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Objective:To investigate the cytotoxic effects of CTL cell induced by DCs loaded with exosomes derived from hepatoma Huh-7 cells(T-exo).Methods: Exosomes derived from hepatoma Huh-7 cells were isolated and purified by combination of ultrafiltration centrifugation and sucrose density gradient centrifugation.Morphology of exosomes was observed under transmission electron microscopy and the expression of CD 9,CD63,HSP70 and AFP was detected by Western blot.DCs were induced with peripheral blood monocytes isolated from healthy donors.Flow cytometry was used to analysis surface markers of the DCs loaded with T-exo.WST-1 light absorption measurement was adopted to evaluate the T cell proliferation ability.Annexin-V/PI Flow cytometry were respectively used to examined cytotoxicity against the tumor cells.Results:Exosomes isolated and extracted from culture supernatant of Huh-7 cells presented as circular or elliptical vesicle with bilayer membrane , unequal in size , and with diameter of 50 to 100 nm.Western blot showed that the T-exo expressed CD9,CD63,HSP70 and AFP molecules.DCs loaded with T-exo caused significantly higher T cell pro-liferation and cytotoxic effect against AFP positive Huh-7 cells as compare to gainst AFP negative SMMC 7721 cells and un-loaded control group ( P<0.05 ).Conclusion: T-exosome loaded-DC can promote proliferation and induce significant cytotoxic effect of CTL against Huh-7 cells.
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Objective: To explore the mechanism of immunomodulatory activity of Polyporus umbellatus polysaccharides (PPS) on murine bone marrow dendritic cells (BMDC). Methods: BMDC phenotype and the function indexes were observed by 3H-TdR incorporation, ELISA, and flow cytometry. Results: Compared with the negative group, PPS could increase the co-expression of CD11c and CD86 molecules on dendritic cells (DC) surface and the production of IL-12 and IL-10 in a dose-dependent manner. PPS also enhanced matured BMDC capacity of T cell initial activation and decreased phagocytosis of BMDC. Anti-Toll-like receptor 4 (TLR4), but not anti-TLR2 or complement receptor 3 (CR3) monoclonal antibodies inhibited PPS-induced production of IL-12 p40 and blocked the combination between fluorescence-labeled PPS (f-PPS) and BMDC. Conclusion: The data demonstrate that PPS could promote the activation of murine BMDC via TLR4 and maturation of immunomodulationy activity.
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Objective:To investigate the effects of 17β-Estradiol (E2) on mesenchymal stem cells (MSC) and to evaluate the effects of MSC treated with E2 on the maturation and function of dendritic cells (DC).Methods: We first isolated and cultured MSC from the human fetal lung.The MSC were treated with E2 for 24 hours at various concentrations ( 10~(-9),10~(-8) and 10~(-7) mol/L).After cell counting,proliferation,adherent ability and immunophenotypes of MSC were detected by flowcytometry.The gene expressions of cytokine (IL-6,TGF-βand VEGF) were measured by RT-PCR.The effects of MSC treated with E2 on the maturation and function of DC were determined.Results:After treated with E2,the proliferation and adherent ability of MSC were increased,while the immunophenotypes of MSC were not affected.When MSCs co-cultured with DC,MSC could inhibit the immuophenotypes and function of DC.However,when DC co-cultured with E2-pretreated MSC,the immunophenotypes (MHC Ⅱ,CD80 and CD86) of DC had been reconstructed.After treated with the high concentration of E2 for 24 hours,MSC secreted lower level of TGF-β than that in the control group,while IL-6 and VEGF expressions were increased compared with those in the control group.Conclusion: Estrogen may alter the immuno-suppressive effects of MSC on DC via modulating the cytokine secretion of MSC.
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BACKGROUND: Immunization of dendritic cells (DCs) pulsed with tumor antigen can activate tumor-specific cytotoxic T lymphocytes (CTL) that are responsible for protection and regression. In this study, we examined whether the uptake of necrotic tumor cells could modulate DC phenotypes and whether the immunization of necrotic tumor cell-loaded DCs could elicit efficient tumor specific immune responses followed by a regression of established tumor burdens. METHODS: We prepared necrotic tumor cell-pulsed DCs for the therapeutic vaccination and investigated their phenotypic characteristics, the immune responses induced by these DCs, and therapeutic vaccine efficacy against colon carcinoma in vivo. Several parameters including phagocytosis of tumor cells, surface antigen expression, chemokine receptor expression, IL-12 production, and NK as well as CTL activation were assessed to characterize the immune response. RESULTS: DCs derived from mouse bone marrow efficiently phagocytosed necrotic tumor cells and after the uptake, they produced remarkably increased levels of IL-12. A decreased CCR1 and increased CCR7 expression on DCs was also observed after the tumor uptake, suggesting that antigen uptake could induce DC maturation. Furthermore, co-culturing of DCs with NK cells in vitro enhanced IL-12 production in DCs and IFN-gamma production in NK cells, which was significantly dependent on IL-12 production and cell-to-cell contact. Immunization of necrotic tumor cell-loaded DCs induced cytotoxic T lymphocytes as well as NK activation, and protected mice against subsequent tumor challenge. In addition, intratumoral or contra-lateral immunization of these DCs not only inhibited the growth of established tumors, but also eradicated tumors in more than 60% of tumor-bearing mice. CONCLUSION: Our data indicate that production of IL-12, chemokine receptor expression and NK as well as CTL activation may serve as major parameters in assessing the effect of tumor cell-pulsed DC vaccine. Therefore, DCs loaded with necrotic tumor cells offer a rational strategy to treat tumors and eventually lead to prolonged survival.