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Objective To explore the possible role and mechanism of the Kupffer cells (KCs) in liver allo-geneic transplantation at the early stage. Methods In vitro cell contact coculture system was established. Culture supernatants were collected respectively on the 1st, 2nd, 4th, 6th d after cocul-ture and the KCs and PBMCs were harvested on the 6th day after culture. The expression of HLA-G on the membrane of the KCs and PBMCs was detected with immunochemistry. Nitrate reduction test was used to determine the concentration of nitric oxide. IFN-γ, IL-10, TGF-β1 cytokine levels in the supernatants were also measured with ELISA. The proliferation of lymphocytes was evaluated with MTT. Results six days later, no HLA-G molecules were detected on the membrane of the KCs and PBMCs. In the experimental group containing KCs, the levels of NO, IL-10 and TGF-β1 was signifi-cantly increased(P<0. 05), while the levels of IFN-γ was relatively lower(P<0. 05) as compared to the experimental group without KCs. No IL-10 and IFN-γ were detected in the control group, and on-ly few NO and TGF-β1 was found in the control group with KCs. MTT test showed that the value of optical density was lower in the experimental group with KCs than that in any other group(P<0. 05).Conclusion No HLA-G is expressed on the membrane of KCs and PBMCs after contact coculture.KCs may participate in regulating production of NO and Th2/Th3-like cytokines and suppressing the proliferation of lymphocytes, through which KCs probably take part in inducing immunotolerance of liver transplantation in early stage.
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Objective To evaluate the alteration of CD4+ regulatory T cells in peripheral blood from patients with ankylosing spondylitis(AS). Methods Seventy-eight AS patients and 50 healthy individuals were included in this study. The proportion of CD4+CD25+CD127lo/- T cell population in CD4+ T cells as well as that cytotoxic T lymphocytes(CTL) and NK cells in lymphocytes was evaluated by flow cytometry. Serum TGF-β and TNF-α were measured by ELISA. The inhibitory function of CD4+CD25+ regulatory T cells was measured by mixed lymphocyte culture. Results The population of CD4+CD25+CD127lo/- T cells in peripheral blood of AS patients accounted for (4.36±1.21)% of CD4+ T lymphocytes, which was significantly lower than that in healthy individuals (P<0.05). The CD4+CD25+CD127lo/- T cells population in AS patients was positively correlated with TGF-β level, but negatively with TNF-α. Compared with healthy individuals, the function of CD4+CD25+ regulatory T cells in the inhibition of alloreactive T cells was lower in AS patients, which was related to the decreased secretion of TGF-β. Conclusion The CD4+ regulatory T cells in peripheral blood of AS patients are significantly decreased and its function is defective, which leads to immune regulatory dysfunction in vivo. It may be one of immune pathogenesis mechanisms of AS.
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Objective To construct vector expressing soluble mPDL1-hIgGFc and study its effect on the proliferation and apeptosis of cells in vitro. Methods The extrncellular domain of mPDL1 gene was amplified from pmPDL1 vector by PCR and inserted into phIgGFc vector. The recombinant pmPDL1-hIgGFc was transfected into CHO cells by LipofectAMINETM2000, and the transfected cells were named as CHOp. The expression of mPDL1-hIgGFc in the culture supernatants of CHOp was assayed by ELISA and Western blot. The effects of CHOp culture supernatants on mixed lymphocyte culture(MLC) was analysed by Flowm-etry. Results The extracellular domain of mPDL1 gene were obtained from PCR. DNA sequencing and the identification of digestion by HindⅢ and KpnⅠ indicated the recombinant plasmid pmPDL1-hIgGFc was suc-cessfully constructed. ELISA and Western blot analysis proved that the CHOp could express mPDL1-hIgG-Fc. CHOp culture supernatants could inhibit lymphocyte proliferation and induce the apoptosis of the activa-ted T cells in MLC in vitro in a dose-dependent manner. Conclusion The mPDL1-hIgGFc protein could in-hibit lymphocyte proliferation and induce the apoptosis of the activated T cells.
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Objective To investigate the effect of mesenchymal stem cell(MSC) on the balance of Th1/ Th2 with different stimulations.Methods To inoculate MSC in 24-well tissue culture plates.after 3 days, added T lymphocytes co-stimulated by PMA and Ionomycin,or the MSC were added to the two-way mixed lymphocyte culture according to different proportion.The subsets and cytokines of T lymphocytes were analyzed by flow cytomety.Results Differentiation into Th1 of T lymphocytes activated by co-stimulation can be inhibited MSC;In MLC,CD8~+T cell subsets and Th1 were evidently decreased.But Th2 cells were slightly increased.Conclusion MSC can significantly suppress CD8~(+)T cell,Th1,increase Th2.MSC has potentialities of alleviating acute graft-versus-host disease(aGVHD) and maintaining graft versus leukemia(GVL).
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Triptolide (TL) is a major active component extracted from Chinese traditional herb Tripteryginm wil-fordii Hook. In this article, the effects of TL on mixed lymphocyte culture (MLC),suppressor T cell(Ts) activity, delayed type hypersensitivity (DTH) reaction, IL - 2 secretion activity and Th/Ts ratio were evaluated, In vitro TL 0. 5~10 ?g ? L-1suppressed one-way MLC,Lymphocytes induced in first MLC with TL of 5,10 ?g ? L-1 suppressed the second MLC after irradiation with 3000 rad 60Co source. This suggests that TL may induce Ts cell-s. In vivo, DTH reaction sensitized to dinitrofluo-robenzene (DNFB) monitored by the increase in weight of the ears challenged with antigen was suppressed by TL at doses of 0. 12 to 0. 50 mg ? kg-1(ip,qd?5) and the IL -2 activity secreted by spleen cells of these mice was inhibited at doses of 0. 25 and 0. 5 mg ? kg-1. TL 0. 25, 0. 5 mg ? kg-1(ip,qd?8) also had prominent suppres-sive effects on enhanced DTH reaction which was induced by cyclophosphamide(250 mg ? kg-1,ip). Th/Ts ratio of mouse thymus cells were reduced after given 0. 25 mg ? kg-1 of TL for 5 consecutive days. The above-mentioned data suggest that TL has suppressive effects on cellular immunity function and the suppressive mechanism may be related to the suppression of Th cells and IL-2 secretion activity and the induction of Ts cells.
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The efficacy of different inducers used to induce immune interferon (IFN-?)from ionsillar cells of onsillectomized normal children was sfudied.Tonsillar cells were made in suspension of 1?107 cells/ml treated with antibiotics,and cultured for 2-3 days and then supernants were assayed for IFN-r.The results show that cells of individual human tonsil could produce IFN-? spo ntaneously owing to the induction by pharyngeal normal flora.Esculentoside (Es) alone or .pokeweed mitogen (PWM) alone,their combinatian or with other inducers were capable of inducing IFN-?.Among them,PWM was more effective in inducing IFN-? than Es,and its efficacy was similar to that of PWM+ scoppolamine,but higher than that of PWM+C.parvum or PWM+Con A.Mixed lymphocyte culture (MLC) of human tonsils,with or without PWM or Es,could produce IFN-?,but slightly higher titer was obtained with MLC+PWM.The possible reasons for the difference of efficacy of various methods in inducing IFN-? from human tonsillar cells are discussed.