amplification of genes in MDS and AURKB, future studies aimed at assessing the level of expression of these enzymes in patients with or without cytogenetic alterations may contribute to the understanding of the involvement or not of this gene with the disease. This study can not associate with profound anemia cytogenetic changes.

Myelodysplastic syndrome (MDS) is a heterogeneous group of diseases characterized by ineffective hematopoiesis which is caused by damage of differentiation of pluripotent haematopoietic stem cells. TP53 gene mutations are identified approximately in 10% of MDS and represent a negative prognostic factor. Altered TP53 gene expression may have similar effect as the mutation. Mutations or deregulated expression of this gene have an impact on many cellular processes including apoptosis, DNA repair, cell growth and angiogenesis. In this work, the expression mRNA levels of genes involved in p53 signalling pathway were studied in CD34+ pluripotent haematopoietic cells from bone marrow of patients with low- risk MDS. MDS patients showed increased expression of genes involved in apoptosis induction, regulation of cell cycle and DNA repair (BAX, BBC3, CCNE1, CDC25A, CDKN1A, FAS, GADD45A) as compared to healthy subjects. The patients with TP53 mutation had decreased expression of apoptotic genes (BAX, PIDD, TRAF2) and increased gene expression of apoptotic inhibitor (BCL2A1), indicating a reduced activity of apoptotic pathways and that way the pathological cell clone may gain a growth advantage. Deregulation of 21 genes (BAX, BBC3, EGR1, KAT2B, MDM2 etc.) was observed in patients with del (5q) compared to...

A síndrome mielodisplásica (SMD) é uma doença hematológica heterogênea, caracterizada por hematopoese anormal, displasia e instabilidade genômica, portanto, a análise citogenética é determinante no diagnóstico, prognóstico e acompanhamento evolutivo da doença. Considerando que as células hematopoéticas (CHs) e as estromais mesenquimais multipotentes (CTMs) estão em estreita associação, estudos que visem à caracterização destas poderão contribuir para elucidar os mecanismos que governam a progressão tumoral e identificar novos alvos terapêuticos. Objetivo: Caracterizar e comparar as CHs e CTMs derivadas de pacientes através da citogenética convencional e molecular para os genes aurora cinase A e B. Avaliar as propriedades biológicas das CTMs derivadas de SMD e controles saudáveis. Métodos: o estudo iniciou-se com a avaliação clinica de 25 pacientes e 8 controles saudáveis doo HCFMRP-USP e HAC-Jaú. Em seguida, foi realizada a análise cariótipica das CHs e CTMs da medula óssea pelo bandamento G e por FISH para os genes aurora A e B e o perfil imunofenotípico, bem como potencial de diferenciação em adipócito e osteócito das CTMs de pacientes portadores de SMD e controles saudáveis. Resultados: A avaliação clínica mostrou plaquetopenia (76%), neutropenia (100%), hemoglobina baixa (16%). A análise citogenética das CHs revelou cariótipo alterado em 13 pacientes (52%), com cariótipo complexo resultando em alterações numéricas e estruturais. Ao contrário, nas CTMs, o cariótipo se mostrou alterado em sete pacientes (28%) e um padrão de menor complexidade, apenas quatro pacientes apresentaram alterações nas duas populações celulares, porém, diferentes. Foram encontradas apenas alterações numéricas (sendo 86% monossomia e 14% ganho de cromossomo). As CHs e CTMs dos controles apresentaram cariótipos 100% normais. Na análise de FISH não foi evidenciada amplificação dos genes AURKA e AURKB. As CTMs dos pacientes e controles apresentaram-se semelhantes quanto à morfologia e potencial de diferenciação. Entretanto, as CTMs de pacientes mostraram-se alteradas para dois antígenos de superfície, CD90 e CD146, os quais mostraram níveis de expressão mais elevados nas amostras dos pacientes (p= 0,04, p = 0,001 respectivamente). Conclusão: Observou-se que as CTMs se encontram alteradas embora em menor frequência e diferindo das alterações encontradas nas CHs. Esses dados sugerem que as CTMs devem exercer importante papel na progressão tumoral e devem ser consideradas como alvos na busca de novas terapias e melhor esclarecimento dos mecanismos que governam a progressão tumoral. Apesar de não ter evidenciado amplificação dos genes AURKA e AURKB em SMD, estudos futuros que visem avaliar o nível de expressão dessas enzimas em pacientes portadores ou não de alterações citogenéticas poderão contribuir para a compreensão do envolvimento ou não desse gene com a evolução da doença. Além disso, não foi evidenciada associação de anemia profunda e citogenética alterada. / The myelodysplastic syndrome (MDS) is a heterogeneous hematologic disease characterized by abnormal hematopoiesis, dysplasia and genomic instability, therefore, cytogenetic analysis is crucial in the diagnosis, prognosis and monitoring of disease evolution. Whereas hematopoietic cells (CHs) and stromal multipotent mesenchymal (MSCs) are in close association studies aimed at the characterization of these may help to elucidate the mechanisms that govern tumor progression and identify novel therapeutic targets. Objective: To characterize and compare the CHs and MSCs derived from patients by conventional cytogenetics and molecular genes aurora kinase A and B. To evaluate the biological properties of MSCs derived from MDS and healthy controls. Methods: The study began with the clinical evaluation of 25 patients and eight healthy controls HCFMRP dooUSP and CH-Jau. Next, we performed a karyotypic analysis of CHs and MSCs from bone marrow by G-banding and FISH for aurora A and B genes and immunophenotypic profile and potential to differentiate into adipocytes and osteocytes of MSCs in patients with MDS and controls healthy. Results: The clinical evaluation showed thrombocytopenia (76%), neutropenia (100%), low hemoglobin (16%). The cytogenetic analysis revealed karyotype of CHs changed in 13 patients (52%), resulting in complex karyotype with numerical and structural changes. In contrast, in MSC, the karyotype was abnormal in seven patients (28%) and a pattern of lower complexity, only four patients had changes in both cell populations, however, different. Were found only numerical changes (monosomy being 86% and 14% gain in chromosome). The CHs and MSCs controls showed 100% normal karyotypes. In FISH analysis there was no evidence of gene amplification and AURKA AURKB. The MSCs of patients and controls were similar regarding the morphology and differentiation potential. However, the CTMs of patients proved to be changed to two surface antigens, CD90 and CD146, which showed higher expression levels in samples of patients (p = 0.04, p = 0.001 respectively). Conclusion: Furthermore, it was observed that the MSCs are changed although less frequently and differing from changes found in CHs. These data suggest that MSCs should play an important role in tumor progression and should be considered as targets in the search for new therapies and better explain the mechanisms that govern tumor progression. Although not shown AURKA amplification of genes in MDS and AURKB, future studies aimed at assessing the level of expression of these enzymes in patients with or without cytogenetic alterations may contribute to the understanding of the involvement or not of this gene with the disease. This study can not associate with profound anemia cytogenetic changes.

Les syndromes myélodysplasiques (SMD) sont dus à une atteinte oligoclonale de la cellule souche hématopoïétique aboutissant à une dysplasie des lignées myéloïdes, des cytopénies sanguines et une évolution fréquente vers la leucémie aiguë. De nombreuses mutations décrites dans des gènes contrôlant la régulation épigénétique sont responsables de la genèse des SMD. Mais des travaux récents montrent également que des anomalies du microenvironnement médullaire, notamment des cellules stromales mésenchymateuses (CSM), peuvent induire et propager un SMD suggérant l’idée d’une communication intercellulaire étroite entre la niche et les cellules hématopoïétiques. L’invalidation du gène Dicer1 (RNASE de type III impliquée dans le processing des microARN) dans les progéniteurs ostéoblastiques murins induit un véritable SMD avec dysmyélopoïèse.Nous avons confirmé la sous-expression de Dicer1 dans les CSM SMD à partir de prélèvements primaires de moelle totale et dans les CSM en expansion. La sous-expression de Dicer1 s’accompagne d’une dérégulation du profil des microARN au sein de CSM SMD mise en évidence par étude transcriptomique des CSM SMD vs CSM témoins. Nous avons découvert une possible cible thérapeutique : le miR-486-5p que nous avons retrouvé constamment surexprimé dans les CSM SMD. Un des moyens pour les CSM d’influer sur les cellules souches hématopoïétiques peut se faire par la sécrétion de vésicules extracellulaires (EVs). Ces EVs sont hétérogènes et peuvent être définies par leur taille. Nous nous sommes plus particulièrement intéressés aux petites vésicules extracellulaires (sEVs) contenant la fraction exosomale qui est connue comme pouvant transporter des microARN, mARN et des protéines entre les cellules. Nous avons retrouvé ce miR-486-5p transporté comme cargo dans les sEVs sécrétées des CSM, des CSM vers les CD34+. De plus, nous montrons dans un modèle de co-incubation (sEVs avec CD34+ de sujets sains), que sur le plan fonctionnel, les sEVs provenant de CSM SMD induisent plus d’apoptose, plus de stress oxydatif ainsi que plus de dommage à l’ADN.Par ailleurs, la surcharge martiale observée chez les patients SMD est également responsable d’un stress oxydatif. Le déférasirox (DFX), un chélaleur de fer, a montré dans le cadre d’études rétrospectives une amélioration de l’érythropoïèse chez des patients SMD. Grâce à un modèle de différenciation érythroïde avec surcharge martiale, nous avons montré que de faibles doses de DFX induisent une meilleure prolifération des progéniteurs érythroïdes (moins d’apoptose et plus de cellules en cycle) via une activation de NF-κB. Cette activation est due à une diminution du niveau de dérivés réactifs de l’oxygène (ROS) en rapport avec une diminution du fer labile et est contrôlée de manière très fine par le niveau de ROS.Enfin, nous avons utilisé les propriétés du microenvironnement médullaire pour établir un modèle murin de SMD humain. En effet, la relative incapacité des cellules souches myélodysplasiques humaines de greffer et de reconstituer une hématopoïèse pathologique dans des souris immunodéprimées suggère que ces cellules souches SMD doivent avoir besoin d’un support extrinsèque du microenvironnement. Nous avons réalisé un modèle de souris humanisées en co-injectant des CSM et des CD34+ en intratibial. Une prise de greffe a été observée chez toutes les souris injectées et avons pu étudier l’évolution clonale au fil des générations dans les différentes sous-populations de progéniteurs myéloïdes (common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP) and megakaryocyte–erythroid progenitor (MEP)). Notre modèle est stable au cours des générations avec persistance du clone fondateur initial.En conclusion, ce travail confirme le rôle prépondérant du microenvironnement médullaire dans la genèse et la physiopathologie des syndromes myélodysplasiques et ouvre la voie à de nouvelles possibilités thérapeutiques. / Myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) oligoclonal diseases leading to dysplasia, blood cytopenia and evolution to acute leukemia. Numerous mutations in genes involved in epigenetic regulation are responsible of MDS genesis. But recently, studies show that medullar microenvironment, particularly mesenchymal stromal cells (MSC), could induces and propagates a truly MDS suggesting a narrow communication between HSC and this niche. Dicer1’s (type III RNAse implicating in microRNA processing) invalidation in murine osteoblastic progenitors induces a MDS with sign of dysplasia.In this work, we have confirmed the under expression of Dicer1 in MDS mesenchymal stromal cells from total bone marrow and cultured MSC. Dicer1 down regulation leads to a deregulation of miRNome profile in MDS MSC highlighted by transcriptomic approaches. We found a potential therapeutic target: miR-486-5p which is constantly overexpressed in MDS MSC. Extracellular vesicles (EVs) could be a possible way for MSC to influence HSC fates. Those EVs are heterogeneous are could be characterized by their sight. We mainly focused on small EVs (sEVs) containing the exosomal fraction known to be able to carry miRNA, mRNA and proteins. We found that miR-486-5p is carry from MSC to the HSC. Transcriptomic analyses of HD HSC overexpressing miR-486-5p are ongoing. Moreover, in a co-incubation model (sEVs and healthy donor (HD) HSC), sEVs coming from MDS MSC induced apoptosis, oxidative stress and DNA damages.Moreover, iron overload seen in MDS patients is also able to induce DNA damages and oxidative stress. Deferasirox (DFX), an iron chelator, has shown an erythropoiesis improvement in MDS patients. Using an erythroid differentiation model with iron overload, we have observed that low dose of DFX induce a better proliferation of erythroid progenitors (less apoptosis and more cycling cells) due to NF-κB activation. This activation is due to a decrease of reactive oxygen species level in relation to a decrease of the labile iron pool.Finally, we have used medullar microenvironment properties to establish a murine model of MDS. Indeed, MDS HSC incapacity to reconstitute a pathological hematopoiesis in immunocompromised mice suggests that MDS HSC need an extrinsic support from the microenvironment. We have engineered a MDS patient derived xenograft (PDX) model by intra-tibial co-injection CD34+ cells with MSC. All mice engrafted et we have follow the clonal evolution over mice generation in the different subset of myeloid progenitors. (common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP) and megakaryocyte–erythroid progenitor (MEP)). Our model is stable over generations with persistence of the initial founding clone.In conclusion, this work confirms the preponderant role of the medullary microenvironment in the genesis and physiopathology of myelodysplastic syndromes and opens the way to new therapeutic possibilities.

No description available.