Inhibitive effect of IL-35 on cardiac allograft rejection in mice / 中华器官移植杂志

Objective To investigate the action mechanism of IL-35 gene transfection ameliorating cardiac allograft rejection and prolonging allograft survival.Methods pEBI3-L-p35-Fc plasmid was amplified by polymerase chain reaction.In vitro plasmid DNA pEBI3-L-p35-Fc or pSec-L-Fc was,respectively,transfected into HEK293 cells using Lipofectamine 3000.At 48 and 72 h after transfection,IL-35 concentration in culture supernatant of transfected HEK293 cells was detected by ELISA.Balb/c and C57BL/6 splenocytes treated with mitomycin (MMC) served as the stimulators,those not treated with MMC as responders,and they were subjected to one-way mixed lymphocyte culture (MLC).In the presence or absence of IL-35,the percentage of CD4+ CD25+ Tregs was detected by flow cytometry.Abdominal heterotopic heart transplantation model was established by using inbred male Balb/c mice as donors and C57BL/6 as recipients respectively.In experimental group,recipients were intravenously administrated with IL-35 plasmid (50μg) on the day 1 to day 3 post-transplantation.The control mice were treated with normal saline.The IL-35 expression in the blood,CD4+ CD25+ Tregs proportion in the blood and spleen,and the survival and the histopathologic changes of the cardiac grafts were also observed.Results In vitro the transfected HEK293 cells expressed IL-35.IL-35 enhanced the proliferation of CD4+ CD25+ Tregs of MLC in vitro.The median survival time of the cardiac grafts in experimental group (16 days) was significantly longer than in control group (7 days) (P＜0.01).As compared with control group,CD4+ CD25+ Tregs proportion was significantly increased (P＜0.01),CD8+ T cells proportion was decreased (P＜0.01) and the proliferation of lymphocytes and monocytes infiltration was inhibited in the experimental group.Conclusion IL-35 could alleviate cardiac allograft rejection and prolong cardiac allograft survival via the induction of proliferation and differentiation of CD4+ CD25+ Tregs and inhibition of proliferation of CD8 + effector T cells.

Study on the immunoregulatory and lethal effects of natural killer T cells on melanoma cells in vitro / 国际检验医学杂志

Objective To explore the immunoregulatory and lethal effects of natural killer T lymphocytes(NKTs) in vitro .Meth‐ods The mixed lymphocyte cultured(MLC) system was established ,in which the B16F10‐luc‐G5 cells were set as target cells ,the total lymphocyte cells were set as effector cells .(1)In the experiment on immunoregulatory effects ,NKT lymphocytes or CD4+CD25+ T lymphocytes were set as regulating cells ,there was three groups ,including the NKT group ,CD4+CD25+ T group and pure target cell control group .Otherewise ,the 1640 blank control group was set by only adding RPMI1640 solution .(2)In the ex‐periment on antitumor effects ,the NKT or natural killer(NK) lymphocytes were set as killer cells ,there was three groups ,inclu‐ding the NKT group ,NK group and pure target cell control group .Mixed culturing 24 ,48 and 72 hours ,the bioluminescence of target cells in MCL system was detected by using the in vivo imaging system .Results (1)In the experiment on immunoregulatory effects ,there were statistically significant differences in measured average photon numbers between NKT group ,CD4+ CD25+ T group and the two control groups(P<0 .05) .The statistically significant differences were also found in the NKT group between 24 hours and 72 hours (P<0 .05) .(2)In the experiment on antitumor effects ,there were statistically significant differences in meas‐ured average photon numbers ,when the NKT group and NK group were compared to the pure target cell control group(P<0 .05) . After culturing 24 and 72 hours ,statistically significant differences were found between NKT group and NK group(P<0 .05) .Con‐clusion The NKT cells could inhibit the lethal effects of lymphocyte cells on target cells ,and the inhibitory effects are changed by the length of culturing .Compared with the CD4+CD25+ T lymphocytes ,NKT lymphocytes have strongger regulatory effects .Addi‐tionally ,the NKT cells have lethal effects on target cells ,which might be weaker than that of NK cells .

Evaluation of CD4+CD25+ T lymphocyte response time kinetics in patients with chronic Chagas disease after in vitro stimulation with recombinant Trypanosoma cruzi antigens

Introduction CD4+CD25+ T lymphocytes have been implicated in the regulation of host inflammatory response against Trypanosoma cruzi, and may be involved in the clinical course of the disease. Methods Peripheral blood mononuclear cells from patients with chronic Chagas disease were cultured in the presence of T. cruzi recombinant antigens and assayed for lymphocytes at distinct time points. Results It was possible to differentiate clinical forms of chronic Chagas disease at days 3 and 5 according to presence of CD4+CD25+ T cells in cell cultures. Conclusions Longer periods of cell culture proved to be potentially valuable for prospective evaluations of CD4+CD25+ T lymphocytes in patients with chronic Chagas disease. .

Prolongation of cardiac allograft survival in mice by Decitabine-induced upregulation of Foxp3 expression / 中华器官移植杂志

Objective To investigate the effect of Foxp3 gene expression induced with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (Aza) in recipients on ameliorating allograft rejection and prolonging allograft survival and the possible mechanisms.Method BALB/c and C57BL/6 splenocytes treated with mitomycin (MMC) served as the stimulators,those not treated with MMC as responders,and they were subjected to one-way mixed lymphocyte culture (MLC).In the presence or absence of Aza,the percentage of CD4+ CD25+ Tregs was detected by flow cytometry.Abdominal heterotopic heart transplantation model was established by using inbred male Balb/c mice as donors and C57BL/6 as recipients respectively.In experimental group,recipients were intravenously administrated with decitabine (1.5 mg/kg/day) on the day 1 to day 3 post-transplantation.The control mice were treated with normal saline.CD4+ CD25+ Tregs proportion in the blood and Foxp3 mRNA in cardiac grafts and spleen of recipients were measured respectively,and the survival and the histopathologic changes of the cardiac grafts were also observed.Result Decitabine enhanced the proliferation CD4 + CD25 + Tregs of MLC in vitro.The median survival time of the cardiac grafts in experimental group (11 days) was longer than that in control group (7 days) (P＜0.01).As compared with control group,Foxp3 mRNA expression in the cardiac grafts and spleen in recipient mice was significantly up-regulated,and CD4+ CD25 + Tregs proportion was increased (P＜0.01) and the proliferation of lymphocytes and monocytes infiltration was inhibited.Conclusion Up-regulation of Foxp3 gene expression induced with decitabine in recipients could alleviate cardiac allograft rejection and prolong cardiac allograft survival via the induction of proliferation and differentiation of CD4+ CD25 + Foxp3 + Tregs.

Change and significance of the regulatory T lymphocytes expression in rats model of chronic abacterial prostatitis / 中华泌尿外科杂志

Objective To investigate the change and significance of regulatory T lymphocytes in peripheral blood of rats model of chronic abacterial prostatitis.Methods Twelve Wistar rats with weight of approximate 390 g were randomly divided into two groups,model group and control group.Rats in the model group was injected subcutaneously 17β-estradiol(0.25 mg/day,for 30 days) after castration to establish rat model of chronic abacterial prostatitis.Flow cytometry was applied to detect the frequency of CD+4 CD+25 cells and CD+8CD-28 cells in peripheral blood of rats after model establishment.Results Compared with control group (11.63±1.36)％,the proportion of CD+4 CD+25T lymphocytes in model group (7.90±1.74)％ significanlty decreased (P＜0.01).Compared with control group (24.64±4.76)％,the proportion of CD+8CD-28T lymphocytes in model group (17.18±2.83) ％ also significantly decreased (P＜0.01).Conclusions The changes of the ratio of CD+4 CD+25T lymphocytes and CD+8CD-28T lymphocytes in peripheral blood of rats model of chronic abacterial prostatitis provided evidences for pathogenic mechanism of regulatory T lymphocytes participating in the development of chronic abacterial prostatitis.

Therapeutic effect of 4-1BB monoclonal antibodies and glucocorticosteroid on immune hepatitis induced by ConA / 中华风湿病学杂志

Objective To investigate the therapeutic effect of 4-1BB monoclonal antibodies (4-1BBmAb) and glucocorticosteroid and the affect of the expression of CD4+CD25+T lymphocytes on the mouse hepatitis induced by ConA.Methods Mouse model of hepatic injury was induced by the application of ConA and checked by hepatic function tests and hepatic pathology.Mter the animal models were constructed,the mice in the group of therapeutic alliance were treated with glucocorticosteroid and 4-1BBmAb.In contrast,mice in the control group were treated with 4-1BBmAb or glucocorticosteroid alone.The groups were then compared.After blood was collected respectively from the control group,the model group and the therapy group,flow cytometry was used to examine CD4+CD25+T lymphocytes.Chi-square test and t-test were used for statistical analysis.Results Compared with the control group,the conditions of the mice were improved after being disposed with 4-1BBmAb.The conditions became even better when 4-1BBmAb were used combined with glucocorticosteroid.ALT and AST of the control group were (140±22) U/L and (131±16) U/L respectively,that of 4-1BBmAb group were (98±14) U/L and (89±11) U/L respectively.The ALT and AST of the glucocorticosteroid treatment group were (76±11) U/L and (71±10) U/L respectively,ALT was (61±8) U/L and AST was (55±7) U/L in the combination treatment group.Differences among groups was statistically significant (P＜0.01).The expression of CD4+CD25+T lymphocytes was (3.0±0.8)％ in the control group,(8.5±2.9)％ in the gluco-corticosteroid treatment group,(8.4±3.5)％ in the 4-1BBmAb treatment group and was (11.2±3.5)％ in the combination treatment group.The difference was significant among the groups (P＜0.05).Conclusion 4-1BB-mAb have therapeutic effect for hepatic injury.The effectiveness will become even more evident when 4-1BB monoclonal antibodies are used together with glucocorticosteroid.During the course of treatment,4-1BBmAb and glucocorticosteroid can impact the expressions of CD4+CD25+T lymphocytes and this is the mechanism for the effectiveness in treating immune-mediated hepatic injury.

Effect of Argon-Helium Cryosurgery on Regulatory T Lymphocytes in the Peripheral Blood of Patients with Advanced Renal Carcinoma / 中国肿瘤临床

Objective: To analyze the effect of Argon-Helium cryosurgery (AHCS) on CD4+ CD25+ regulatory T cells (Treg) and its implication in patients with advanced renal carcinoma.Methods:,Peripheral venous blood samples were ob-tained from 32 patients with advanced renal cell carcinoma before and after AHCS.The proportions of Treg cells and T lym-phocyte subsets (CD3+ T, CD4+ T, CD8+ T, CD4+ T/CD8+ T, and NK cells) in the peripheral blood were measured by flow cytometry.Enhanced CT or enhanced MRI was used to observe the necrosis of tumor at 1 month after AHCS.The areas with no imaging enhancement in tumor were regarded as tumor necrosis.The necrosis rate was measured by Cavalieri method and the tumor burden was evaluated.Results: At 3 months after AHCS, the percentages of Treg cells were gradual-ly decreased from 4.18%±1.58% to 1.96%±0.54%, with a significant difference (P=0.001).At 3 months after AHCS, the pro-portions of CD3+ T, CD4+ T, NK and CD4+ T/CD8+ T were gradually increased from 19.26%±7.52%, 43.54%±12.99%, 1.15%±0.57%, and 17.49%±8.36% to 30.83%±5.69%, 49.58±10.76%, 1.84%±0.12%, and 27.63%±8.20%, with a statistical significance (P=0.000, P=0.003, P=0.02, and P=0.001).The proportion of CD8 + T was decreased from 40.86%±8.89% to the lowest ratio (26.74%±4.29%) at 3 months after AHCS, with a significant difference (P=0.000).At 3～6 months after cryo-therapy, there was only a slight change in the proportions of CD3 + T, CD4 + T, CD4 + T/CD8 + T, NK, CD8 + T, and Treg cells, with no significant difference (P>0.05).Correlation analysis showed that the decrease in tumor burden was positively correlated with the decrease of the proportion of Treg cells (r=0.793, P<0.01).Conclusion: After AHCS, the distribution of T-lymphocyte subsets can be improved and the anti-tumor immune response was strengthened.The percentage of Treg cells is correlated with tumor burden.

Changes of CD4+CD25+Regulatory T Cells in Patients with Acute Coronary Syndrome and the Effects of Atorvastatin / 华中科技大学学报（医学）（英德文版）

The function of CD4+CD25+regulatory T lymphocytes (Treg) in patients with acute coronary syndrome (ACS) and the effects of atorvastatin were investigated. Forty-eight patients with ACS were randomly divided into two groups: group C receiving conventional therapy (n=24), and group C+A receiving conventional therapy+atorvastatin (10 mg/day, n=24). T lymphocytes from ACS patients (before and 2 weeks after the treatment) or 18 healthy subjects were separated and the flow cytometry was used to measure the percentage of Treg. The inhibitory ability of Treg on effector T cells was determined by mixed lymphocyte reaction (MLR). ELISA was used to measure the serum levels of cytokines (IL-10, TGF-β1 and IFN-γ) before and after treatment. The results showed that as compared with normal control group, Treg percentage was decreased significantly (P<0.01), the in- hibitory ability of Treg on the T lymphocytes proliferation was reduced (P<0.01), IFN-γ, levels were increased and IL-10 and TGF-β1 levels were lowered in ACS patients. After treatment with atorvas- tatin, Treg percentage and the inhibitory ability of Treg on T lymphocytes proliferation were signifi- cantly increased in ACS patients. Serum IFN-γ, was decreased significantly, while IL-10 and TGF-β1 were elevated significantly as compared with the non-atorvastatin group. The number of Treg was positively correlated with serum TGF-β1, but negatively with serum IFN-γ and CRP. It was concluded that ACS was associated with decreased number and defected function of Treg, which may play an important role in initiating immune-inflammatory response in ACS. The inhibitory ef- fects of atorvastatin on inflammation in ACS may be due to its beneficial effects on Treg and restora- tion of immune homeostasis.

Association between CD4~+CD25~+ regulative T cells and systemic lupus erythematosus disease active stage / 中国免疫学杂志

Objective: To investigate the ratios of peripheral blood CD4+CD25+,CD4+CD8+ regulative T cells of systemic lupus erythematosus(SLE) patients, and explore the association with disease active stage,nephropathy,serum anti-ds-DNA antibody,and both IgG and C3 levels. Methods: The percentage of CD4+CD25+T cells and CD4+CD8+T cells of peripheral blood from patients with systemic lupus erythematosus(SLE)(30 females and 7 males),30 rheumatism controls and 30 normal individuals were measured by flowcytometry. Results: Patients with active disease had statisitically lower levels of CD4+CD25+T cells than did normal controls(P