Expressão de genes relacionados as subpopulações de linfócitos t reguladores e th17 em sangue periférico de pacientes submetidos ao transplante de células hematopoéticas: importância prognóstica na doença do enxerto contra hospedeiro.
Data
2013
Autores
Cabral, Camila Marca De Veiga 
Orientadores
Kerbauy, Fábio Rodrigues
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: Apesar de todos os avancos relacionados ao Transplante de celulas hematopoeticas (TCH), a doenca do enxerto contra hospedeiro (DECH) continua a ser uma complicacao frequente, repercutindo diretamente na morbimortalidade relacionada ao transplante. Os linfocitos T reguladores (Treg) desempenham papel fundamental na manutencao da auto-tolerancia imunologica e sua recuperacao adequeda em numero e funcao apos o TCH, seria fator importante para o efetico controle de linfocitos T citotoxicos e ausencia de DECH. Mais recentemente, as celulas Th17 parecem contribuir para o desenvolvimento da DECH atraves do aumento da producao de citocinas pro-inflamatorias e recrutamento de linfocitos Th1 em orgaos linfoides na fase inicial apos o TCH. Entretanto, grande parte do conhecimento a respeito do papel das celulas Treg e, principalmente Th17 na DECH vem de modelos experimentais em camundongos e os resultados em humanos ainda sao bastante divergentes. Objetivos: 1) Avaliar a expressao de gene especifico para caracterizacao da subpopulacao de linfocitos Treg (FOXP3) e linfocitos Th17 (RORt) em amostras de sangue periferico obtidas de pacientes submetidos a TCH alogenico de doador HLA-identico aparentado. 2) Correlacionar o perfil de expressao dos genes FOXP3 e RORt com a presenca ou ausencia de DECH na populacao estudada. Pacientes e Metodos: cinquenta e um pacientes submetidos ao transplante de celulas hematopoeticas (TCH) alogenico HLA-identicos foram incluidos no estudo. Amostras de sangue periferico foram coletadas pre-TCH, no momento da DECH aguda, no momento da DECH cronica e no D+90 dias pos-TCH naqueles que nao tiveram sinais clinicos de DECH aguda. A expressao dos genes FOXP3 e RORt, como principais representantes das subpopulacoes de linfocitos Treg e Th17, respectivamente, foi avaliada por PCR quantitativo em tempo real (qPCR). Resultados: Foram avaliados 51 pacientes neste estudo, sendo todos os pacientes submetidos a TCH alogenico aparentado HLA-identico, com idade mediana de 36 anos (16-69). As leucemias agudas/Sindrome mielodisplasica foram as principais indicacoes de TCH (61%), assim como predominaram a utilizacao de fonte periferica de celulas hematopoeticas (77%), condicionamento mieloablativo (82%) e como regime preparatorio para o TCH a combinacao de bussulfano e ciclofosfamida (51%). Durante o tempo de seguimento, o tempo mediano de Sobrevida Global e Sobrevida Livre de Progressao foi de 43.3 meses e 33 meses, respectivamente. A mortalidade nao relacionada a recaida foi de 31.4% em 1 ano, sendo a DECH cronica a principal causa de obito (31%). A incidencia cumulativa de DECH aguda em 100dias foi de 45.1% e de DECH cronica de 58.8% em 1 ano. O acometimento de dois ou mais orgaos na DECH-A (96%) e DECH-C (71%) predominou. Quanto a classificacao, 48% dos pacientes com DECH-A foram Grau II e a maioria dos pacientes com DECH-C (74%) foram classificados como grave. Foi observado menor expressao do gene FOXP3 no momento da DECH-A (p=0.003) e DECH-C (p= 0.039) ao realizar analise pareada com suas respectivas amostras do pre-TCH. Nao houve alteracao da expressao do gene RORt no momento da DECH aguda e cronica. Nao foram encontradas diferencas estatisticamente significantes da expressao dos genes FOXP3 e RORt nos diferentes graus de severidade da DECH aguda ou cronica. Da mesma forma que nao demonstraram ser uma ferramenta na predicao de desfechos clinicos na DECH-A. Conclusoes: Houve diminuicao da expressao do gene FOXP3 nas amostras no momento da DECH-A e DECH-C comparativamente as suas respectivas amostras pre-TCH em pacientes submetidos ao TCH alogenico aparentado HLA-identico. Nao houve alteracao na expressao do gene RORt no momento da DECH, tanto aguda quanto cronica, quando comparado ao grupo controle e ao grupo de pacientes sem sinais clinicos de DECH submetidos ao TCH alogenico aparentado HLA-identico. A expressao de FOXP3 e de RORt nao se correlacionou ao prognostico da DECH, nao predizendo mortalidade.
Introduction: Despite all the advances related to hematopoietic cell transplantation (HCT), graft versus host disease (GVHD) remains a common complication with direct impact on the morbidity and mortality related to transplantation. Regulatory T lymphocytes (Treg) play a important role in the maintenance of immunological selftolerance and adequate recovery in number and function after HCT would be an important factor for effective control of cytotoxic T lymphocytes and absence of GVHD. More recently, Th17 cells appear to contribute to the development of GVHD by increasing the production of proinflammatory cytokines and recruitment of Th1 lymphocytes in lymphoid organs in early stage after TCH. However, much of the knowledge about the role of Treg and Th17 in GvHD mainly comes from experimental models in mice and in humans the results are still quite divergent.Objectives: 1) To evaluate the expression of specific gene to characterize the subpopulation of Treg (FOXP3) and Th17 lymphocytes (RORt) in peripheral blood samples obtained from patients undergoing allogeneic HCT from HLAidentical related donor. 2) To correlate the expression profile of genes FOXP3 and RORt with the presence or absence of GVHD in the study population. Patients and Methods: Fiftyone patients undergoing hematopoietic cell transplantation (HCT) allogeneic HLAidentical were included in the study. Peripheral blood samples were collected beforTCH, at the time of acute GVHD (AGVHD) at the time of chronic GVHD (CGVHD) and 90 days postHCT those who did not have clinical signs of acute GVHD. The gene expression of FOXP3 and RORt, as representatives of the major subpopulations of Treg and Th17 cells, respectively, was assessed by realtime PCR (qPCR). Results: There were 51 patients in the study, all patients underwent allogeneic HCT related HLAidentical, with a median age of 36 years (1669. Acute leukemias / myelodysplastic syndrome were the most common indications for HCT (61%), as well as the use predominant source of hematopoietic cells from peripheral (77%), myeloablative conditioning (82%) and as a preparative regimen for HCT the combination of busulfan and cyclophosphamide (51%). During the followup period, the median time of overall and Progression Free Survival was 43.3meses and 33 months, respectively. The nonrelapse mortality was 31.4% at 1 year, and chronic GVHD was the leading cause of death (31%). The cumulative incidence of acute GVHD in 100days was 45.1% and 58.8% of chronic GVHD at 1 year. Involvement of two or more organs in AGVHD (96%) and CGVHD (71%) predominated. Regarding the classification, 48% of patients with AGVHD were Grade II and most patients with CGVHD (74%) were classified as severe. We found a lower expression of the FOXP3 gene at the time of AGVHD (p = 0.003) and CGVHD (p = 0.039) when analyzing samples paired with their respective samples before HCT. There was no change in gene expression RORt the time of GVHD. There were no significant differences in the expression of genes FOXP3 and RORt in different degrees of severity of acute or chronic GVHD. Likewise not shown is a tool for the prediction of clinical outcomes in AGVHD. Conclusions: There was a decrease of FOXP3 gene expression in the samples at the time of GVHD and acute and chronic GVHD benchmark its respective samples before HCT in patients undergoing allogeneic HCT related HLAidentical. There was no change in gene expression RORt the time of GVHD, both acute and chronic, when compared to the control group and the group of patients without clinical signs of GVHD undergoing allogeneic HCT related HLAidentical. The expression of FOXP3 and RORt is not correlated with the prognosis of GVHD, not predicting mortality.
Introduction: Despite all the advances related to hematopoietic cell transplantation (HCT), graft versus host disease (GVHD) remains a common complication with direct impact on the morbidity and mortality related to transplantation. Regulatory T lymphocytes (Treg) play a important role in the maintenance of immunological selftolerance and adequate recovery in number and function after HCT would be an important factor for effective control of cytotoxic T lymphocytes and absence of GVHD. More recently, Th17 cells appear to contribute to the development of GVHD by increasing the production of proinflammatory cytokines and recruitment of Th1 lymphocytes in lymphoid organs in early stage after TCH. However, much of the knowledge about the role of Treg and Th17 in GvHD mainly comes from experimental models in mice and in humans the results are still quite divergent.Objectives: 1) To evaluate the expression of specific gene to characterize the subpopulation of Treg (FOXP3) and Th17 lymphocytes (RORt) in peripheral blood samples obtained from patients undergoing allogeneic HCT from HLAidentical related donor. 2) To correlate the expression profile of genes FOXP3 and RORt with the presence or absence of GVHD in the study population. Patients and Methods: Fiftyone patients undergoing hematopoietic cell transplantation (HCT) allogeneic HLAidentical were included in the study. Peripheral blood samples were collected beforTCH, at the time of acute GVHD (AGVHD) at the time of chronic GVHD (CGVHD) and 90 days postHCT those who did not have clinical signs of acute GVHD. The gene expression of FOXP3 and RORt, as representatives of the major subpopulations of Treg and Th17 cells, respectively, was assessed by realtime PCR (qPCR). Results: There were 51 patients in the study, all patients underwent allogeneic HCT related HLAidentical, with a median age of 36 years (1669. Acute leukemias / myelodysplastic syndrome were the most common indications for HCT (61%), as well as the use predominant source of hematopoietic cells from peripheral (77%), myeloablative conditioning (82%) and as a preparative regimen for HCT the combination of busulfan and cyclophosphamide (51%). During the followup period, the median time of overall and Progression Free Survival was 43.3meses and 33 months, respectively. The nonrelapse mortality was 31.4% at 1 year, and chronic GVHD was the leading cause of death (31%). The cumulative incidence of acute GVHD in 100days was 45.1% and 58.8% of chronic GVHD at 1 year. Involvement of two or more organs in AGVHD (96%) and CGVHD (71%) predominated. Regarding the classification, 48% of patients with AGVHD were Grade II and most patients with CGVHD (74%) were classified as severe. We found a lower expression of the FOXP3 gene at the time of AGVHD (p = 0.003) and CGVHD (p = 0.039) when analyzing samples paired with their respective samples before HCT. There was no change in gene expression RORt the time of GVHD. There were no significant differences in the expression of genes FOXP3 and RORt in different degrees of severity of acute or chronic GVHD. Likewise not shown is a tool for the prediction of clinical outcomes in AGVHD. Conclusions: There was a decrease of FOXP3 gene expression in the samples at the time of GVHD and acute and chronic GVHD benchmark its respective samples before HCT in patients undergoing allogeneic HCT related HLAidentical. There was no change in gene expression RORt the time of GVHD, both acute and chronic, when compared to the control group and the group of patients without clinical signs of GVHD undergoing allogeneic HCT related HLAidentical. The expression of FOXP3 and RORt is not correlated with the prognosis of GVHD, not predicting mortality.
Descrição
Citação
CABRAL, Camila Marca de Veiga. Expressão de genes relacionados às subpopulações de linfócitos T reguladores e Th17 em sangue periférico de pacientes submetidos ao transplante de células hematopoéticas: importância prognóstica na doença do enxerto contra hospedeiro. 2013. 127f. Tese (Doutorado em Hematologia) – Escola Paulista de Medicina, Universidade Federal de São Paulo. São Paulo, 2013.