AIM: To investigate the effects of uric acid sodium salt (UANa) as adjuvant on humoral and cellular immune response in BALB/c mice. METHODS: BALB/c mice were immunized with trichosanthin (TCS) as antigen together with UANa suspension as adjuvant. The antibody titers of IgG were detected by enzyme-linked immunosorbent assay. Dendritic cells (DC) were induced in vitro, the phenotypes of DC were analyzed by flow cytometry and the effect of UANa on DC maturity was evaluated. A delayed-type hypersensitivity (DTH) model was used to analyze the effect of UANa on cellular immune responses in vivo. The in vitro proliferation of lymphocytes was determined by ConA stimulation. RESULTS: Freunds adjuvant greatly enhanced the antibody response of mice to TCS, while UANa adjuvant failed to promote the antibody response but significantly reduced the antibody response as compared to TCS only. No effect of UANa on the expression of CD11c and CD83 in DC was observed by flow cytometry analysis. However, UANa significantly enhanced the expression of MHC II molecule. In the DTH model, UANa enhanced the degree of allergen-induced ear swelling and promoted the ability of lymphocyte proliferation in vitro. CONCLUSION: UANa suspension as adjuvant significantly enhances the cellular immune response but inhibits the humoral immune response to a certain degree, suggesting that UANa has potential application in the vaccine research.

AIM:To investigate the effects of uric acid sodium salt (UANa) as adjuvant on humoral and cellular immune response in BALB/c mice. METHODS:BALB/c mice were immunized with trichosanthin (TCS) as antigen together with UANa suspension as adjuvant. The antibody titers of IgG were detected by enzyme-linked immunosorbent assay. Dendritic cells (DC) were induced in vitro,the phenotypes of DC were analyzed by flow cytometry and the effect of UANa on DC maturity was evaluated. A delayed-type hypersensitivity (DTH) model was used to analyze the effect of UANa on cellular immune responses in vivo. The in vitro proliferation of lymphocytes was determined by ConA stimulation. RESULTS:Freund's adjuvant greatly enhanced the antibody response of mice to TCS,while UANa adjuvant failed to promote the antibody response but significantly reduced the antibody response as compared to TCS only. No effect of UANa on the expression of CD11c and CD83 in DC was observed by flow cytometry analysis. However,UANa significantly enhanced the expression of MHC II molecule. In the DTH model,UANa enhanced the degree of allergen-induced ear swelling and promoted the ability of lymphocyte proliferation in vitro. CONCLUSION:UANa suspension as adjuvant significantly enhances the cellular immune response but inhibits the humoral immune response to a certain degree,suggesting that UANa has potential application in the vaccine research.

AIM: To investigate the anti-HIV-1 activity of five anthraquinone derivatives (emodin,rhein,chrysophanol,physcion and aloe-emodin) in vitro.METHODS: Viral replication was estimated by observation of cytopathogenesis and measurement of HIV-1 p24 antigen production in HIV-1ⅢB acutely infected C8166 cells. The anti-HIV-1 activity was evaluated by the 50% effective concentrations (EC50) and selective indexes (SI) of these derivatives.RESULTS: These anthraquinone derivatives inhibited HIV-1ⅢB replication on syncytia formation induced by HIV-1ⅢB infection with EC50 mean values of (11.44±0.93)μmol/L (emodin),(51.28±2.86)μmol/L (rhein),(90.58±2.30)μmol/L (chrysophanol),(8.59±0.38)μmol/L (physcion) and (0.89±0.08)μmol/L (aloe-emodin),respectively. The p24 antigen production with EC50 mean values were (11.61±0.56)μmol/L (emodin),(12.35±4.73)μmol/L (rhein),(39.63±2.87)μmol/L (chrysophanol),>250 μmol/L (physcion) and (2.75±0.20)μmol/L (aloe-emodin) respectively. CONCLUSION: These structurally-related chemicals show different anti-HIV-1 activity in vitro. Among them,aloe-emodin is the most potent inhibitor to HIV-1 replication.

AIM: To investigate the amount and patterns of expressing CD69 , IL-4 and IFN-? on TCRV?24 +NKT cells, and compare with that of CD3 +T cells from human peripheral blood in response to in vitro stimulation. METHODS: The whole blood was stained with three-color immunofluorescence directly or after cultured with PDB+ionomycin(Ion) for 6 h, then the mononuclear cells were separated by lysing red blood cells. The expression rates of CD69, IL-4 and IFN-? on TCRV?24 +NKT cells and CD3 +T cells were estimated by flow cytometer. RESULTS: As a proportion of mature T cells, the ratio of TCRV?24 + NKT cells to CD3 +T cells was about(1.34?0.42)%. The expression rates of CD69 on TCRV?24 + NKT cells and CD3 +T cells in response to PDB + Ion for 6 h were (96.71?1.33)% and (98.60?0.47)%, respectively, while the ratio were (11.47?2.86)% and (1.07?0.45)% in the unstimulated group, and there were significant difference between them. The expression rates of IL-4 and IFN-? on TCRV?24 +NKT cells stimulated with PDB+Ion for 6 h were (48.62?2.44)% and (46.65?8.91)%, respectively ,which were significantly higher than that of unstimulated group [(31.57?3.31)%, (13.45?6.29)%] and that of stimulated CD3 +T cells, though the expression rates on stimulated CD3 +T cells were significantly higher than that of unstimulated CD3 +T cells. CONCLUSIONS: There is small amount of NKT cells in adult human peripheral blood. The expression rates of IFN-? and IL-4 on these lymphocytes are higher than CD3 +T cells, suggesting that NKT cells are important immunomodulatory cells in special microvironments.

AIM: To investigate the effect of berberine (Ber) on the activation and proliferation of T lymphocytes and its mechanism of action. METHODS: Whole peripheral blood from normal subjects was stimulated with phytohemagglutinin (PHA) or phorbol ester (PDB) plus ionomycin (Ion) and the expression levels of CD69 and CD25 were evaluated with flow cytometry after the staining with appropriate fluorescent monoclonal antibody. The distribution of cell cycles was analyzed by propidium iodide staining and dead cells by 7-aminoactinomycin live staining. RESULTS: 100 ?mol/L and 50 ?mol/L of Ber had significant inhibition of the expression of CD69 on T cells stimulated with PDB plus Ion or PHA, while effect of 25 ?mol/L Ber was not significant. And as time of action extended, the extent of inhibition decreased. For the expression of CD25, Ber at the concentrations as above all exerted significant inhibitory effect in a dose-dependent manner. Moreover, Ber could block lymphocytes cell cycle progression from G_0/G_1 phase to S and G_2/M phase without phase specificity. Besides, live staining analysis revealed that Ber did not have significant cytotoxicity on lymphocytes. CONCLUSIONS: Ber significantly inhibits the expression of early and mid activation antigens of T cells and also blocks the progression of lymphocytes cell cycles. These results suggest that Ber exerts immunosuppression effect through inhibiting the activation and proliferation of T cells.

AIM: To investigate the inhibitory effect of quercetin on in vitro activation of T lymphocytes by polyclonal activators with CD69 expression as an activation marker. METHODS: After being separated from lymphoid nodes of a C57BL/6 mouse, the lymphocytes were exposed to polyclonal activators (PDB or Con A) with or without quercetin. Then they were harvested at 2 h, 6 h and 24 h, respectively. The expressional rates of CD69 on T lymphocytes were assessed by two-color immunofluorescent staining and flow cytometry, and the inhibitory rates of quercetin at different time points were estimated. RESULTS: Quercetin had no effect on the expressional rate of CD69 on T lymphocytes under resting states. After the stimulation with PDB or Con A, the expressional rates of CD69 on T lymphocytes in the present of quercetin (10 ?mol/L) showed significant decrease compared with those of control groups at different time points (P

AIM: To investigate the details of age-related changes of T lymphocytes in order to seek for sensitive biomarker for immunosenesence. METHODS: Heparin anticoagulated venous blood was collected freshly from young (20-35 years) and elderly (50-75 years) volunteers and three or four color immunofluorescence staining was performed. The nucleated cells were acquired and the phenotypes of T lymphocyte subpopulations were analyzed with flow cytometry. RESULTS: There was no significant difference in percentages of pan-T (CD3 +), helper T (CD4 +) and cytotoxic (CD8 +) T subsets between young and elderly, whereas the density of CD3 molecule (MFI) on T cells in elderly group decreased significantly. It was also found that the rates of CD44 + and CD62L + T cell subsets in young group did not have statistical difference from elderly. However,the rates of CD95 + pan-T, helper T and cytotoxic T subsets of elderly group were all markedly higher than that in young group. CONCLUSIONS: The relative rates of T cell and its subsets displayed no age-related changes while the density of CD3 was down-regulated during aging in these groups investigated. Moreover, the expression percentage of CD95 (Fas) on T cells increased as aging, suggesting that it is a potential biomarker for evaluating immunosenescence.

AIM: To study the effect of genistein on activation and proliferation of T cells,and explore the molecular mechanism of genistein. METHODS: Fluorescence conjugated monoclonal antibodies and flow cytometry were used to detect the express of CD69 and CD25 by activated T cells in vitro in response to Concanavalin (ConA )and Phorbol 12,13-dibutyrate(PDB) or T cell proliferation stained by CFSE in response to PDB / Ionomycin or ConA. RESULTS: Genistein inhibited the expression of CD69 and CD25 in activated T cells in response to Con A in a concentration-dependent manner and in response to PDB in a high concentration. Genistein inhibited proliferation of T cells in both groups in a concentration-dependent manner. CONCLUSION: Genistein inhibited activation and proliferation of T cells in vitro in response to polyclonal stimulus,and it may hold potential as a new immunosuppressant.

AIM: To construct the mammalian cell expression vector for enhanced green fluorescent protein (EGFP) and HLA-A*0201 fusion protein and analyze its expression and subcellular localization in the transfected K562 cells. METHODS: The HLA-A*0201 cDNA was cloned by RT-PCR and the gene was inserted into pEGFP-N1 to construct a vector for the fusion protein. The expression of the fusion protein in K562 cells transfected with the vector was evaluated by flow cytometry and its subcellular localization was investigated by confocal microscopy. RESULTS: The full-length encoding region of HLA-A*0201 cDNA was cloned from two HLA-A2 positive donors and the expression vector for the HLA-A*0201-EGFP fusion protein was constructed by PCR using a primer pair to introduce a Kozak sequence before ATG and the stop codon was deleted. Five hours after K562 cells was transfected with the vector, the expression percentages of HLA-A*0201 and EGFP were 25.12?2.26 and 27.37?3.59, respectively and no significant increase was observed after 24 h. The fusion protein was predominantly located on the membrane with low level distribution within the cells. In contrast, no HLA-A*0201 but only EGFP was detected in the empty vector transfected K562 cells and the EGFP was dispersed within the cells. CONCLUSIONS: The expression vector for HLA-A*0201-EGFP fusion protein was constructed and the fusion protein expressed in K562 cells was primarily distributed on the membrane. The results suggest that the transfected K562 cells are potential antigen-presenting cells.

AIM: To study the effect of natural killer(NK)-cells on graft-versus-host disease(GVHD), graft rejection, engraftment and reconstituting of hematopoiesis in mouse allogeneic bone marrow transplantation. METHODS: Lethally irradiated BALB/c(H-2 d) mice were transplanted with C57/6j(H-2 b) bone marrow containing donor peripheral T cells and/or NK cells. Recipients CD 34 + cell counts and the expression of H-2K b+ cell were detected by flow cytometry, peripheral white blood cell(WBC) was detected by auto-cytometry, and the recipients survival rates, GVHD, engraftment and hematopoiesis recovery were then observed. RESULTS: In the group of transplanted with NK cells infusion, the incidence of GVHD was evidently lessened, the survival rates, WBC and CD 34 + cell counts and the expression of H-2K b+ cell were significantly high than that without NK cells infusion. CONCLUSION: In mouse allogeneic bone marrow transplantation, alloreactivity NK cells prevent GVHD, reduce graft rejection, and promote engraftment and reconstituting of hematopoiesis. [