Expressão de Fas e Fas-ligante em pacientes com hemoglobinúria paroxística noturna
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Data
2009
Tipo
Tese de doutorado
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INTRODUÇÃO: A deficiência e a exacerbação do sistema Fas/FasL está associada ao
aparecimento de diversas doenças hematológicas ou não. Na HPN, existem
evidências de que o sistema Fas/FasL possa atuar na seleção e proliferação do clone
mutante através do aumento da apoptose das células normais. Nosso objetivo foi
determinar a expressão dos marcadores de apoptose Fas e FasL em neutrófilos e
monócitos de pacientes com HPN e tentar estabelecer relação entre estes
marcadores de apoptose e os subtipos clínicos da doença.
MATERIAL E MÉTODOS: Foram estudados 16 pacientes em acompanhamento nos
Serviços de Hematologia da Universidade de São Paulo (USP) e da Escola Paulista de
Medicina (UNIFESP). Os pacientes foram divididos clinicamente em HPN clássica
(HPN: 9 indivíduos) e HPN associada a quadro de falência medular (HPN/FM: 7
indivíduos). Foi avaliada a expressão de Fas (CD95) e de FasL em neutrófilos (CD59)
e monócitos (CD14) de portadores de HPN, através de citometria de fluxo
(FACScalibur, programa Cell Quest - BDB). Nos indivíduos HPN, as populações de
neutrófilos foram divididas segundo sua positividade para o antígeno CD59 (59+/PC
ou 59-/PC), e nos monócitos para o CD14 (14+/PC ou 14-/PC). Também foi
analisada a expressão de Fas/FasL em neutrófilos (59+/GC) e monócitos (14+/GC)
de 10 indivíduos normais (grupo controle).
RESULTADOS: Quando analisado o grupo total de pacientes, não foi vista diferença
estatística na expressão do Fas quando comparados granulócitos (59+/PC, 59-/PC,
59+/GC) e monócitos (14+/PC, 14-/PC, 14+/GC). A expressão de FasL nos
granulócitos 59-/PC foi significantemente menor do que nos 59+/PC (p=0,008). Não
foi observada diferença estatística na expressão de FasL em monócitos. Quando
analisados os subtipos clínicos, pacientes com HPN/FM apresentaram menor
expressão de Fas nos granulócitos 59-/PC (p=0,041) e nos monócitos 14-/PC
(p=0,014) do que os indivíduos com HPN. A expressão de FasL não mostrou
alterações em granulócitos e monócitos entre os grupos HPN e HPN/FM. Nos
indivíduos com HPN/FM, entretanto, as células 59-/PC apresentaram expressão de
FasL significantemente diminuídas em relação às 59+/PC (p=0,031).
DISCUSSÃO E CONCLUSÕES: Relatos anteriores sugerem que ocorra maior
expressão de Fas em células-tronco CD59+ em comparação às células-tronco CD59-.
Neste estudo, pacientes com falência medular associada apresentaram menor
expressão de Fas em neutrófilos 59-/PC e monócitos 14-/PC do que os pacientes com
HPN clássica, sugerindo que nestes pacientes as células 59+/PC e 14+/PC estariam
mais sujeitas à apoptose, concordando com a hipótese de vantagem da sobrevida do clone HPN. A expressão de FasL foi maior em neutrófilos 59+/PC em comparação
com 59-/PC, fato que se confirmou especialmente no grupo com falência medular
associada. Este dado poderia sugerir um excesso de FasL na medula óssea levando à
apoptose das células-tronco, o que explicaria a alterações medulares características
da HPN. Apesar da importância do FasL na apoptose, este é o primeiro estudo a
analisar a expressão de FasL em fagócitos de pacientes com HPN.
INTRODUCTION: The Fas/FasL system dysfunction has been associated with hematological and non hematological diseases. In PNH, there are evidences that this system could select and maintain the proliferation of the mutational clone through the promotion of normal cells apoptosis. The aim of this study was to analyze the expression of Fas and FasL in neuthophils and monocytes from PNH patients. We tried also to detect a possible relation between these expressions and clinical subtypes of this disease. METHODS: We analyzed 16 patients followed in 2 institutions: Sao Paulo State University (USP) and Federal University of Sao Paulo (UNIFESP). The patients were clinically classified as classic PNH (PNH/CL: 9 patients) and PNH in the setting of another bone marrow failure syndrome (PNH/BF: 7 patients). The expression of Fas (CD95) e Fas ligant (FasL) in neutrophils (CD59) and monocytes (CD14) was analyzed by flow citometry (FACScalibur, Cell Quest software - BDB). In PNH individuals, the neutrophils were divided according CD59 positivity (59+/Pt or 59- /Pt), and in monocytes according CD14 positivity (14+/Pt or 14-/Pt). There was analyzed the Fas/FasL expression in neutrophils (59+/CG) and monocytes (14+/CG) in 10 normal individuals (control group). RESULTS: When we analyzed all patients, no statistical difference was found in Fas expression among the neutrophils 59+/Pt, 59-/Pt and 59+/CG. The same result was observed among monocytes 14+/Pt, 14-/Pt and 14+/CG. FasL expression on 59-/Pt neutrophils was significantly less than on 59+/Pt (p=0.008). FasL expression on monocytes showed no statistical difference. When we analyzed the data according with clinical subtypes, PNH/BF patients had less Fas expression on 59-/Pt neutrophils (p=0.041) and on 14-/Pt monocytes (p=0.014) than PNH/CL. FasL expression did not showed difference on neutrophils and monocytes between PNH/CL and PNH/BF patients. The group of PNH/BF individuals, however, had 59-/Pt with less FasL expression than 59+/Pt cells (p=0.031). DISCUSSION: Previous studies had been show higher expression of Fas in CD59+/stem cells than CD59-/stem cells. In this study, PNH/BF patients had less Fas expression on 59-/Pt and on 14-/Pt cells than PNH/CL patients. This data suggest that 59+/Pt and 14+/PT cells could be more susceptible to apoptosis, with is in agreement with the PNH clone survival advantage hypothesis. FasL expression was higher on 59+/Pt than on 59-/Pt, especially in the PNH/BF group. We could speculate, with this data, that FasL in excess could act in bone marrow, initiating the stem cells apoptosis process, what explain the characteristics bone marrow alterations found in PNH. Although of the importance of FasL in apoptosis, this is the first study to analyze its expression on PNH.
INTRODUCTION: The Fas/FasL system dysfunction has been associated with hematological and non hematological diseases. In PNH, there are evidences that this system could select and maintain the proliferation of the mutational clone through the promotion of normal cells apoptosis. The aim of this study was to analyze the expression of Fas and FasL in neuthophils and monocytes from PNH patients. We tried also to detect a possible relation between these expressions and clinical subtypes of this disease. METHODS: We analyzed 16 patients followed in 2 institutions: Sao Paulo State University (USP) and Federal University of Sao Paulo (UNIFESP). The patients were clinically classified as classic PNH (PNH/CL: 9 patients) and PNH in the setting of another bone marrow failure syndrome (PNH/BF: 7 patients). The expression of Fas (CD95) e Fas ligant (FasL) in neutrophils (CD59) and monocytes (CD14) was analyzed by flow citometry (FACScalibur, Cell Quest software - BDB). In PNH individuals, the neutrophils were divided according CD59 positivity (59+/Pt or 59- /Pt), and in monocytes according CD14 positivity (14+/Pt or 14-/Pt). There was analyzed the Fas/FasL expression in neutrophils (59+/CG) and monocytes (14+/CG) in 10 normal individuals (control group). RESULTS: When we analyzed all patients, no statistical difference was found in Fas expression among the neutrophils 59+/Pt, 59-/Pt and 59+/CG. The same result was observed among monocytes 14+/Pt, 14-/Pt and 14+/CG. FasL expression on 59-/Pt neutrophils was significantly less than on 59+/Pt (p=0.008). FasL expression on monocytes showed no statistical difference. When we analyzed the data according with clinical subtypes, PNH/BF patients had less Fas expression on 59-/Pt neutrophils (p=0.041) and on 14-/Pt monocytes (p=0.014) than PNH/CL. FasL expression did not showed difference on neutrophils and monocytes between PNH/CL and PNH/BF patients. The group of PNH/BF individuals, however, had 59-/Pt with less FasL expression than 59+/Pt cells (p=0.031). DISCUSSION: Previous studies had been show higher expression of Fas in CD59+/stem cells than CD59-/stem cells. In this study, PNH/BF patients had less Fas expression on 59-/Pt and on 14-/Pt cells than PNH/CL patients. This data suggest that 59+/Pt and 14+/PT cells could be more susceptible to apoptosis, with is in agreement with the PNH clone survival advantage hypothesis. FasL expression was higher on 59+/Pt than on 59-/Pt, especially in the PNH/BF group. We could speculate, with this data, that FasL in excess could act in bone marrow, initiating the stem cells apoptosis process, what explain the characteristics bone marrow alterations found in PNH. Although of the importance of FasL in apoptosis, this is the first study to analyze its expression on PNH.
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Citação
PAVANI, Rodrigo. Expressão de Fas e Fas-ligante em pacientes com hemoglobinúria paroxística noturna. 2009. 116 f. Tese (Doutorado em Ciências) - Escola Paulista de Medicia, Universidade Federal de São Paulo, São Paulo, 2009.