Effects of glucocorticoid on miRNA-155 expression in CD4+T cells of asthmatic mice

AIM:To investigate the effects of glucocorticoid on the regulation of microRNA-155 (miRNA-155) expression in the CD4⁺ T cells of asthmatic mice. METHODS:The ovalbumin (OVA)-induced asthma mouse model was established and the mice were treated with glucocorticoid. The effects of glucocorticoid on the pulmpnary histopathological changes, the expression of miRNA-155 in the lung tissues and CD4⁺T cells, and the levels of cytokines in the bronchoal-veolar lavage fluid (BALF) were evaluated. RESULTS:The results of RT-qPCR showed that the expressions of miRNA-155 in the lung tissues and CD4⁺T cells from the spleen of asthmatic mice were significantly increased, and the level of miRNA-155 in the CD4⁺T cells was significantly increased with the increase in the allergen exposure time (P<0.01). HE and PAS staining showed that OVA significantly increased inflammatory cell infiltration as compared with control group, and the peribronchial and perivascular inflammation and mucus secretion of proliferative goblet cells were significantly reduced after glucocorticoid treatment. Glucocorticoid treatment inhibited the increase in the proportion of CD4⁺ CD8^- cells in the spleen and decreased the accumulation of CD4⁺ T cells in the lung tissues of asthmatic mice (P<0.01). After glucocorticoid treatment, the levels of interleukin-4 (IL-4), IL-5 and IL-13 in BALF were decreased, while the level of interferon-γ was increased significantly (P<0.01). CONCLUSION:Glucocorticoid reduces the accumulation of CD4⁺ T cells and inhibits the expression of miRNA-155 in the lung tissues and spleen CD4⁺ T cells of asthmatic mice.     

Analysis of activation of T cells from draining lymph node and peripheral blood in allotransplantation mice

AIM:To study the activation of T cells from local lymph node and peripheral blood early after allotransplantation.METHODS:Transplant of myocardio-tissue into mouse forearm subcutaneously was used as a model to analyze the expression of CD69 by T subpopulations from draining lymph node and peripheral blood by flow cytometry.RESULTS:The expression rates of CD69 by both CD4⁺T cells and CD8⁺T cells from the draining lymph node were raised (P＜0.01) 72 h after allotransplantation, and it was higher on CD8⁺T cells than on CD4⁺T cells (P＜0.01). No significant difference in CD69 expression was found on CD4⁺T and CD8⁺T cells from peripheral blood among the groups, topical complete Freund's adjuvant (CFA) and systemic cyclosporin(CsA) enhanced and inhibited expression of CD69 by both CD4⁺T cells and CD8⁺T cells after allotransplantation, respectively (P＜0.05 or P＜0.01).CONCLUSION:To detect the expression of CD69 by T cells from draining lymph node can keep insight to the allorecognition early after transplantation.     

Study on polarized Th1/Th2 cell in vitro from adult human peripheral blood

AIM: To investigate the details of CD4⁺ T cell polarized to Th1/Th2 in vitro. METHODS: After isolated the PBMCs and blood-plasma from adult human peripheral blood by Ficoll-Hypaque centrifugation, the PBMC culture procedure with or without the self-blood -plasma was applied to polarize T cells in vitro, these cells were polarized by PHA(20 mg/L),non-PHA respectively. The polarized rates of Th cell after 24 h,48 h,72 h were estimated respectively by flow cytometry following two-color immunofluorescent staining. RESULTS: CD4⁺T cell would polarize to Th1/Th2 two subsets after self-cytokines and PHA activation in vitro. The polarized rates of T cell after cultured for 24 h,48 h and 72 h were (13.28%±1.59%)/(12.70%±1.65%),(17.19%±1.03%)/(17.50%±1.30%),(19.49%±2.87%)/(18.58%±1.49%) respectively, but the polarized rates of T cell were very low if without self-blood-plasma. The difference between them was significant. The ratios of Th1/Th2 cells were about 1. CONCLUSION: CD4⁺T cell from adult human peripheral blood would polarize to Th1/Th2 two subsets in the presence of self-blood-plasma and PHA(20 mg/L) in vitro, and the cell number of Th1 and Th2 would be in balance.     

Effect of pachyman polysaccharides on Th17/Treg balance in systemic lupus erythematosus patients

AIM: To investigate the immunomodulatory effect of pachyman polysaccharides (PPS) on T helper 17 cell (Th17)/regulatory T cell (Treg) balance in the peripheral blood of systemic lupus erythematosus (SLE) patients. METHODS: The CD4⁺ T cells were isolated from the peripheral blood samples obtained from 45 SLE patients and 35 healthy controls enrolled in our study using magnetic bead separation method. The proportions of Th17 and Treg cells were measured by flow cytometry. The CD4⁺ T cells from SLE patients and healthy controls were treated with PPS. The cytoto-xicity of PPS was evaluated by detecting cell viability with MTT assay. The contents of interleukin-17 (IL-17), IL-6, IL-10 and transforming growth factor-β (TGF-β) were measured by ELISA. The expression of retinoid-related orphan receptor γt (RORγt) and forkhead box protein P3 (Foxp3) at mRNA and protein levels was determined by RT-qPCR and Western blot, respectively. RESULTS: The Th17 cells were significantly elevated, while Treg cells were obviously decreased in the SLE patients compared with the healthy control group (P<0.05). Compare with control group, the contents of IL-17 and IL-6 were decreased, while the contents of IL-10 and TGF-β were increased (P<0.05). The expression of RORγt at mRNA and protein levels was down-regulated and the expression of Foxp3 was up-regulated (P<0.05). The ratio of Th17/Treg was decreased in 100 μg/L nontoxic PPS-treated CD4⁺ T cells isolated from the SLE patients (P<0.05). CONCLUSION: PPS treatment inhibits Th17 cells and elevates Treg cells in the CD4⁺ T cells isolated from SLE patients, which may have a therapeutic effect on SLE patients.     

The characteristics of TCRVα24+ NKT cells in response to in vitro stimulation

AIM: To investigate the amount and patterns of expressing CD69, IL-4 and IFN-γ on TCRVα24⁺ NKT cells, and compare with that of CD3⁺ T cells from human peripheral blood in response to in vitro stimulation. METHODS: The whole blood was stained with three-color immunofluorescence directly or after cultured with PDB+ionomycin (Ion) for 6 h, then the mononuclear cells were separated by lysing red blood cells. The expression rates of CD69, IL-4 and IFN-γ on TCRVα24⁺ NKT cells and CD3⁺ T cells were estimated by flow cytometer. RESULTS: As a proportion of mature T cells, the ratio of TCRVα24⁺ NKT cells to CD3⁺ T cells was about (1.34±0.42)%. The expression rates of CD69 on TCRVα24⁺ NKT cells and CD3⁺T cells in response to PDB+Ion for 6 h were (96.71±1.33)% and (98.60±0.47)%, respectively, while the ratio were (11.47±2.86)% and (1.07±0.45)% in the unstimulated group, and there were significant difference between them. The expression rates of IL-4 and IFN-γ on TCRVα24⁺ NKT cells stimulated with PDB+Ion for 6 h were (48.62±2.44)% and (46.65±8.91)%, respectively, which were significantly higher than that of unstimulated group [(31.57±3.31)%, (13.45±6.29)%] and that of stimulated CD3⁺ T cells, though the expression rates on stimulated CD3⁺ T cells were significantly higher than that of unstimulated CD3⁺ T cells. CONCLUSION: There is small amount of NKT cells in adult human peripheral blood. The expression rates of IFN-γ and IL-4 on these lymphocytes are higher than CD3⁺ T cells, suggesting that NKT cells are important immunomodulatory cells in special microvironments.     

Adipose-derived stem cells mediate immunosuppression of Th17 in multiple sclerosis

AIM: To investigate how human adipose-derived stem cells (hASCs) regulates the differentiation of Th17 cells in multiple sclerosis. METHODS: hASCs were isolated from the adipose tissues. Magnetic-activated cell sorting (MACS) kit was used to isolate CD4⁺ T cells from peripheral blood mononuclear cells (PBMCs) which were isolated by density gradient centrifugation. The percentage of CD4⁺ T cells was detected by flow cytometry. The activated CD4⁺ T cells were co-cultured with hASCs for about 4 d at different ratios of hASCs to CD4⁺ T cells (1:4 and 1:10) in a Th17 polarised condition. Another group adding anti-leukemia inhibitory factor (LIF) antibody was set up. Th17 cell proportion of the CD4⁺ T cells was determined by flow cytometry. The level of LIF in the supernatant of co-cultured system was measured by ELISA. The mRNA expression of retinoid-related orphan receptor γt (RORγt), interleukin-6 receptor (IL-6R), interleukin-23 receptor (IL-23R), LIF and leukemia inhibitory factor receptor (LIFR) was detected by real-time PCR. RESULTS: The result of flow cytometry suggested there were mainly hASCs, and the percentage of CD4⁺ T cells in the PBMCs were above 90% after MACS. The Th17 cell proportion decreased in 1:4 and 1:10 co-cultured groups in a dose-dependent manner. The mRNA expression of IL-6R, IL-23R and RORγt was downregulated and the expression of LIFR and LIF was up-regulated. When the anti-LIF was added into the co-cultured system, the ratio of Th17 cells increased and reached to the control level. The protein level of LIF obviously increased after co-cultured. After anti-LIF added, the mRNA expression of RORγt and IL-6R was up-regulated. CONCLUSION: hASCs inhibits the differentiation of Th17 cells from multiple sclerosis patients through the competitive inhibition of LIF/IL-6 by secreting LIF.     

Changes of CD28, CD56 and CD57 expression on CD8+T cells in the peripheral blood of healthy elderly individuals

AIM:The expression of CD28, CD56 and CD57 on CD8⁺T cells in the peripheral blood of young (age range 20-35) and elderly(age range 60-75) healthy donors were compared to explore the change of the cellular immune function with aging.METHODS:Three-color fluorescent flow cytometry was performed to analyze the differences in percentage of CD8⁺CD28⁺, CD8⁺CD56⁺ and CD8⁺CD57⁺T cells in the peripheral blood between the young and elderly groups.RESULTS:CD8⁺CD28⁺T cells in the peripheral blood of the elderly group was significantly lower than those in the young group, with percentage of 34.07±5.28 and 49.84±7.43,respectively (P＜0.05). Conversely, CD8⁺CD56⁺T cells and CD8⁺CD57⁺T cells in the peripheral blood of the elderly group were significantly higher than those in the young group, and the percentage was 6.60±2.40 vs 2.10±0.35,41.82±6.01 vs 22.89±2.80, respectively(P＜0.05).CONCLUSION:The expression of CD28, CD56 and CD57 on the CD8⁺T cells in the peripheral blood are changed significantly with aging. The decrease in CD28 expression may play an important role in the immunosenescence, while the increase in CD56 and CD57 expression seems to be a compensatory adaptation for the immune dysfunction.     

Effect of adoptive transfer of CD4+ T cells with miR-7 knockdown on acute liver injury in mice

AIM:To study the effect of adoptive transfer of CD4⁺ T cells with microRNA-7 (miR-7) knockdown (KD) on mouse acute liver injury model and to investigate its significance. METHODS:CD4⁺ CD62L⁺ T cells were purified from the spleen of normal wild-type (WT) mice and miR-7KD mice by magnetic bead sorting, and were stained with CFSE. These 2×10⁶ CFSE-labeling cells were injected into normal mice via tail vein, and then the mouse acute liver injury model was induced by intraperitoneal injection of 30 mg/kg concanavalin A. After 72 h, the appearance, weight and weight index of the liver were investigated. The pathological change of the liver tissues was observed by HE staining. Real-time PCR was used to examine the mRNA expression of Bax and P53. The expression levels of CD62L, interleukin-4 (IL-4) and interferon-γ (IFN-γ) in the CD4⁺ T cells were analyzed by flow cytometry. RESULTS:We found that the liver tissue became lighter, and the weight (P<0.01) and weight index (P<0.05) were changed significantly in miR-7KD mice compared with control group. Moreover, HE staining showed that the liver cell damage was increased in the liver of miR-7KD mice. Meanwhile, the expression levels of Bax and P53 were significantly increased in miR-7KD group (P<0.05). The percentage of CD62L in CD4⁺ T cells was significantly decreased (P<0.01) in miR-7KD mice, with high expression of IFN-γ (P<0.05) and low expression of IL-4 (P<0.01) in CD4⁺T cells. CONCLUSION:These findings suggest that miR-7 knockdown significantly promotes the pathology of CD4⁺ T cell-mediated acute liver injury, which provides a preliminary experimental basis for further exploration on the mechanism of acute liver injury occurrence.     

Effects of magnesium on apoptosis and expression of Foxp3 in peripheral CD4+CD25+ regulatory T cells from asthma patients

AIM:To observe the apoptosis and the expression of forkhead box protein 3(Foxp3) induced by magnesium in CD4⁺CD25⁺ regulatory T cells isolated from healthy and asthmatic human peripheral blood. METHODS:Peripheral blood from healthy volunteers and asthma patients was collected. CD4⁺CD25⁺ T cells were separated by Percoll centrifugation and magnetic separation. The cells were cultured for 72 h and treated with magnesium(10 mmol/L) or control solution. The apoptotic rate and the expression of Foxp3 in the cells were analyzed by flow cytometry. RESULTS:The purity of CD4⁺CD25⁺T cells was 77.4%~92.3% in health group, and was 75.2％~93.8％in asthma group. The proportion of CD4⁺CD25⁺ T cells in CD4⁺T cells was 4.12%~7.98% in healthy adults, and 4.51%~8.68% in asthma patients. No significant difference between the 2 groups was observed. Magnesium at concentration of 10 mmol/L up-regulated the apoptotic rate of CD4⁺CD25⁺ T cells(P<0.05) and did not affect the Foxp3 expression in the cells in both health and asthma groups. CONCLUSION:Magnesium plays therapeutic effects on asthma by inducing the apoptosis of peripheral CD4⁺CD25⁺ regulatory T cells.     

IL-1β promotes differentiation of naïve T cells into Th22 cells and its expression in non-small cell lung cancer

AIM:To investigate the mechanism of interleukin-1β (IL-1β) promoting the transformation of naïve T cells into Th22 cells and the correlation of its peripheral blood expression in non-small cell lung cancer patients. METHODS:CD4⁺ naïve T cell magnetic bead sorting kit was used to isolate the peripheral blood mononuclear T cells from healthy people. Transforming growth factor-β (TGF-β) and IL-2 were added to promote differentiation and proliferation. IL-1β was used to induce differentiation into Th22 cells. The proportion of CD4⁺ IL-22⁺ T cells was analyzed by flow cytometry, and the expression of IL-22 was detected by ELISA. We selected 60 cases of non-small cell lung cancer patients in our hospital, including 18 in I phase, 20 in Ⅱ phase, 13 in Ⅲ phase and 9 in IV phase, as well as 25 healthy persons. The proportion of Th22 (CD4⁺ IL-22⁺) cells in peripheral blood was detected by flow cytometry, and the serum levels of IL-1β and IL-22 were measured by ELISA. RESULTS:IL-1β induced the transformation of naïve T cells into Th22 cells and promoted the secretion of IL-22 (P<0.05). The proportion of Th22 cells and the IL-22 and IL-1β levels in peripheral blood of the patients with non-small cell lung cancer were higher than those in healthy subjects, and correlated with the clinical stage. CONCLUSION:IL-1β induces the differentiation of Th22 cells and the expression of IL-22. The levels of IL-1β and IL-22 are related to the progression of non-small cell lung cancer, which may be involved in immunosuppression and promote the occurrence of non-small cell lung cancer.     

Elevation of blood Th17 cells and CD3+CD8-IL-21+ T cells in patients with cervical cancer

AIM: To investigate the role of Th17 cells in the patients with cervical cancer.METHODS: We measured the peripheral levels of Th17 cells and CD3⁺CD8^-IL-21⁺ T cells in 37 cervical cancer (CC) patients, 25 cervical intraepithelial neoplasia (CIN) patients and 18 healthy controls by flow cytometry. The percentages of Th17 cells and CD3⁺CD8^-IL-21⁺ T cells in total CD4⁺ cells were calculated.RESULTS: Compared with controls, the patients with CC or CIN had higher proportions of Th17 cells (all P<0.01) and CD3⁺CD8^-IL-21⁺ T cells (all P<0.05). Notably, in CC patients, the increased percentages of Th17 cells and CD3⁺CD8^-IL-21⁺ T cells were independently associated with the clinical stage(all P<0.05), lymph node metastasis (P<0.01,P<0.05) and vasoinvasion (all P<0.01), while the elevated percentage of CD3⁺CD8^-IL-21⁺ T cells was also associated with the tumor size(P<0.01). Furthermore, the percentage of Th17 cells was positively correlated with that of CD3⁺CD8^-IL-21⁺ T cells in healthy controls and CC patients, but not in CIN patients.CONCLUSION: Our results indicates a possible role of Th17 cells in CC patients correlated with CD3⁺CD8^-IL-21⁺T cells, and the elevated percentage of circulating Th17 cells may be involved in the development and progression of cervical cancer.     

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.     

TL1A promotes development of intestinal fibrosis associated with chron?ic experimental colitis by regulating IL-17 and IFN-γ

AIM To explore how tumor recrosis factor ligand-related molecule 1A (TL1A) promotes the development of intestinal fibrosis associated with chronic experimental colitis by regulating interleukin-17 (IL-17) and interferon-γ (IFN-γ). METHODS Aexperimental colitis-associated wild-type (WT) and TL1A (L-Tg) transgenic intestinal fibrosis model was established by dextran sulfate sodium (DSS) induction.The severity of colitis was evaluated by detecting the disease activity index (DAI). HE staining was used to observe the histopathological changes and pathological score of the colitis.Myeloperoxidase (MPO) was measured in each group. The collagen deposition was detected by Masson’s trichrome staining and Sirius red staining. The lamina propria, spleen and mesenteric lymph nodes(MLN) mononuclear cells were isolated and counted, and the levels of IL-17 and IFN-γ weremeasured by ELISA, andthe percentages of CD4⁺IFN-γ⁺T cells and CD4⁺IL-17⁺T cells were analyzed by flow cytometry. RESULTS After drinking DSS water,the body weight of the mice in DSS/Tg group was decreased significantly as compared with WT group(P<0.05). The DAI score, histology score and MPO activity were significantly increased(P<0.05). Thelevels of IL-17 and IFN-γ, LPMC, spleen and MLN were significantly increased. The percentages of CD4⁺IFN-γ⁺T cells and CD4⁺IL-17⁺T cells were significantlyincreased.The thickness and collagen deposition of the colon were increased inTg group. CONCLUSION TL1A promotes the development of intestinal fibrosis associated with chronic experimental colitis by regulating IL-17 and IFN-γ.     

Dynamic changes of mouse cardiac functions in early stage of sepsis induced by CLP

AIM: To detect the changes of cardiac functions of septic mice in the early stage of sepsis. METHODS: Health male Kunming mice were used in the study. The techniques of 2D, M-mode and Doppler echocardiography were applied to evaluate the cardiac functions before cecal ligation and puncture(CLP) as baseline and at time points of 12 h, 24 h, 36 h, 48 h and 168 h after CLP. The mice survived for 168 h(7 d) were considered as survivals. RESULTS: Compared to the baseline at the time point of 24 h after CLP, the blood volumes of heart return decreased significantly in the early stage of sepsis induced by CLP. LVEDV reduced by 46%. Notable compensatory responses of the hearts in septic mice were observed, especially the systolic functions, in which LVEF and LVFS increased by 27% and 39%, respectively. However, the compensatory responses of diastolic function were weaker than the systoles. E/A ratio and EDT decreased by 30% and 25% respectively at the time point of 24 h. CONCLUSION: The strong compensatory cardiac functions are one of the factors for supporting the septic animal to survive. Protection of the cardiac functions especially the diastoles is important in the treatment of septic patients.     

Balance of Treg/Th17 in synovium of collagen-induced arthritis rats and impact of TNF-α blockage therapy

AIM: To investigate the balance of Treg/Th17 in synovium of collagen-induced arthritis (CIA) and the impact of tumor necrosis factor α(TNF-α) blockage therapy. METHODS: Rat CIA model was established by bovine II collagen injection. The pathological score was evaluated by HE staining and toluidine blue staining. The TNF-α level in plasma was measured by ELISA. The expression of Treg/Th17 in synovium was detected by double staining immunofluorescence. RESULTS: The plasma level of TNF-α in CIA group was significantly higher than that in control group and TNFR-Fc treatment group (P<0.01), whereas no significant difference was found between TNFR-Fc treatment group and control group (P>0.05). No significant difference between CIA group and control group in the ratio of CD4⁺Foxp3⁺Treg cells/CD4⁺ cells in synovium (23.12%±4.93% vs 24.66%±5.82%, P>0.05) was observed, whereas the ratio in TNFR-Fc treatment group was significantly increased(33.07%±5.14%). The ratio of CD4⁺RORγt⁺Th17 cells/CD4⁺ cells in CIA group was significantly higher than that in control group and TNFR-Fc treatment group (9.74%±2.23% vs 1.00%±0.59%, 5.63%±1.76%, P<0.01). CONCLUSION: Differentiation disturbance of Treg/Th17 exists in the synovium of CIA rats. TNFR-Fc may restore the balance of Treg/Th17 by inhibiting Th17 cell differentiation and inducing the production or accumulation of Treg.     

Effect of heterogenic corneal stroma transplantation on peripheral T cells of rats

AIM: To Compare immunogenicity of three kinds of heterogenic corneal stroma. METHODS: 36 SD rats were randomized into 4 groups, each group consisting of 9 rats. Group 1 was control group. Three kinds of heterogenic corneal stroma: porcine, rabbit and chicken corneal stroma were heterotopically transplanted to subcutaneous layer of 27 (group 2-4) SD rats, respectively. The expression of CD4⁺, CD8⁺, CD25⁺, CD71⁺ on peripheral T cells was identified and analyzed by dual fluorescence flow cytometry at 7, 14, 28 days after operation. RESULTS: Compared with control group, the expression of CD4⁺, CD8⁺, CD25⁺, CD71⁺ was no significant change in porcine corneal stroma group(P＞0.05), the expression of CD4⁺ was increased in rabbit corneal stroma (P＜0.05), CD4⁺, CD4⁺ CD71⁺ markedly higher in the chicken corneal stroma (P＜0.01) at 7 days after operation. CONCLUSION: The immunogenicity of porcine stroma is the lowest in three kinds of heterogenic corneal stroma (chicken, rabbit and porcine).     

Calreticulin expression is necessary for protective immune effect of T-cell vaccine in EAE mice

AIM:To study the role of cell membrane ectopic calreticulin (CALR) expression on the protective immunie effect of T-cell vaccine (TCV) on experimental autoimmune encephalomyelitis (EAE). METHODS:EAE model was established by myelin oligodendrocyte glycoprotein 35-55 (MOG_35-55) immunization in C57BL/6 mice, and the mice were immunized with MOG_35-55-specific CALR⁺ and CALR^- T-lymphocytes. Symptomatic scores were compared at the maximum of the disease. On the 15th day after immunization, the proportion of CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells (Treg) in the spleen, and the expression of interferon-γ (IFN-γ), interleukin-4 (IL-4), IL-10 and IL-17A in the serum were measured. RESULTS:Increased expression of CALR in activated T cells after γ-irradiation was observed. Blockade of CALR on the vaccinating T-cell surface reduced the protective effect of TCV. Furthermore, blockade of CALR reduced the number of Treg in the spleen and up-regulated pro-inflammatory cytokines. CONCLUSION:CALR expression in the T cells is necessary for the protective immunity induced by TCV in EAE mice.     

Changes of immune function and mortality in patients with sepsis-induced stress hyperglycemia

AIM: To investigate the role of stress hyperglycemia on condition assessment and predicting prognosis in patients with sepsis. METHODS: The study included 44 patients with sepsis, divided into three groups according to their blood glucose profile within 24 h after admission: patients with stress hyperglycemia (group SH, n=15), diabetes mellitus type 2 (group DM, n=10), and normal glucose levels (group NG, n=19). CD4+/CD8+ ratio, Th1/Th2 ratio and HLA-DR% of the patients were measured within 24 h after admission by flow cytometry as assessment of their immune function. The sepsis-related organ failure assessment (SOFA) scores and acute physiology and chronic health evaluation II (APACHE II) scores of patients were recorded at the same time to analyze whether stress hyperglycemia affects the immune function and the 28 d mortality in patients with sepsis. RESULTS: A higher mortality rate of septic patients with stress hyperglycemia was observed compared to diabetic patients (53.3% vs 10.0%) and group NG (53.3% vs 21.1%). SOFA score and APACHE II score were higher in group SH than those in group DM and group NG while lower levels of human leucocyte antigen DR (HLA-DR) expression and CD4+/CD8+ ratio was found in group SH than those in group DM and group NG. No difference in the levels of Th1/Th2 among the three groups was observed. Non-survivors had higher levels of SOFA score, APACHEⅡ score, Th1/Th2 ratio and HLA-DR%. No difference was detected for CD4+/CD8+ ratio, mean glucose values and age. CONCLUSION: Stress hyperglycemia is associated with decreased immune function and an adverse clinical outcome in patients with sepsis.     

Association between development of CD4+CD25+ regulatory T cells and thymus CD4-CD25+ cells

AIM: To explore the correlation between development of CD4⁺CD25⁺ regulatory T cells (CD4⁺CD25⁺ Tr) and thymus CD4^-CD25⁺ cells. METHODS: The ratios of CD4⁺CD25⁺ regulatory T cells to CD4+ T cells in thymus, spleen, lymph node and peripheral blood of mice from birth to mature and also the ratios of CD4^-CD25⁺ cells to CD4^- T cells in thymus were measured by flow cytometry. Purified CD4⁺CD25⁺ T cells and CD4⁺CD25^- T cells were labeled with CFDA-SE, and then stimulated with various kinds of stimulators. RESULTS: The percentages of CD4⁺CD25⁺ Tr in mouse spleen, lymph nodes and peripheral blood increased gradually, but not in thymus, from day one to week 10 of the age with rapid rising from day one to week 1. The percentages of CD4^-CD25⁺ cells in mouse thymus were quite high on day one after birth, and decreased rapidly from day one to week 1. Both CD4⁺CD25⁺ Tr and CD4⁺CD25^- T cells showed no proliferation in response to ConA, while CD4⁺CD25⁺ Tr showed a transient enlargement of cell size. Both CD4⁺CD25⁺ Tr and CD4⁺CD25^- T cells underwent proliferation in response to PDB plus ionomycin. CD4⁺CD25^- T cells, but not CD4⁺CD25⁺ Tr, showed a proliferative response to the stimulation of coated anti-CD3 plus soluble anti-CD28 antibody, however, CD4⁺CD25⁺ Tr showed significant proliferation and CD4⁺CD25^- T cells showed a stronger response in addition of high dose of IL-2. CONCLUSION: The thymus CD4^-CD25⁺ cells are probably the precursor of CD4⁺CD25⁺ Tr during cell development.     

Effects of berberine and yohimbine on splenocyte apoptosis in septic mice

AIM: To observe the effects of berberine and yohimbine on splenocyte apoptosis in septic mice and underlying mechanisms. METHODS: The mice were subjected to cecal ligature and puncture (CLP). The drugs or vehicle were given intragastrically 2 h after the surgery according to the following 5 groups: sham, CLP, CLP+berberine, CLP+yohimbine, and CLP+berberine+yohimbine. The apoptosis of splenocytes stained by TUNEL was observed under laser scanning confocal microscope 20 h after CLP. The splenic lymphocytes were isolated and observed using flow cytometry. The activities of caspase-3, caspase-8 and caspase-9 in splenic lymphocytes were detected, and the expression of Fas, Bim, Bcl-2 and Bax in the splenocytes was also determined by Western blotting. RESULTS: The TUNEL staining showed that the apoptotic rate of the splenocytes in septic mice 20 h after CLP was significantly higher than that in sham and CLP+yohimbine groups (P＜0.05). Compared with CLP group, the proportion of apoptotic cells was decreased in septic mice in CLP+berberine+yohimbine and CLP+yohimbine groups (P＜0.05). Flow cytometry analysis demonstrated the similar results in the apoptosis of splenocytes and T lymphocytes. However, only yohimbine treatment reduced the apoptosis of B lymphocytes in the spleen of sepsis-challenged mice. Compared with CLP group, caspase-9 activity was significantly reduced in CLP+berberine group (P＜0.05), the activities of caspase-3, caspase-8 and caspase-9 were all statistically reduced (P＜0.05) in CLP+yohimbine group and CLP+yohimbine+berberine group. CLP significantly increased the expression of cytosolic Fas, Bim and mitochondrial Bax in the splenocytes, and decreased Bcl-2 expression compared with sham group. Compared with CLP group, the expression of cytosolic Bim and mitochondrial Bax in CLP+berberine group were reduced (P＜0.05). Fas expression decreased only in CLP+yohimbine group (P＜0.05). Berberine combined with yohimbine reduced the expression of cytosolic Fas, Bim and mitochondrial Bax in the septic mouse splenocytes (P＜0.05).CONCLUSION: Yohimbine reduces sepsis-induced splenic lymphocyte apoptosis in mice by inhibiting Fas expression and in turn blocking both extrinsic and intrinsic apoptosis pathways. Berberine reduces Bim expression and inhibits caspase-9 activation, but not caspase-3 activation and apoptosis in the septic mouse splenocytes. Berberine combined with yohimbine reduces splenocyte apoptosis in the septic mice by inhibiting both extrinsic and intrinsic apoptotic pathways.