Influence of GPIF on the expression of costimulatory molecules lineaged T cells in mice with immunodeficiency in vivo

AIM:To investigate the effects of goat placenta immunoregulating factor (GPIF) on the expression of costimulatory molecules lineaged T cells in BALB/c mice. METHODS:Animal model for immunodeficiency made from BALB/c mice with whole-body irradiation by 5 Gy 60Coγ-ray was applied for research. The immunosuppressive mice were injected with GPIF for seven days continuously. FACS was applied to analyze the rate of CD28+, CD152+, CD4+CD28+, CD8+CD28+, CD4+CD152+ and CD8+CD152+ cells in splenic lymphocytes and ELISA method was employed to measure the amount of IL-2 and IFN-γ in serum of mice. RESULTS:GPIF increased the percentage of CD28+, CD4+CD28+ and CD8+CD28+ cells (P<0.05, P<0.01), and decreased the percentage of CD152+ (P<0.05, P<0.01), CD4+CD152+ cells (P<0.05, P<0.01) in splenic lymphocytes of immunosuppressive mice significantly. GPIF increased the content of IL-2 and IFN-γ in serum of mice simultaneously (P<0.01). CONCLUSION:Immuno-enhancing effect of GPIF facilitates the costimulation of CD28 pathway, which can activate T cells and accelerate the course of renewing T cell activity. The function of GPIF may have close relationship with an immune network formed by the secretion of IL-2 and IFN-γ and the expression of CD28 and CD152.     

Effect of hepatitis virus B protein on the functions of PBMCs isolated from patients with chronic HBV infection

AIM: To study the effect of hepatitis virus B proteins on peripheral blood mononuclear cells (PBMCs) from patients among various types of chronic hepatitis B virus (HBV) infection.METHODS: 80 patients of various types of chronic HBV infection were observed, including 40 HBeAg positive with abnormal alanine aminotransferase (ALT) (A group), 20 HBeAg positive with persistent normal ALT(B group), 20 HBeAg and HBV-DNA negative with persistent normal ALT level(C group). IL-10, IFN-γ in CD8+CD28+T cells, after stimulation with PHA, HBeAg and HBcAg for 48 h, were inspected respectively in PBMCs.RESULTS: IFN-γ was significantly lower in HBeAg positive patients. IL-10 was significantly higher in HBeAg positive with normal ALT. CD8+CD28+T were significantly lower than others. CONCLUSION: In HBeAg positive group, secretion of cytotoxic T lymphocyte (CTL) and Th1 type cellular immunologic reaction is decreased, Th2 type cellular immunologic reaction is enhanced.     

Relationship between Th1/Th2 balance and CD28/CTLA-4 expression on peripheral blood T cells in patients with systemic lupus erythematosus

AIM: To investigate the pattern of Th1/Th2 balance in systemic lupus erythematosus(SLE) patients and the relationship between CD28/CTLA-4(cytotoxic T-lymphocyte antigen-4) molecule expression and Th1/Th2 balance.METHODS: Eighteen SLE patients met the ARA 1997 updated SLE criteria were selected in the study. According to Bombardier's SLEDAI criteria, all patients were classified into two groups: active group(12 cases) and static group(6 cases). Fourteen normal individuals, matched for age and sex of the patients, served as controls. The peripheral blood mononuclear cells(PBMCs) were isolated by density gradient centrifugation and cultured in RPMI-1640 culture medium. After treated with PMA(5 μg/L) and ionomycin(500 μg/L) for 72 h, the PBMCs were collected, the contents of IFN-γ and IL-10 in the supernatant of cultured PBMCs were detected using enzyme linked immunosorbent assay(ELISA). The expression of CD28 and CTLA-4 molecules on T cells were detected by flow cytometric technique with double staining by FITC or PE labeled monoclonal antibodies. RESULTS: The level of IL-10 was higher in the PBMCs of active and static SLE patients(351.29 ng/L±153.31 ng/L and 319.37 ng/L±153.39 ng/L) than that in controls(254.48 ng/L±120.69 ng/L), but the difference did not reach statistical significance(P>0.05). The level of IFN-γ was significantly lower in the PBMCs of active SLE patients(25.76 ng/L±16.09 ng/L) than that in controls(50.71 ng/L±27.92 ng/L, P＜0.05). The ratio of IL-10/IFN-γ was significantly higher in active SLE patients(18.74±13.77) than that in controls(6.66±4.95, P<0.05). Either before or after culture, the expression of CD28 molecule on CD3⁺and CD8⁺ T cells from all SLE patients was not remarkably different from that in the cells of controls. Before culture, the expression of CTLA-4 molecule on CD3⁺T cells of active SLE patients(0.79%+0.37%) was significantly lower than that in the cells of controls(1.31%+0.61%, P<0.05). After culture, the expression of CTLA-4 molecule on CD3⁺ T cells of SLE patients was still lower than that in the cells of normal controls without statistical significance(P＞0.05).The expression level of CD28 molecule on CD3⁺ or CD8⁺ T cells in active SLE patients and controls was not correlated with the levels of IFN-γ and IL-10 in the supernatants(P＞0.05). The level of CTLA-4 molecule expression on CD3⁺ T cells of active SLE patients was positively correlated with IFN-γ level(r=0.681, P<0.05), while was negatively correlated with IL-10 levels(r=-0.624,P＜0.05) and the ratio of IL-10/IFN-γ(r=-0.738, P<0.01). The level of CTLA-4 molecule expression on CD3⁺ or CD8⁺ T cells of controls showed no correlation with IFN-γ levels, while showed negative correlations with IL-10 level(r=-0.587, P<0.05; r=-0.563, P<0.05, respectively).CONCLUSION: There is a bias in the differentiation of Th0 cells towards Th2 in SLE patients. CTLA-4 probably plays an important role in this mechanism through suppressing the signal transmitted by CD28.     

Chronic exposure to house dust mite induces airway remodeling related to Th1 inflammation in mice

AIM: To investigate the inflammatory characteristics in the airway of mice with chronic exposure to dust mite. METHODS: The α-SMA-Cre/R26R transgenic reporter mice were intranasally exposed to dust mite extract for 60 d (DME group), and then subjected to the measurement of lung resistance. The performance of bronchoalveolar lavage, pathological changes of the lung tissues and splenocytes isolation 24 h after the last challenge were observed. The protein extracts from the lungs were subjected to the detection of α-smooth muscle actin (α-SMA) by Western blotting. The supernatants of the lung homogenate were collected for testing the levels of interleukin-4 (IL-4) and interferon-γ (IFN-γ) with enzyme-linked immunosorbent assay. CD4⁺ T-cell subsets of the splenocytes were analyzed by flow cytometry.RESULTS: The mice chronically exposed to dust mite extract demonstrated severe airway hyperresponsiveness. The pulmonary pathological sections with HE staining manifested strong evidence of airway remodeling in DME group, corresponding to an enhanced X-gal staining that is related to α-SMA activation in the subepithelial basement membrane of bronchia. Total cell and lymphocyte counts were increased in the lungs of DME group compared to control group. No difference was found in eosinophil count of mice between DME and control groups. There was an elevated level of IFN-γ in the lungs of DME challenged mice coordinated with an increased proportion of IFN-γ-producing CD4⁺ T cells in the splenocytes.CONCLUSION: Chronic exposure to dust mite in the mice induces Th1-dominant inflammation with an airway hyperresponsiveness and the development of airway remodeling.     

Altered T cell signaling events caused by anti-CD4 monoclonal antibody in suppressing experimental autoimmune cardiomyopathy

AIM:We examined the efficacy of anti-L3T4 McAb in the T cell signaling pathway in treating experimental autoimmune cardiomyopathy in BALB/c mice, as a model of the autoimmune mechanism involved in human dilated cardiomyopathy (DCM). METHODS:ADP/ATP carrier peptides were used to induce autoimmune cardiomyopathy in BALB/c mice. After 3 months, anti-L3T4 McAb was administered to deplete CD4+ T cells in the mice. Real-time PCR were used to detect the expression of intracellular signaling molecules (p56lck, p59fyn and Zap-70) and cytokine production (IFN-γ, IL-2 and IL-4) in T cells. The expression of CD45 was determined by immunohistochemistry analysis. RESULTS:Reduced expression of p56lck, p59fyn and Zap-70 and the reduced cytokine production of IFN-γ, IL-2 and IL-4 in T cells of anti-L3T4-treated DCM mice were found. Also, the expression of CD45 in spleen T cells was significantly decreased in the anti-L3T4-treated group. In contrast, immunization with irrelevant Ab did not protect the mice, the expression of T cell signaling molecules, CD45, and cytokine were not inhibited. CONCLUSION:These studies provide direct evidence that anti-L3T4 McAb can be an effective immunomodulator to T cell signal molecules and subsequent cytokine production events in ADP/ATP carrier-induced DCM in BALB/c mice.     

Regulatory T cells regulate function of effector T lymphocytes in tumor-draining lymph node of mouse hepatocelluar carcinoma

AIM: To investigate the roles of regulatory T cells (Tregs) on the function of effector T lymphocytes in tumor-draining lymph nodes (TDLNs). METHODS: The number expansion of Foxp3+ Tregs and CD4+ or CD8+ T cells in the TDLNs from mouse hepatocellular carcinoma model was detected by immunohistochemical staining and flow cytometry. Foxp3 mRNA expression was determined by real-time quantitative PCR. The ability of IFN-γ secretion in CD8+ T cells in the TDLNs was measured by enzyme-linked immunosorbent spot technique(ELISPOT). RESULTS: The expansions of Tregs and effector T cells were significantly increased in the TDLNs during tumor development. Tregs diffusely distributed in the CD8+ T cells occupancy area. The level of Foxp3 mRNA expression was significantly higher in the TDLNs than that in the inguinal lymph node (P<0.01) and spleen (P<0.01) from the same mouse inoculated Hepa1-6 cells. Tregs trended to accumulate within the TDLNs exclusively, but not in other peripheral lymph nodes(LNs) of the same host. Foxp3 mRNA expression was significantly higher in the spleen from the tumor mice than that from mice injected with LPS (P<0.01). Tregs suppressed the CD8+ T cells primed in the TDLNs that retained the ability to secrete IFN-γ via anti-CD3 stimulation. CONCLUSION: Tregs play an important role in regulating the function of CD8+ T cells. Deletion of Tregs could be crucial for establishment of tumor-specific immunotherapy.     

Effect of mesenchymal stem cells on secreting function of T lymphocytes from patients with idiopathic thrombocytopenic purpura in vitro

AIM: To analyze the effect of mesenchymal stem cells (MSCs) on secreting cytokines by T lymphocytes from patients with idiopathic thrombocytopenic purpura (ITP) in vitro.METHODS: Human bone marrow-derived MSCs were isolated by Ficoll Hypaque and cultured for proliferating to passage cells. Allogeneic T lymphocytes of ITP were isolated from peripheral blood by Ficoll Hypaque and nylon cotton column. Then the stromal feeder layers of different numbers (2×103, 1×104, 5×104 per well) of MSCs treated with mitomycin were co-cultured with above-mentioned T lymphocytes. The supernatant were respectively collected on day 2, 4 and 6 after co-culture, then the levels of IL-2, IFN-γ, IL-4, IL-10 secreted by T lymphocytes were measured by enzyme linked immunosorbent assay (ELISA) dynamically.RESULTS: The levels of IL-2 and IFN-γ secreted by T cells from ITP were higher than those from normal control (P<0.05, respectively). Inversely, IL-4 and IL-10 were lower than those in normal control (P<0.05, respectively). After co-cultured with T lymphocytes, MSCs significantly inhibited the cytokine levels of IL-2 and IFN-γ secreted by T lymphocytes from ITP or health adults (P<0.05, respectively) in a dose dependent manner (P<0.05, respectively), and the effect was more obvious when co-cultured for 4 days or 6 days than that for 2 days (P<0.05, respectively). However, MSCs significantly promoted the releases of IL-4 and IL-10 by T lymphocytes from ITP patients (P<0.05, respectively) in a dose dependent manner (P<0.05, respectively), and the effect on IL-10 was in a time dependent way (P<0.05), while the effect on IL-4 had no obvious difference among 2 d, 4 d and 6 d(P>0.05). As for health control group, when cell numbers exceeded above 1×104, MSCs obviously promoted IL-4 and IL-10 levels secreted by T lymphocytes (P<0.05) in a dose dependent manner (P<0.05), and both of the effects were more noticeable when co-cultured for 4 d or 6 d than that for 2 d(P<0.05, respectively).CONCLUSION: MSCs regulate the balance between Th1 and Th2 reaction and partly correct ITP Th1 polarization in vitro.     

Cytokine production in mice with experimental cardiomyopathy treated with anti-L3T4 monoclonal antibody at different stages

AIM: To clarify the mechanism of treating autoimmune cardiomyopathy at different stages with anti-L3T4 monoclonal antibody. METHODS: Mice immunized with human mitochondria ADP/ATP peptides were used as the cardiomyopathy (DCM) group, and the sham-immunized mice were regarded as the controls. Mice receiving early treatment were immunized with the same peptides, followed by the injection of 400 μg of anti-L3T4 on day 0, 1 and 2 post-immunization. Mice in the late treatment group were immunized as of the early treatment group but anti-L3T4 was administered 3 months post-immunization. The cytokine expression was measured with three-color flow cytometry to quantitate the splenic Th1/Th2 cell subsets in the different groups of mice. In addition, serum and myocardial cytokines were measured by enzyme-linked immunosorbent assay and real-time PCR. RESULTS: Th1 and Th2 subsets in the early treatment group were similar to those in control group, but were drastically lower than those in DCM group. Mice in the late treatment group showed an increased level of Th1-related cytokines, while the Th2 level was between the DCM and early treatment group. IFN-γ and IL-6 levels in early treatment group were similar to those in control group. In the early treatment group, IL-4 level was higher than that in control and lower than that in DCM group, whereas IL-2 and TNF-α contents were lower than those in control and DCM group. In the late treatment group, IFN-γ and IL-2 levels were higher than those in DCM group and lower than those in the early treatment group, while IL-6 and IL-4 levels were lower than those in DCM group. CONCLUSION: These results suggest that the cytokine production in cardiomyopathic mice may be repressed by treatment with anti-L3T4 at different stages. Early treatment with anti-L3T4 has better inhibitory function than treatment in late stage of autoimmune cardiomyopathy.     

TCRVβ7.1 transfection activates signal protein ERK and insreases IFN-γ expression in PBMCs

AIM: To investigate the effect of tumor-specific T cell receptor (TCR) gene transfection on production of cytokine and signaling activation in T cells.METHODS: TCRVβ7.1 gene was transferred into peripheral blood mononuclear cells (PBMCs) obtained from healthy adults, and the expression of Vβ7.1 was detected by flow cytometry before and after transfection. The total quantities of protein and phosphorylation of ERK1/2 were detected by Western blotting. The expressions of IL-4 and IFN-γ were detected by ELISA.RESULTS: The results of flow cytometry showed that TCRVβ7.1 protein was efficiently expressed after transfection. The phosphorylation level of ERK increased significantly in TCRVβ7.1-modified PBMCs, and was related with the activation of T cells. The expression of IFN-γ was significantly higher in TCR-transfected cells than that in non-transfected cells. The expression of IL-4, however, has no distinct difference between groups.CONCLUSION: The transfection of TCRVβ7.1 induces phosphorylation of ERK1/2 and production of IFN-γ, and activates T lymphocytes.     

Ovarian carcinoma cells inhibit ζ chain expression and the secretion of Tc1/Tc2 type cytokines in CD8+ T cells

AIM: To investigate the effect of ovarian carcinoma cells on ζ chain expression and the secretion of Tc1/Tc2 type cytokine in CD8+ T cells, and its role in the ovarian carcinoma induced immunosuppression.METHODS: The supernatants of human ovarian carcinoma cell lines of OVCAR3, CAOV3 and SKOV3 and RPMI-1640 were added into CD8+ T cells (groups I, II, III, and control), which were isolated from the peripheral venous blood of healthy persons. The expression of ζ chain was analyzed by Western blotting. Thiazolyl blue(MTT) method was used to detect the effects of those cell line supernatants on the growth of CD8+ T cells. The secretion of the Tc1 type cytokine interferon (IFN)-γ mRNA and the Tc2 type cytokine interferon (IL)-10 mRNA were detected by RT-PCR. RESULTS: The expression of ζ chain was significantly lower in groups I, II, and III in comparison with that in control group. The absorbance at the wavelength 570 nm of CD8+ T cells culture in the group I, II, and III was all significantly lower than that in the control group. The IFN-γ expression was significantly lower in groups I, II, and III in comparison with that in control group, while the expression of IL-10 was significantly higher. CONCLUSION: Ovarian carcinoma may suppress CD8+ T cell proliferation and secretion of the Tc1/Tc2 type cytokine through inhibition of ζ chain, which may play an important role in the ovarian carcinoma induced immunosuppression.     

CTLA4Ig induces immune tolerance of T cells to oxidized-low density lipoprotein in vitro

AIM: Recently,it is widely accepted that atherosclerosis (AS) is an auto-immune related disease and the oxidized-low density lipoprotein (ox-LDL) is the most important AS-related antigen.In order to prevent immune injuries in AS and find new strategies to prevent AS,the immune tolerance of T cells to ox-LDL in vitro was induced in this study.METHODS: Human monocytes were separated from peripheral blood to induce dendritic cells (DCs).DCs were treated with LPS (30 μg/L),ox-LDL (10 mg/L) and LDL (10 mg/L) for 48 h.Then DCs were mixed with allogenic T lymphocytes to carry out mixed lymphocytes reaction (MLR).CTLA4Ig in different concentrations was added in the MLR of ox-LDL group.MTT method was used to assay the proliferation of T cells and expressed in stimulation index (IS).The CD25 expression and apoptosis of T cells in MLR were tested by flow cytometry.The excretion of IL-2,IFN-γ and IL-4 was assayed by ELISpot method.RESULTS: SI in ox-LDL group was higher than that in LDL group significantly (P<0.05) and CTLA4Ig inhibited the SI in ox-LDL group with dose-dependent effect (P<0.05,P<0.01).CTLA4Ig decreased the CD25 expression (P<0.05,P<0.01) and induced apoptosis of T cells in MLR (P<0.05,P<0.01).CTLA4Ig decreased the ELISpot counts of IL-2 and IFN-γ (P<0.01),while increased that of IL-4 (P<0.05).CONCLUSION: CTLA4Ig induces T cells tolerance to ox-LDL in vitro.CTLA4Ig inhibits T cells activation,promotes T cells apoptosis and Th1/Th2 immune deviation,which is the important mechanism in it′s induction of tolerance.     

Establishment of T-lymphocytes that express CD20scFv-IgGFc-CD28-ζ and CD20scFv-IgGFc and their killing activity of B-lymphoma cells

AIM: To investigate the target killing effect of T lymphocytes with chimeric CD20scFv gene on Daudi cells and the activation of T lymphocytes. METHODS: Two kinds of plasmids were transfected into retrovirus-packed PA317 cell lines. The supernatant was collected from successfully transfected PA317 culture and was used to infect peripheral blood T lymphocytes. After one-week screening with G418, the cells were used to kill Daudi and K562 cells. The positive rates of AnnexinⅤ in Daudi cells were measured at different times points respectively by flow cytometry. Meanwhile, the level of IL-2 and IFN-γ were determined by ELISA. RESULTS: The Annexin V positive rate was significant higher in Daudi cells compared to control K562 cell lines at 24 h. No difference of AnnexinV in Daudi cells was observed in CD20 modification T lymphocyte groups. The secretions of IL-2 and IFN-γ in CD20scFv-CD80-IgGFc-CD28-ζ gene modified T cells co-cultured with Daudi cells were dramatically higher than that in CD20scFv-IgGFc group at 72 h. CONCLUSION: ① The two kinds of genetic modified specific T cells have no significant difference in inducing early apoptosis of Daudi cells. CD28-ζ cant affect Daudi cell early apoptosis at the CD20scFv target killing. ② The increase in the secretions of IL-2 and IFN-γ is more obvious in CD20scFv-IgGFc-CD28-ζ group, indicating that the self-activation takes place in CD3ζ and CD28 modified T cells without MHC restriction and then increases the activation and killing function of T cells.     

Effects of Foxp3 transfection on splenocyte functions in asthma mice

AIM: To study the expression and the effects of Foxp3 on the immunologic functions by transfecting the Foxp3 eukaryotic expression plasmid into the splenocytes of the asthma mice. METHODS: The mice were sensitized and challenged by ovalbumin to make asthma model. The splenocytes were harvested and cultured. The Foxp3 expression vector pcDNA3.1(-)-Foxp3 was transfected into the splenocytes with electroporation. The splenocytes transfected with empty vector and control splenocytes (non-transfected) were also set up. The expression of Foxp3 at mRNA and protein levels was detected by RT-PCR and Western blotting, respectively. The proportion of CD4⁺CD25⁺ Treg cells/CD4⁺ cells was measured by flow cytometry. Proliferation of the splenocytes was analyzed with MTT assay. ELISA was used to determine the levels of interleukin 4 (IL-4) and interferon γ (IFN-γ) in the supernatant of the splenocytes. RESULTS: The expression of Foxp3 at mRNA and protein levels in transfection group was significantly higher than that in empty vector group and control group. The proportion of CD4⁺CD25⁺Treg cells/CD4⁺ cells in transfection group was higher than that in empty vector group and control group. The proliferation of transfected cells was markedly inhibited compared with empty vector group and control group. The levels of IL-4 and IFN-γ were significantly lower in transfection group than those in empty vector group and control group. CONCLUSION: The transfected Foxp3 gene overexpresses in the splenocytes of asthma mice. Foxp3 increases the number of CD4⁺CD25⁺ T cells and inhibits the proliferation and production of Th1/Th2 cytokines in splenocytes.     

Effect of glucocorticoid-treated dendritic cells on asthmatic Th2 response

AIM: To investigate the effect of dexamethasone-treated dendritic cells (DCs) on Th2 cytokine production from autologous T cells in asthmatic patients and explore the mechanisms by studying the effect of dexamethasone on differentiation, maturation and function of DCs from patients with asthma. METHODS: Human peripheral blood monocyte-derived DCs generated from asthmatic patients and healthy subjects were cultured in the absence or presence of dexamethasone. The phenotypic characterization of DCs was analyzed by flow cytometry. The mature DCs were harvested, washed, and then cocultured in vitro with autologous T cells purified by a nylon cotton column. The DC-T coculture supernatants were collected after 72 h incubation and analyzed for levels of IL-5 and IFN-γ by ELISA. RESULTS: The concentrations of IL-5 in the culture supernatants of DC-T coculture were significantly up-regulated in patients with asthma compared with that in healthy controls ［(145.13±89.76) ng/L vs (50.28±22.37) ng/L, P<0.01］. The level of IFN-γ in the DC-T coculture supernatants tended to be decreased in asthmatic patients than that in healthy controls, although this difference did not achieve statistical significance ［(197.58±76.32) ng/L vs (220.46±65.34) ng/L, P>0.05)］. There were significantly decreased levels of IL-5 by autologous T cells primed by dexamethasone-treated mature DCs from asthmatic patients ［(45.39±19.61) ng/L vs (145.13±89.76) ng/L, P<0.01］, alterations not observed from healthy controls (P>0.05). IFN-γ production was decreased by autologous T cells primed by dexamethasone-treated mature DCs from both asthmatic patients and healthy controls ［asthma group: (40.21±22.89) ng/L vs (197.58±76.32) ng/L, P<0.01; healthy controls: (56.78±20.37) ng/L vs (220.46±65.34) ng/L, P<0.01］. Dexamethasone-treated DCs exhibited decreased expression of CD83 (P<0.01) and increased expression of CD14 (P<0.01) in both asthmatic patients and healthy controls. CONCLUSION: DCs of asthmatic patients induce a Th2-skewed cytokine production from autologous T cells. Dexamethasone-treated DCs inhibit the Th2 reactions, and this effect is probably mediated through the pathway that dexamethasone inhibits DCs maturation and skews the macrophage/DC balance towards the macrophage side and thus directs the development more towards the macrophage lineage.     

Effects of dexamethasone on intracellular expression of TH1/TH2 cytokines in experimental asthma mice

AIM: To study the effects of dexamethasone (DXM) on intracellular expression of T_H1/T_H2 cytokines and the mechanism of that during the development of asthma. METHODS: Eighteen BALB/c mice were divided into 3 groups at random: control group, asthma group, and DXM treated group, with 6 mice in each. The expressions of T-box expressed in T cells (T-bet) and GATA-binding protein-3 (GATA-3) in lung tissue were detected by Western blotting. The expressions of intracellular cytokines interleukin-4 and interferon-γ in CD4⁺ T cell were measured by flowcytometry.RESULTS: The results of flow cytometry indicated that the ratio of intracellular cytokines IL-4/IFN-γ in CD4⁺ T cells in asthma group was much higher than that in control group (P<0.01), the ratio of intracellular IL-4/IFN-γ in T cells in DXM group was lower than that in asthma group significantly (P<0.01). The expression of T-bet in lung tissue in asthma group was lower than that in control group significantly (P<0.01), while GATA-3 was higher than that in control group significantly (P<0.01). The expressions of T-bet and GATA-3 in DXM group were much lower than those in asthma group (P<0.01), but the decreased degree of GATA-3 was more than that of T-bet. CONCLUSION: With pathological process of asthma, to reverse the ratio of IL-4/IFN-γ in CD4⁺ T cell by regulating T-bet and GATA-3 expression can improve the inflammatory reaction and may be one of the mechanisms of DXM in treating asthma.     

Intracellular expression of cytokines in placenta cells from NOD/SCID mice and its relationship with pregnancy outcomes

AIM: To investigate the relationship between the intracellular expression of Th1 and Th2 type cytokines in placental lymphocytes and the pregnancy outcomes in NOD/SCID mice. METHODS: The resorption rate of embryos (RR) was compared between BALB/c and NOD/SCID mice. In addition, the expression rates of Th1 type (TNF-α and IL-2) and Th2 type (IL-10) cytokines were detected intracellularlly in placental lymphocytes by four-color flow cytometry. RESULTS: Although multiple-immunodeficiency was confirmed in the NOD/SCID, no significant difference was observed in RR between BALB/c and NOD/SCID mice. At the same time, a dramatically elevated level of CD8+IL-10+/CD8+ cell percentage was found at the feto-maternal interface in NOD/SCID×NOD/SCID, as compared with BALB/c×BALB/c mice, while no significant difference was observed for TNF-α and IL-2 expression in CD4+ and CD8+ cell subsets isolated from these mice. CONCLUSION: The spontaneous elevation of CD8+IL-10+/CD8+ cell percentage at the feto-maternal interface may be related to the roughly normal fertility in NOD/SCID mice.     

Effects of inactivated HIV-1 particles on human CD4+T cell activation and Th1/Th2 cytokine secretion in whole blood

AIM: To investigate the effects of AT-2-inactivated HIV-1 particles on human CD4+T cell activation and cytokine secretion in whole blood (WB) in vitro. METHODS: HIV-1ⅢB particles were inactivated by AT-2 chemical and the concentration of p24 antigen was determined by p24 ELISA. AT-2-inactivated HIV-1ⅢB particles were added to human WB culture system in serial concentrations to stimulate the cells. PHA was used as positive control. After 24 h, all the cultural supernatants were harvested and the concentrations of Th1 (IL-2, IFN-γ and TNF-α) and Th2 (IL-4, IL-6 and IL-10) cytokines released to the supernatants were detected by cytometric bead array (CBA). The percentage of CD69 expression on CD4+T cells from WB was detected by immuno-fluorescence staining plus flow cytometry. RESULTS: The concentration of p24 antigen in the AT-2-inactivated specimen was 85.5 μg/L. 24 h later, the percentage of CD69 expression on CD4+T cells from control group was (1.62±0.63) %, whereas it was (38.82±6.00)%, (3.83±1.07)%, (5.94±0.85)% and (9.30±1.22)% in PHA group, HIV-1 (1/500) group, HIV-1 (1/50) group and HIV-1 (1/5) group, respectively. Cytokines secreted by WB in control group were mainly TNF-α and IL-6. However, all the six cytokines tested were strikingly increased in PHA group, as well as in HIV-1ⅢB groups. CONCLUSION: AT-2-inactivated HIV-1ⅢB particles activate CD4+T cells from WB, and up-regulate both Th1 and Th2 cytokine secretion in WB. Besides the effects of viral proteins, other mechanisms may be proposed that HIV-1 particles act as antigen presenting cell (APC) because many host-derived immune molecules are incorporated into HIV-1 envelop when it is released from infected cells by budding, and exert immune modulation.     

Abnormal T-cell receptor signal pathway in murine autoimmue cardiom yophy induced with adenine nucleotide translocase

AIM: To explore the molecular mechanism in the pathogenesis of dilated cardiomyopathy (DCM) by analyzing the expression of T cell signaling molecules in mice with autoimmune DCM. METHODS: Mouse DCM model was induced by immunizing the animals with adenine nucleotide translocase (ANT) synthetic peptides. P56lck in T cells was detected with real-time fluorescent quantitative PCR in both DCM-group and the sham-immunized controls. At the same time, flow cytometry was used for quantity of Th cell intracellular cytokine IFN-γ and IL-4, ELISA for examining the level of serum anti-ANT antibody, immune histochemistry for investigating the expression of CD45 in Th cells. RESULTS: The mRNA expression of P56lck (1 369.51±874.05 vs 47.93±10.21, P<0.01), the percentage of IFN-γ and IL-4 (especially IL-4) (8.27±1.29 vs 5.58±0.59, P<0.01; 9.93±1.53 vs 2.05±0.21, P<0.01), the level of anti-ANT autoantibody (0.105±0.015 vs 0.006±0.002, P<0.01) and expression of CD45 (0.154±0.021 vs 0.026±0.008, P<0.01) were all elevated significantly in DCM-group compared with the controls. CONCLUSION: Impairment of T cell receptor (TCR) signal transduction pathway plays an important role in the development of DCM induced with ANT synthetic peptides in mice.