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.

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

Background: The high incidence of mutations and cytogenetic abnormalities in patients with myelodysplastic syndrome (MDS) suggests the involvement of DNA repair mechanism defects in the pathogenesis of this disorder. The first part of this work was focused on monitoring of gene expression of DNA repair genes in MDS patients and on their alterations during disease progression. In the second part, next generation sequencing was used to detect single nucleotide polymorphisms (SNPs) and mutations in DNA repair genes and their possible association with MDS development was evaluated. Methods: Expression profiling of 84 DNA repair genes was performed on bone marrow CD34+ cells of patients with MDS. Screening cohort consisted of 28 patients and expression of selected genes was further validated on larger cohort of 122 patients with all subtypes of MDS. Paired samples were used for monitoring of RAD51 and XRCC2 gene expression during disease progression. Immunohistochemical staining for RAD51 recombinase protein was done on samples acquired by trephine-biopsy. Targeted enrichment resequencing of exonic parts of 84 DNA repair genes was performed on the screening cohort of MDS patients. Real-time PCR was used for genotyping of selected SNPs in the population study. Results: RAD51 and XRCC2 genes showed...

No description available.

The myelodysplastic syndrome (MDS) is a group of hematopoietic clonal disorders resulting in the inefficient production of myeloid lineage blood cells, with the prevalence of patients older than 65 years. One of the possible treatment options for MDS is 5- azacytidine and 5-aza-2'-deoxycytidine therapy. These compounds have been shown to cause the induction of cell-cycle arrest, cell differentiation and/or apoptosis. The in vitro experiments with 5-aza-2'-deoxycytidine indicated that this compound causes the premature cellular senescence, a state of the irreversible cell-cycle arrest. We have asked, whether 5-azacytidine, as a molecule with similar structure, is capable of causing the same effect. This treatment strategy could be beneficial in case that the negative pro- inflammatory effect of senescent cells on their surroundings can be nullified. In this thesis we have shown that 5-azacytidine induces DNA damage response, which is described as a fundamental event for the onset of the cell senescence. We tested 5- azacytidine treated HeLa cells for several markers of the cell senescence - the increase of the β-galactosidase activity, the PML and PML nuclear bodies and the formation of persistent DNA damage signaling lesions - albeit all these markers were positive, it was the very low increase in...

Im Rahmen dieser Dissertation wurde retrospektiv die Minimale Resterkrankung von Patienten mit akuter myeloischer Leukämie und Myelodysplastischen Syndrom nach allogener Stammzelltransplantation mit minimaler Konditionierung untersucht. Hierfür wurden vier unterschiedliche Methoden zur Detektion der Minimalen Resterkrankung analysiert. Nach Etablierung einer quantitativen Real-Time PCR für das Wilms Tumor Gen 1 (WT1) im peripheren Blut wurden diese Ergebnisse mit bereits routinemäßig erhobenen Daten des Chimärismus im Gesamtknochenmark und in CD34+ Zellen sowie der Fluoreszenz-in-situ-Hybridisierung (FISH) krankheitsspezifischer chromosomaler Aberrationen von insgesamt 88 Patienten verglichen und statistisch ausgewertet. Es konnte gezeigt werden, dass die Genexpressionanalysen des WT1 sowie die Chimärismusanalysen ein Rezidiv im Gegensatz zu den FISH Analysen vier Wochen im Voraus detektieren können. In Reiceiver Operating Curve Analysen wurden eine WT1 Expression von > 24 WT1/10.000 ABL1 Kopien und der Abfall des CD34+ Spenderchimärismus von ≥ 5% als diagnostisch stärkste Methoden identifiziert. In uni- und multivariaten Analysen von insgesamt 20 Parametern wurden die beiden Methoden als unabhängige Variablen für ein frühes Rezidiv, progressionsfreies Überleben und Gesamtüberleben bestätigt. Kombiniert man beide Methoden, so kann bei jeweiligem negativen Testergebnis ein Rezidiv innerhalb der nächsten vier Wochen nahezu ausgeschlossen werden.

Το Μυελοδυσπλαστικό Σύνδρομο (ΜΔΣ) αποτελεί νόσημα με διαταραχή σε επίπεδο αρχέγονου αιμοποιητικού κυττάρου (stem cell) που χαρακτηρίζεται από μη αποδοτική αιμοποίηση και κυτταροπενίες του περιφερικού αίματος που περιλαμβάνουν μία ή περισσότερες αιμοποιητικές σειρές. Διάφορες ανοσολογικές διαταραχές των ασθενών με ΜΔΣ, όπως, αυξημένη ευαισθησία σε βακτηριακές λοιμώξεις, αυτοάνοσα φαινόμενα και υψηλή συχνότητα κακοηθειών του λεμφικού ιστού, υποδεικνύουν αδυναμία των ασθενών με ΜΔΣ για ανοσολογική απάντηση, οι αιτίες των οποίων παραμένουν άγνωστες μέχρι σήμερα. Τα Δενδριτικά Κύτταρα (ΔΚ) είναι κύτταρα του ανοσολογικού μηχανισμού που προέρχονται από το μυελό των οστών. Ως αντιγονοπαρουσιαστικά κύτταρα (APC), είναι εξειδικευμένα για τη πρόσληψη, επεξεργασία, μεταφορά και παρουσίαση του αντιγόνου στα Τ λεμφοκύτταρα. Στη παρούσα μελέτη πραγματοποιήθηκε ανάλυση διαφορετικών ποσοτικών και λειτουργικών παραμέτρων των ΔΚ από ασθενείς με Μυελοδυσπλαστικό Σύνδρομο, in vivo ή in vitro. Αρχικά διερευνήθηκε ο αριθμός, ο φαινότυπος, η ικανότητα ενδοκύττωσης και η αλλογενής διεγερτική δυνατότητα των ΔΚ, προερχόμενων από μονοκύτταρα του περιφερικού αίματος (ΜοΔΚ) ασθενών με ΜΔΣ και υγιών μαρτύρων, σε διαφορετικά στάδια διαφοροποίησης. Τα μονοκύτταρα των ασθενών με ΜΔΣ χαρακτηρίστηκαν από μειωμένη ικανότητα διαφοροποίησης σε ΔΚ, λόγω του μειωμένου αριθμού των διαφοροποιημένων κυττάρων και τη χαμηλή έκφραση του CD1a αντιγόνου επιφανείας. Τα ΜοΔΚ των ΜΔΣ ασθενών παρουσίασαν χαμηλή έκφραση του υποδοχέα της μανόζης και μειωμένη ικανότητα ενδοκύττωσης. ΜοΔΚ των ΜΔΣ ασθενών επέδειξαν μειωμένη απάντηση ύστερα από διέγερση με TNF-α, καθώς η έκφραση των CD83, CD80 και CD54 αντιγόνων και η αλλοδιεγερτική ικανότητα ήταν μειωμένη, ενώ η επίδραση με LPS είχε ως αποτέλεσμα να εμφανίσουν φαινοτυπικά χαρακτηριστικά και ικανότητα διέγερσης των Τ-κυττάρων, όμοια με τα ΜοΔΚ των φυσιολογικών μαρτύρων. Σε δύο από τους ασθενείς με σύνδρομο 5q-, σχεδόν όλα τα μονοκύτταρα και τα ΜοΔΚ περιείχαν τη χρωμοσωμική διαταραχή, υποδηλώνοντας την προέλευσή τους από τον παθολογικό κλώνο. Στη συνέχεια διερευνήθηκε το δυναμικό πολλαπλασιασμού και διαφοροποίησης των CD34+ προγονικών κυττάρων του μυελού ασθενών με ΜΔΣ σε δενδριτικά κύτταρα (CD34-ΔΚ) σε υγρή καλλιέργεια παρουσία κυτοκινών. Παράλληλα, έγινε ανάλυση των κυκλοφορούντων ΔΚ περιφερικού αίματος στους ίδιους ασθενείς. Τα CD34+ προγονικά κύτταρα παρουσίασαν χαμηλή δυνατότητα ανάπτυξης ΔΚ in vitro, καθώς ο αριθμός των παραγόμενων ΔΚ ανά CD34+ κύτταρο ήταν χαμηλότερος συγκριτικά με τα δείγματα των υγιών μαρτύρων. Παρά την αυξημένη απόπτωση των προγονικών κυττάρων του μυελού των ΜΔΣ ασθενών, η επιβίωση και ο πολλαπλασιασμός των CD34+ κυττάρων στην καλλιέργεια, δεν συσχετίστηκε με την απόπτωση και αποτελεί αξιοσημείωτη παρατήρηση. Φαινοτυπικά, τα CD34-ΔΚ των ΜΔΣ ασθενών δεν διέφεραν από τα ΔΚ που παρήχθησαν από τα CD34+ κύτταρα του μυελού των φυσιολογικών μαρτύρων καθώς επέδειξαν όμοια έκφραση των CD83, CD80, CD40, HLA-DR και CD54 αντιγόνων. Κυτταροεπιλεγμένα CD1a+ κύτταρα ασθενών είχαν όμοια διεγερτική ικανότητα αλλογενών Τ κυττάρων με τα CD34-ΔΚ των φυσιολογικών ατόμων. Το ποσοστό των κυκλοφορούντων μυελοειδών- και πλασματοκυτταροειδών- ΔΚ στους ασθενείς με ΜΔΣ ήταν σημαντικά μειωμένο συγκριτικά με τους υγιείς μάρτυρες. Στους ασθενείς με 5q έλλειψη, τόσο τα CD34-ΔΚ, όσο και τα ΔΚ του αίματος, είχαν τη χρωμοσωμική ανωμαλία. Τα παραπάνω αποτελέσματα υποδηλώνουν ότι η διαδικασία παραγωγής δενδριτικών κυττάρων από το μυελό (‘δενδριτοποίηση’) των ασθενών με ΜΔΣ, είναι μέρος της κλωνικής διαταραχής με αποτέλεσμα την μη αποδοτική παραγωγή ΔΚ από τα προγονικά κύτταρα του μυελού και το χαμηλό ποσοστό των κυκλοφορούντων πρόδρομων ΔΚ. Όλες οι ΔΚ υποομάδες προέρχονται από τον παθολογικό κλώνο και χαρακτηρίζονται από ποσοτικές και ποιοτικές ανωμαλίες. Το σύνολο αυτών των διαταραχών που παρατηρήθηκαν στα ΔΚ πολύ πιθανόν να συμβάλει στη διαταραγμένη ανοσολογική απάντηση έναντι παθογόνων οργανισμών, στην επιβίωση και στην επικράτηση του παθολογικού κλώνου, όπως επίσης και στην εμφάνιση αυτοάνοσων φαινομένων, που παρατηρούνται στους ασθενείς με ΜΔΣ. / Myelodysplastic syndrome (MDS) is a stem cell disorder characterized by ineffective hematopoiesis and blood cytopenias involving one or several myeloid lineages. Various immune disturbances in MDS such as increased susceptibility to bacterial infections, autoimmune phenomena and high incidence of lymphoid malignancies reveal an underlying defect of the immune response in MDS patients, the reasons for which still remain unclear. Dendritic cells (DCs) are bone marrow derived cells. As the most potent antigen presenting cells (APC), they are specialized for the uptake, processing, transport and presentation of Ag to T cells. In the present study different quantitative and functional parameters of DCs in patients with MDS were analyzed either in vivo or in vitro. The number, phenotype, endocytic ability, and allostimulatory capacity of DCs derived from peripheral blood monocytes (MoDCs) were investigated in patients with MDS and healthy controls at different stages of differentiation using the maturation stimuli-TNF-á and LPS. Monocytes in MDS showed low potential to differentiate into DCs, as determined by low cell yield and CD1a expression. MDS-MoDCs exhibited low expression of Mannose receptor and reduced endocytic capacity. When stimulated with TNF-á, MoDCs obtained from MDS patients showed a diminished response with low CD83, CD80 and CD54 expression and allostimulatory capacity, whereas in the presence of LPS MDS-MoDCs acquired phenotypic characteristics and ability to stimulate T-cells similar to MoDCs derived from controls. In two patients with 5q- syndrome the vast majority of both monocytes and MoDCs were positive for the 5q deletion, suggesting that they originate from the malignant clone. Second, we investigated the potential of bone marrow CD34+ progenitors in patients with MDS to proliferate and differentiate into DCs in a liquid cytokine supplemented culture system and also analyzed the status of blood DC subsets in those patients. CD34+ progenitors had low potential to generate DCs in vitro, as the number of DCs obtained from one CD34+ cell was significantly lower compared to controls. Interestingly, although the increased apoptotic level of bone marrow progenitors in MDS, the survival and proliferation of CD34+ cells in culture was not correlated to the degree of apoptosis. Phenotypically the MDS CD34-DCs did not differ from DCs obtained from normal BM CD34+ cells, exhibiting similar expression of CD83, CD80, CD40, HLA-DR, and CD54. FACsorted CD1a+ cells from MDS patients were as efficient stimulators of allogeneic T cells as normal CD34-DCs. The percentage of both circulating DC subsets, MDCs and PDCs in MDS patients was extremely diminished compared to controls. In cases with the 5q deletion both CD34-DCs and blood DCs harbor the cytogenetic abnormality. The results indicate that “dendritopoiesis” in MDS is affected by the transformation process resulting in ineffective production of DCs from bone marrow progenitors with low circulating blood precursors. All DC subsets were derived from the malignant clone and exhibited quantitative and qualitative abnormalities. This constellation of DCs defects probably contribute to the defective immune response against pathogens, escape and expansion of the malignant clone, as well as autoimmune phenomena, observed in MDS patients.

Το Μυελοδυσπλαστικό Σύνδρομο (ΜΔΣ) αποτελεί μια ετερογενή ομάδα κλωνικών αιματολογικών διαταραχών με κοινό χαρακτηριστικό τη μη αποδοτική αιμοποίηση που οδηγεί σε ανθεκτικές κυτταροπενίες και συχνή εκτροπή προς Οξεία Λευχαιμία (ΟΛ). Η παράδοξη συνύπαρξη ενός πληθωρικού ή νορμοκυτταρικού μυελού των οστών και κυτταροπενιών στην περιφέρεια έχει αποδοθεί στην αυξημένη απόπτωση προγονικών και ώριμων αιμοποιητικών κυττάρων του μυελού των ασθενών αυτών. Παρ'όλ'αυτά, αιτιοπαθογενετική σχέση της υπέρμετρης απόπτωσης με την ανεπαρκή κλωνογόνο ικανότητα των προγονικών αιμοποιητικών κυττάρων του μυελοδυσπλαστικού μυελού δεν τεκμηριώνεται επαρκώς από τα υπάρχοντα ερευνητικά δεδομένα. Αντίθετα, αδιαμφισβήτητη είναι η συσχέτιση της κατάργησης των μηχανισμών του προγραμματισμένου κυτταρικού θανάτου στο προχωρημένο ΜΔΣ με την εκτροπή της νόσου προς ΟΛ. Η αυξημένη έκφραση αντιαποπτωτικών πρωτεϊνών όπως οι Bcl-2, Bcl-xL, IAPs, survivin έχει προταθεί ότι υποστηρίζει την πρόοδο της νόσου και την επακόλουθη λευχαιμική εξέλιξη. Οι Heat Shock Proteins αποτελούν θεμελιώδεις πρωτεΐνες, ως προς τις αποφάσεις των κυττάρων σχετικά με την επιβίωση ή το θάνατό τους. Η Hsp73, ένα σταθερά εκφραζόμενο μέλος, και οι Hsps 72 και 27, δύο ισχυρά επαγόμενες από ποικίλα στρεσσογόνα ερεθίσματα πρωτεΐνες, είναι τρεις από τις πιο σημαντικές Hsps εκδηλώνοντας πολυδιάστατη αντι-αποπτωτική δράση και παρέχοντας ισχυρή κυτταροπροστασία. Σε αρκετά είδη συμπαγών όγκων αλλά και αιματολογικών κακοηθειών έχει δειχθεί υπερέκφραση των Hsps, ενώ έχει προταθεί ακόμη και αιτιοπαθογενετική σχέση της διαταραχής της έκφρασης των πρωτεϊνών αυτών με την ογκογένεση. Σκοπός της παρούσας εργασίας ήταν η εκτίμηση του βαθμού της απόπτωσης στα προγονικά και ώριμα κύτταρα μυελού ασθενών με ΜΔΣ και η περαιτέρω διερεύνηση της ουσιαστικής συνεισφοράς του φαινομένου στην ανεπαρκή κλωνογόνο ικανότητα των προγονικών κυττάρων και, κατ' επέκταση, στη μη αποδοτική αιμοποίηση που χαρακτηρίζει το σύνδρομο. Εν συνεχεία, διερευνήθηκαν οι αποπτωτικές μεταβολές κατά την πρόοδο της νόσου οι οποίες συσχετίσθηκαν με την έκφραση των αντι-αποπτωτικών Heat Shock Proteins. Η έκφραση των Hsps 72, 27 & 73 μελετήθηκε υπό συνθήκες ηρεμίας (βασική έκφραση, ΒΕ), ενώ ελέγχθηκε η επαγωγιμότητα των Hsps 72 και 27 κατόπιν εφαρμογής θερμικού shock ή ο-αποπτωτικής διέγερσης με συνδυασμό κυτταροκινών (TNFα+IFNγ). Η προσδιοριζόμενη έκφραση των Hsps συσχετίσθηκε, κατόπιν, με την απόπτωση και το στάδιο της νόσου. Η παρούσα μελέτη επιβεβαιώνει την παρουσία αυξημένης απόπτωσης τόσο στα ώριμα CD34- όσο και στα προγονικά CD34+ κύτταρα του μυελού ασθενών με ΜΔΣ με αξιοσημείωτη ετερογένεια να διέπει τα αποτελέσματα ακόμη και μεταξύ ασθενών της ίδιας κατά FAB κατηγορίας νόσου. Τα επίπεδα ανιχνευόμενης απόπτωσης υπερείχαν σημαντικά στο μυελό των ασθενών με "πρώιμη" (RA και RARS) έναντι εκείνων με "προχωρημένη" (RAEB και RAEB-t) νόσο, ενισχύοντας τη θεωρία της κατάργησης των αποπτωτικών μηχανισμών κατά την πρόοδο της νόσου. Μετά από 24 ώρες υγρής καλλιέργειας, το ποσοστό των ανιχνευόμενων πρώιμων αποπτωτικών CD34+ κυττάρων μυελού ασθενών με ΜΔΣ μειώθηκε σημαντικά. Προκειμένου να διερευνηθεί η πραγματική συμβολή της απόπτωσης στη μη αποδοτική αιμοποίηση του ΜΔΣ in vitro, αποπτωτικά και μη αποπτωτικά προγονικά CD34+ φρέσκα κύτταρα μυελού διαχωρίστηκαν με τη μέθοδο του Κυτταροδιαχωρισμού ενεργοποιούμενου από φθορισμό και τοποθετήθηκαν σε βραχείας διάρκειας ημιστερεές καλλιέργειες, όπου ελέγχθηκε η σχετική κλωνογόνος ικανότητά τους. Τόσο τα αποπτωτικά όσο και τα μη αποπτωτικά CD34+ κύτταρα των ασθενών με ΜΔΣ επέδειξαν εξίσου ανεπαρκή ανάπτυξη in vitro, υποδεικνύοντας ότι η παρουσία απόπτωσης δεν επηρεάζει σημαντικά τη συμπεριφορά των κυττάρων στην καλλιέργεια. Η βασική ενδοκυττάρια έκφραση των αντιαποπτωτικών Hsps 27, 72 και 73 βρέθηκε σημαντικά αυξημένη στο μυελό ασθενών με ΜΔΣ. Η υπερέκφραση των Hsps αφορούσε κυρίως τα ολικά μονοπύρηνα κύτταρα του μυελού των ασθενών, ενώ τα προγονικά CD34+ κύτταρα δεν επέδειξαν σημαντικά υψηλότερα επίπεδα των υπό μελέτη πρωτεϊνών. Επιπλέον, η εφαρμογή θερμικού shock οδήγησε επιτυχώς στην επαγωγή των Hsps 27 και 72 στο μυελό ασθενών και μαρτύρων, προσεγγίζοντας όμως υψηλότερα τελικά επίπεδα έκφρασης στο μυελό των ασθενών. Tόσο η βασική όσο και η εκ θερμικού stress επαγόμενη έκφραση της Hsp72 βρέθηκε σημαντικά ενισχυμένη στα μονοπύρηνα κύτταρα του μυελού ασθενών με "προχωρημένου τύπου" ΜΔΣ, ενώ σημαντικά θετική συσχέτιση τεκμηριώθηκε μεταξύ της έκφρασης ηρεμίας των τριών Hsps και του ποσοστού των βλαστών του μυελού των ασθενών. Τα ευρήματα αυτά υποδηλώνουν ενδεχόμενο ρόλο της υπερέκφρασης των Hsps στην ευνοϊκή επιλογή και εξάπλωση των κλωνικών κυττάρων και, συνεπακόλουθα, στην πρόοδο της νόσου. Η συνδυασμένη επίδραση κυτταροκινών σε κύτταρα φυσιολογικών μυελών οδήγησε σε σημαντική μείωση της έκφρασης της Hsp72, ενώ στο 36% των ασθενών παρατηρήθηκε αύξηση της έκφρασης της Hsp72. Τέλος, αρνητική ήταν η συσχέτιση της βασικής ή επαγόμενης έκφρασης των Hsps με την αυτόματη ή προκλητή απόπτωση σε ολικά ή προγονικά κύτταρα μυελού ασθενών με ΜΔΣ, επιβεβαιώνοντας τον κυτταροπροστατευτικό τους ρόλο. Συνοψίζοντας, τα ευρήματα της παρούσας μελέτης επιβεβαιώνουν την παρουσία αυξημένης απόπτωσης στο μυελό των ασθενών με ΜΔΣ, ιδιαίτερα πρώιμου τύπου. Τα πρώιμα αποπτωτικά προγονικά κύτταρα επιδεικνύουν δυνατότητα διαφυγής από τον κυτταρικό θάνατο και σχετική λειτουργική ανάνηψη κατά την απομάκρυνσή τους από το προαποπτωτικό μικροπεριβάλλον του μυελού. Εναλλακτικά, πρόκειται για κύτταρα που, εξ'αιτίας αποτυχίας του αποπτωτικού μηχανισμού να περατωθεί, υφίστανται "ατελή απόπτωση" και διασώζονται, φέροντα όμως δυνητικά ογκογόνες μεταλλάξεις. Η γενική αντίληψη, πάντως, που καθιστά την απόπτωση ως την αιτία της μη αποδοτικής αιμοποίησης του συνδρόμου κλονίζεται, καθώς αποπτωτικά και μη αποπτωτικά προγονικά κύτταρα διαθέτουν παρόμοια κλωνογόνο ικανότητα, in vitro; φαίνεται ότι επιπρόσθετες ενδογενείς ανωμαλίες περιορίζουν το κλωνογόνο δυναμικό αποπτωτικών και μη προγονικών κυττάρων. Η υπερέκφραση Hsps που τεκμηριώθηκε στο μυελό ασθενών με ΜΔΣ ενδεχομένως υποστηρίζει την διαδικασία κακοήθους εξαλλαγής μέσω της σιωπηρής συσσώρευσης μεταλλάξεων στα κύτταρα του κλώνου. Επιπλέον, τα αυξημένα επίπεδα των αντι-αποπτωτικών αυτών πρωτεϊνών θα μπορούσε να ευθύνεται για την προτεινόμενη ατελή απόπτωση των κυττάρων του μυελοδυσπλαστικού μυελού. Όπως προκύπτει από τη σημαντική συσχέτιση της έκφρασης των Hsps τόσο με το ποσοστό βλαστών όσο και με το στάδιο της νόσου, τα υπερεκφράζοντα ώριμα με τα αντίστοιχα προγονικά τους παθολογικά κύτταρα, ενέχοντας πλεονέκτημα επιβίωσης έναντι των φυσιολογικών στο προ-αποπτωτικό μικροπεριβάλλον του δυσπλαστικού μυελού, φαίνεται ότι επιλέγονται και επικρατούν κατά την πρόοδο της νόσου. Σε ένα τέτοιο υπόστρωμα αντίστασης στην απόπτωση ένα επιγενετικό συμβάν που αναστέλλει τη διαφοροποίηση ενδέχεται να πυροδοτήσει τη λευχαιμική εκτροπή. / Myelodysplastic syndrome comprises a heterogeneous group of clonal stem cell disorders characterized by ineffective hematopoiesis leading to refractory cytopenias and frequent evolution to acute myeloid leukemia (AML). The existing inconsistency between normal or hypercellular bone marrow and peripheral blood cytopenias remains a paradox that has been attributed to excessive intramedullary apoptosis. However, a causative relationship of apoptosis to the progenitor’s defective clonogenic growth has not been sufficiently demonstrated. On the other hand, it is widely accepted that abrogation of apoptotic control in advanced MDS favours the expansion of the malignant clone and contributes to the frequently observed evolution to acute myeloid leukaemia (AML). Enhanced expression of antiapoptotic proteins Bcl-2, Bcl-xL, IAPs, survivin has been proposed to contribute to elimination of apoptosis, disease progression and subsequent leukemic evolution. Heat Shock Proteins (Hsps) are fundamental for cell life and death decisions and essential for the coordination between proliferation, differentiation and apoptosis. Hsp73, one of the constitutively expressed Hsps, and Hsps 72 and 27, two strongly inducible by several stress stimuli proteins, are three of the most important Hsps. These three Hsps regulate programmed cell death by interfering with multiple key-regulatory points of the apoptotic cascade, acting as apoptosis inhibitors and conferring strong cytoprotection. Hsps 72 and 27 are overexpressed in a number of human cancers and haematological malignancies, while a causative relationship of these proteins to oncogenesis has even been proposed. The aim of our study was to determine the degree of programmed cell death in progenitor and mature bone marrow cells of MDS patients. Moreover, apoptosis' actual contribution to defective clonogenic capacity of MDS progenitors and, subsequently, to ineffective hematopoiesis in MDS was assessed. Furthermore, apoptosis modifications during disease progression were detected and correlated to the expression of antiapoptotic Hsps. Hsps 72, 27 and 73 intracellular expression was studied under conditions of tranquillity in bone marrow and progenitor cells of MDS patients, while the inducibility of Hsps 27 and 72 was further tested after application of an environmental type of stress (heat shock) and pro-apoptotic stimulation under combined cytokine treatment (TNFα+IFNγ). Finally, we determined an existing link between Hsps' levels, apoptosis and disease progression. Apoptosis was significantly augmented in both progenitor and mature bone marrow cell fractions from MDS patients. Apoptosis determined in the bone marrow of patients with "early" MDS (RA and RARS) significantly exceeded cell death levels detected in those with "advanced" (RAEB and RAEB-t) disease, supporting the theory of apoptosis' abrogation as the disease evolves. We should remark, however, that even inside the same FAB-category, a great heterogeneity existed in results. After 24 hours of liquid culture the percentage of early apoptotic progenitors in MDS BM significantly decreased. In order to determine apoptosis' actual contribution to defective clonogenic capacity of MDS progenitors, “apoptotic” and “non-apoptotic” bone marrow CD34+ cells were sorted by FACS (Fluorescence Activated Cell Sorting) and their differential clonogenic capacity was assessed in a short-term semisolid culture system. There was no correlation between apoptosis’ existence and culture performance, since non-apoptotic as well as apoptotic CD34+ bone marrow cells both exhibited similar defective growth. Basal intracellular expression of antiapoptotic Hsps 27, 72 and 73 was found significantly elevated in bone marrow cells of MDS patients. Hsps overexpression mainly involved total BMMC, while higher protein levels were also detected within patients’ progenitor bone marrow cells, but the differences noted did not attain statistical significance. Moreover, HS treatment provoked the effective induction of Hsps 27 and 72 in both MDS and normal subjects leading to the achievement of even higher final protein levels in the patients’ marrow, despite the already enhanced basal expression. Both basal and HS-induced Hsp72 expression were significantly enhanced in BMMC of MDS patients with advanced disease, while a positive correlation between all three Hsps basal expression and blast percentage was established in the patients' marrow. These findings suggest a probable role of Hsps overexpression in the favored selection and expansion of clonal cells, further hematopoiesis depression and disease progression. Combined pro-apoptotic treatment of normal BM cells caused a significant downregulation of Hsp72. On the contrary, BM cells from MDS patients responded by elevating the expression of cytoprotective Hsp72 in about 36% of cases. Finally, in accordance to the well-demonstrated cytoprotective role of Hsps, an inverse correlation was noted between spontaneous or induced apoptosis and Hsps basal or induced expression. In conclusion, our findings support the previous observations of increased apoptosis in MDS marrow, especially in "early" disease. Interestingly, early apoptotic MDS progenitors exhibit the potential to escape from apoptosis and even recover functionally, as shown in CFU-assays, when separated from the bone marrow microenvironment, the main source of pro-apoptotic signalling. Alternatively, these cells, due to intrinsic defects in cell death activated pathways, may undergo incomplete apoptosis and get rescued possibly carrying, though, potentially transforming mutations. However, apoptosis does not seem to be the only cause of impaired clonogenic growth in MDS, as apoptotic and non-apoptotic progenitors exhibit similar patterns of growth, both defective compared to normal. Apparently, additional intrinsic abnormalities limit the clonogenic potential of apoptotic and non-apoptotic progenitors. Augmented Hsps expression established in MDS marrow may support the underlying transformation process through the suppression and silent gathering of mutations, probably promoting viability and growth of otherwise mutant cells. Moreover, increased levels of anti-apoptotic Hsps may account for the proposed incomplete apoptosis of MDS marrow cells. Hsps appear to provide resistance against spontaneous or induced apoptosis to the overexpressing cell population. Hsps' positive correlation to blast count and disease stage implies that the overexpressing mature cells along with their abnormal progenitors, encompassing a survival advantage over normal cells in the pro-apoptotic MDS marrow, get selected and expanded during disease progression. On such a background of non-susceptibility to apoptosis a secondary event blocking differentiation could lead to leukaemic transformation.

CCCTC-binding factor (CTCF) can both activate as well as inhibit transcription by forming chromatin loops between regulatory regions and promoters. In this regard, Ctcf binding on the non-methylated DNA and its interaction with the Cohesin complex results in differential regulation of the H19/Igf2 locus. Similarly, a role for CTCF has been established in normal hematopoietic development; however its involvement, despite mutations in CTCF and Cohesin complex were identified in leukemia, remains elusive. CTCF regulates transcription dependently on DNA methylation status and can if bound block interactions of enhancers and promoters. Here, we show that in hematopietic cells CTCF binds to the imprinting control region of H19/Igf2 and found that chromatin remodeller Smarca5, which also associates with the Cohesin complex, facilitates Ctcf binding and regulatory effects. Furthermore, Smarca5 supports CTCF functionally and is needed for enhancer-blocking effect at imprinting control region. We identified new CTCF-recognized locus near hematopoietic regulator SPI1 (PU.1) in normally differentiating myeloid cells together with members of the Cohesin complex. Due to DNA methylation, CTCF binding to the SPI1 gene is reduced in AML blasts and this effect was reversible by DNA methylation inhibitor 5-azacitidine.