Lethal effect of mononuclear cells derived from human umbilical cord blood differentiating into dendritic cells after in vitro induction of cytokines on neuroblastoma cells

BACKGROUND： Dendritic cell is the most major antigen presenting cell of organism. It is proved in recent studies that human umbilical cord blood mononuclear cells induced and cultured in vitro by recombinant human granulocyte-macrophage colony stimulating factor （rhG-MCSF） and recombinant human interleukin-4 （rhlL-4） can generate a great many dendritic cells and promote the lethal effect of T cells on human neuroblastoma, but it is unclear that whether the lethal effect is associated with the most proper concentration of dendritic cells. OBJEETIVE： To investigate the lethal effect of human umbilical cord blood mononuclear cells induced in vitro by cytokines differentiating into dendritic cells on human neuroblastoma, and its best concentration range. DESIGN ： Open experiment SEI-FING： Department of Pediatrics, the Medical School Hospital of Qingdao University MATERIALS ： The study was carried out in the Shandong Provincial Key Laboratory （Laboratory for the Department of Pediatrics of the Medical School Hospital of Qingdao University） during September 2005 to May 2006. Human umbilical cord blood samples were taken from the healthy newborn infants of full-term normal delivery during October to November 2005 in the Medical School Hospital of Qingdao University, and were voluntarily donated by the puerperas. Main instruments： type 3111 CO2 incubator （Forma Scientific, USA）, type 550 ELISA Reader （Bio-Rad, USA）. Main reagents： neuroblastoma cell line SK-N-SH （Shanghai Institute of Life Science, Chinese Academy of Sciences）, RPMI-1640 culture fluid and fetal bovine serum （Hyclone）, rhlL-4 （Promega, USA）, rhG-MCSF （Harbin Pharmaceutic Group Bioengineering Co.Ltd）, rat anti-human CDla monoclonal antibody and FITC-labeled rabbit anti-rat IgG （Xiehe Stem cell Gene Engineering Co.Ltd）. METHODS： ① Human umbilical cord blood mononuclear cells obtained with attachment methods differentiated into human umbilical cord blood dendritic cells, presenting typical morphology of dendritic cells after in vitro induction by rhG-MCSF and rhlL-4. ② Different concentrations of dendritic cells[ dendritic cells： neuroblastoma cells=20：1,50：1,100：1 （2×10^8 L^-1,5×10^8 L^-1,1×10^9 L^-1）], 1×10^9 L^-1 T cells and 1×10^7 L^-1 neuroblastoma cells were added in the experimental group. 1 ×10^9 L^-1 T cells and 1 ×10^7 L^-1 neuroblastoma cells were added in the control group. ③ Main surface marker CDla molecules of dendritic cells were detected with indirect immunofluorescence, and the percent rate of dendritic cells was counted with ultraviolet light and expressed as the expression rate of CD1a^＋ cells. ④Single effector cells and target cells were respectively set in the experimental group and control group to obtain the lethal effect. The lethal effect of dendritic cells on neuroblastoma cells was indirectly evaluated by detecting cellular survival with MTT assay. The lethal effect（%）= （1-A experimentat well-A effector cell /A target cell well）×100%.⑤The expenmental data were presented as Mean ±SD, and paired t test was used. MAIN OUTCOME MEASURES： ① Morphological characters of dendritic cells in the process of induction and differentiation. ②CD1a^＋ cellular expression rate. ③Lethal effect of dendntic cells on neuroblastoma cells. RESULTS： ①Morphological characters of dendritic cells in the process of induction and differentiation： On the 15^th day after human umbilical cord blood mononuclear cells were induced by rhG-MCSF and rhlL-4, typical morphology of dendritic cells could be seen under an inverted microscope. ②Expression rate of CD1a^＋ cells was （43.12±5.83）%. ③Lethal effect of dendritic cells on neuroblastoma cells： Lethal effect of dendritic cells stimulated T cells in each experimental group （ dendritic cells： neuroblastoma cells=100：1,50：1, 20：1 respectively） on neuroblastoma cells was significantly higher than that in control group[（31.00 ±4.41 ）%, （30.92±5.27）%,（33.57±5.35）%,（26.23±5.20）%, t=3.51,2.98,4.24, P〈 0.01 ）; But the lethal effect of dendntic cells on neuroblastoma was significantly lower when their ratio was 100：1 and 50：1 in comparison with 20：1 （t=2.01,2.36, P 〈 0.05）, and no significant difference in lethal effect existed between the ratio at 100：1 and 50：1 （t=0.06,P 〉 0.05）. CONCLUSION： Dendritic cells differentiated from human umbilical cord blood mononuclear cells after in vitro induction of cytokines can promote the lethal effect of T cells on neuroblastoma cells. The lethal effect is associated with the concentration of dendritic cells within some range.

Polysaccharide from Astragalus membranaceus promotes the activation of human peripheral blood and mouse spleen dendritic cells

Astragalus membranaceus(A.membranaceus)is a widely used traditional herb in China and Korea.A.membranaceus polysaccharides(AMP),which make up a major part of the root extract,have been shown to modulate immune modulations,especially activation of bone marrow-derived dendritic cells(BMDCs)and T cells.However,the immune stimulatory effect of AMP in the mouse in vivo and human peripheral blood DCs(PBDCs)has not been well investigated.In this study,we found that intravenous(i.v.)injection of AMP in C57 BL/6 mice induced remarkable elevations in co-stimulatory and MHC class I and II molecule levels in the splenic DCs and its subsets.The stimulatory effect of DCs by AMP was elevated 6 h after treatment,which rapidly decreased 18 h after injection.Furthermore,AMP promoted intracellular production of pro-inflammatory cytokines in spleen DC subsets,which contributed elevation of serum cytokine levels.Finally,the AMP promoted PBDC activation.Thus,these results demonstrate that AMP can be used as an immune stimulatory molecules in human and mouse.

Peroxisome proliferator-activated receptor a agonist attenuates oxidized-low density lipoprotein induced immune maturation of human monocyte-derived dendritic cells

Accumulating evidence suggests that the Thl immune .response induced by various antigens such as oxidized low density lipoprotein （ox-LDL） and heat shock proteins （HSPs） play a key role in the process of atherosclerosis.1Dendritic cells （DCs） are the most potent antigen-presenting cells （APCs） in the body with the unique ability to initiate a primary immune response to certain antigens by the activation of ＂naive＂ T cells.2 The maturation of DC with the upregulation of costimulatory molecules such as CD83, CD40, CD86, and major histocompatibility complex （MHC） class molecules such as human leukocyte antigen （HLA）-DR, is required for DC to activate T cells. Pathologic studies have shown that immature DCs are present in normal arterial while abundant mature DCs clustered with T cells could be visualized in atherogenic vessels suggesting that DC 3 maturation is linked to the progression of atherosclerosls. Peroxisome proliferator-activated receptors （PPARs） a, one member of the family of PPARs, was found to have favorable effects on slowing the progression of atherosclerosis and reducing the risk of coronary heart disease in high-risk patients independent from their metabolism effects.4＇5 Furthermore, PPAR-α is also expressed on monocytes and monocyte-derived DCs.6 The effects of PPAR-α on DCs maturation and immune function remain unknown now, we therefore observed the effects of fenofibrate, a PPAR-α agonist, on the maturation and immune function of oxidized LDL-treated DCs in this study.