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Objective To investigate mechanisms underlying the prevention of experimental autoimmune encephalomyelitis (EAE) in mice by intraperitoneal infusion of myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) (MOG i.p.).Methods C57BL/6 mice were immunized with MOG35-55 to establish the model of EAE and then were intraperitoneally injected daily with MOG35-55 or ovalbumin (OVA, served as control) from day 6 to day 16.EAE was evaluated daily using a general clinical scoring system and histological analysis.Numbers of lymphocytes in peripheral blood and central nervous system (CNS) were detected at different time points.Effects of MOG i.p.on the migration of MOG-T cells in vivo were analyzed by an adoptive transfer experiment.Maturation of splenic antigen-presenting cells (APCs) and migration of MOG-T cells in vitro were examined by fluorescence activated cell sorting (FACS) and a Transwell system, respectively.Results MOG i.p.protected the mice from development of EAE by blocking the lymphocyte recruitment to CNS.More effector T cells were trapped in the periphery of EAE and naive mice in adoptive transfer experiment after MOG i.p.treatment.MOG i.p.induced the maturation of splenic APCs and enhanced the expression of CD80, CD86 and major histocompatibility complex class Ⅱ (MHCⅡ) molecules.Mature APCs blocked the recruitment of effector T cells to CNS.Conclusion MOG i.p.protects mice from EAE by inducing the maturation of splenic APCs.
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Objective:To study the relationship between atrophy of the thymus and disease severity in EAE.Methods:MOG35-55 peptide induced EAE in C57BL/6 mice and analyzed the relationship between the severity of EAE and thymic atrophy,Flow cytometry analysis was used to evaluate thymic CD4+CD8+DP cells,CD4+CD8-,CD4-CD8+SP cells in relation to the severity of the disease.Results:The number of thymocytes in mice with decreased tail tone was (20.25 ±3.49) ×106 ,hindlimb weakness(4.93 ± 0.85)×106,complete hindlimb paralysis(1.8 ±0.19) ×106,and forelimb and hindlimb paralysis(0.52 ±0.07) ×106,there were statistically significant differences between groups ( P<0.05 ).As the disease progresses, CD4+CD8+DP cells ratio decreased, CD4+CD8-,CD4-CD8+SP cell ratio increased,different disease groups was statistically significant difference (P<0.05).Conclusion: The atrophy of thymus was closely related to the severity of EAE.Migration of activated T cells in EAE may cause atrophy of thymus.
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Objective:To investigate the role of alloreactive T cell in clonal deletion and regulatory T cells ( Treg) in transplant tolerance.Methods:F1 mice were bred by crossing female BALB/c mice and male C57BL/6 mice.Within 24 h,newborn C57BL/6 mice were inoculated with F1 spleen cells via the orbital branch of the anterior facial vein.Six weeks later,the mice were subjected to F1 skin grafting to evaluate their tolerance.Proliferation,flow cytometry and adoptive transfer assay were used to analyze clonal deletion of alloreactive T cells and the expression of CD4+Foxp3+T cells in neonatal treated mice.Results: Newborn C57BL/6 mice injected with F1 splenic cells could induce transplantation tolerance,the level of tolerance was associated with the dose of splenic cells.3×107 splenic cells from F1 mice could induce long-term skin graft acceptance in C57BL/6 mice ,1×107splenic cells significantly prolonged the survival of F1 skin grafts,but the grafts completely rejected within 50 days.The mixed lymphocyte reaction ( MLR) experiment in vivo showed that alloreactive T cell in long-term tolerant mice was deleted completely,but a certain amount of reactive T cells existed in the low-dose group mice.Flow cytometry ( FCM) analysis showed that the expression of CD4+Foxp3+T cell in the high-dose group and low-dose group mice had no obvious difference compared with the naive mice.When alloreactive T-cells were injected into tolerant mice,the skin graft rejection was observed,and Treg cells upregulated in graft-rejected mice.Conclusion:The degree of transplantation tolerance depended on the clonal deletion of alloreactive T cells,instead of on the expression of CD4+Foxp3+Treg cells.CD4+Foxp3+regulatory T cells upregulated in graft rejected mice,which may be served as a negative feedback mechanism to control the intensity of rejection.
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Objective:To investigate the best preservation conditions of N-terminal pro-brain natriuretic peptide( NT-proBNP) and provide detection references with stable performance for detection kits.Methods: ELISA was used to quantitatively detect the changes in NT-proBNP contents in various preservation solutions.The effects of basic buffer system, preservative Proclin300 and antibiotics on the preservation of NT-proBNP were analyzed using univariate analysis.The combination of various factors was then optimized using orthogonal experiments, to identify the best preservation system for NT-proBNP.Results: The univariate analysis determined that the basic buffer system for NT-proBNP was 0.02 mol/L phosphate buffered saline(PBS) at pH7.2,the addition of pre-servative Proclin300 could extend the preservation time of NT-proBNP at 37℃ by one day, the combined addition of penicillin and streptomycin prolonged the preservation time of NT-proBNP by one day compared with individually adding penicillin or streptomycin.The orthogonal experiments identified a preservation solution for NT-proBNP as 20%calf serum,1/1 000 Proclin300,120 U/ml penicillin and 80 U/ml streptomycin in a basic buffer system of 0.02 mol/L PBS at pH7.2.This solution was used to preserve an NT-proBNP reference sample at 37℃.Seven days later,the calibrated fixed-value of the sample at 37℃was only 1.3%lower than that at 4℃.Conclusion:Optimized NT-proBNP serum preservation solution could preserve NT-proBNP standard sample at 37℃ for seven days.
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Objective To investigate the role of oridonin in preventing skin graft rejection.Methods BALB/c mice were transplanted with skin grafts from C57BL/6 mice.Grafted mice were treated daily with oridonin,CsA and PBS,respectively.The survival of grafts was inspected daily and evaluated by histological analysis.On day 7 after transplantation,the percentage of CD4+ CD25+ Foxp3+ cells (Treg) in the spleen was determined by flow cytometry.The effect of oridonin on MLR and apoptosis was examined in vitro.Naive BALB/c mice were intraperitonealy injected with oridonin (15 mg/kg/day).At different time points,the number of T cells and macrophages in peripheral blood mononuclear cells (PBMCs) as well as the spleen was examined.Results The survival of skin grafts in the oridonin group (15.8 ± 1.5 days) was significantly longer than that in the control group (12.3 ± 1.2 days) and the CsA group (13.3 ± 1.1 days).Oridonin reduced inflammatory cell infiltration in grafts.The expression of Tregs was higher in the oridonin group (17.6 ± 3.6％) than in the control group (14.8 ± 2.3％).In vitro oridonin inhibited MLR and induced apoptosis in a dose-dependent manner.The number of T cells in PBMCs was rapidly decreased following oridonin treatment.With the depletion of T cells in PBMCs,high frequency of granulocytes was observed.On day 8,the number of T cells in the spleen was decreased,which was accompanied by increased phagocyte number.Conclusion Oridonin could suppress allograft rejection and prolong survival of skin grafts.The mechanism may be attributed to upregulation of Tregs and clearance of T cells.
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ObjectiveTo investigate the mechanisms of neonatal transplantation tolerance,especially the role of immature immune system and chimerism in tolerance.MethodsF1 ( or GFP-F1 ) mice were bred by crossing male C57BL/6 (or GPF transgenic C57BL/6) and female BALB/c mice. Within 24 h,newborn C57BL/6 mice were inoculated with different doses of splenocytes from F1 or GFP-F1 mice,irradiated spleen cells were used as control.Six weeks later,the mice were subjected to F1 skin grafting,and mixed-lymphocyte reaction was performed to determine their tolerance.Flow analysis was used to detect chimerism.ResultsLiving F1 spleen cells could induce chimerism and neonatal transplantation tolerance,but irradiated cells not.The chimerism in long-term tolerant mice is higher than that in chronic rejected mice,with 6.48％ ±4.02％ vs 1.57％ ±0.89％,the difference is significant in statistical analysis.The degree of neonatal transplantation tolerance is determined by the dosage of donor cells,the mice induced with 3 × 107 F1 spleen cells have 80％ long-term tolerance,but the dose of0.7×107 F1 spleen cells could only prolong allografts survival.ConclusionNeonatal transplantation tolerance is dependent on chimerism,the chimerism of donor cells leads to clonal deletion of alloreactive T cells.
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Objective The therapeutic effect of epigallocatechin-3-gallate (EGCG) on the collageninduced arthritis model (CIA) was observed and its immunological mechanism was analyzed. Methods EGCG was administered to CIA mice and PBS was admitted as negative control. The severity of CIA was evaluated by clinical scores and histopathological assessment (H-E staining). Immunological mechanisms inv-suppressive effect on IL-17 secretion of CD4+T cells (EGCG group: 0.41%; PBS group: 4.05% ) and inhibitive activity of C Ⅱ -reactive splenocytes proliferation. There was statistical significant difference between IKB expression and down-regulate phosphorylated IKB expression in lymph node cells of CIA mice.Conclusion EGCG can significantly ameliorate the severity of CIA. The therapeutic mechanisms may be related to inhibition of C Ⅱ -reactive splenocyte proliferation and IL-17 secretion and via inhibiting the activity of NF-κB by inducing the expression of IKB and by suppressing the expression of phosphorylated IKB in CIA mice.