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AIM:To evaluate the ability of dendritic cells (DCs) loaded with tumor lysate to initiate cell mediated immune responses by stimulating naive T cells, and the efficiency of activated T cells to kill autologous tumor cells in vitro. METHODS: The peripheral blood lymphocytes and monocytes were obtained from the advanced renal cell carcinoma patient by eonglutination method. The immature dendritic cells were generated in the presence of interleukin -4(IL-4) and granulocyte/macrophage colony-stimulating factor (GM-CSF) from monocytes of healthy individuals.These cells were pulsed with tumor lysate or not. Induction of tumor-specific cytotoxic T lymphocytes(CTLs) response by mature dendritic cells (mDCs) was evaluated by the CD95(Fas) expression assay through FCM and the cytotoxic assay a gninst autolognns human tumor cells. RESULTS: Human immature dendritic cells and T cells obtained from healthy donors were stimulated with tumor- pulsed dendritic cells. The immature dendritic cells were applied to the cytotoxicity assay a gainst target autologons tumor cells. The CD95 (Fas) expression, IFN-γ, and TNF -α secreted by the CTLs in tumor lysate-plused DC group were higher than those of other groups. The capacity of the CTLs to kill autolognns tumor cells was significantly different(P<0. 05). Antigen-specific DCs vaccine can induce T cells activation and proliferation, thus we can obtain higher proportion of tumor specific cytotoxic T cells(CTLs), and enhance the CTLs to secret IFN-γ and TNF-α. CONCLUSION: Our results indicate that monocyte-derived human dendritic cells pulsed with tumor lysate could in duce the specific antitumor effect against autologons tumors. This in vitro model offers a new and simple approach to the development of DC + CTL - based immunotherapy.
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Objective To investigate the effect of cimetidine on the clonal expansion of TCR V? subfamily of T cells in cord blood after the stimulation by K562 cells in vitro.Methods Cimetidine(1?10-5 mol/L) or K562 cells(1?106/ml) or both of them were respectively cultured with mononuclear cells(MNC) isolated from normal human cord blood for 2 weeks.After the induction,specific cytotoxicity of the proliferated T cells were detected with K562 cells as the target cells.The selective usages and clonal expansion of TCR V? subfa-mily of T cells were analyzed by RT-PCR and genescan technique.Results After induction for 2 weeks,the 3 groups showed the increased cell proliferation,in which specific cytotoxicity of T cells induced by both cimetidine and K562 cells against K562 cells was enhanced significantly compared with the other 2 groups(P
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Objective To study the changes of tissue plasminogen activator (t-PA), plasminogen activator inhibitor 1 (PAI-1), and soluble thrombomodulin (sTM) induced by dengue 2 virus (D 2V) infection of vascular endothelial cells. Methods The effects of D 2V infection on the production of t-PA, PAI-1, and sTM of human umbilical cord vein cells were studied. Results D 2V infection significantly induced the secretion of sTM and t-PA but showed no such effects on PAI-1 of human endothelial cells. Antibody against IL-6 inhibited D 2V-induced t-PA production of endothelial cells. A close correlation between serum levels of IL-6 and t-PA was found in dengue hemorrhagic fever (DHF) but not in dengue fever (DF) patients. Conclusion IL-6 can regulate D 2V-induced t-PA production of endothelial cells, suggesting that endothelial cells can be the target for D 2V infection and that D 2V-induced t-PA, TM, and IL-6 production of endothelial cells may contribute to the pathogenic development of dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS).
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AimTo study the effect of interferon gamma( IFN-γ) and tumor necrosis factor alpha(TNF-α) in the immune pathogenesis of dengue virus infection. Method The serum levels of IFN-γ and TNF-α were measured with emzyme linked immunosorbent assay (ELISA) method in 30 cases of the patients with the dengue virus infection in Guangzhou district. The results were treated with t-test of two sample mean. ResultsThe serum levels of IFN-γ and TNF-α in patients with the dengue virus infection were much higher than healthy controls( P ＜ 0.05、 P ＜ 0.01 ). IFN-γ was detectable on the first day of postinfection. Level of IFN-γ reached their peaks on the second day, then declined . The level of TNF-α had an obvious rise from the second day and reached their peaks on the third day, then declined. ConclusionThe data suggest that the IFN-γ and TNF-α may play an important role in the dengue virus pathogenicity and immunity.
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AIM:To investigate the effects of rhTGF-?1 and TGF-?1 gene transfection on the proliferation of cultured rabbit corneal endothelial cells in vitro.METHODS:Cell growth induced by various concentrations of rhTGF-?1 was determined by MTT proliferation assay.Under the induction of liposomes,recombinant pSecTag2-TGF-?1MP vectors were transferred into the corneal endothelial cells.Morphological changes of transfected cells were observed by HE staining.The expression levels of TGF-?1 were assessed by ELISA.Cell cycle analysis was assessed by flow cytometry.DNA fragment analysis was used to confirm the presence of apoptosis.RESULTS:rhTGF-?1 in concentrations of 5-20 ?g/L showed a significant suppressive effect on the proliferation of corneal endothelial cells,0.5-1 ?g/L had no effect,0.05-0.1 ?g/L facilitated cell growth,as compared with negative controls.The morphous of transfected corneal cells had no significant abnormality compared with normal cells.According to the result of ELISA,the concentration of TGF-?1 in the supernatant was calculated to be(98?3)ng/L.Flow cytometry assay showed that S and G2/M phase of transfected cells decreased significantly compared with that of control group,but the cell cycle recovered normally after adding 10 ?g/L EGF into the culture medium.Agarose electrophoresis didn't show marked ladders in transfected group.CONCLUSION:Effects of rhTGF-?1 on the proliferation of corneal endothelial cells are different with various concentrations.TGF-?1 gene transfection shows suppressive effect on the proliferation of cultured corneal endothelial cells,but does not induce cell apoptosis.EGF is the antagonist of this suppressive effect.
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AIM: Humanized-NOD/SCID(hu-NOD/SCID) mouse model was established and the level of immune reconstitution was assessed in this model. METHODS: Mononuclear cells (MNC) and CD34+ cells were isolated or sorted from cord blood(CB). Human CD45, CD19, CD3 markers on cells from NOD/SCID murine peripheral blood(PB), bone marrow(BM), thymus were detected by FCM from 4 to 10 weeks after hematopoietic stem cell transplantation. After 10 weeks, the gene expressions of the human ?2M and RAG2 were detected by RT-PCR in PB or bone marrow of mice model. RESULTS: Human CD45, CD19, CD3 cells populations in PB and BM were found by flow cytometry in mice model transplanted with CD34+ cells or CB MNC from 4 to 10 weeks. The highest positivity of human lymphocytes was at 8 week after transplantation. The levels of human cell engraftment in mice transplanted with CD34+ cells were higher than those in mice transplanted with CB MNC. The mRNA of human ?2M and RAG2 were found by RT-PCR in BM.CONCLUSION: The higher level of human lymphocyte engraftment is established in NOD/SCID mouse model transplanted with CD34+ compared with CB MNC. The maturation of T lymphocytes could be happened in bone marrow of mice model.
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AIM: To study the dynamic levels of tumor necrosis factor alpha (TNF-?), interleukin-6 (IL-6) and gamma interferon (IFN-?) produced by human dendritic cells infected with Dengue virus. METHODS: Monocytes isolated from healthy human peripheral blood were incubated in medium with GM-CSF and IL-4 for more than 7 days. DCs were then collected and identified by scanning electron microscope, immunohistochemistry and lymphocytes stimulatory ability. Human dendritic cells (DC) were infected with Dengue-2 virus (DV-2) in vitro, culture supernatants were collected in different time postinfection (6 h, 12 h, 24 h, 48 h and 72 h), DV antigen in human dendritic cells were demonstrated by an indirect immunofluorescent assay (IFA), production of TNF-?, IL-6 and IFN-? in the culture supernatants were evaluated by ELISA. RESULTS: After 7 days, typical dendritic cells could be obtained. Virus antigen were detected in infected DC by IFA. Dengue virus induces TNF-? and IL-6 secretion from DC and does not induce IFN secretion. CONCLUSION: Human dendritic cells are target of dengue virus infection. TNF-?, IL-6 production from DC are increased with DV infection. Dendritic cells may play an important role in DV pathogenicity and immunity.
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AIM:To evaluate the ability of dendritic cells (DCs) loaded with tumor lysate to initiate cell-mediated immune responses by stimulating naive T cells, and the efficiency of activated T cells to kill autologous tumor cells in vitro. METHODS: The peripheral blood lymphocytes and monocytes were obtained from the advanced renal cell carcinoma patient by conglutination method. The immature dendritic cells were generated in the presence of interleukin-4(IL-4) and granulocyte/macrophage colony-stimulating factor(GM-CSF) from monocytes of healthy individuals. These cells were pulsed with tumor lysate or not. Induction of tumor-specific cytotoxic T lymphocytes (CTLs) response by mature dendritic cells (mDCs) was evaluated by the CD95(Fas) expression assay through FCM and the cytotoxic assay against autologous human tumor cells. RESULTS: Human immature dendritic cells and T cells obtained from healthy donors were stimulated with tumor-pulsed dendritic cells. The immature dendritic cells were applied to the cytotoxicity assay against target autologous tumor cells. The CD95(Fas) expression, IFN-? and TNF-? secreted by the CTLs in tumor lysate-plused DC group were higher than those of other groups. The capacity of the CTLs to kill autologous tumor cells was significantly different(P
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AIM: The aim of this study is to elucidate the effects of lipopolysaccharide (LPS) on tissue plasminogen activator(tPA) and plasminogen activator inhibitor type-1(PAI-1) expression and secretion in endothelial cells. METHODS: Cultured human umbilical vein endothelial cells (HUVECs) were induced by LPS for different times. Cell viability was then determined by cell counting kit-8. tPA and PAI-1 activities in the media were assayed by fibrin overlay and reverse fibrin autograph, respectively. Cytoplasmic RNA was prepared using the Trizol method and was assayed for PAI-I and tPA mRNA levels by reverse transcript-polymerase chain reaction(RT-PCR). RESULTS: LPS(10 mg/L) did not produce cell toxicity according to LDH determination in culture media. PAI-1 activity in LPS group was high (P0.05). CONCLUSION: LPS (10 mg/L) did not show signs of cell toxicity, but promoted the expression of PAI-1 mRNA and induced an increase activity of PAI-1. However, LPS (10 mg/L) did not change tPA mRNA expression. The time-dependent increase in PAI-1 mRNA expression and activity shifts the local balance toword increased anti-fibrinolytic capacity, which can amplify the extent of acute thrombosis after plaque rupture. This is one of the possible reasons that cause thrombus,blood coagulation and disseminated intravascular coagulation (DIC) during septicemia.
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0.05), the expression of CD4+ was increased in rabbit corneal stroma (P
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AIM: To study effects of tumour necrosis factor alpha(TNF?) and interleukin-10(IL-10) on human cytomegalovirus AD169 (HCMV AD169) infection in human embryonic lung fibroblasts (HEL),and the ability of the infected HEL to produce TNF?. We have attempted to understand the effect of cytokines in the immune of HCMV infection. METHODS: TNF??IL-10 were added separately with different concentrations before 24 h HCMV infection of HEL to study the effect of TNF? and IL-10 on multiplication of HCMV. HCMV was incubated with HEL, TNF? in culture supernatants were measured at 4, 24, 48, 72, 96 h postinfection. The level of TNF? was measured by enzyme-linked immunosorbent assay( ELISA). RESULTS: The addition of TNF? with the concentration of 10-100 ?g/L or IL-10 with the concentration of 1-10 ?g/L before 24 h HCMV infection of HEL could remarkably inhibited the multiplication of HCMV in HEL. Level of TNF? didn't increase in infected cell supernatants. CONCLUSION: TNF? and IL-10 play an important role in the immune of HCMV infection.
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Objective:To explor the effect of gp120mAb on synaptic transmission and plasticity changed by gp120 in the CA 1 region of rat hippocampal slices.Methods:We recorded the excitatory postsynaptic potentials (EPSP) and investigated effect of gp120mAb on long-term potentiation inhibited by gp120 in rat hippocampus in vitro.Results:The results showed that HIV-1 gp120 can inhibit the LTP induced by high-frequence stimulation schaffer in CA 1,gp120 had no effect on basal synaptic transmission.HIV-1 gp120(200 pmol/L) can inhibit the LTP maintance,gp120mAb of gp120-specific antibodies can reverse the inhibition effect,But IgG can't reverse it.Conclusion:The reverse effect of gp120-specific antibody gp120mAb on long-term potentiation inhibited by HIV-1 gp120 may contribute to the HAD pathogenesis.