RESUMO
Clonotype analysis is essential for complete characterization of antigen-specific T cells. Moreover, knowledge on clonal identity allows tracking of antigen-specific T cells in whole blood and tissue infiltrates and can provide information on antigenic specificity. Here, we developed a next generation sequencing (NGS)-based platform for the highly quantitative clonotype characterization of T cells and determined requirements for the unbiased characterization of the input material (DNA, RNA, ex vivo derived or cell culture expanded T cells). Thereafter we performed T cell receptor (TCR) repertoire analysis of various specimens in clinical settings including cytomegalovirus (CMV), polyomavirus BK (BKV) reactivation and acute cellular allograft rejection. Our results revealed dynamic nature of virus-specific T cell clonotypes; CMV reactivation was linked to appearance of new highly abundant antigen-specific clonalities. Moreover, analysis of clonotype overlap between BKV-, alloantigen-specific T cell-, kidney allograft- and urine-derived lymphocytes provided hints for the differential diagnosis of allograft dysfunction and enabled appropriate therapy adjustment. We believe that the established approach will provide insights into the regulation of virus-specific/anti-tumor immunity and has high diagnostic potential in the clinical routine.
Assuntos
Infecções por Citomegalovirus/diagnóstico , Citomegalovirus/imunologia , Rejeição de Enxerto/genética , Sequenciamento de Nucleotídeos em Larga Escala , Infecções por Polyomavirus/diagnóstico , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/patologia , Infecções Tumorais por Vírus/diagnóstico , Vírus BK/genética , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Diagnóstico Diferencial , Humanos , Transplante de Rim/efeitos adversos , Infecções por Polyomavirus/genética , Infecções por Polyomavirus/virologia , Estudos Retrospectivos , Linfócitos T/imunologia , Linfócitos T/virologia , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/virologia , Ativação ViralRESUMO
Resveratrol, a phytochemical present in grapes, has been demonstrated to inhibit tumourigenesis in animal models. However, the specific mechanism by which resveratrol exerts its anticarcinogenic effect has yet to be elucidated. In the present study, the inhibitory effects of resveratrol on cell proliferation and apoptosis were evaluated in the human leukaemia cell line HL-60 and the human hepatoma derived cell line HepG2. We found that after a 2 h incubation period, resveratrol inhibited DNA synthesis in a concentration-dependent manner. The IC50 value was 15 microm in both HL-60 and HepG2 cells. When the time of treatment was extended, an increase in IC50 value was observed; for example, at 24 h the IC50 value was 30 microm for HL-60 cells and 60 microm for HepG2 cells. Flow cytometry revealed that cells accumulated in different phases of the cell cycle depending on the resveratrol concentration. Furthermore, an increase in nuclear size and granularity was observed in the G1 and S phases of HL-60 treated and HepG2-treated cells. Apoptosis was also stimulated by resveratrol in a concentration-dependent manner in HL-60 and HepG2 cells. In conclusion, resveratrol inhibits cell proliferation in a concentration- and time-dependent manner by interfering with different stages of the cell cycle. Furthermore, resveratrol treatment causes stimulation of apoptosis as well as an increase in nuclear size and granularity.