ABSTRACT
Interferon-γ (IFN-γ) is an important cytokine produced by natural killer (NK) cells and T cells in response to various stimuli. The levels of IFN-γ secreted after stimulation of NK cells using a recombinant cytokine is represented as one of functions of NK cells. Recently, a method for evaluating NK cell activity in whole blood samples was developed. The levels of IFN-γ secreted after NK cell stimulation with PROMOCA™ (ATGen, Korea) and T cell stimulation with phytohemagglutinin (PHA) were compared using two different commercial kits: NK Vue Gold (ATGen, Korea) and QuantiFERON-TB Gold In-Tube (Cellestis, Australia). Participants included 43 healthy individuals. Whole blood samples were incubated with either PROMOCA, a recombinant cytokine that specifically activates NK cells, or with PHA. IFN-γ levels in the supernatants were measured by ELISA. The level of IFN-γ by PROMOCA stimulation (PROMOCA IFN-γ) was more varied than that by stimulation with PHA (PHA IFN-γ) (median 1,544.4 pg/mL [ range 193.7–2,530.9] vs. median 2,470.1 pg/mL [ 2,250.1–2,874.4] P=0.0001). The median of PHA IFN-γ/PROMOCA IFN-γ ratio was 1.9 (1.1–12.4). There was a significant difference in levels of IFN-γ secreted after stimulation with PROMOCA or PHA in the healthy population.
Subject(s)
Enzyme-Linked Immunosorbent Assay , Healthy Volunteers , Interferon-gamma , Killer Cells, Natural , Methods , T-LymphocytesABSTRACT
DNA damage response (DDR) in different cell cycle starus of human peripheral blood lymphocytes (PBLs) and the role of H2AX in DDR were investigated.The PBLs were stimulated into cell cycle with phytohemagglutinin (PHA).The apoptotic ratio and the phosphorylation H2AX (S139)were flow cytometrically measured in resting and proliferating PBLs after treatment with camptothecin (CPT) or X-ray.The expressions of γH2AX,Bcl-2,caspase-3 and caspase-9 were detected by Western blotting.DDR in 293T cells was detected after H2AX was silenced by RNAi method.Our results showed that DNA double strand breaks (DSBs) were both induced in quiescent and proliferating PBLs after CPT or X-ray treatment.The phosphorylation of H2AX and apoptosis were more sensitive in proliferating PBLs compared with quiescent lymphocytes (P＜0.05).The expression levels of anti-apoptotic proteins Bcl-2 were reduced and cleaved caspase-3 and caspase-9 were increased.No significant changes were observed in CPT-induced apoptosis in 293T cells between H2AX knocking down group and controls.It was concluded that proliferating PBLs were more vulnerable to DNA damage compared to non-stimulated lymphocytes and had higher apoptosis rates.γH2AX may only serve as a marker of DNA damage but exert no effect on apoptosis regulation.
ABSTRACT
Objective To explore the difference of immunosuppresive effect between the expanded Stro-1+ and Stro-1-subgroups of human mesenchymal stem cells in vitro. Methods Mononuclear cells (MNCs) were isolated from bone marrow (BM) samples and seeded in a T-75 cm2 tissue culture flask contained with Dexter medium. When 50% confluence was obtained, adherent cells were collected and incubated with anti-stro-1 antibody, and the Stro-1+ and Stro-1-MSCs were further seeded for expansion. The total culture time (median) was 15 days. Cells were then analyzed by flow cytometry. One-way mix lymphocyte reaction (MLR) (1?105 responding cells and an equal number of stimulating cells/well were co-cultured in 96-well plates) and nonspecific mitogenic stimuli phytohemagglutinin (PHA) plus interleukin-2 (IL-2) induced lymphocytes proliferation (PBL 1?105/well were mixed with PHA 10?g/ml and IL-2 500U/ml in 96-well culture plates) were established in vitro. 1?103-3?104 irradiated Stro-1+ MSCs and Stro-1-MSCs were added into the two systems at the beginning of reaction. Immunosuppressive actions of both Stro-1+ or Stro-1-MSCs were compared. Results Adherent cells contained a median of 9% (range 5%-26%) Stro-1+ cells, which expressed higher immunophenotype of MSCs. In both reaction systems, suppressive actions occurred in a dose-dependent fashion when whatever Stro-1+ MSC or Stro-1-MSC were added. However, that the addition of 1?103 Stro-1-cells enhanced rather than inhibited the lymphocyte proliferation in one-way MLR. In the presence of various concentrations of MSCs, Stro-1+ MSCs always showed a significantly increased inhibitory effects in comparison to Stro-1-MSCs (P
ABSTRACT
Objective:To research PHA-CIK cells' phenotype and cytotoxicity. Methods: Using phytohemagglutinin(PHA)to stimulate peripheral blood mononuclear cells(PBMNCs) for 24 h,then cultured like incubating cytokine induced killers by traditional method. On 15th day examined its immunophenotype by flow cytometry and using MTT method to evaluate cytotoxicity. Results: The ratio of CD3+,CD8+, CD3+ CD8+ and CD3+ CD56+ cells were high.PHA-CK cells cytotoxicity was strong in some degree.Conclusion:PHA-CIK cells had strong cytotoxicity.The result provides an experimental basis for biotherapy.