Effect of mycophenolic acid on T lymphocyte proliferation and cytokine expression in vitro

AIM: To investigate the effect of mycophenolic acid (MPA) alone or in combination with anti B7-1 mAb on proliferation of T lymphocytes and the possible mechanisms. METHODS: The proliferation of T lymphocytes was detected by BrdU incorporation method. The expressions of IL-2, IFN-γ and IL-10 in mRNA and protein levels were detected by RT-PCR and ELISA, respectively. RESULTS: (1) MPA markedly inhibited the T lymphocyte proliferation as compared with control (P<0.01). (2) MPA significantly inhibited the levels of IL-2 and IFN-γ (42.73 ng/L±14.64 ng/L vs 99.70 ng/L±9.15 ng/L, P<0.01; 7.87 ng/L±4.22 ng/L vs 82.42 ng/L±25.55 ng/L, P<0.05), and significantly increased content of IL-10 compared with control (770.95 ng/L±126.85 ng/L vs 545.71 ng/L±22.45 ng/L, P<0.05). MPA in combination with anti B7-1 mAb obviously enhanced the content of IL-10 compared with MPA alone (941.90 ng/L±56.61 ng/L vs 770.95 ng/L±126.85 ng/L, P<0.05). (3) The expression levels of IL-2 and IFN-γ mRNA in the MPA group were obviously lower than those in control (0.74±0.10 vs 1.17±0.15, 0.52±0.05 vs 0.75±0.12, P<0.01). MPA in combination with anti-B7-1 mAb showed a statistically significant increase in IL-10 mRNA expression (1.28±0.06 vs 0.84±0.09, P<0.01) as compared with MPA alone. CONCLUSION: MPA induces the changes of cytokine expressive spectrum and the Th1 and Th2 shift might be involved in the immunosuppressive effect. The combination of MPA with anti B7-1 mAb might have a synergic effect.     

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.     

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.     

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.     

Immune function of dendritic cells in patients with hepatocellular carcinoma

AIM: To investigate the immune function of dendritic cells (DCs) in patients with hepatocellular carcinoma(HCC). METHODS: The DCs were cultured from human peripheral blood mononuclear cells (PBMC) by using GM-CSF and IL-4 for 7 days. Surface molecules CD86, HLA-DR of DCs were detected by flow cytometry. IL-12 production by DCs and IFN-γ production by T cells was measured with ELISA and ELISPOT, respectively. Allogenic mixed lymphocyte reaction was detected by MTT assay. RESULTS: CD86 expression in DCs in HCC patients were markedly lower than that in health control (91.7％ vs 83.5%, P<0.05). IL-12 production by DCs had no significant difference between the HCC patients and the health control ［(324.6±171.0)ng/L vs (436.5±142.7)ng/L, P>0.05］. However, after stimulated DCs with LPS, IL-12 production in HCC patients was significantly lower than that in health control ［(478.6±142.7)ng/L vs (630.0±151.9)ng/L, P<0.05］. IFN-γ production by T cells and T cell proliferation index (PI) in the HCC patients were all significantly lower than those in health control ［(IFN-γ: 133.4±51.2)103U/L vs (183.0±60.2)103U/L, P<0.05; PI: 2.3±0.7 vs 3.5±0.8, P<0.01］. CTL killing activity assay indicated that DC-induced CTL killing activity in HCC group was significantly lower than that in health group when the E/T ratio was 10∶1 and 20∶1, respectively. CONCLUSION: The immune function of DCs and T cells in the patients with HCC are significantly decreased. The CTL killing activity of HCC group is also markedly decreased.     

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.     

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.     

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.     

Effects of pentoxifylline on ventricular remodeling and cardiac function of dilated cardiomyopathy rats

AIM: To explore the effects of pentoxifylline (PTX) on ventricular remodeling and cardiac function in dilated cardiomyopathy (DCM) rats.METHODS: Lewis rats were randomly allocated to a myocin-induced dilated cardiomyopathy (DCM) group receiving saline (n=10), a DCM group receiving PTX (PTX group; 25 mg·kg^-1·d^-1, ip, for 30 days, n=10) or healthy control group (n=10). The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-10 in the blood plasma were analyzed by ELISA. The extent of fibrosis was estimated using Massons staining and immunohistochemistry analyses. Cardiac structure and function were measured by echocardiography.RESULTS: PTX decreased plasma levels of TNF-α and IL-6, and increased IL-10 level in DCM animals compared with DCM group ［TNF-α: (7.21±0.24) μg/L vs (19.30±1.31) μg/L, P<0.01; IL-6: (119.60±36.58) ng/L vs (189.50±13.25) ng/L, P<0.05; IL-10: (41.26±3.27) μg/L vs (32.45±4.32) μg/L, P<0.05］. Collagen volume fraction (CVF), perivascular collagen area (PVCA) and collagen Ⅰ/Ⅲ ratio were lower in PTX group than those in DCM group ［CVF: (16.45±3.01)% vs (23.33±4.43)%, P<0.05; PVCA: 4.58±2.10 vs 13.74±4.29, P<0.05; Ⅰ/Ⅲ ratio: 2.84±0.67 vs 4.22±0.54, P<0.01］. Left ventricular end-diastolic dimension reduced ［(6.11±0.51) mm vs (6.46±0.28) mm, P<0.05］ and left ventricular ejection fraction elevated ［(77.29±5.20)% vs (62.73±10.11)%, P<0.01］ by PTX compared with DCM.CONCLUSION: PTX modulates plasma levels of inflammatory cytokines, delays the ventricle remodeling and improves the heart function in DCM rats.     

Expansion capacity and cytokine releasing characteristics of TCRVα24+Vβ11+NKT cells in peripheral blood of patients with lymphoma

AIM: To analyze the quantity of TCRVα24+Vβ11+ natural killer T (NKT) cells and cytokines production induced by α-galactosylceramide (α-Galcer) in vitro in lymphoma patients. METHODS: Flow cytometry was utilized to enumerate TCRVα24+Vβ11+NKT cells in peripheral blood mononuclear cells (PBMNCs) in 30 cases of lymphoma patients and 30 cases of age- and gender-matched healthy controls. NKT cells were activated with α-Galcer and interleukin-2 (IL-2) after expansion in vitro. The percentages of positive NKT cells which expressed intracellular interleukin-4 (IL-4), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) were then determined by flow cytometry. RESULTS: The rates of NKT cells in PBMNCs were 0.17%±0.10% and 0.28%±0.18%(P<0.05) in lymphoma patients and controls, respectively. Seven days after expansion and activation with α-Galcer and IL-2, the fold of expansion of NKT cells in two groups was 101.37±44.61 and 129.66±56.31(P<0.05), respectively. The ratio of TCRVα24+NKT cells that secreted IFN-γ or TNF-α in lymphoma patients was significantly lower than that in controls (41.96%±15.06% vs 52.48%±18.85%, P<0.05; 46.30%±16.03% vs 71.37%±17.28%, P<0.05). While the ratio of TCRVα24+NKT cells that secreted IL-4 was not significantly different between the two groups (36.19%±11.74% vs 33.12%±12.95%, P>0.05). There was no significant difference in above parameters among different groups of lymphoma patients subdivided by pathology and clinical stage. CONCLUSION: The quantity of NKT cells in PBMNCs in lymphoma patients is lower than that in controls. The expansion capacity and the function of producing cytokines IFN-γ and TNF-α of NKT cells stimulated with α-Galcer are decreased. This decrease is independent of lymphoma pathology type or clinical stage.     

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.     

Mechanism responsible for pulmonary fibrosis induced by concomitant chronic smoke exposure and pentoxifylline administration

AIM: To investigate the impact of long-term administration of pentoxifylline (PTX) on morphology and inflammation of the lung in mouse models with chronic exposure of cigarette smoke. METHODS: Male BALB/c mice were randomized into the following four study groups: smoke-exposure only, shamed smoke-exposure, smoke-exposure and PTX administration, shamed smoke-exposure and PTX administration. Animals assigned to smoke-exposure were put inside a chamber twice a day for cigarette smoke exposure. The oral dose of PTX allocated to each mouse was about 20 mg·kg-1·d-1. Animals were sacrificed anaesthetically at day 120. Slices of lung were stained with H&E for pathological analysis. Modified ashcroft pulmonary fibrosis score (mAPFS) was estimated, and IFN-γ (a Th1 cytokine), IL-4 (a Th2 cytokine) in broncho-alveolar lavage fluid (BALF) and hydroxyproline in mouse lung tissue were measured by commercial kits of ELISA assay. RESULTS: Lungs in smoke-exposure only group exhibited emphysema-like morphology, low mAPFS (median 1.50, 95%CI 1.25-3.75), lowest hydroxyproline (2.43±0.11) mg/L and lowest ratio of IL-4 to IFN-γ (20.3±25.5), whereas lungs in smoke-exposure and PTX interference group exhibited interstitial fibrosis-like morphology, highest mAPFS (4.75, 4.09-5.71), highest hydroxyproline (5.57±0.55) mg/L and highest ratio of IL-4 to IFN-γ (70.7±59.9) among the four study groups (P<0.01). CONCLUSION: Interference of pulmonary inflammation induced by chronic smoke-exposure with PTX leads to the development of pulmonary fibrosis, which may relate to the turnover of Th1 polarized inflammation into Th2 polarized inflammation of the lungs.     

Protective effects and mechanism of astragalus injection on asthmatic rats

AIM: To explore the protective effects and mechanism of astragalus injection on asthmatic rats.METHODS: OVA was injected intraperitoneally and inhaled to produce the asthmatic model.Forty rats were randomly divided into five groups: control group,asthma group and astragalus groups of high,medium and low dose.The concentrations of IL-4,IFN-γ in BALF,the expression of IL-4 mRNA,IFN-γ mRNA and phospho-p38 MAPK in lung tissues were respectively measured by ELISA,RT-PCR and Western blotting.The number of inflammatory cells in BALF and histropathology changes were observed.RESULTS: In asthmatic group,the number of inflammatory cells and the concentrations of IL-4 in BALF and the expression of IL-4 mRNA,phospho-p38 MAPK in lung tissue were higher,but IFN-γ and IFN-γ mRNA were lower than those in normal control rats (P<0.01).In astragalus group,the number of inflammatory cells,the concentrations of IL-4 in BALF and the expression of IL-4 mRNA,phospho-p38 MAPK in lung tissue were lower,but IFN-γ and IFN-γ mRNA were higher than those in normal control rats (P<0.01),and histropathology damage was alleviated significantly.The efficacies in the astragalus groups of high,medium and low dose were similar,which no significant difference was observed among them.There were positive correlations between the expression of 〖JP3〗phospho-p38 MAPK and the number of eosinophil,the concentration of IL-4,IL-4 mRNA (r=0.63,r=0.69,r=0.71,〖JP〗 P<0.01),and negative correlations between the expression of phospho-p38 MAPK and IFN-γ and IFN-γ mRNA (r=-0.65,r=-0.68,P<0.01).CONCLUSION: p38 MAPK may play a role in pathological process of asthma.Astragalus effectively treats asthma by inhibiting the expression of phospho-p38 MAPK,correcting the inbalance of IFN-γ/ IL-4 and decreasing the number of inflammatory cells.     

Foam cells can be induced by oxidized low density lipoprotein in human monocyte-derived dendritic cells

AIM: The purpose of this study is to investigate the mechanisms related to oxidized low-density lipoprotein (ox-LDL) and dendritic cells (DCs) in the process of atherosclerosis.METHODS: Human DCs were prepared from human CD14+ peripheral blood monocytes using rhGM-CSF (100 μg/L) and rhIL-4 (40 μg/L).Cells were incubated with 100 mg/L native or oxidized LDL for 72 h.The formation of foam cells was investigated by electron microscopy and oil red O staining.Phenotypic and immune functional assays were used with FACS, FITC-dextran phagocytosis, allogeneic mixed T lymphocytes reaction and secretion of Th1/Th2 (IL-12/IL-2) cytokines were also conduced.RESULTS: DCs treated with ox-LDL, but not native LDL were induced into foam cells after cultured for 72 h.Compared with native LDL, ox-LDL-treated DCs were less potent in FITC-dextran phagocytosis.ox-LDL promoted allogeneic T cells proliferation.Moreover, ox-LDL upregulated CD80 (72.4± 9.6 vs 89.5±10.1, P<0.01), CD86 (67.2±8.8 vs 80.2±11.6, P<0.01), HLA-DR (80.6±9.8 vs 86.6±10.8, P<0.01) and CD1a (40.2±10.3 vs 60.2±9.3, P<0.01) expressions, increased IL-12 secretion ［(44.3±8.9)ng/L vs (65.1±10.4)ng/L, P<0.05］ in DCs.However, the secretion of IL-2 was decreased ［(43.6±7.8)ng/L vs (10.0±4.5 )ng/L, P<0.01］ significently.CONCLUSION: DCs were induced into foam cells by ingesting ox-LDL with some functional characteristic of mature DC.DCs seem to be a new source of foam cells and play a key role in immunopathogenesis of atherosclerosis.     

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.     

Association of IFN-γ and IL-4 gene polymorphisms with childhood bronchial asthmatic susceptibility and the levels of plasma IFN-γ,IL-4 and IgE

AIM: To investigate the gene polymorphisms of interferon-γ(IFN-γ) and interleukin-4(IL-4) and the association with asthmatic susceptibility and the levels of plasma IFN-γ, IL-4 and IgE of asthmatic children. METHODS: 100 asthmatic children and 122 control children were enrolled the study. The genotypes of IFN-γ gene-179G/T polymorphism, IL-4 gene-33C/T and-589C/T polymorphisms were tested by PCR-RFLP.The genotype of IFN-γ gene +874A/T polymorphism was tested by AS-PCR.The CA repeat polymorphism of IFN-γ gene was detected by capillary electrophoresis technique.The levels of serum IFN-γ, IL-4 and IgE were measured by ELISA. RESULTS: 100 asthmatic children and 122 control children were all GG homozygotes at -179 locus of IFN-γ gene.-179 locus of IFN-γ gene has no mutation. The genotypes and allele frequency of IFN-γ gene +874A/T and CA repeat polymorphisms showed no significant difference between asthmatic children and the control(P>0.05). An association was revealed between IFN-γ gene +874A/T polymorphism and the level of plasma IFN-γ.The level of IFN-γ was lower in AA genotype than in AT genotype(P<0.05). The genotypes and allele frequency of IL-4 gene -33C/T and -589C/T polymorphisms showed significant difference between asthmatic children and the control(P<0.05).The levels of plasma IL-4 and IgE were higher in TT genotype at -33 locus and -589 locus than those in CT genotype, but only -33C/T polymorphism was associated with the level of plasma IL-4(P<0.05). CONCLUSION: The IFN-γ gene +874A/T and CA repeat polymorphisms were not correlated with asthmatic susceptibility, but there is significant correlation between the level of IFN-γ and +874A/T polymorphism. TT genotype of IL-4 gene -33 locus and -589 locus maybe the susceptible genotype of asthma in children, and the -33 locus polymorphism is associated with the level of IL-4.     

Effect of injecting immature dendritic cell intratumorally after microwave ablation under different temperature on the activity of cytotoxic T lymphocytes

AIM: To investigate whether the injection of immature dendritic cell (iDC) after ablation induce and upregulate tumor specific cytotoxic T lymphocytes (CTL).METHODS: The model of hepatoma was established and the tumors were ablated with microwave under (45±2) ℃, (50±2) ℃, (55±2) ℃, (60±2) ℃ and void ablation, after which the bone marrow derived iDC was injected intratumorally. The activity of CTL was detected, and the levels of IFN-γ and IL-4 secreted by activated spleic lymphocytes were measured.RESULTS: Compared with iDC injection intratumorally after the ablation of (45±2) ℃, (55±2) ℃, (60±2) ℃ and void ablation, the cytotoxic effect of CTL towards Hepa l-6 was heightened by injecting iDC intratumorally 72 hours after ablation of (50±2) ℃ (P<0.05), so was the secretion of IFN-γ (P<0.05). The level of IL-4 decreased after ablation of (50±2) ℃ subsequently with injecting iDC intratumorally (P<0.05). The cytotoxic effect of CTL towards Hepa l-6 was higher than that towards B16 lymphadenoma cell after iDC injection intratumorally and ablation of (50±2) ℃ and (55±2) ℃ (P<0.05).CONCLUSION: Thermal ablation of (50-55) ℃ and subsequent injection of iDC intratumorally may induce tumor specific CTL by the way of promoting the antigenicity of ablated tumor tissue and augmenting the presentation of TAA to effector cells.     

Expression of Th cytokines in peripheral blood before and after splenectomy in immune thrombocytopenia patients

AIM: To investigate the changes of Th cytokines before and after splenectomy in immune thrombocytopenia (ITP) patients. METHODS: The QuantiGene Plex method was used to measure the mRNA expression of Th1, Th2 (IL-4, IL-5, IL-6 and IL-10), Th3 (transforming growth factor β₁,TGF-β₁), and Th17 (IL-17) cytokines in peripheral blood of ITP patients before and after laparoscopic splenectomy and those in peripheral blood of healthy controls. RESULTS: The mRNA level of IL-2 was significantly decreased in ITP patients before operation compared with the healthy controls, whereas IL-17 was obviously over-expressed. No significant difference of the other cytokines between preoperative group and the normal controls was found. After splenectomy, the expression levels of both IL-2 and TGF-β₁ were significantly higher than those in preoperative group and the normal controls. IL-2 was also significantly increased after operation, but was still lower than that in the normal controls. No significant difference of other cytokines between postoperative group and healthy controls was observed. In addition, The Th1 cytokines (IL-2 and IFN-γ) were found to be positively correlated (r=0.647, P<0.01) in preoperative patients, while no correlation was found between the other cytokines. There was a positive correlation between IL-2 and IFN-γ (r=0.787, P<0.01) in postoperative patients. IL-17 also had positive correlations with IL-2 (r=0.554,P<0.01) and IFN-γ (r=0.461,P<0.05) in ITP patients after operation, respectively. CONCLUSION: There is an imbalance of Th cytokines in ITP patients. The mechanism of splenectomy for treating ITP may be associated with the balance regulation of Th cytokines.     

Expression and significance of thrombospondin-1 in myocardium of type 2 diabetic cardiomyopathy rats

AIM: To investigate the expression and significance of thrombospondin-1 (TSP-1) in left ventricular myocardium of type 2 diabetic cardiomyopathy (DCM).METHODS: The rat model of DCM was established by eating a high-fat diet together with injection of low dose streptozocin (30 mg/kg) intrapertoneally.After 12 weeks,the content of collagen was quantified by Masson staining.The mRNA level of TSP-1 was determined by quantification real-time RT-PCR,while the protein level of TSP-1 was analyzed by Western blotting and immunohistochemistry.RESULTS: Compared with the control group,the content of collagen in the DCM group was increased greatly (11.01±3.05 vs 16.92±3.18,P<0.01).The mRNA and protein expressions of TSP-1 were significantly higher than those in control group (0.0089±0.0034 vs 0.0141±0.0037,P<0.05；96.38±16.80 vs 129.98±16.96,P<0.05).In DCM group,the mRNA and protein expressions of TSP-1 showed significantly positive correlations with the levels of fasting blood glucose and collagen (r=0.762,P<0.01; r=0.717,P<0.05; r=0.735,P<0.01; r=0.750,P<0.01).There was a significantly positive correlation of TSP-1 mRNA level with LVEDP (r=0.658,P<0.05).In contrast,there was a significantly negative correlation of TSP-1 protein with LVSP and -dp/dtmax (r=-0.605,P<0.05; r=-0.694,P<0.05).There was a significantly positive correlation of TSP-1 protein with LVEDP (r=0.716,P<0.05).There was a significantly negative correlation of TSP-1 protein with LVSP and -dp/dtmax (r=-0.633,P<0.05; r=-0.669,P<0.05).CONCLUSION: The increased expression of TSP-1 may play an important role in the development of myocardial interstitial fibrosis in DCM.