A fisiopatologia das síndromes de falência da medula óssea (FMO) está relacionada a mecanismos adquiridos de destruição das células-tronco hematopoeiticas na medula ou a defeitos constitucionais em genes fundamentais para o reparo do DNA e manutenção dos telômeros. A anemia aplástica (AA), o protótipo das doenças de FMO, pode ter etiologia adquirida ou constitucional. A avaliação genética de pacientes com AA adquirida tem como objetivo a detecção de mutações somáticas que possam ser usadas como marcadores de resposta ao tratamento imunossupressor. Diferentemente, em pacientes com AA constitucional, a avaliação genética é fundamental para detecção de mutações etiológicas na doença do paciente, sendo essencial para o tratamento e seleção de doadores de medula óssea. Contudo, o papel das mutações constitucionais na fisiopatologia e modulação imunológica da AA adquirida ainda não é conhecido. Neste estudo, nós sequenciamos pacientes com AA de duas coortes independentes utilizando diferentes painéis de sequenciamento de genes alvos. A primeira coorte, composta por 13 pacientes com AA adquirida, foi sequenciada utilizando um painel com 165 genes relacionados à FMO, neoplasias hematológicas, reparo de DNA, manutenção dos telômeros e vias de resposta imune. A segunda coorte, composta por 59 pacientes investigados para doença constitucional, foi sequenciada com um painel de sequenciamento comercial com 49 genes relacionados à FMO hereditária. Foram identificadas alterações potencialmente patogênicas em três dos cinco pacientes com AA adquirida que não responderam à imunossupressão: dois pacientes com variantes em TERT e um com uma variante em DHX36. Não foram identificadas variantes funcionalmente relevantes nos pacientes que responderam ao tratamento imunossupressor. Em contraste, foram identificadas variantes potencialmente patogênicas em RTEL1 em 8 pacientes com AA constitucional. Variantes em RTEL1 foram associadas tanto ao encurtamento telomérico quanto à erosão excessiva do 3\' overhang, independentemente do comprimento dos telômeros. Desse modo, apenas a medida do comprimento dos telômeros não foi suficiente para identificar todos ospacientes com disfunções teloméricas. As plataformas de sequenciamento de nova geração diminuíram o custo e o tempo para a avaliação genética dos pacientes com FMO. Em nosso estudo, os pacientes com AA adquirida não apresentaram um padrão genético associado à sua resposta ao tratamento com imunossupressores, no entanto, o sequenciamento da coorte com suspeita de AA constitucional foi capaz de identificar o defeito genético associado à doença do paciente em 40% dos casos. O uso de dados clínicos, investigação familiar, análises in silico e testes funcionais foram essenciais para uma correta interpretação da patogenicidade de novas variantes identificadas por sequenciamento de nova geração. / The pathophysiology of bone marrow failure (BMF) can be immune, as in acquired aplastic anemia (AA), or constitutional, due to germline mutations in genes critical for DNA repair and telomere maintenance. The genetic screening of patients with constitutional AA is performed to detect germline mutations that are etiologic in patients\' disease. That is critical for treatment decisions and to identify a donor for a bone marrow transplant. In acquired AA, the genetic screening has been used to detect somatic mutations that can predict patients\' outcomes after treatment, as the role of germline mutations in this disease is yet not clear. To investigate the role of germline variants in AA, we screened two independent cohorts with two different targeting sequencing panels; a first cohort composed by 13 patients with acquired AA that was screened using a panel with 165 genes related to BMF, hematologic malignancies, DNA repair, telomere maintenance, and immune response pathways. A second cohort composed of 59 patients suspected to have a constitutional disease screened by a commercial Inherited Bone Marrow Failure Sequencing panel. In our first cohort, while patients without functional relevant germline variants responded to immunosuppression treatment (n=8), three out of 5 nonresponder patients were identified with variants in telomere biology genes. We found patients carrying TERT and DHX36 variants. In our constitutional AA cohort, we identified 8 patients carrying variants in the RTEL1 gene, a helicase critical to telomere maintenance. RTEL1 variants associated with both patients\' overall telomere shortening and single-stranded 3\' overhang erosion independent of telomere length. Also, 3\' overhang erosion was associated with patients\' predisposition to clonal evolution. In this context, the variants identified in the helicases genes DHX36 and RTEL1 were both associated with patients\' normal telomere length and poor outcomes. Also, telomere length measurement alone was insufficient to identify all primary telomere defects. The platforms of next-generation sequencing decreased the cost and time for the genetic screening of patients with BMF. In our study, acquired AA patients did not display a clear genetic pattern associated with their immunosuppressive treatment response. In contrast, the sequencing of the cohort selected based on their suspicion to have an inherited diseaseidentified a molecular defect that might be pathogenic in up to 40% of patients, including the RTEL1 variants. Pathogenicity assessment of genetic variants requires a combination of clinical, in silico, and functional data required to avoid misinterpretation of common variants.

A anemia aplástica (AA) é uma doença hematológica rara caracterizada pela hipocelularidade da medula óssea, o que provoca pancitopenia. Esta pode ser de origem genética (associada a encurtamento telomérico) ou adquirida (não-associada a desgaste excessivo dos telômeros). Na forma adquirida, a ativação anormal de linfócitos T provoca a destruição das células hematopoéticas. O mecanismo que leva a essa destruição ainda não foi elucidado. Um dos tratamentos mais eficazes para repovoar a medula óssea hipocelular é o transplante com célulastronco hematopoéticas (CTHs). Porém, uma grande porcentagem de pacientes não se beneficia de nenhum tratamento, fazendo-se necessário o desenvolvimento de novas alternativas para terapia. A geração de células-tronco pluripotentes induzidas (iPSCs) a partir de células somáticas (reprogramação) representa uma ferramenta promissora para o estudo de doenças e para o desenvolvimento de possíveis terapias paciente-especificas, como transplantes autólogos. Neste trabalho, avaliamos a capacidade de reprogramação de fibroblastos e eritroblastos de pacientes com AA adquirida. Metodologias de reprogramação utilizando lentivírus ou plasmídeos epissomais não integrativos foram testadas em células de quatro pacientes e de um controle saudável. Eritroblastos dos quatro pacientes e do controle foram reprogramados utilizando os plasmídeos não integrativos. As iPSCs geradas apresentaram-se similares a células-tronco embrionárias quanto à morfologia, expressão dos marcadores de pluripotência OCT4, SOX2, NANOG, SSEA-4, Tra-1-60 e Tra-1-81, e capacidade de diferenciação in vitro em corpos embrioides (EBs). A dinâmica telomérica das células pré- e pós-reprogramação foi avaliada em diferentes passagens utilizando a técnica de flow-FISH. O comprimento telomérico foi aumentado nas iPSCs quando comparado às células parentais o que indica que a célula foi completamente reprogramada. No presente trabalho, células de pacientes com AA adquirida foram reprogramadas a um estado de pluripotência por meio de um método não integrativo. As iPSCs geradas serão essenciais para futuros ensaios de diferenciação hematopoética, o que poderá contribuir para o entendimento dos mecanismos envolvidos no desenvolvimento dessa doença. Além disso, a diferenciação dessas células livres de transgenes poderá servir como uma alternativa terapêutica para os pacientes com AA como, por exemplo, em transplantes autólogos / Aplastic anemia (AA) is a rare hematological disease characterized by bone marrow hypocellularity that leads to pancytopenia. Its origin can be genetic (associated with telomere shortening) or acquired (non-associated with telomere shortening). The acquired form exhibit T lymphocytes abnormal activation, which leads to hematopoietic cells destruction. The mechanisms behind this phenomenon are still unclear. One of the most effective treatments for hypocelullar bone marrow repopulation is hematopoietic stem cell (HSCs) transplantation. However, a large percentage of patients do not benefit from any of the available treatments. This highlights the need to develop new therapeutic strategies. The generation of induced pluripotent stem cells (iPSCs) from somatic cells (reprogramming) represents a powerful tool for disease modeling and for the development of patient-specific therapies such as autologous transplants. In this study, we evaluate the capacity of reprogramming acquired AA patients\' fibroblasts and erythroblasts. Reprogramming methods using lentivirus or non-integrative episomal plasmids were tested in four patients\' cells and in cells from one healthy donor. Erythroblasts from these four patients and healthy donor were reprogrammed using non-integrative plasmids. The iPSCs resembled human embryonic stem cells in morphology, in the expression of pluripotent markers such as OCT4, SOX2, NANOG, SSEA-4, Tra-1-60 and Tra-1-81, and in in vitro differentiation (capacity to form embryoid bodies). The telomere dynamics of the cells before and after reprogramming was assessed along passaging using flow-FISH. The telomere length in the iPSCs was increased when compared to the parental cells. Thus, acquire AA patients\' cells could be reprogrammed to a pluripotent state by a nonintegrative method. The iPSCs will be essential for future hematopoietic differentiation assays that could contribute to the understanding of the mechanisms involved in the disease development. Furthermore, the differentiation of transgene-free cells may serve as an alternative therapy for patients with AA such as autologous transplants

Introdução: A anemia aplástica (AA) adquirida é uma doença grave, caracterizada por pancitopenia e medula óssea hipocelular sem que haja associação com aumento de reticulina ou infiltração anormal na medula. Embora o mecanismo fisiopatológico não esteja totalmente elucidado, atribui-se a uma resposta imunomediada dos linfócitos T no ambiente medular. A trombopoetina (codificada pelo gene THPO) é um hormônio glicoproteico produzido pelo fígado e responsável pelo estímulo de crescimento de megacariócitos, desenvolvimento plaquetário e de demais linhagens e, quando disfuncional, contribui para o desenvolvimento da AA adquirida. Objetivos: Investigar a presença de variantes genéticas no THPO em amostras de sangue periférico e medula óssea de pacientes com AA adquirida (grupo caso) e de indivíduos saudáveis (grupo controle) e verificar a presença de alterações no número de plaquetas durante o seguimento dos pacientes com AA adquirida. Métodos: O gene THPO foi sequenciado em amostras de DNA de medula óssea de 92 pacientes com AA adquirida e no DNA de sangue periférico de 92 controles, cujas amostras haviam sido previamente armazenado no Laboratório de Hematologia da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (FMRP-USP). O sequenciamento foi realizado pelo método de Sanger. Realizou-se também a associação entre a presença (ou ausência) de variantes em THPO e o número de plaquetas em 83 pacientes utilizando o teste ANOVA Para outras análises estatísticas, foram utilizados os testes t e qui-quadrado com nível de significância de 5%. Resultados: Foram encontrados três polimorfismos de nucleotídeo único (SNPs) nos pacientes com AA adquirida (rs956732, rs6141 e rs3804618). Os mesmos três SNPs foram observados nos indivíduos do grupo controle (p>0,05). Não houve associação entre o número de plaquetas e a presença de SNPs nos pacientes (p>0,05). Conclusões: Três SNPs foram encontrados em frequências alélicas semelhantes tanto no grupo de pacientes quanto nos controles, sugerindo que a trombopoetina não apresenta alterações genéticas que possam ser associadas à fisiopatologia da AA adquirida nessa coorte. / Introduction: Acquired aplastic anemia (AA) is a severe illness, characterized by pancytopenia and hypocellular bone marrow without increased reticulin or abnormal infiltration of the bone marrow. Although the physiopathological mechanism has not been completely understood, an immune-mediated T-lymphocyte response has been attributed to the bone marrow environment. Thrombopoietin (encoded by THPO), a glycoprotein hormone produced by the liver and responsible for stimulating the growth of megakaryocytes, development of platelets and other lineages that when dysfunctional, contributes to the progress of acquired AA. Objectives: To screen the THPO gene for genetic variants in bone marrow of acquired AA patients and in the peripheral blood of controls, and to verify the correlation between the THPO status and platelet counts in the patients during the treatment. Method: Sanger sequencing of the THPO gene was carried out in 92 acquired AA patients (case group) and 92 controls, in DNA samples previously stored in the Hematology Laboratory of the Ribeirão Preto School of Medicine at the University of São Paulo. The association between the THPO status and the platelet counts was performed in 83 patients through the ANOVA test. The Chi-squared test and t-test were also applied for statistical analysis with a 5% significance level. Results: Three single nucleotide polymorphisms (SNPs) were found in the AA patients (rs956732, rs6141, and rs3804618), as well as in the healthy subjects (p>0,05). No association was verified between the platelet counts and the presence of SNPs in the AA patients (p>0,05). Conclusion: Three SNPs were found in both groups, suggesting that thrombopoietin does not harbor genetic variants that could be etiological for the acquired AA in our cohort.

AA é uma doença grave caracterizada por pancitopenia e hipocelularidade medular, que pode levar a morte. Em casos severos os tratamentos são restritos ao transplante alogênico de MO e/ou uso de imunossupressores. Para isso, deve ser tipado o HLA entre doador e beneficiário. Com isso, as CTM são fontes celulares candidatas para este propósito, pois são praticamente não imunogênicas devido à sua ação parácrina de imunomodulação. Além disso, as CTM possuem um importante papel na hematopoiese. Para induzir AA utilizamos inicialmente ciclofosfamida (n=10) e radiação ionizante a 6 Gy (n=40), que foi o modelo experimental adotado. Após a radiação ocorreram três transplantes de CTIPDh com intervalo de 15 dias via IP. A resposta clínica do transplante foi monitorada pela avaliação da massa corpórea, hemograma e histopatologia da MO. O enxerto das CTIPDh na MO e seu efeito na hematopoiese, também foi verificado. Os resultados demonstraram que os animais irradiados e tratados com CTIPDh apresentaram benefícios clínicos em relação aos animais não tratados. Observamos enxertia das CTIPDh na MO e baço dos camundongos e recuperação dos componentes medulares em comparação aos animais não tratados. Assim, o transplante das CTIPDh são uma fonte terapêutica em potencial. / AA is a several disease characterized by peripheral pancytopenia and bone marrow hypoplasia, which can lead to death. In severe cases, treatments are limited to allogeneic BM transplant and/or immunosuppressants administration. For this purpose, the donor blood must be typed to identify their HLA. Thereby, MSC are appropriate candidates for therapeutic use, because they present low immunogenicity and provide immunomodulation effect in tissue repair. Furthermore, MSC have important role in supporting hematopoiesis. Thus, AA was induced using cyclophosphamide (n = 10) and 6 Gy ionizing radiation (n = 40), which caused BM ablation in mice. Thus ionizing radiation of AA model was selected. After radiation, the mice received three equal doses of hDPSC each 15-day via intraperitoneal injection. Clinical response was monitored by body mass, blood cells counting and histopathology of BM. The influence of hDPSC infusion on hematopoiesis and engraftment capacity of this cell was also investigated. Our data show the hDPSC transplantation in irradiated mice improves clinical conditions and the transplant intraperitoneal showed hDPSC grafting in BM and recovery of medullary components when compared to untreated group. The results indicate that hDPSC transplantation is a potential tool for cell-based therapeutics.

Introdução: A anemia aplástica (AA) adquirida é uma doença grave, caracterizada por pancitopenia e medula óssea hipocelular sem que haja associação com aumento de reticulina ou infiltração anormal na medula. Embora o mecanismo fisiopatológico não esteja totalmente elucidado, atribui-se a uma resposta imunomediada dos linfócitos T no ambiente medular. A trombopoetina (codificada pelo gene THPO) é um hormônio glicoproteico produzido pelo fígado e responsável pelo estímulo de crescimento de megacariócitos, desenvolvimento plaquetário e de demais linhagens e, quando disfuncional, contribui para o desenvolvimento da AA adquirida. Objetivos: Investigar a presença de variantes genéticas no THPO em amostras de sangue periférico e medula óssea de pacientes com AA adquirida (grupo caso) e de indivíduos saudáveis (grupo controle) e verificar a presença de alterações no número de plaquetas durante o seguimento dos pacientes com AA adquirida. Métodos: O gene THPO foi sequenciado em amostras de DNA de medula óssea de 92 pacientes com AA adquirida e no DNA de sangue periférico de 92 controles, cujas amostras haviam sido previamente armazenado no Laboratório de Hematologia da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (FMRP-USP). O sequenciamento foi realizado pelo método de Sanger. Realizou-se também a associação entre a presença (ou ausência) de variantes em THPO e o número de plaquetas em 83 pacientes utilizando o teste ANOVA Para outras análises estatísticas, foram utilizados os testes t e qui-quadrado com nível de significância de 5%. Resultados: Foram encontrados três polimorfismos de nucleotídeo único (SNPs) nos pacientes com AA adquirida (rs956732, rs6141 e rs3804618). Os mesmos três SNPs foram observados nos indivíduos do grupo controle (p>0,05). Não houve associação entre o número de plaquetas e a presença de SNPs nos pacientes (p>0,05). Conclusões: Três SNPs foram encontrados em frequências alélicas semelhantes tanto no grupo de pacientes quanto nos controles, sugerindo que a trombopoetina não apresenta alterações genéticas que possam ser associadas à fisiopatologia da AA adquirida nessa coorte. / Introduction: Acquired aplastic anemia (AA) is a severe illness, characterized by pancytopenia and hypocellular bone marrow without increased reticulin or abnormal infiltration of the bone marrow. Although the physiopathological mechanism has not been completely understood, an immune-mediated T-lymphocyte response has been attributed to the bone marrow environment. Thrombopoietin (encoded by THPO), a glycoprotein hormone produced by the liver and responsible for stimulating the growth of megakaryocytes, development of platelets and other lineages that when dysfunctional, contributes to the progress of acquired AA. Objectives: To screen the THPO gene for genetic variants in bone marrow of acquired AA patients and in the peripheral blood of controls, and to verify the correlation between the THPO status and platelet counts in the patients during the treatment. Method: Sanger sequencing of the THPO gene was carried out in 92 acquired AA patients (case group) and 92 controls, in DNA samples previously stored in the Hematology Laboratory of the Ribeirão Preto School of Medicine at the University of São Paulo. The association between the THPO status and the platelet counts was performed in 83 patients through the ANOVA test. The Chi-squared test and t-test were also applied for statistical analysis with a 5% significance level. Results: Three single nucleotide polymorphisms (SNPs) were found in the AA patients (rs956732, rs6141, and rs3804618), as well as in the healthy subjects (p>0,05). No association was verified between the platelet counts and the presence of SNPs in the AA patients (p>0,05). Conclusion: Three SNPs were found in both groups, suggesting that thrombopoietin does not harbor genetic variants that could be etiological for the acquired AA in our cohort.

Στην επίκτητη απλαστική αναιμία (ΑΑ) ο υποκυτταρικός μυελός και η πανκυτταροπενία στο περιφερικό αίμα είναι αποτέλεσμα βλάβης των αρχέγονων αιμοποιητικών κυττάρων. Προηγούμενες μελέτες έχουν δείξει ότι κύριο χαρακτηριστικό γνώρισμα της νόσου είναι η ποσοτική αλλά και ποιοτική διαταραχή της stem cell δεξαμενής. Κλινικά και εργαστηριακά ευρήματα προτείνουν το σημαντικό ρόλο του ανοσοποιητικού συστήματος και ειδικά των Τ λεμφοκυττάρων στην ανάπτυξη της απλαστικής αναιμίας. Σήμερα, πλέον, είναι ευρύτερα αποδεκτό ότι η καταστολή του μυελού, που παρατηρείται στην ιδιοπαθή απλαστική αναιμία, είναι αποτέλεσμα της υπερπαραγωγής των μυελοκατασταλτικών κυτταροκινών IFN-γ και TNF-α από διεγερμένα CD8+ κυτταροτοξικά λεμφοκύτταρα, τα οποία συναντούμε τόσο στο περιφερικό αίμα όσο και στο μυελό ασθενών με απλαστική αναιμία. Οι κυτοκίνες αυτές παρουσιάζουν μάλλον προσθετική αντί συνεργική δράση, η οποία σχετίζεται με την IFN-γ-εξαρτώμενη αύξηση της έκφρασης του Fas στα CD34+ κύτταρα και από την IFN-γ-επαγώμενη έκκριση του TNF-α από τα μακροφάγα. Μελέτες έχουν δείξει ότι τόσο τα CD34+ όσο και τα BMMNC κύτταρα του μυελού των ασθενών με ΑΑ είναι περισσότερα αποπτωτικά σε σχέση με φυσιολογικούς μυελούς. Στόχος της διατριβής ήταν η περαιτέρω μελέτη των μοριακών μηχανισμών που εμπλέκονται στην ανοσολογικής προέλευσης απλαστική αναιμία. Λόγω, όμως, του ότι η απλαστική αναιμία είναι μια σπάνια νόσος, η παρουσία ενός εύκολα αναπαραγώγιμου in vitro μοντέλου μυελικής απλασίας θα βοηθούσε περισσότερο στη μοριακή μελέτη αυτής. Στη μελέτη μας, η αναπαραγωγή του καταλληλότερου μοντέλου μυελικής απλασίας επιτεύχθηκε με την προσθήκη των μυελοκατασταλτικών κυτταροκινών IFN-γ και TNF-α σε φυσιολογικό σύστημα μακράς διάρκειας καλλιέργειας μυελού των οστών. Στο μοντέλο αυτό έγινε διερεύνηση των μοριακών μονοπατιών των σχετιζομένων με την Fas και TRAIL επαγόμενη απόπτωση. Παράλληλα, έγινε σύγκριση των δεδομένων από το in vitro μοντέλο με τα αποτελέσματα της παράλληλης μελέτης των αντίστοιχων μοριακών παραμέτρων σε κύτταρα μυελού ασθενών με απλαστική αναιμία. Στο IFN-γ/ΤNF-α μοντέλο παρατηρήθηκε σημαντική μείωση τόσο των πιο άωρων LTC-IC όσο και των πιο δεσμευμένων προγονικών κυττάρων, σε σχέση με τις καλλιέργειες-μάρτυρες. Επιπλέον, τα αποτελέσματα των πρωτογενών και δευτερογενών καλλιεργειών βραχείας διάρκειας σε μυελικά κύτταρα ασθενών με ενεργό νόσο, επιβεβαίωσαν το χαρακτηριστικό γνώρισμα της απλαστικής αναιμίας, δηλαδή, το μειωμένο αριθμό προγονικών αιμοποιητικών κυττάρων. Η επώαση φρέσκων φυσιολογικών μυελικών κυττάρων, σε 5-6 εβδομάδων LTBMC σύστημα, με συνδυασμό των TNF-α/IFN-γ παραγόντων οδηγεί σε αύξηση της Fas mRNA έκφρασης στα CD34+ κύτταρα, κάτι που δεν παρατηρείται στα, αντίστοιχα, φρέσκα και στα 5-6 εβδομάδων κύτταρα-μάρτυρες. Το εύρημα αυτό σε συνδυασμό με τη χαμηλή mRNA έκφραση της caspase 3 καθώς και την απουσία έκφρασης των Bcl-2, Bax και της caspase 8 στον ίδιο πληθυσμό, προτείνουν το σημαντικό ρόλο του external μονοπατιού επαγωγής της απόπτωσης, όπως αυτό ρυθμίζεται από την δράση των μυελοκατασταλτικών κυτοκινών TNF-α/IFN-γ. Παράλληλα, η παρουσία χαμηλής Bcl-2 mRNA έκφρασης, στα CD34+ κύτταρα, τονίζει τη σημασία της αναλογίας προ-αποπτωτικών/αντι-αποπτωτικών σημάτων στη κυτταρική έκβαση. To σημαντικότερο, πάντως, εύρημα του TNF-α/IFN-γ μοντέλου είναι η συνεχής TRAIL mRNA έκφραση, στα CD34+ κύτταρα αυτού, κάτι το οποίο δεν έχει αναφερθεί, ως τώρα, στη βιβλιογραφία. Η μοριακή ανάλυση των μυελικών κυττάρων των ΑΑ ασθενών απεκάλυψε, εκτός της Fas mRNA έκφρασης στα BMMNC και/ή στα CD34+ κύτταρα, την αυξημένη TRAIL mRNA έκφραση στο CD34+ κυτταρικό πληθυσμό των ασθενών με ενεργό νόσο. Αντίθετα, στους ασθενείς σε ύφεση, απουσιάζει η έκφραση και των δύο γονιδίων στον ίδιο πληθυσμό. Ενώ, στα BMMNC η έκφραση του TRAIL mRNA παραμένει, ένα συνεχές εύρημα, ακόμη και στους ασθενείς σε ύφεση. Επιπρόσθετα, η μειωμένη έκφραση των αντι-αποπτωτικών γονιδίων Bcl-xl και/ή Bcl-2 στα BMMNC όλων των ασθενών και του Bcl-xl στα CD34+ κύτταρα των ασθενών με ενεργό νόσο, δείχνει «ανίκανη» να αναστείλλει το μηχανισμό της απόπτωσης στους AA ασθενείς. Το γεγονός ότι η έκφραση του TRAIL mRNA είναι συνεχής στο CD34+ κυτταρικό πληθυσμό του TNF-α/IFN-γ μοντέλου και μόνο στα CD34+κύτταρα ΑΑ ασθενών με ενεργό νόσο, δείχνει τη σημαντικότητα του συγκεκριμένου μορίου στην απόπτωση των προγονικών κυττάρων στη μυελική απλασία. Συμπερασματικά, το μοντέλο μυελικής απλασίας που τελικά επιλέξαμε επιβεβαιώνει προηγούμενη γνώση της συμμετοχής των TNF-α και IFN-γ στη παθοφυσιολογία της απλαστικής αναιμίας. Παράλληλα, τα μοριακά δεδομένα, τόσο του μοντέλου όσο και των ασθενών με απλαστική αναιμία, ενισχύουν την εμπλοκή του Fas/FasL στη παθογένεση της νόσου και προτείνουν την πιθανή συμμετοχή του TRAIL/Apo2L στην όλη διαδικασία. / In aplastic anemia (AA) the hypocellular bone marrow and blood pancytopenia occur as a result of damage to hematopoietic stem cells . Previous studies have shown that a profound quantitative and qualitative defect in the stem cell compartment is a common feature in most patients with AA. Clinical and laboratory data suggest that the immune system, especially T lymphocytes, have an important role in the development of AA. It is well established that IFN-γ and TNF-α mediate hematopoietic stem cell suppression in aplastic anemia. These proinflammatory cytokines exhibit additive rather than synergistic effect, which may be mediated by the IFN-γ-dependent increase in Fas expression on CD34+ progenitor cells and by the IFN-γ-inducible secretion of TNF-α by macrophages. Bone marrow total mononuclear and progenitor cells from aplastic anemia patients are more apoptotic than cells from normal donors, indicating that apoptosis may be a major mechanism of stem cell loss in aplastic anemia. The aim of our study was to investigate the molecular mechanisms involved in the immune-mediated pathology of aplastic anemia. Since aplastic anemia is a rare disease the existence of an easily reproducible model of in vitro hematopoietic cell suppression can facilitate studies concerning the molecular pathways of this disease. In our study, we reproduced such a model with the addition of the myelosuppressive cytokines IFN-γ and TNF-α in a normal long term bone marrow culture system. In this model, we examined the Fas mediated pathway of apoptosis and especially the correlation between TRAIL expression and myelosuppression. We, also, studied these parameters in marrow cells from aplastic anemia patients. The IFN-γ and TNF-α inhibitory cytokines appeared to affect both immature LTC-ICs and more commited progenitor cells capable of lineage-specific colony formation (CFCs). In addition our progenitor cell assays results in patients, supported this unifying feature of reduced haematopoietic progenitor cells in aplastic anemia. TNF-α and IFN-γ treatment up-modulated Fas expression and induced apoptosis of 5-6 weeks cultured normal CD34+ cells, while normal freshly isolated and 5-6 weeks untreated cultured CD34+ cells showed no Fas mRNA expression. This finding, along with the low mRNA expression of caspase 3 and the absence of Bcl-2, Bax and caspase 8 expression, proposes the major role for activation of the extrinsic apoptosis pathway due to treatment of BMMNC with TNF-α and IFN-γ. In parallel, the existence of the low Bcl-xl mRNA expression in the same cell compartment points to the importance of the ratio of pro-apoptotic to anti-apoptotic signals, in cell fate. The most interesting finding is the constant TRAIL mRNA expression on CD34+ cells in TNF-α/IFN-γ treated LTBMC, something not mentioned before. Molecular analysis of patients’ marrow cells revealed, apart from Fas mRNA expression in BMMNC and/or CD34+ cells, TRAIL mRNA expression in CD34+ cell population in active disease. Ιn contrast, in patients in remission, both Fas and TRAIL mRNA expression does not exist. Instead, in BMMNC’s cell compartment TRAIL mRNA expression remains a constant finding even in patients in remission. Additionally, the decreased expression of anti-apoptotic bcl-xl and/or bcl-2 in all patients’ BMMNCs and bcl-xl expression in CD34+ cells from patients with active disease, seems unable to inhibit the mechanism of apoptosis in aplastic anemia patients. In our study, the fact that the expression of TRAIL was constant on CD34+ cells in TNF-α/IFN-γ treated LTBMC and only in CD34+ cells of patients with active disease, points out its significance in apoptosis of progenitor cells. In conclusion, our in vitro model of hematopoietic suppression confirmed previous knowledge for participation of TNF-α and IFN-γ in the pathophysiology of marrow failure in aplastic anemia. In parallel, the molecular data both from the in vitro model, as well as from patients with aplastic anemia, reinforce the implication of Fas/FasL pathway in the pathogenesis of this disease and propose a probable role for TRAIL/Apo2L in the process.

2,4,6-Trinitrotoluene, TNT, is an explosive used in munitions production that is known to cause both aplastic and hemolytic anemia in exposed workers. Deaths have been reported secondary to both varieties of anemia. Studies have shown that TNT systemic absorption is significant by both the respiratory and dermal routes. A literature review revealed that the most recent review article on TNT exposure arguing for a TWA drop from the PEL to the TLV was in 1977 -- this article cited anemia issues in addition to other untoward effects of TNT. No studies encountered looked at hemoglobin change or anemia cases in respiratory protected workers, this present effort may be the first. TNT PEL (1.5mg/m^3), REL (0.5mg/m^3), and TLV (0.1 mg/m^3) 8 hr TWAs all with skin notations (based on animal models and TNT urine metabolite extrapolation in TNT workers suggesting important role of skin absorption). The earliest effects of systemic TNT poisoning involve hgb and hematocrit drop. The investigator hypothesized that respiratory protection alone is insufficient to protect TNT workers from the risk of anemia development and hemoglobin concentration drop. A retrospective observational study design was incorporated utilizing a records review of TNT vapor air concentration values and worker Hgb values for 8 sets of workers in respiratory protection at a demilitarization operation from October 2006 to April 2007 in order to observe whether or not respiratory protection provided adequate protection against anemia development and hemoglobin change; and to help characterize the probable role of TNT skin absorption on hemoglobin change and anemia risk. Worker baseline hgbs were compared with their exposure hgbs for statistically significant hgb concentration changes (two-tailed paired t-tests), and anemia cases were recorded. Mean hgb changes within each of the 8 groups of workers were then regressed on mean TNT air concentrations (10 hr TWAs) using air sampling levels that were performed closest in time to exposure hgbs. Statistically significant hgb concentration drops and anemia cases were apparent at values about the REL and PEL in respiratory protected workers. There were no anemia cases or statistically significant hgb drops at values about the TLV, however. For mean TNT air concentrations from 0.12/m 3 to 0.31/m 3 there was strong positive linear association with regard to magnitude of hgb change (r=0.996). The results appear to confirm the necessity of the skin notation for TNT. However, the TLV seems to be protective against the possibility of anemia risk principally by the dermal route in workers who are respiratory protected. A question does still remain, however, as to anemia risk in workers who are below the TLV who may not be using respiratory protection. The absence of a continued linear association between mean TNT air concentrations and mean hgb change above the 0.31 mg/m 3 TNT level most likely reflects a marrow response, as the TNT levels evident in the study are reported to be mainly associated with extravascular hemolysis with a minimal, or non-existent, aplastic component assumed. This study adds evidence to the argument that the TLV should be adopted as the new PEL.

No description available.

A anemia aplástica severa (AAS) pode ser tratada com sucesso na maioria dos casos com terapia imunossupressora (IS) ou transplante alogenêico de medula óssea (TMO). Os principais fatores que determinam a escolha da modalidade terapêutica são a idade e a disponibilidade de um doador HLA-histocompatível. Em pacientes mais jovens, o TMO de um doador aparentado é preferível, enquanto que em pacientes acima de 40-50 anos, a terapia IS é a modalidade terapêutica de escolha. Resposta hematológica é obtida em 60-75% dos casos com terapia IS na AAS, o que correlaciona com melhor sobrevida. Recidivas ocorrem em aproximadamente um terço dos respondentes e evolução clonal para mielodisplasia em 10-15% ao longo termo. A doença do enxerto-versus-hospedeiro (GVHD) agudo ocorre em 30-40% dos casos sendo a forma crônica presente em 40-50%. Infecções são frequentes e podem complicar o transplante. Portanto, a refratariedade à terapia IS, recidivas e evolução clonal limitam o sucesso da terapia IS na AAS, enquanto rejeição do enxerto, GVHD, e infecções limitam o sucesso do TMO na clínica. Fatores preditivos dessas complicações seriam de grande valor na clínica, uma vez que poder-se-iam realizar decisões terapêuticas com base mais racional, onde pacientes fossem alocados a diferentes tratamentos com base no seu perfil de risco. Ou seja, pacientes com alta probabilidade de resposta e baixo risco de recidiva e evolução clonal se beneficiariam de terapia IS, enquanto àqueles com baixa probabilidade de resposta e alto risco de recidiva e/ou evolução clonal teriam mais benefícios do TMO, por exemplo. Com base nessa premissa, desenvolvemos estudos para investigar fatores que pudessem estar associados ao sucesso da terapia IS na AAS. Os principais achados de 3 análises distintas sobre o tema evidenciou: 1) crianças (< 18 anos) apresentam alta taxa de resposta à terapia IS (em torno de 75%) com uma excelente sobrevida geral em pacientes respondentes; 2) o número absoluto de reticulócitos e de linfócitos pré-tratamento correlaciona com resposta hematológica aos seis meses após terapia IS; e 3) o comprimento telomérico não está associado à resposta hematológica, porém, está associado a probabilidade de recidiva, evolução clonal, e sobrevida geral após terapia IS. Esses parâmetros identificados nesses estudos podem servir de base em algoritmos futuros onde faz-se estratificação de risco de cada paciente, a fim de alocar a modalidade terapêutica mais apropriada com base no perfil individual de risco. No que diz respeito ao comprimento telomérico, é provável que esse marcador biológico não só esteja associado ao processo de evolução clonal na AAS, mas que também participe na biologia da instabilidade genômica de células na medula óssea levando a aberrações cromossômicas e o desenvolvimento de mielodisplasia e leucemias. / Severe aplastic anemia (SAA) can be treated successfully in the majority of cases with immunosuppressive therapy (IST) or allogeneic bone marrow transplantation (BMT). The principal factors that determine the choice of treatment modalities are age and availability of an HLA-histocompatible donor. In younger patients, BMT from a related donor is preferred, while in patients over 40-50 years of age, IST is often employed. Hematologic response is achieved in 60-75% of cases with IST, which correlates with better survival. Relapses occur in approximately one third of responders and clonal evolution to myelodysplasia occurs in 10-15% of cases long-term. Acute graft-versus-host disease (GVHD) occurs in 30-40% of cases and chronic GVHD in 40-50%. Infections are common and complicate transplant outcomes. Therefore, refractoriness, relapses and clonal evolution limit the success of IST in SAA, while graft rejection, GVHD, and infections limited the success of BMT in the clinic. Predictors for these complications would be of great value in the clinic since one could make more rational treatment decisions where patients were allocated to different treatment modalities based on their risk profile. For example, patients with high probability of response and low risk of relapse and clonal evolution would benefit more from IST, while those with low probability of hematologic response and high risk of recurrence and/or clonal evolution most likely to benefit from BMT. Based on this premise, we developed studies to investigate factors that could be associated with the success of IST in SAA. The main findings of three separate analysis on the subject showed: 1) children ( < 18 years) have a high response rate to IST (around 75%) with an excellent long-term survival rate among responders; 2) the absolute number of reticulocytes and lymphocytes pre-treatment correlates with hematologic response at 6 months after IST, and 3) telomere length is not associated with hematologic response, but, associated with the likelihood of relapse, clonal evolution, and overall survival after IST. These parameters may serve as a basis for future algorithms allowing for risk stratification for each individual patient allowing for better treatment allocation. With respect to the telomere length, it is likely that it not only represents a biological marker but that it is involved in the process of clonal evolution contributing to genomic instability in bone marrow cells leading to the development of myelodysplasia and leukemia

A anemia aplástica severa (AAS) pode ser tratada com sucesso na maioria dos casos com terapia imunossupressora (IS) ou transplante alogenêico de medula óssea (TMO). Os principais fatores que determinam a escolha da modalidade terapêutica são a idade e a disponibilidade de um doador HLA-histocompatível. Em pacientes mais jovens, o TMO de um doador aparentado é preferível, enquanto que em pacientes acima de 40-50 anos, a terapia IS é a modalidade terapêutica de escolha. Resposta hematológica é obtida em 60-75% dos casos com terapia IS na AAS, o que correlaciona com melhor sobrevida. Recidivas ocorrem em aproximadamente um terço dos respondentes e evolução clonal para mielodisplasia em 10-15% ao longo termo. A doença do enxerto-versus-hospedeiro (GVHD) agudo ocorre em 30-40% dos casos sendo a forma crônica presente em 40-50%. Infecções são frequentes e podem complicar o transplante. Portanto, a refratariedade à terapia IS, recidivas e evolução clonal limitam o sucesso da terapia IS na AAS, enquanto rejeição do enxerto, GVHD, e infecções limitam o sucesso do TMO na clínica. Fatores preditivos dessas complicações seriam de grande valor na clínica, uma vez que poder-se-iam realizar decisões terapêuticas com base mais racional, onde pacientes fossem alocados a diferentes tratamentos com base no seu perfil de risco. Ou seja, pacientes com alta probabilidade de resposta e baixo risco de recidiva e evolução clonal se beneficiariam de terapia IS, enquanto àqueles com baixa probabilidade de resposta e alto risco de recidiva e/ou evolução clonal teriam mais benefícios do TMO, por exemplo. Com base nessa premissa, desenvolvemos estudos para investigar fatores que pudessem estar associados ao sucesso da terapia IS na AAS. Os principais achados de 3 análises distintas sobre o tema evidenciou: 1) crianças (< 18 anos) apresentam alta taxa de resposta à terapia IS (em torno de 75%) com uma excelente sobrevida geral em pacientes respondentes; 2) o número absoluto de reticulócitos e de linfócitos pré-tratamento correlaciona com resposta hematológica aos seis meses após terapia IS; e 3) o comprimento telomérico não está associado à resposta hematológica, porém, está associado a probabilidade de recidiva, evolução clonal, e sobrevida geral após terapia IS. Esses parâmetros identificados nesses estudos podem servir de base em algoritmos futuros onde faz-se estratificação de risco de cada paciente, a fim de alocar a modalidade terapêutica mais apropriada com base no perfil individual de risco. No que diz respeito ao comprimento telomérico, é provável que esse marcador biológico não só esteja associado ao processo de evolução clonal na AAS, mas que também participe na biologia da instabilidade genômica de células na medula óssea levando a aberrações cromossômicas e o desenvolvimento de mielodisplasia e leucemias. / Severe aplastic anemia (SAA) can be treated successfully in the majority of cases with immunosuppressive therapy (IST) or allogeneic bone marrow transplantation (BMT). The principal factors that determine the choice of treatment modalities are age and availability of an HLA-histocompatible donor. In younger patients, BMT from a related donor is preferred, while in patients over 40-50 years of age, IST is often employed. Hematologic response is achieved in 60-75% of cases with IST, which correlates with better survival. Relapses occur in approximately one third of responders and clonal evolution to myelodysplasia occurs in 10-15% of cases long-term. Acute graft-versus-host disease (GVHD) occurs in 30-40% of cases and chronic GVHD in 40-50%. Infections are common and complicate transplant outcomes. Therefore, refractoriness, relapses and clonal evolution limit the success of IST in SAA, while graft rejection, GVHD, and infections limited the success of BMT in the clinic. Predictors for these complications would be of great value in the clinic since one could make more rational treatment decisions where patients were allocated to different treatment modalities based on their risk profile. For example, patients with high probability of response and low risk of relapse and clonal evolution would benefit more from IST, while those with low probability of hematologic response and high risk of recurrence and/or clonal evolution most likely to benefit from BMT. Based on this premise, we developed studies to investigate factors that could be associated with the success of IST in SAA. The main findings of three separate analysis on the subject showed: 1) children ( < 18 years) have a high response rate to IST (around 75%) with an excellent long-term survival rate among responders; 2) the absolute number of reticulocytes and lymphocytes pre-treatment correlates with hematologic response at 6 months after IST, and 3) telomere length is not associated with hematologic response, but, associated with the likelihood of relapse, clonal evolution, and overall survival after IST. These parameters may serve as a basis for future algorithms allowing for risk stratification for each individual patient allowing for better treatment allocation. With respect to the telomere length, it is likely that it not only represents a biological marker but that it is involved in the process of clonal evolution contributing to genomic instability in bone marrow cells leading to the development of myelodysplasia and leukemia