Construction and identification of recombinant adenovirus vector containing CTLA4Ig gene

AIM:To construct a recombinant adenovirus expression vector containing CTLA4Ig gene.METHODS:The CTLA4Ig gene derived from the plasmid PCDNA3.0/CTLA4Ig by using polymerase chain reaction (PCR) was inserted into the backward position of cytomegalovirus (CMV) immediate early promoter of the shuttle plasmid (pAdTrack-CMV). After being identified by endonuclease, PCR and sequencing, the recombinant shuttle plasmid pAdTrack-CTLA4Ig was co-transformed into E.coli. BJ5183 cells with the adeoviral backbone plasmid pAdEasyl-1 to obtain the homologous recombination. The adenovirus was generated in 293 cells. A series methods such as PCR and fluorescence microscope was employed to identify the generated recombinant adenovirus.RESULTS:Recombinant CTLA4Ig adenoviruses were constructed and the titer of virus was generally up to 1.65×10¹² phaque forming units per liter (PFU/L).CONCLUSION:Success in constructing recombinant pAdTrack-CTLA4Ig will be the base of the further research on its expression in the mammalian cells, and be potenially used in the prevention of transplant rejection and autoimmunity diseases.     

Inhibitory effect of berberine on the activation and proliferation of T lymphocytes

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 μmol/L and 50 μmol/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 μmol/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 G₀/G₁ phase to S and G₂/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.     

IL-12 induces T lymphocytes apoptosis and influences the expression and signal transduction of Fas/FasL

AIM: IL-12 acts upon Tlymphocytes and activates its receptor complexes of β1/β2,and so IL-12 can regulate TH1/TH2 balance. Our study is aimed at IL-12-inducing apoptosis of Tcells and expression and signal transduction of Fas/FasLduring Tcell apoptosis. METHODS: The apoptosis of Tcells was detected by Annexin Vstaining cytometry and the expression of Fas/FasLunder different inhibitors were detected by semi-quantitative PCR. RESULTS: IL-12 induced the human leukemic Tcell line(TIB-152) and the human lymphoma Tcell line(HTB-176) and the normal human Tcells to undergo apoptosis. The FasLexpression at 6 hours after treatment with IL-12 increased apparently, and reached the max at 24 hours, and FasLexpression induced by IL-12 was inhibited by PKCinhibitor. But IL-12 did not influence Fas expression. CONCLUSIONS: IL-12 can induce Tcells to undergo apoptosis which is characterized by early membrane changes, the inducing effect is correlated with the concentration of IL-12 and the maturation of Tcells. FasLparticipates in the progression of Tcell apoptosis as a apoptosis mediator, and the effect of IL-12 on FasLexpression may be related with PKCpathway.     

Effect of sodium nitroprusside on activation of nuclear factor κB

AIM: To investigate the effect of sodium nitroprusside (SNP) on activation of nuclear factor κB. METHODS: The techniques of culture of human T lymphocytes, Western blot and RT-PCR were applied. The effects of NO donor sodium nitroprusside (SNP) at different concentrations on mRNA and protein expression of IκB_α in human T lymphocytes at 30 min or 120 min after stimulating with phytohaemagglutinin (PHA-P) were observed. RESULTS: SNP at middle or high concentrations reduced the degradation of κIB_αprotein 30 min after stimulating with PHA-P,and increased the re-expression of κIB_αmRNA 120 min after stimulating with PHA-P significantly.CONCLUSION: The mechanism of inhibitory effect of SNP at middle or high concentrations may be due to the decrease in degradation and the increase in re-synthesis of κIB_α.The regulatory mechanism of SNP at low concentration may not be through κIB_α.     

锌对雏鸭外周血T-淋巴细胞的影响

15 0只 1日龄天府肉鸭健雏随机分为 3组 ,分别喂给缺锌 (每千克日粮含 Zn 2 2 .9mg)、对照 (每千克日粮含 Zn10 0 mg)和锌中毒 (每 kg日粮含 Zn130 0 mg)日粮 7周或 4周 ,以酸性 - α-醋酸萘酯酶 (ANAE)染色法观测外周血 T-淋巴细胞的动态变化。结果锌缺乏组和锌中毒组雏鸭外周血 T-淋巴细胞的 ANAE阳性率显著低于对照组 (P<0 .0 1) ,表明锌缺乏或锌中毒可抑制 T-淋巴细胞的生成 ,降低其在外周血中的数量。本试验还对锌缺乏和锌中毒引起的外周血 T-淋巴细胞减少的机理进行了探讨     

Effects of psychological stress on in vitro expression of activated surface molecules on T cells of peripheral blood from healthy persons

AIM: To study cellular and molecular mechanism involved in increasing susceptibility of infection in psychological stress persons. METHODS: Comparative studies were performed with double staining and flow cytometry analysis on immunophenotyping and in vitro expression of early activating surface molecule CD69 in response to mitogens on T cells from peripheral blood of 20 healthy college student volunteers before and after psychological stress. A series of term final examinations was defined as psychological stress. RESULTS: Immunophenotyping analysis showed no statistically significant difference in the percentage of CD2, CD3, CD4, CD8, CD19, CD20, CD16 and CD56 positive lymphocyte populations before and after psychological stress. There was a statistically significant decrease in the in vitro expression of CD69 in response to polyclonal stimulators on the T cells from persons after psychological stress than those before psychological stress. The percentage of CD69 expression (CD69⁺CD3⁺/CD3⁺%) in response to PHA and PDB in the whole blood culture for 72 hours decreased respectively from 28.1±4.1 and 80.7±6.8 on the T cells obtained before psychological stress to 17.6±3.8 and 65.8±7.9 on those obtained after psychological stress, while there was no statistically significant difference between the CD69 expression rates without stimulators on the T cells obtained before and after psychological stress. CONCLUSIONS: The effects of psychological stress to immune system is not on the level of changing proportions of the sub-populations within peripheral blood lymphocytes. Psychological stress can decrease the activating response of T cells in healthy persons, which may be responsible for the increase of susceptibility to infection in the psychological stress persons.     

The expression of IFN-γ and IL-4 on T lymphocytes that infiltrate in nasal polyps

AIM:To investigate the expression of Th1-typed cytokine IFN-γ and Th2-typed cytokine IL-4 on T lymphocytes that infiltrate in nasal polyps for searching the pathogenesis of nasal polyps. METHODS:Nasal polyps tissue samples and peripheral blood were obtained from 21 patients. Normal human inferior turbinate mucosa and peripheral blood were obtained as well. Flow cytometry was adopted to detect the expression of IFN-γ and IL-4 of T lymphocytes. RESULTS: Th cytokines were rarely detected in inferior turbinate from normal human. Nasal polyps tissue consisted of abundant T lymphocytes. The expression of IL-4 and IFN-γ increased in peripheral blood from patients compared with normal human (P<0.05). The expression of IL-4 increased but the expression of IFN-γ decreased in nasal polyps compared with that of peripheral blood from the same patient (P＜0.05). CONCLUSION:There were generous of T lymphocytes infiltrating in nasal polyps. There was abnormal immune status in the local nasal mucosa from the patients, and the predomination of Th cytokine secretion changed compared with peripheral blood from the same patients, which resulted in the change of microenvironment of nasal mucosa and possibly close related to the formation of nasal polyps.     

Distribution and identification of immunocytes in humanized SCID mouse model

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 β₂M 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 β₂M 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.     

The effect and mechanism of H. polygyrus infection in T-cell-mediated inflammatory bowel disease in mice

AIM: To investigate the effect and mechanism of Heligmosomoides polygyrus (H. polygyrus) infection in mouse inflammatory bowel disease (IBD) mediated by CD4⁺ helper T-cells. METHODS: Ovalbumin (OVA) -specific CD4⁺ helper T-cells were transferred into SCID (severe combined immunodeficiency) mice to establish an IBD model. The IBD mice were infected by H. polygyrus and sacrificed 14 days later. The histological changes of the colon were observed, and the expression of interferon-gamma (IFN-γ) and interleukin-4 (IL-4) in mesenteric lymph nodes was detected by ELISA and flow cytometry. Additionally, IL-4 monoclonal antibody was intraperitoneally injected into the H. polygyrus-infected IBD mice to block the secretion of IL-4. The IL-4-blocking IBD mice were sacrificed 9 days later and the above indexes were also determined.RESULTS: Compared with the non-infection group, the H. polygyrus-infected IBD mice had more severe colonic lesions, higher level of IL-4 and lower level of IFN-γ in mesenteric lymph nodes (all P<0.05). Compared with the non-blocking group, the H. polygyrus-infected IBD mice with IL-4 blockage had less colonic lesions, lower IL-4 level and higher IFN-γ level (all P<0.05).CONCLUSION: H. polygyrus infection in CD4⁺ T-cell-mediated IBD model promotes inflammation in the early stage probably by inducing the secretion of Th2 cytokine and inhibiting the secretion of Th1 cytokine. The finding suggests that using worms for treatment of IBD needs to be cautious.     

Balance of Treg/Th17 in synovium of collagen-induced arthritis rats and impact of TNF-α blockage therapy

AIM: To investigate the balance of Treg/Th17 in synovium of collagen-induced arthritis (CIA) and the impact of tumor necrosis factor α(TNF-α) blockage therapy. METHODS: Rat CIA model was established by bovine II collagen injection. The pathological score was evaluated by HE staining and toluidine blue staining. The TNF-α level in plasma was measured by ELISA. The expression of Treg/Th17 in synovium was detected by double staining immunofluorescence. RESULTS: The plasma level of TNF-α in CIA group was significantly higher than that in control group and TNFR-Fc treatment group (P<0.01), whereas no significant difference was found between TNFR-Fc treatment group and control group (P>0.05). No significant difference between CIA group and control group in the ratio of CD4⁺Foxp3⁺Treg cells/CD4⁺ cells in synovium (23.12%±4.93% vs 24.66%±5.82%, P>0.05) was observed, whereas the ratio in TNFR-Fc treatment group was significantly increased(33.07%±5.14%). The ratio of CD4⁺RORγt⁺Th17 cells/CD4⁺ cells in CIA group was significantly higher than that in control group and TNFR-Fc treatment group (9.74%±2.23% vs 1.00%±0.59%, 5.63%±1.76%, P<0.01). CONCLUSION: Differentiation disturbance of Treg/Th17 exists in the synovium of CIA rats. TNFR-Fc may restore the balance of Treg/Th17 by inhibiting Th17 cell differentiation and inducing the production or accumulation of Treg.