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.     

Correlation between CD4+CD28-T cells and lymphocytic apoptosis in rheumatoid arthritis patients

AIM: To investigate the fraction of CD4⁺CD28^-T cells and its correlation with lymphocytic apoptosis in peripheral blood of rheumatoid arthritis (RA) patients.METHODS: The RA patients and age-matched health controls were selected in the study. The lymphocytes were isolated from peripheral blood. CD4⁺ T cells without CD28 expression (CD4⁺ CD28^-) were analyzed by flow cytometry. The incidence of apoptosis in the cells cultured with or without PHA for 24 h was determined by flow cytometry with Annexin V-FITC/PI di-staining. The correlation between the fraction of CD4⁺CD28^-T cells and lymphocytic apoptosis was also observed.RESULTS: The fraction of CD4⁺CD28^-T cells was significantly higher in RA group than that in healthy control group (7.79%±3.52% vs 1.89%±1.78%, P<0.05). The apoptotic level in PHA cultured lymphocytes was significantly lower in RA group than that in healthy controls (11.38%±5.73% vs 19.46%±6.32％, P<0.05). Spearman analysis showed that there was a negative correlation between the fraction of CD4⁺CD28^-T cells and apoptotic level of activated lymphocytes (r=-0.433,P<0.01).CONCLUSION: The increased CD4⁺CD28^-T cells contribute to prolong the lifespan of activated lymphocytes in peripheral blood of RA patients, and the persistence of activated lymphocytes may contribute to the pathogenesis of RA.     

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.     

Proliferation of CD4+CD25+ T cells from PBMCs of the gastric cancer patients and inhibitory effect on CD4+CD25- T cells in vitro

AIM:To investigate the proliferation of CD4+CD25+ T cells from PBMCs of the gastric cancer patients and the inhibitory effect on CD4+CD25- T cells in vitro. METHODS:Magnetic activated cell sorting (MACS) method was used to separate CD4+CD25+T and CD4+CD25-T cells from peripheral blood monocytic lymphocytes in the gastric cancer patients, and then the purity and activity of CD4+CD25+T cells were analyzed with flow cytometer. After stimulated with anti-CD3 Ab, anti-CD28 Ab and rh IL-2, CD4+CD25- and CD4+CD25+ T cells were cocultured. The inhibitory effect of CD4+CD25+T on CD4+CD25-T cells was assayed by ［3H］ thymidine proliferation experiment. RESULTS:(1)After sorting, CD4+CD25+ T cells purity in healthy control and gastric cancer patients were 83.80%±1.84% and 84.13%±2.77%, respectively. No significant difference between the two groups (P>0.05) was observed. (2)The activity of CD4+CD25+ and CD4+CD25- T cells in healthy control and the gastric cancer patients after sorting were 98.52%±0.72% and 97.80%±0.95%. There was no significantly difference between the two groups (P>0.05). (3) CD4+CD25+ T cells obviously inhibited the CD4+CD25-T cell proliferation in vitro. The inhibition achieved to maximum in coculture of CD4+CD25+ T cells together with CD4+CD25- T cells (ratio of 1∶〖KG-*2〗1). CONCLUSION:The MACS system can effectively isolate CD4+CD25+ and CD4+CD25- T cells. After sorting, CD4+CD25+T cells obviously inhibit the proliferation of CD4+CD25- T cells in vitro and the inhibitory effect display an effect-target ratio relationship.     

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.     

Establishment of experimental autoimmune encephalomyelitis model in mice induced by MOG and its effect on CD4+CD25+ T cells

AIM: To investigate the feasibility of inducing experimental autoimmune encephalomyelitis(EAE)model in C57BL/6 mice by TrxA-extracellular immunoglobulin domain of MOG(MOGIgd-TrxA)fusion protein produced by molecular cloning in our laboratory. Also to investigate the role of CD4+CD25+ T cells in the pathogenesis of EAE. METHODS: (1)The MOGIgd-TrxA fusion protein was induced and produced by molecular cloning and purified by metal chelate affinity chromatography and concentrated through ultrafiltration. The concentration of the protein was measured by Bradford method at last. (2)Animal experiment: C57BL/6 mice(12 mice in each group)were used. The mice in group MOG were immunized with MOGIgd -TrxA fusion protein. The mice in group GPSCH were received emulsion of spinal cord homogenate of guinea pigs(GPSCH), and mice in group TrxA or normal control group(group NC)were received the same volume emulsion of TrxA or saline/adjuvant, respectively. Clinical scores and histopathology were measured to value the models quality. (3)The percentages of CD4+CD25+ regulatory T cells in EAE mice were tested through flow cytometric analysis. RESULTS: (1)The purity of purified MOGIgd -TrxA fusion protein was about 98%, and its concentration was 2.3 g/L. (2)No significant difference between group MOG and group GPSCH in the clinical score was observed(P>0.05). Histologic sections of the brain and spinal cord taken from affected animals in both groups showed pathological change of different level throughout the central nervous system(CNS). (3)Percentages of CD4+CD25+ T cells in group MOG and group GPSCH were(4.71±1.61)%and(1.44±0.65)%,respectively, both of which were significantly lower than those in group NC(9.22±1.24)%and TrxA group(8.97±1.20)％(P<0.01). CONCLUSION: (1)The animal model of EAE in C57BL/6 mice induced by MOGIgd fusion protein produced through molecular cloning in our laboratory is stable and with high incidence. Thus, the author finds a good way to study the immune mechanisms of MS further and to search for the effective treatments as well. (2)The reduction of CD4+CD25+ T cells in EAE mice may have some relationship with the clinic.     

Effect of acupuncture on CD4+ CD25+ Foxp3+ regulatory T-cells in rats of embryo implantation failure

AIM：To observe the effect of acupuncture on CD4+ CD25+ Foxp3(forkhead box P3)+ regulatory T-cells(Treg cells) in rats with embryo implantation failure. METHODS：One hundred and forty-four pregnant rats were randomly divided into control group(N), mifepristone treatment group(M), mifepristone+acupuncture treatment group(A) and mifepristone+progestin treatment group(W). The rats in groups M, A and W were treated with mifepristone-sesame oil solution on day 1, while the rats in group N were injected with the same amount of sesame oil. The Housanli(ST36) and Sanyinjiao(SP6) points were selected for acupuncture. From day 1 to the time of death, the rats in group A were fasten up and then the acupuncture was performed. Accordingly, the rats in group N and group M were only fixed, and the rats in group W were given progestin. Implanted embryos in each group were counted. The proportions of CD4+ CD25+ Foxp3+ Treg cells in peripheral blood and CD4+ Foxp3+ Treg cells in the endometrium were detected by flow cytometry. The mRNA and protein levels of Foxp3 were determined by real-time PCR and Western blotting, respectively. RESULTS：Compared with group N, the number of implanted embryos, the percentages of CD4+ CD25+ Foxp3+ Treg cells in peripheral blood and CD4+ Foxp3+ Treg cells in the endometrium, and the expression of Foxp3 protein and mRNA in the endometrium were significantly decreased in group M(P<005). Compared with group M, the above indexes in group A and group W were significantly increased. CONCLUSION: The effect of acupuncture in rats with embryo implantation failure may be closely correlated with the modulation of CD4+ CD25+ Foxp3+ Treg cells.     

Effects of splenectomy on CD4+ , CD8+ and spleen function in cirrhotic rats with portal hypertension

AIM:To investigate the effect of subtotal splenectomy on the expression of CD4+、CD8+ and tuftsin in cirrhosic rats with portal hypertension (PHT) . METHODS:Rats liver cirrhosis was induced by subcutaneous injection of 40% CCl4. Fifty rats were randomly divided into five groups (n=10). Group A:control rats;group B:PHT rats;group C:normal rats with total splenectomy;group D:PHT with total splenectomy and group E:PHT with subtotal splenectimy. The hepatic function, the expression of CD4+, CD8+, the ratio of CD4+ to CD8+ and tuftsin were analyzed at the fourth week after treatment. RESULTS:The expression of tuftsin ,the ratio of CD4+ to CD8+ was significantly decreased in PHT rats with total splenectomy compared with PHT rats ［（171±21） ng/L vs （433±44）ng/L,P<0.01;(2.01±0.22 vs 1.12±0.12),P<0.01］. In the group of PHT rats with subtotal splenectomy, the expression of tuftsin, the ratio of CD4+ to CD8+ was higher than those in the PHT rats with total splenectomy ［（434±42） ng/L vs （171±21） ng/L,P<0.01;（1.97±0.18 vs 1.12±0.12,P<0.01］, however, the hepatic function was not show difference（P>0.05）. CONCLUSION:Spleen and immune function is significantly improved in PHT rats after subtotal splenectomy, but the hepatic function is not changed significantly.     

Mechanisms underlying the induction of IL-2 secretion by PDB pl us ionomycin in CD4+CD25+ T cells from cord blood and adult peripheral blood

AIM:To confirm that CD4+CD25+ regulato ry T cells don't have an instinctive defection in IL-2 secretion,and to have an insight into the maturation state of CD4+CD25+ T cells in cord blood.METHODS:CD4+CD25+ and CD4+CD25- T cells were purified f rom cord blood of term infants (CB) and adult peripheral blood (PB) by autoMACS,and stimulated with PDB plus ionomycin.After 45 hours of culture,cells were d etected for expression of CD69 and CD25 by flow cytometry,and the supernatants were measured for 7 kinds of cytokines by Luminex.RESULTS:CD4+CD25+ T cells from both CB and PB proliferated comparably with CD4+CD25- T cells when stimulated with PDB plus ionomycin.A fter 45 hours of culture,however,the CD4+CD25+ T cells underwent a tendenc y of cell death.Expression of CD25 was further upregulated when CD25+ cells w ere activated.Under stimulation of PDB plus ionomycin,both CD4+CD25+ and C D4+CD25- T cells in PB secreted high levels of IFN-γ,IL-2 and TNF-α,with CD25+ cells secreted much higher level of IL-5,IL-4 and IL-10 than those in CD25- cells;CD4+CD25+ and CD4+CD25- T cells in CB also secreted high level of IL-2 and TNF-α but much lower level of IFN-γ than those in PB,and no secretion of IL-5,IL-4 and IL-10 was observed.CONCLUSION:CD4+CD25+ regulatory T cells don't have an i nstinctive defection in IL-2 secretion,otherwise there may be a different TCR signaling pattern in CD4+CD25+ T cells from traditional T cells.The CD4+C D25+ T cells in cord blood have not fully matured in function.     

Effect of proliferating cell nuclear antigen specific antisense oligonucleotide on differentiation of cord blood CD34+ cells

AIM:To study the effect of proliferating cell nucler antigen antisense oligonucleotide on ex vivo expansion of cord blood CD34+ hematopoietic stem/progenitor cells. METHODS:CD34+ cells were purified from fresh cord blood by immunomagnetic beads. CD34+ cells were incubated in liquid culture system with different concentrations of PCNA-ASODN. Using flow cytometry, the number of different kinds of stem/progenitor cells and PCNA expression were measured after CD34+ cell incubation. RESULTS:PCNA was lowly expressed in low experiential group, with a positive rate of (27.2±3.6)% and (19.0±1.5)%, the positive rate of control group was (53.8±8.3)% (P<0.01), a high significant difference was observed. Just in low concentration of PCNA-ASODN, the percentage of CD34+cells were increased to (33.4±3.2)%, CD34+CD38-cells expanded (57.8±9.9) folds, and the percentage of CD34+cells were (25.2±2.6)% (P<0.01), the CD34+CD38-cells expanded (43.5±7.4) folds (P<0.05), it has significant difference compared with the control group. CONCLUSION:Low concentration of PCNA-ASODN decreases PCNA expression effectively, slows down differentiation of CD34+cells during ex vivo expansion procedure, and improves the expansion efficiency.     

CD45+CD86+ cells isolated from para-aortic lymph nodes in a murine abortion-prone model

AIM: To address whether the analysis of CD45+CD86+ cells isolated from para-aortic lymph nodes (PLNs) is valuable in assessment of the status of local immunity at the feto-maternal interface. METHODS: CBA/J×DBA/2, virgin CBA/J, and CBA/J×BALB/c mice were used as an abortion-prone model (group A), non-pregnant controls (group N), and fertile controls (group F), respectively. The percentage of CD45+CD86+ cell in the CD45+ cell group (CD45+CD86+ percentage for short) and the absolute number of these cells were determined with flow cytometry (FCM), using mononuclear cells isolated from PLNs collected on day 5.5, 9.5, and 13.5 of gestation, respectively, and mononuclear cells from placentas on day 13.5 of gestation. To clarify the identity of these CD86+ cells, FCM was also performed with CD3, CD19 and DX5 as markers for T cells, B cells, and NK cells, respectively. RESULTS: Both resorption rate and absolute number of resorption were significantly higher in group A (29.3%, 1.8±1.0) than those in group F (4.8%, 0.3±0.5, P<0.01, respectively). Similarly, both cell percentage and absolute number of CD45+CD86+ cells in PLNs collected on day 13.5 of gestation were significantly higher in group A than that in group F (27.5%±14.0% vs 12.3%±7.1%, and 1 362±687 vs 615±353, P<0.01, respectively). The CD45+CD86+ percentage was around 7.5% in virgin CBA/J mice, similar to the 10.6% in CBA/J×DBA/2 mice on day 5.5 of gestation, but increased dramatically to 23.9% by day 9.5 (P<0.01 vs virgin mice and P<0.05 vs CBA/J×DBA/2 mice on day 5.5), and remained at a higher level (27.5%) until day 13.5. However, this trend was not observed in group F during pregnancy. CONCLUSIONS: The increased CD45+CD86+ percentage on day 9.5, when resorption begins, may support the assumption that CD45+CD86+ cells play a role in the course of embryo resorption. Lymphocyte phenotypic analysis in the lymph nodes that drain the pregnant uterus may be helpful to assess the status of local immunity at the feto-maternal interface.     

CD4+CD25+FOXP3+ Tregs and HBV-specific CTLs in peripheral blood from chronic hepatitis B patients

AIM: To investigate the roles of CD4⁺CD25⁺FOXP3⁺ regulatory T cells (Tregs) and HBV-specific cytotoxic T-lymphocytes (CTLs) in peripheral blood from the patients with chronic hepatitis B (CHB).METHODS: Peripheral blood mononuclear cells from 28 patients with CHB and 15 healthy controls were analyzed for Treg frequency using flow cytometry and for HBV-specific CTLs using enzyme-linked immunospot assay (ELISPOT).The clinical data of HBV-infected patients were considered.RESULTS: The frequency of CD4⁺CD25⁺FOXP3⁺Tregs was higher in the patients with CHB than that in the patients of healthy controls (3.14%±0.97% vs 1.95%±0.68%, P<0.05), and a positive correlation was found between Tregs and the DNA levels of HBV (r=0.831, P<0.01).HBV-specific CTLs were detected by ELISPOT in CHB patients and a negative correlation was observed between Tregs and CTLs (r=-0.540, P<0.01).CONCLUSION: Peripheral blood CD4⁺CD25⁺FOXP3⁺ Tregs in CHB patients are increased and closely correlated with the DNA replication of HBV and CTLs, suggesting that the clearance of HBV can be influenced by the inhibition of cellular immunoreaction through Tregs.     

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.     

Relationship of CTCs with CD4+/CD8+, NLR,TTV and tumor stage in patients with primary hepatocellular carcinoma

AIM: To explore the relationship of circulating tumor cells (CTCs) with CD4⁺/CD8⁺, neutrophil-to-lymphocyte ratio (NLR), total tumor volume (TTV) and tumor stage in the patients with primary hepatocellular carcinoma.METHODS: We selected 80 cases of histologically diagnosed primary hepatocellular carcinoma in the study. The method of CanPatrol^TM was used, which was developed by SurExam biotechnology company to identify CTCs in the blood. CD4⁺ T cells and CD8⁺ T cells were counted by flow cytometry. The patients were divided into 2 groups according to the levels of CD4⁺/CD8⁺ ratio, NLR and TTV. The patients were also divided into Ⅰ＋ Ⅱstage group and Ⅲ＋ Ⅳ stage group according to the seventh edition of TMN staging criteria.RESULTS: The numbers of peripheral blood CTCs and mesenchymal CTCs in high CD4⁺/CD8⁺ ratio group were significantly lower than those in low CD4⁺/CD8⁺ ratio group (P < 0.05). The numbers of peripheral blood CTCs, mesenchymal CTCs and hybrid CTCs in high TTV group were significantly higher than those in low TTV group (P < 0.05). The numbers of peripheral blood CTCs, mesenchymal CTCs and hybrid CTCs in I＋ II stage group were significantly lower than those in III＋ IV stage group (P < 0.05). The numbers of peripheral blood CTCs, mesenchymal CTCs, hybrid CTCs and epithelial CTCs between high NLR group and low NLR group had no statistical difference.CONCLUSION: CTCs exist in the peripheral blood of the patients with primary hepatocellular carcinoma. The peripheral blood CTCs have significant correlations with TTV, tumor stage and T-cell immunity.The worse cell immune function, the larger TTV and the later tumor stage a patient has, the more the peripheral blood CTCs are.     

Role of Kv1.3 channel of CD4+T lymphocytes in the formation of atherosclerosis in rat spleen

AIM: To investigate the function of voltage-gated potassium channel Kv1.3 and its possible role in CD4⁺ T lymphocytes in the formation of atherosclerosis (AS) in rat spleen. METHODS: The rat atherosclerosis model was established by feeding high-fat diet. The proportion of lymphocytes was determined by flow cytometry. The CD4⁺ T lymphocytes were separated using immunomagnetic bead. The mRNA expression of Kv1.3 in CD4⁺ T lymphocytes was detected. The concentrations of intracellular calcium and cytokines were also measured. RESULTS: (1) The proportion of CD4⁺ T lymphocytes in AS group was significantly higher than that in control group (74.93%±2.15% vs 67.80%±2.54%, P<0.05). (2) After stimulated with concanavalin A (ConA), the proliferation of CD4⁺ T lymphocytes in AS group was significantly higher than that in control group (1.1321±0.1750 vs 0.7971±0.0955, P<0.05). (3) After stimulated with ConA, the concentration of intracellular calcium in AS group was higher than that in control group. (4) In AS group, the releases of cytokines of IL-2 and TNF-α in AS group were significantly higher when stimulated with ConA for 48 h than that for 24 h. (5) The mRNA expression of Kv1.3 in CD4⁺ T lymphocytes was greatly higher in AS group than that in control group (3.670±1.579 vs 1). CONCLUSION: In AS rats, the increase in CD4⁺ T lymphocytes as well as the augmentation of Kv1.3 mRNA expression in the cells suggest that up-regulation of Kv1.3 mRNA expression in CD4⁺ T lymphocytes may be involved in the mechanism of atherosclerotic formation in rat spleen.     

Role of CD163/TWEAK pathway in atherosclerosis

AIM:To investigate the effect of CD163/tumor necrosis factor-like weak inducer of apoptosis (TWEAK) pathway on atherosclerosis in mice. METHODS:APOE^-/- mice and wild-type (WT) C57BL/6 mice were divided into 4 groups (8~10 weeks, n=10):APOE^-/- +normal diet (ND) group, APOE^-/- +western diet (WD) group, WT+ND group, and WT+WD group. Detection of blood lipid levels and oil red O staining of aorta artery were performed to confirm whether the atherosclerotic model was well established in 16 weeks after feeding. The aortic tissues were harvested to measure CD163 and TWEAK protein levels by Western blot, and immunohistochemical staining was also performed to localize CD163 and TWEAK protein expression on atherosclerotic plaque in each group. The cell experiments were conducted to study whether CD163 regulated TWEAK expression in M1 macrophages and foam cells, and the possible downstream pathway was investigated. RESULTS:The blood lipid levels and aorta oil red O staining showed that the animal model of atherosclerosis was successfully established in APOE^-/- +ND group and APOE^-/- +WD group. The protein level of CD163 was significantly increased in the aortic tissue in APOE^-/- mice (P<0.05) as compared with C57BL/6 mice (P<0.05). Consistently, the protein level of TWEAK was also markedly higher in APOE^-/- +ND group and APOE^-/- +WD group than that in WT+ND group and WT+WD group (P<0.05). Immunohistochemical staining showed that CD163 was mainly expressed in the parts away from the lipid core, and TWEAK was found in all parts of the atherosclerotic plaque. CD163 significantly inhibited the protein expression of TEWAK in the M1 macrophages, and also significantly down-regulated the level of nuclear factor-κΒ (NF-κB) in the M1 macrophages and foam cells (P<0.05). CONCLUSION:The protein levels of CD163 and its ligand TWEAK are significantly increased in atherosclerotic mice. The CD163 positive macrophages are mainly located at the site far away from the lipid core, and CD163 may play an anti-atherosclerotic effect by inhibiting TWEAK/NF-κB pathway.     

Proportion of CD14+CD16+ monocytes in peripheral blood from patients with type 2 diabetic mellitus and effect of LPS and IL-15 on expression of STAT5 in monocytes

AIM: To characterize the proportion of CD14⁺CD16⁺ monocytes in peripheral blood from type 2 diabetes (T2DM) patients and to observe the response of CD14⁺CD16⁺ monocytes to lipopolysaccharide (LPS) and interleukin-15 (IL-15) for further exploring the potential mechanism of inflammatory immune response in the pathogenesis of T2DM. METHODS: Twenty-eight patients with T2DM and 20 healthy volunteers were enrolled in the study. The peripheral blood was collected for determining the percentage of CD14⁺CD16⁺ monocytes by flow cytometry. The peripheral blood mononuclear cells (PBMC) were isolated and subject to stimulation with LPS and IL-15 for 4 h. The protein expression of STAT5 was detected by Western blotting and the phosphorylated (p)-STAT5 was determined by Western blotting and immunofluorescence. Serum levels of 25-hydroxyvitamin D₃ and IL-6, and the concentrations of IL-6 and monocyte chemoattractant protein-1(MCP-1) in the culture supernatants were assessed by ELISA. Serum level of C-reactive protein (CRP) was measured by immunoturbidimetry. RESULTS: There were positive correlations between the quantity of CD14⁺CD16⁺ monocytes and serum levels of CRP and IL-6 (r=0.394, P<0.05 and r=0.741, P<0.01), while serum 25 (OH) D₃ was negatively correlated with the quantity of CD14⁺CD16⁺ monocytes (r=-0.409, P<0.01), serum CRP(r=-0.479,P<0.01) and serum IL-6 (r=-0.774,P <0.01). After stimulated with LPS and IL-15, PBMC showed significant up-regulation of p-STAT5 protein expression, and significant increases in the supernatant levels of IL-6 and MCP-1 were observed (P<0.05). The expression of p-STAT5 existed in the nucleus.CONCLUSION: These findings suggest that the functional disturbance in monocytes occurs in T2DM, which may be related to insufficiency of vitamin D₃. The aberrant activation of STAT5 signaling pathway underlies the functional abnormalities of the monocytes in T2DM.     

Effects of intravenous immunoglobulin on IL-4 levels and CD20+ cells in peripheral blood and recurrent rate of infants with wheezing

AIM: To investigate the effects of intravenous immunoglobulin on IL-4 levels and CD20⁺ cells in peripheral blood and the recurrence rate of infants with wheezing. METHODS: IL-4 levels and CD20⁺ cells in peripheral blood of 30 normal infants and 66 infants with wheezing were tested by flow cytometry and ELISA, respectively. The relief time of wheezing and recurrent rate were also recorded. RESULTS: The IL-4 levels and CD20⁺ cells in the wheezing infants were higher than those in controls(P<0.01). The IL-4 levels and CD20⁺ cells in the wheezing infants were decreased after routine treatment but were still higher than those in control infants after treatment. The IL-4 levels and CD20⁺ cells in the wheezing infants were decreased after immunoglobulin treatment and were almost the same as controls after treatment. The relief time of wheezing in the infants with immunoglobulin treatment was shorter than that in the infants with routine treatment(P<0.01), and recurrent rate of immunoglobulin treatment was lower than that of routine treatment(P<0.05). CONCLUSION: The IL-4 levels and CD20⁺ cells in peripheral blood are increased more significantly in infants with wheezing than those in control infants. The mechanisms of wheezing relief and decreasing the recurrent rate by intravenous immunoglobulin are associated with the down-regulation of IL-4 levels and CD20⁺ cells.     

Suppression of allergic airway inflammation in a mouse model by Der p2 recombined BCG-induced adaptive CD4+CD25+ Treg

AIM: The bacillus calmette-guerin (BCG) vaccine is the most widely used Th1-inducing vaccine. In recent years, some studies argued that mycobacterium vaccae can be used as adjuvant to induce regulatory T cells (Treg) and then suppress asthmatic airway inflammation. We previously have engineered recombined BCG that expressed Der p2 of house dust mites (Der p2 rBCG) on the cell wall. The aim of this study is to investigate the immune regulatory mechanisms of Der p2 rBCG. METHODS: Mice were vaccined with PS, BCG or rBCG. The relative proportion and the absolute numbers of related Tregs in spleen cells were analyzed. The suppressive activity of Der p2 rBCG-induced CD4+CD25+ T cells was detected both in vitro and in vivo. RESULTS: (1) Der p2 rBCG induced a CD4+CD25+Foxp3+ T cell subtype. (2) Der p2 rBCG-induced CD4+CD25+ T cells suppressed the proliferation of Th2 effector cells in vitro in an antigen-specific way. (3) Der p2 rBCG-induced CD4+CD25+ T cells mediated Der p2 specific suppression of airway allergy in vivo. CONCLUSION: Der p2 rBCG induces a CD4+CD25+Foxp3+ T cell subtype, which suppresses inflammation in allergic airway in a mouse model.