Τα μυελοδυσπλαστικά σύνδρομα (ΜΔΣ) χαρακτηρίζονται από μη αποδοτική αιμοποίηση, αναστολή ωρίμανσης των προγονικών κυττάρων του μυελού και αυξημένη ενδομυελική απόπτωση. Η συσχέτιση του ΜΔΣ με την παρουσία αυξημένου οξειδωτικού στρες και οξειδωτικά τροποποιημένων πρωτεϊνών στα προγονικά αιμοποιητικά κύτταρα του μυελού έχει προηγουμένως αναφερθεί. Πρόσφατα βιβλιογραφικά δεδομένα δηλώνουν τη δυνατότητα διάκρισης δύο υποπληθυσμών της μυελικής σειράς βάσει σκεδαστικών χαρακτηριστικών και της έκφρασης του CD45, CD45dim (Neutrophil granulocytic subpopulation-1, NGS1) και CD45high (Neutrophil granulocytic subpopulation-2, NGS2). Ο υποπληθυσμός CD45dim φέρει άωρα κύτταρα, που στην πλειοψηφία τους δεν ωριμάζουν έως τελικού σταδίου[1]. Ο πλήρης χαρακτηρισμός των πληθυσμών αυτών, καθώς επίσης, και η συμμετοχή τους στη μη αποδοτική αιμοποίηση των ΜΔΣ και στην εξέλιξη της νόσου είναι στοιχεία άγνωστα. Σκοπός της παρούσης εργασίας ήταν η διερεύνηση δεικτών απόπτωσης και οξειδωτικού φορτίου (ελεύθερες ρίζες οξυγόνου-ROS) στους υποπληθυσμούς της μυελικής σειράς CD45dim και CD45high. Μελετήθηκαν 17 δείγματα μυελού ασθενών με ΜΔΣ μετά από ανάλυση συνδυασμού 5 χρωμάτων με κυτταρομετρία ροής, για τους δείκτες CD11b/CD16, annexin-V/7-AAD και 2,7-dichlorodihydrofluo-rescein-diacetate(DCF). Η στατιστική ανάλυση των αποτελεσμάτων πραγματοποιήθηκε με τη χρήση του κατάλληλου προγράμματος με μη παραμετρικά κριτήρια (Wilcoxon & Mann-Whitney). Το ποσοστό των αποπτωτικών 7-AAD/annexin-V+ κυττάρων ήταν σημαντικά αυξημένο στα CD45high/ CD11b+/CD16+ της μυελικής σειράς συγκριτικά με τα CD45dim/CD11b+/CD16- κύτταρα. Τα CD45dim και CD45high κύτταρα μυελού με τη χρήση του ειδικού ανιχνευτή των επιπέδων των ελεύθερων ριζών οξυγόνου DCF αναλύθηκαν σε ROSLow και ROSHigh πληθυσμούς. Η ανάλυση με τη χρήση των παραπάνω κριτηρίων έδειξε σημαντική αύξηση του ποσοστού των ROSHigh κυττάρων στα CD45high κύτταρα, συμπεραίνοντας έτσι ότι ο υποπληθυσμός CD45dim έχει χαμηλότερα επίπεδα ενδοκυττάριων ROS εν συγκρίσει με τον CD45high υποπληθυσμό. Ο CD45high υποπληθυσμός της μυελικής σειράς χαρακτηρίζεται από μεγαλύτερα ποσοστά έκφρασης αποπτωτικών δεικτών καθώς επίσης και από τη παρουσία αυξημένου οξειδωτικού φορτίου συγκριτικά με τα CD45dim κύτταρα. Η περεταίρω διερεύνηση του ρόλου των πληθυσμών αυτών στις διαδικασίες ανάπτυξης του ΜΔΣ και στην εξέλιξη της νόσου είναι υπό εξέλιξη. / --

Η Οξεία λευχαιμία (ΟΛ) αποτελεί νεοπλασματικό, αιμοποιητικό νόσημα, που οφείλεται στον πολλαπλασιασμό και την επέκταση κυττάρων, που προέρχονται από τους λευχαιμικούς βλάστες. Η φυσική εξέλιξη του νοσήματος είναι η αντικατάσταση των φυσιολογικών κυττάρων του αιμοποιητικού ιστού από τους απόγονους των λευχαιμικών βλαστών και θάνατος, λόγω των επιπλοκών της έλλειψης των ώριμων αιμοποιητικών κυττάρων, όπως λοιμώξεις, αναιμία και αιμορραγία. Θεραπευτικά για την αντιμετώπιση της ΟΛ χρησιμοποιούνται σχήματα χημειοθεραπείας και ακτινοθεραπείας, που έχουν σαν σκοπό την καταστροφή των λευχαιμικών βλαστών και την αποκατάσταση της φυσιολογικής αιμοποίησης. Συχνά η ΟΛ, ιδιαίτερα σε ασθενείς μεγάλης ηλικίας, εμφανίζεται ταυτόχρονα με δυσπλαστικές διαταραχές των αιμοποιητικών κυττάρων που ωριμάζουν ή εξελίσσεται σε μυελοδυσπλαστικό σύνδρομο μετά από χημειοθεραπεία. Από την βιβλιογραφία είναι γνωστό ότι, τόσο η ΟΛ μπορεί να είναι στάδιο εξέλιξης των μυελοδυσπλαστικών συνδρόμων και κατά συνέπεια μετά τη θεραπεία της ΟΛ επανέρχεται η δυσπλαστική κατάσταση της αιμοποίησης, όσο ότι η χημειοθεραπεία καθαυτή μπορεί να προκαλέσει μυελοδυσπλασία. Είναι σημαντική η διάγνωση των πρωτοπαθών μυελοδυσπλαστικών συνδρόμων που εξελίσσονται σε ΟΛ, ως προς την πρόγνωση των ασθενών, αλλά και τη θεραπευτική τους αντιμετώπιση. Επίσης, είναι σημαντική η διάκριση ομάδων ασθενών που θα αναπτύξουν δυσπλασία μετά από χημειοθεραπεία, σε σχέση με αυτούς που δεν θα αναπτύξουν και ως προς την πρόγνωση και ως προς τη θεραπευτική αντιμετώπιση. Μέχρι σήμερα, κατά την εμφάνιση της ΟΛ η διάγνωση υποκείμενης μυελοδυσπλασίας είναι δύσκολη και στηρίζεται σε μορφολογικά χαρακτηριστικά των ώριμων κυττάρων και στην παρουσία ορισμένων κυτταρογενετικών διαταραχών. Η διάκριση ομάδων που θα αναπτύξουν δυσπλασία μετά από χημειοθεραπεία είναι αδύνατη. Σκοπός της μελέτης ήταν να συμβάλλει στην ανάπτυξη νέων πρωτοκόλλων στο λογισμικό της κυτταρομετρίας ροής, που θα διευκολύνουν τη διάγνωση πρωτοπαθών δυσπλαστικών συνδρόμων κατά την εμφάνιση της ΟΛ, αλλά και θα διακρίνουν τις ομάδες που μπορούν να αναπτύξουν δυσπλασία μετά από θεραπεία. Για τον σκοπό αυτό, παρακολουθήσαμε την έκφραση χαρακτηριστικών αντιγονικών συνδυασμών, που εκφράζονται σε διαφορετικά στάδια ωρίμανσης των φυσιολογικών κυττάρων, παράλληλα με την έκφραση των αντιγόνων των βλαστών. Τα δεδομένα αυτά μελετήθηκαν, τόσο κατά την εμφάνιση της ΟΛ, όσο και κατά τη παρακολούθηση της. Τα δεδομένα αναλύθηκαν με συστήματα ταυτόχρονης ανάλυσης και συσχέτισης 15-20 παραμέτρων, με σκοπό τον καθορισμό συσχετισμών που θα έχουν διαγνωστική και προγνωστική σημασία για τους ασθενείς με ΟΛ. Τα αποτελέσματα του ανοσοφαινοτύπου αναλύθηκαν, επιπλέον, με το λογισμικό πακέτο στατιστικής ανάλυσης SPSS 16.0. Για τους σκοπούς της μελέτης αναλύθηκαν αναδρομικά τα αποτελέσματα της κυτταρομετρίας ροής στο μυελό των οστών 148 ασθενών με ΟΜΛ κατά την εμφάνιση της νόσου, κατά την διάρκεια και μετά από θεραπεία. Αναλύθηκε η έκφραση των αντιγόνων CD11b/CD16/CD13 σε όλα τα στάδια ωρίμανσης της μυελικής σειράς, ενώ η έκφραση των αντιγόνων CD34/CD117 μόνο στα άωρα κύτταρα. Τα ευρήματα του ανοσοφαινοτύπου συγκρίθηκαν με τα μορφολογικά χαρακτηριστικά των αντίστοιχων μυελών των οστών. Συμπερασματικά, τα αποτελέσματα έδειξαν ότι, η έκφραση των αντιγόνων CD11b και CD13 στα μεταμυελοκύτταρα και ουδετερόφιλα των ασθενών διακρίνει αποτελεσματικά τους ασθενείς με de novo ΟΜΛ σε σχέση με αυτούς που εμφάνισαν ΟΜΛ μετά από ΜΔΣ. Στους ασθενείς με de novo ΟΜΛ η έκφραση των αντιγόνων CD11b, CD13, CD16 δεν διέφερε κατά την εμφάνιση της ΟΛ στους υποπληθυσμούς των προμυελοκυττάρων, μυελοκυττάρων, μεταμυελοκυττάρων και ουδετερόφιλων μεταξύ των ασθενών που κατά ή μετά την θεραπεία εμφάνισαν μυελοδυσπλασία, σε σχέση με αυτούς που δεν εμφάνισαν. Επίσης, η θετική συν- έκφραση των αντιγόνων CD34/CD117 στους λευχαιμικούς βλάστες κατά την εμφάνιση δεν συσχετίζονταν με την εμφάνιση ΜΔΣ μετά την θεραπεία. Αντίθετα, η υψηλή έκφραση του λευχαιμικού φαινοτύπου CD34+/CD117- στα άωρα κύτταρα της μυελικής σειράς κατά την εμφάνιση της ΟΜΛ, έδειξε ότι σχετίζεται με την εμφάνιση μυελοδυσπλαστικών χαρακτηριστικών μετά τη θεραπεία. Η ανάλυση των ανοσοφαινοτύπων του μυελού των οστών κατά ή μετά την θεραπεία έδειξε παθολογική έκφραση CD11b, CD13, CD16 στα μεταμυελοκύτταρα και ουδετερόφιλα των ασθενών που εμφάνισαν ΜΔΣ μετά θεραπεία για de novo ΟΜΛ και ασθενών (5/17) που δεν εμφάνισαν ΜΔΣ. Συμπερασματικά, η έκφραση των αντιγόνων CD11b, CD16 και CD13 στα ώριμα κύτταρα της μυελικής σειράς κατά την εμφάνιση της ΟΜΛ διαχωρίζει αποτελεσματικά την de novo ΟΜΛ από την δευτεροπαθή μετά ΜΔΣ. Η έκφραση αυτών των αντιγόνων κατά την εμφάνιση της ΟΜΛ δεν μπορεί, όμως, να προβλέψει την εξέλιξη της de novo ΟΜΛ και την εμφάνιση δυσπλαστικών χαρακτηριστικών μετά τη θεραπεία. Αντιθέτως, η μελέτη των λευχαιμικών φαινοτύπων CD34+/CD117- και CD34+/CD117+ μπορεί να παίξει καθοριστικό ρόλο στην πρόγνωση της εμφάνισης μυελοδυσπλαστικών χαρακτηριστικών κατά τη διάρκεια ή μετά τη θεραπεία του ασθενούς. / --

Orientador: Agnes Cristina Fett Conte / Banca: Cleide Largman Borovik / Banca: Cláudia Regina Bonini Domingos / Resumo: 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... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: 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) / Mestre

A SMD primária hipocelular ocorre com uma frequência de 10-20% dos casos de SMD no adulto, no entanto, é o subtipo mais frequente na infância. O diagnóstico da SMD primária hipocelular é bastante difícil, pois devido à ausência de células na medula óssea esta pode ser confundida com a LMA hipocelular ou AA. O diagnóstico diferencial entre estas entidades hematológicas é de extrema importância devido a maior agressividade da LMA e a possibilidade de evolução da SMD para LMA. Além disso, SMD e AA são indicadas para o TCTH, entretanto, o regime de condicionamento pré-transplante é específico para cada doença. A combinação entre a análise morfológica, realizada através do mielograma e biópsia de medula óssea, e análise citogenética tem desempenhado um papel fundamental no reconhecimento da SMD primária hipocelular. Entretanto, estudos têm sido realizados para tentar melhorar o diagnóstico da doença levando em consideração as características biológicas da SMD como a presença de apoptose. Sendo assim, este estudo teve como objetivo caracterizar o padrão cromossômico da SMD primária hipocelular e correlacionar com aspectos celulares e clínicos. Foram analisados citogeneticamente 86 casos de SMD primária hipocelular, 74 AR, 10 com AREB e 2 com AREB-t. Dentre os pacientes com AR 50% apresentaram cariótipo anormal e todos os pacientes com AREB e AREB-t apresentaram cariótipo anormal. A alteração cromossômica mais frequente foi a del(17p), seguida de alterações envolvendo o cromossomo 7. Nossos resultados sugerem que o padrão cromossômico em SMD primária hipocelular é caracterizado principalmente por perdas parciais e completas de cromossomos (deleções e monossomias). A análise citogenética auxiliou no diagnóstico dos casos com suspeita de SMD primária hipocelular e foi uma importante ferramenta para a escolha do tratamento. O IPSS mostrou ser um bom sistema de escala prognostica para este grupo de pacientes. Alterações envolvendo o cromossomo 17 estiveram associados com o subtipo AR e características displásicas envolvendo o setor granulocítico, no entanto, a del(17p) também pôde ser observada no subtipo AREB. Para análise de apoptose foram utilizadas 42 amostras de pacientes com SMD, 23 com SMD hipocelular, 8 com SMD normocelular e 11 com SMD hipercelular. O índice de apoptose total nos casos de SMD primária hipocelular apresentou uma média de 9,5%, enquanto os pacientes com SMD primária normocelular e hipercelular apresentaram uma média de 12% e 14,1%, respectivamente. Pela análise de linhagens específicas as células já comprometidas com o programa de diferenciação celular parecem ser o principal alvo do programa de apoptose. Apesar dos pacientes com SMD primária hipocelular apresentarem índice de apoptose total mais elevado que os controles eles foram sempre inferiores aos apresentados pela SMD primária normocelular e hipercelular, com exceção dos eritroblastos que foram maiores nos casos de SMD primária hipocelular. O índice de apoptose total foi maior nos estágios iniciais da doença independentemente da celularidade da medula óssea. Os pacientes com del (11q) e del (17p) estiveram associados com a diminuição do índice de apoptose total. Nossos resultados sugerem que a hipocelularidade da medula óssea não é causada pelo processo de apoptose e sim provavelmente por algum defeito no programa de proliferação celular. / Hypocellular primary MDS occurs in a frequency of 10-20% of the adults MDS cases, however it is the most frequent subtype in childhood. Diagnosis of hypocellular primary MDS is very difficult, because the small number of cells in bone marrow and it can be confused with hypocellular AML or AA. The differential diagnosis between these hematologic entities is extremely important because of AML is more aggressive and the possibility of MDS evolve to AML. Besides, MDS and AA are indicated to HSCT, however, conditioning regimens before the transplantation is specific for each disease. The combination between morphologic analysis, carried out through mielogram and bone marrow biopsy, and cytogenetic analysis have been performed a fundamental role on the recognition of hypocellular primary MDS. However, studies have been carried out to try improving the MDS diagnosis considering the biological characteristics as the presence of apoptosis. Thus, the aim of this study was characterize the chromosomal pattern of hypocellular primary MDS and correlate with cellular and clinic aspects. It was analyzed 86 cases of hypocellular primary MDS, 74 RA, 10 RAEB and 2 RAEB-t. Patients with RA presented 50% of abnormal karyotype and all patients with RAEB presented abnormal karyotype as well as RAEB-t patients. The most frequent chromosomal alterations in this study was del(17p) followed by alterations involving chromosome 7. Our results suggest that chromosomal pattern in hypocellular primary MDS is characterized mainly by partial and complete loss of chromosomes (deletion and monosomy). The cytogenetic analysis aided in diagnosis of cases with suspicion of hypocellular primary MDS and it was an important tool for treatment choice. IPSS showed to be a good prognostic scoring system for this group of patients. Alterations involving chromosome 17 were associated with RA subtype and dysplastic characteristics involving granulocytic setor, however, we could see del(17p) in RAEB patients. For apoptosis analysis were used 42 samples of MDS patients, 23 with primary hypocellular MDS, 8 with primary normocelular MDS and 11 with primary hipercelular MDS. The total apoptosis index in cases of hypocellular primary MDS presented a average of 9,5%, whereas patients with primary normocelular MDS and primary hipercelular MDS presented an average of 12% and 14,1%, respectively. For the analysis of specific lineage cells already commited with cellular proliferation program appears to be the main target of apoptosis program. Despite of patients with primary hypocellular MDS presented total apoptosis index more raised than controls they were always lower than primary normocelular MDS and primary hipercelular MDS, except for eritroblasts that were higher in primary hypocellular. The total apoptosis index was higher in initial stage of the disease independently of the bone marrow cellularity. Patients with del(11q) and del(17p) were associated with decreasing of total apoptosis index. Our results suggest that the hypocellularity of bone marrow is not caused by apoptosis process, but probably by probably some defect in cellular proliferation program.

A SMD primária hipocelular ocorre com uma frequência de 10-20% dos casos de SMD no adulto, no entanto, é o subtipo mais frequente na infância. O diagnóstico da SMD primária hipocelular é bastante difícil, pois devido à ausência de células na medula óssea esta pode ser confundida com a LMA hipocelular ou AA. O diagnóstico diferencial entre estas entidades hematológicas é de extrema importância devido a maior agressividade da LMA e a possibilidade de evolução da SMD para LMA. Além disso, SMD e AA são indicadas para o TCTH, entretanto, o regime de condicionamento pré-transplante é específico para cada doença. A combinação entre a análise morfológica, realizada através do mielograma e biópsia de medula óssea, e análise citogenética tem desempenhado um papel fundamental no reconhecimento da SMD primária hipocelular. Entretanto, estudos têm sido realizados para tentar melhorar o diagnóstico da doença levando em consideração as características biológicas da SMD como a presença de apoptose. Sendo assim, este estudo teve como objetivo caracterizar o padrão cromossômico da SMD primária hipocelular e correlacionar com aspectos celulares e clínicos. Foram analisados citogeneticamente 86 casos de SMD primária hipocelular, 74 AR, 10 com AREB e 2 com AREB-t. Dentre os pacientes com AR 50% apresentaram cariótipo anormal e todos os pacientes com AREB e AREB-t apresentaram cariótipo anormal. A alteração cromossômica mais frequente foi a del(17p), seguida de alterações envolvendo o cromossomo 7. Nossos resultados sugerem que o padrão cromossômico em SMD primária hipocelular é caracterizado principalmente por perdas parciais e completas de cromossomos (deleções e monossomias). A análise citogenética auxiliou no diagnóstico dos casos com suspeita de SMD primária hipocelular e foi uma importante ferramenta para a escolha do tratamento. O IPSS mostrou ser um bom sistema de escala prognostica para este grupo de pacientes. Alterações envolvendo o cromossomo 17 estiveram associados com o subtipo AR e características displásicas envolvendo o setor granulocítico, no entanto, a del(17p) também pôde ser observada no subtipo AREB. Para análise de apoptose foram utilizadas 42 amostras de pacientes com SMD, 23 com SMD hipocelular, 8 com SMD normocelular e 11 com SMD hipercelular. O índice de apoptose total nos casos de SMD primária hipocelular apresentou uma média de 9,5%, enquanto os pacientes com SMD primária normocelular e hipercelular apresentaram uma média de 12% e 14,1%, respectivamente. Pela análise de linhagens específicas as células já comprometidas com o programa de diferenciação celular parecem ser o principal alvo do programa de apoptose. Apesar dos pacientes com SMD primária hipocelular apresentarem índice de apoptose total mais elevado que os controles eles foram sempre inferiores aos apresentados pela SMD primária normocelular e hipercelular, com exceção dos eritroblastos que foram maiores nos casos de SMD primária hipocelular. O índice de apoptose total foi maior nos estágios iniciais da doença independentemente da celularidade da medula óssea. Os pacientes com del (11q) e del (17p) estiveram associados com a diminuição do índice de apoptose total. Nossos resultados sugerem que a hipocelularidade da medula óssea não é causada pelo processo de apoptose e sim provavelmente por algum defeito no programa de proliferação celular. / Hypocellular primary MDS occurs in a frequency of 10-20% of the adults MDS cases, however it is the most frequent subtype in childhood. Diagnosis of hypocellular primary MDS is very difficult, because the small number of cells in bone marrow and it can be confused with hypocellular AML or AA. The differential diagnosis between these hematologic entities is extremely important because of AML is more aggressive and the possibility of MDS evolve to AML. Besides, MDS and AA are indicated to HSCT, however, conditioning regimens before the transplantation is specific for each disease. The combination between morphologic analysis, carried out through mielogram and bone marrow biopsy, and cytogenetic analysis have been performed a fundamental role on the recognition of hypocellular primary MDS. However, studies have been carried out to try improving the MDS diagnosis considering the biological characteristics as the presence of apoptosis. Thus, the aim of this study was characterize the chromosomal pattern of hypocellular primary MDS and correlate with cellular and clinic aspects. It was analyzed 86 cases of hypocellular primary MDS, 74 RA, 10 RAEB and 2 RAEB-t. Patients with RA presented 50% of abnormal karyotype and all patients with RAEB presented abnormal karyotype as well as RAEB-t patients. The most frequent chromosomal alterations in this study was del(17p) followed by alterations involving chromosome 7. Our results suggest that chromosomal pattern in hypocellular primary MDS is characterized mainly by partial and complete loss of chromosomes (deletion and monosomy). The cytogenetic analysis aided in diagnosis of cases with suspicion of hypocellular primary MDS and it was an important tool for treatment choice. IPSS showed to be a good prognostic scoring system for this group of patients. Alterations involving chromosome 17 were associated with RA subtype and dysplastic characteristics involving granulocytic setor, however, we could see del(17p) in RAEB patients. For apoptosis analysis were used 42 samples of MDS patients, 23 with primary hypocellular MDS, 8 with primary normocelular MDS and 11 with primary hipercelular MDS. The total apoptosis index in cases of hypocellular primary MDS presented a average of 9,5%, whereas patients with primary normocelular MDS and primary hipercelular MDS presented an average of 12% and 14,1%, respectively. For the analysis of specific lineage cells already commited with cellular proliferation program appears to be the main target of apoptosis program. Despite of patients with primary hypocellular MDS presented total apoptosis index more raised than controls they were always lower than primary normocelular MDS and primary hipercelular MDS, except for eritroblasts that were higher in primary hypocellular. The total apoptosis index was higher in initial stage of the disease independently of the bone marrow cellularity. Patients with del(11q) and del(17p) were associated with decreasing of total apoptosis index. Our results suggest that the hypocellularity of bone marrow is not caused by apoptosis process, but probably by probably some defect in cellular proliferation program.

Oxidative stress is an important factor in carcinogenesis of oncohematological diseases. However its role in the pathogenesis of myelodysplastic syndromes (MDS) remains unclear. In this study, we have determined the oxidative status and evaluated proteomic changes in plasma of MDS patients as a consequence of oxidative dysbalance (oxidative modifications, protein-protein interaction and complex forming). We measured the levels of total cysteine, homocysteine, cysteinyglycine, glutathione, nitrites and nitrates in the plasma from 61 MDS patients and 23 healthy donors using high performance liquid chromatography. Glutathione and nitrites levels reduced significantly while other aminothiols levels increased significantly in plasma of MDS patients. This association with oxidative stress did not correlate with iron overload. We also found enhanced levels of asymmetric dimethylarginine in serums of middle aged patients with MDS that correlate to posttranslational modifications of proteins arginyl residues. Furthermore, carbonylated proteins level was significantly elevated in MDS patients compared to healthy donors. Using mass spectrometry, 5 S-nitrosylated blood platelets proteins were identified in plasma and blood platelets of MDS patients and set of 16 plasma proteins with high probability of...

Myelodysplastic syndromes (MDS) represent a group of clonal stem cell disorders characterized by ineffective hematopoiesis, peripheral cytopenia, morphological dysplasia and the risk of transformation to acute myeloid leukemia (AML). MDS belongs to one of the most common hematological diseases in patients over 60 years old. MDS incidence is still increasing. Appropriate therapy of MDS remains challenging. There is no curative approach besides peripheral stem cells transplantation, which is regretfully appropriate only for a small group of patients due to a higher median age of the MDS population. This is why the search for therapeutic alternatives remains paramount. MDS treatment was rather frustrating until the recent introduction of two new therapeutic approaches: immunomodulation therapy with lenalidomide and epigenetic or demethylating therapy with 5-azacytidine. Both new drugs have significantly higher effect than standard therapy. However, the precise mechanism of this effect remains unknown. As a result, we decided to initiate several research projects while introducing this promising treatment to our patients. Our aim is to investigate the mechanism of both agents in relation to disease pathogenesis by examining changes of certain occurrences and factors prior to and during the course of...

Orientador: Sara Teresinha Olalla Saad / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-18T22:22:55Z (GMT). No. of bitstreams: 1 Baratti_MarianaOzello_D.pdf: 9890862 bytes, checksum: f7eb8503519cc264f616a79dc48baf4f (MD5) Previous issue date: 2011 / Resumo: As síndromes mielodisplásicas (SMDs) constituem um grupo heterogêneo de desordens hematopoéticas, caracterizadas por exibirem hematopoese ineficaz com evidências de displasia da medula óssea resultando em citopenias no sangue periférico. Tecnologia de microarranjo tem permitido o refinado mapeamento da atividade transcricional do genoma humano. RNAs não codificadores (ncRNAs) transcritos de regiões intrônicas de genes conhecidos estão envolvidos em vários processos relacionados com controle transcricional e pós-transcricional da expressão gênica, interações com cromatinas, modificação de histonas e também estão se tornando evidentes em vários tipos de cânceres. Caracterização de ncRNAs em células progenitoras e células estromais de pacientes com SMD representa uma estratégia aparentemente importante para o entendimento da regulação gênica nesta doença. Neste estudo, o perfil de expressão gênica de células CD34+ e estromais de pacientes com SMD do subgrupo anemia refratária com sideroblastos em anel (ARSA) foi comparado com o de indivíduos saudáveis, usando oligoarranjos de 44 kilobases contendo íntrons e éxons, o qual incluiu sequências para genes codificadores, RNAs sense e antisense totalmente e parcialmente intrônicos. Em células CD34+ de pacientes com SMD-ARSA, 216 genes foram diferencialmente expressos (q-value ? 0,01) em comparação com indivíduos saudáveis, dos quais 65 (30%) eram transcritos não codificadores. O gene DMT1, um transportador de ferro, foi encontrado hiperexpresso em células CD34+ de SMD-ARSA. Em medula óssea total de 34 pacientes, a expressão foi mais evidente no subgrupo de pacientes com SMD de baixo risco/INT-1. Ensaios de imuno-histoquímica corroboram os dados encontrados na análise de expressão gênica e demonstram que DMT1 se encontra mais expresso nas células eritroblásticas. A hiperexpressão de DMT1 pode estar relacionada com o homeostase do ferro anormal nas SMDs. Em células estromais de SMD-ARSA, 12 genes foram diferencialmente expressos (q-value ? 0,05) em comparação com indivíduos saudáveis, dos quais 3 (25%) eram transcritos não codificadores. O gene SEMA3A, um membro secretado da família das semaforinas, foi encontrado hiperexpresso em células estromais de SMD-ARSA e na medula óssea total de 34 pacientes; sua hiperexpressão foi mais evidente em pacientes com SMD de alto risco/INT-2 e em pacientes com leucemia mieloide aguda (n=19). Ensaios funcionais demonstraram que SEMA3A está envolvido com aumento da adesão, diminuição da diferenciação e apoptose de células leucêmicas cocultivadas com células estromais HS27 hiperexpressando SEMA3A e age de maneira parácrina sobre as células precursoras. Pela primeira vez, o perfil diferencial de ncRNA em células CD34+ e células estromais entre SMD-ARSA e indivíduos saudáveis foi demonstrado, sugerindo que ncRNA pode ter um importante papel durante o desenvolvimento das síndromes mielodisplásicas / Abstract: Myelodysplastic syndromes (MDS) are a group heterogeneous of hematological disorders characterized by ineffective hematopoiesis with morphological evidence of marrow cell dysplasia resulting in peripheral blood cytopenia. Microarray technology has permitted a refined high-throughput mapping of the transcriptional activity in the human genome. Noncoding-RNAs (ncRNAs) transcribed from intronic regions of genes are involved in a number of processes related to post-transcriptional control of gene expression, and chromatins interaction, and histone modification and they are becoming evident in several cancers. Characterization of ncRNAs in progenitor cells and stromal cells of MDS patients could be strategic for understanding gene expression regulation in this disease. In this study, gene expression profiles of CD34+ and stromal cells of MDS patients with refractory anemia with ringed sideroblasts (RARS) subgroup were compared those of healthy individuals, using 44 kilobases combined introns and exons oligoarrays, which included probes for protein-coding genes, for sense and antisense strands of totally and partially intronic noncoding RNAs. In CD34+ cells of MDS-RARS patients, 216 genes were significantly differentially expressed (q-value ? 0.01) in comparison to healthy individuals, of which 65 (30%) were noncoding transcripts. The DMT1 gene, an iron-transporter, was found up-regulated in CD34+ cells of MDS-RARS. In the total bone marrow of 34 patients, the expression of DMT1 was more evident in the subgroup of low risk/INT-1 MDS patients. The immunohistochemistry assay confirms the data obtained in the gene expression assay and show that DMT1 is more expressed in erythroid cells. The higher expression of DMT1 can be related with abnormal iron homeostasis in MDS. In stromal cells of MDS-RARS, 12 genes were significantly differentially expressed (q-value ? 0.05) in comparison to healthy individuals, of which 3 (25%) were noncoding transcripts. The SEMA3A gene, a secreted member of the semaphorins family, was found up-regulated in stromal cells of MDS-RARS and in the total bone marrow of 34 patients; further, the higher expression was more evident in high risk/ INT-2 subgroup of MDS patients and acute myeloid leukemia patients (n = 19). Functional assays demonstrated that SEMA3A is related to adhesion increase, differentiation decrease, and apoptosis of leukemia cells cocultivated with HS27 stromal cells higher expressing SEMA3A, also acting in a paracrine fashion in the precursors cells. These results demonstrated, for the first time, in CD34+ cells and stromal cells the differential ncRNA expression profile between MDSRARS and healthy individuals, suggesting that ncRNAs may play an important role during the development of myelodysplastic syndromes / Doutorado / Medicina Experimental / Doutor em Fisiopatologia Medica

Orientadores: Lilian Maria de Castilho, Jeane Eliete Laguila Visentainer / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T13:14:12Z (GMT). No. of bitstreams: 1 Guelsin_GlauciaAndreiaSoares_D.pdf: 3524338 bytes, checksum: 77312cdc47d244e00235a67d7f52c666 (MD5) Previous issue date: 2014 / Resumo: A síndrome mielosplásica (SMD) correponde a um grupo de distúrbios clonais heterogêneos decorrente de um defeito intrínseco das células progenitoras hematopoéticas, resultando em insuficiência da medula óssea, desencadeando anemia com dependência de transfusões sanguíneas e infecções decorrentes da neutropenia. Embora a terapia transfusional seja segura, muitos desses pacientes correm risco de complicações relacionadas à sobrecarga de ferro e à aloimunização contra antígenos de grupos sanguíneos, que muitas vezes dificulta a busca de sangue compatível para esses pacientes, além estar associada a reações hemolíticas transfusionais tardias e formação de auto-anticorpos. A compatibilidade para antígenos Rh e K tem sido utilizada na tentativa de reduzir a formação de anticorpos em pacientes que recebem transfusões crônicas, mas a fenotipagem estendida, incluindo os antígenos Fya e Jka, também tem sido recomendada. Este estudo teve como objetivos avaliar o perfil transfusional dos pacientes com SMD, propor um protocolo de compatibilidade molecular para seleção de sangue fenótipo compatível e avaliar uma possível associação dos alelos HLA com a susceptibilidade ou proteção a aloimunização eritrocitária. Foram analisados 61 pacientes portadores de SMD, sendo 18 pacientes não transfundidos e 43 pacientes submetidos à terapia transfusional com e sem formação de anticorpos. Realizamos genotipagem para os alelos de grupos sanguíneos RHD, RHCE, FY, DO, CO, DI, SC, GYPA, GYPB, LU, KEL, JK e LW e para os alelos HLA classe I e classe II nas amostras dos pacientes e comparamos os resultados com grupos- controle. Com relação ao perfil transfusional dos pacientes estudados, a maioria recebe transfusões sanguíneas regulares e 44% encontra-se aloimunizada. Os principais aloanticorpos detectados foram contra antígenos Rh e K. Verificamos que a genotipagem é superior a fenotipagem para determinação dos antígenos de grupos sanguíneos e que a compatibilidade molecular para Rh e K seria suficiente para evitar a aloimunização eritrocitária na maioria dos pacientes. Nossos resultados também mostraram uma associação entre o alelo HLA-DRB1*13 e a proteção à aloimunização contra antígenos de grupos sanguíneos em pacientes com SMD / Abstract: The myelodysplastic syndrome (MDS) is a group of heterogeneous clonal disorder caused by an intrinsic stem cell defect with propensity to the bone marrow failure that results in the transfusion dependence and neutropenic infection. Although blood transfusion is generally safe, many of those patients are at risk of transfusion-related complications such as iron overload and RBC alloimmunization that often makes finding compatible RBC products difficult and is also associated with delayed hemolytic transfusion reactions (DHTRs) and autoantibody formation. Matching for Rh and K antigens has been used in an attempt to reduce antibody formation in patients receiving chronic transfusions but an extended phenotyping matching including Fya and Jka antigens has also been recommended. This study was aimed to identify the transfusion profile of the patients with myelodysplastic syndrome (MDS), an efficient transfusion protocol of genotype matching and a possible association of HLA class alleles with susceptibility or protection to RBC alloimmunization. We evaluated 61 patients with MDS, 18 not transfused and 43 undergoing transfusion therapy with and without antibody formation. We performed genotyping for RHD, RHCE, FY, DO, CO, DI, SC, GYPA, GYPB, LU, KEL, JK e LW and for HLA class I and class II alleles in the patient DNA samples and compared the results with a control group. We verified that the majority of patients have regular transfusions and 44% are alloimmunized to RBC antigens. Blood group genotyping was superior to phenotyping to determine the antigen profile in those patients and molecular matching for Rh and K would be enough for most of the patients. Our results also showed a significant association of HLA-DRB1*13 with protection to RBC alloimmunization in patients with MDS / Doutorado / Clinica Medica / Doutora em Clínica Médica

Orientador: Carmen Sílvia Bertuzzo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T12:57:33Z (GMT). No. of bitstreams: 1 Goncalves_ClaudiaEstela_D.pdf: 2258187 bytes, checksum: 7cac2852cc031e31eba1a31c62d9dda2 (MD5) Previous issue date: 2014 / Resumo: A Anemia de Fanconi (AF) é uma alteração genética caracterizada por múltiplas anomalias congênitas, anormalidades hematológicas e predisposição a uma variedade de tumores. A incidência mundial da AF em todo o mundo é de aproximadamente três por milhão e a frequência de heterozigotos é estimada em um para 300 na Europa e Estados Unidos. É uma doença causada por mutações em genes relacionados ao sistema de reparo. Até o momento foram descritos 16 genes que podem estar multados. São eles: FANCA, FANCB, FANCC, FNCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, FANCN, FANCO, FANCP E PANCQ. Os grupos mais frequentes são o FANCA e FANCC. De qualquer modo devido a essa heterogeneidade gênica, o diagnóstico molecular dessa alteração é complexo. Com o intuito de testar uma estratégia diagnóstica, o presente trabalho se propôs a identificar as mutações mais frequentes no gene FANC por PCR e digestão enzimática e investigar mutações no gene FANCA, por meio da Reação em Cadeia da Polimerase seguida de digestão enzimática da mutação Brasileira e posterior sequenciamento dos 43 éxons em 60 pacientes portadores de Anemia de Fanconi DEB positivos. Foram detectados 19 pacientes (27,94%), como sendo do grupo C e 16 pacientes como grupo A (23,53%). A mutação ?3788-3790 do gene FANCA teve uma frequência alélica de 15,4%. Foram encontradas 3 mutações intrônicas, 1 mutação sinônima e 1 mutação de sentido trocado no gene FANCA. Não foram encontradas correlações com as manifestações hematológicas, renais, baixo peso, malformações congênitas de membros, machas e pigmentação de pele, sexo e idade / Abstract: The Fanconi Anemia (FA) is a genetic disorder characterized by multiple congenital and hematological abnormalities and predisposition to a variety of tumors. The worldwide incidence of AF is approximately three per million and the frequency of heterozygotes is estimated at one in 300 in Europe and the United States. It is a disease caused by mutations in genes involved in the repair system. So far have been described 16 genes that may be mutated. They are: FANCA , FANCB , FANCC , FNCD1 , FANCD2 , FANCE , FANCF , FANCG , FANCI , FANCJ , FANCL , FANCM , FANCN , FANCO , FANCP And PANCQ . The most common groups are the FANCA and FANCC. However due to this genetic heterogeneity, molecular diagnosis of this change is complex. In order to test a diagnostic strategy, the present study aimed to identify the most frequent mutations in the FANC gene by PCR and restriction enzyme digestion and investigate mutations in the FANCA gene, using the polymerase chain reaction followed by enzymatic digestion of the mutation Brazilian and subsequent sequencing of the 43 exons in 60 patients with Fanconi Anemia positive DEB. 19 patients (27.94%) were detected as group C and 16 patients as group A (23.53%). The ?3788 - 3790 mutation in the FANCA gene had an allelic frequency of 15.4%. Three intronic mutations, one synonymous mutation and one mutation changed direction in FANCA gene were found. No correlation with hematologic, renal, low weight manifestations of congenital malformations members, butches and skin pigmentation, age and sex were found / Doutorado / Clinica Medica / Doutora em Clínica Médica