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.     

Involvement of caspase-3 and p38MAPK in allogeneic CD8+T cell-induced apoptosis of vascular endothelial cells

AIM: To investigate the function of caspase-3 and mitogen-activated protein kinases (MAPKs) in allogeneic CD8+T cell-induced apoptosis of vascular endothelial cells. METHODS: Allogeneic CD8+T cells were isolated from PBMC by positive selection using magnetic beads coated with anti-CD8 antibody. After cocultured with allogeneic CD8+T cells, apoptosis of human umbilical vein endothelial cells (HUVECs) and human dermal microvascular endothelial cells (HDMECs) were detected by AnnexinV-FITC labeling. Western blotting was used to examine the change of MAPK and caspase-3 expression in the vascular endothelial cells. The influence of SB203580 (inhibitor of p38MAPK), SP600125 (inhibitor of JNK), PD98059 (inhibitor of ERK), Z-DEVD-FMK (a caspase-3-specific peptide inhibitor) on apoptosis was also examined. RESULTS: At 24 h and 48 h time-point, the apoptosis rates of HUVECs were 41.7%±10.1% and 29.4%±8.3%, respectively (P<0.01, vs untreated HUVECs); the apoptosis rates of HDMECs were 28.9%±7.2% and 15.2%±4.8%, respectively (P<0.01, vs untreated HDMECs). These effects were largely prevented by Z-DEVD-FMK and SB203580 (P<0.05). Allogeneic CD8+T cells enhanced cleavage of caspase-3 and led to p38MAPK phospholation. CONCLUSION: Caspase-3 and p38MAPK mediate allogeneic CD8+T cells-induced apoptosis of vascular endothelial cells.     

Effects of aplastic anemia patient serum on the expression of the crucial cyclin D isoform in cord blood CD34+ cells

AIM:To explore the pathogenesis of aplastic anemia (AA), we identified the crucial isoform of cyclin D that determine the proliferation of the cord blood CD34+ cells and observed effects of AA serum on the expression of crucial cyclin D isoform in umbilical cord blood CD34+ cells. METHODS:The CD34+ cells were isolated with MIDI-MACS system. The isoforms of cyclin D were detected by RT-PCR and Western blotting. Methylcellulose culture system was used to measure the formation of CFU-GM. The expression level of crucial cyclin D isoform was assayed by RT-PCR and Western blotting after the CD34+ cells were incubated in AA serum. RESULTS:The crucial cyclin D isoform in CD34+ cells was cyclin D3. The AA serum inhibited the formation of CFU-GM and down-regulated expression level of the cyclin D3 from the mRNA to protein level, respectively. CONCLUSION:The AA serum inhibits the proliferation of CD34+ cells and down-regulates level of cyclin D3, this may be one of hematopoiesis inhibition mechanisms in AA.     

Isolation of endothelial progenitor cells from cord blood with CD133 immunomagnetic sorting

AIM: To isolate, purify and differentiate endothelial progenitor cells from cord blood in vitro and to study their biological characteristics. METHODS: CD133+ cells were selected from fresh cord blood mononuclear cells (MNC) by magnetic activated cell-sorting system (MACS). EPC was studied by flow cytometry, immunocytochemistry and immunofluorescence staining. Isolated cells were cultured in IMDM medium supplemented with or without VEGF, bFGF, SCF. RESULTS: The percentage of CD133+ cells of cord blood MNC was (1.41±1.14)%, and purity was 75%-85% (FACS method). CD133+ cells were grown on fibronectin-coated chamber slides in the presence of VEGF, bFGF, SCF. Within 1-2 hours of culture cells became adherent. On day 7-10, the adherent cells displayed a typical “cobblestone” morphology. After 14 days of culture, the adherent cells revealed a heterogeneous cell population, comprising small-sized round cells, spindle-like cells and formed tube-like structure. Weibel-Palade bodies were shown on the transmission electron microscopy photomicrographs. Compared with the original, cell markers CD133 and CD34 decreased significantly (77.0%±3.3% to 1.6%±2.2% and 93.1%±4.7% to 37.4%±4.9%, P<0.05), while Flk-1 increased significantly (from 22.3%±3.3% to 94.3%±4.1%, P<0.05) after 14 days of culture with VEGF, bFGF, SCF. The vWF was strongly expressed (77.9%±3.3%) on the 14th day later. CONCLUSION: Vascular endothelial progenitor cells were isolated from cord blood with specific expression of CD133/CD34/Flk-1. With the stimulation of the growth factors, seven-ten days after culture EPCs could be turned to endothelial cells.     

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.     

Effects of glucocorticoid inhalation on the levels of CD4+CD25+ regulatory T cells in peripheral blood of asthmatic children

AIM: To investigate the effect of glucocorticoid inhalation on the levels of CD4+CD25+ regulatory T cells in peripheral blood of asthmatic children. METHODS: Glucocorticoid inhalator was inhaled by 70 children with attack asthma. The levels of CD4+CD25+Tr in peripheral blood of asthmatic children were tested by flow cytometry (FCM). RESULTS: The CD4+CD25+Tr levels in peripheral blood of asthmatic children were (5.62%±1.29%) and (7.05%±1.61%) before and after of regulated glucocorticoid inhalation, respectively (P<0.01). The Tr levels were (7.56%±1.88%), (7.09%±1.23%) and (6.11%±1.96%) in the complete control group, part control group and poor control group, respectively (P<0.05). The Tr level in formal treatment group (7.05%±1.61%) was higher than that in irregular treatment group (5.91%±1.76%), P<0.01. CONCLUSION: The level of CD4+CD25+Tr is remarkable increased by regulated glucocorticoid inhalation, and the level of Tr can reflect the effects of glucocorticoid inhalation.     

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.     

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.     

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.     

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 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.     

Biological characteristics of JAK2 transduced CD34+ cells from cord blood during ex vivo expansion

AIM: To explore the feasibility and biological characterization of long-term regulated expansion of JAK2 transduced human CD34⁺ cord blood cells in vitro.METHODS: A retrovirus (RV) vector which contains JAK2 catalytic domain and two binding sites for a chemical inducer,dimerization (AP20187),was cloned (designated MGI-F₂JAK2).CD34⁺cells were enriched from cord blood with a MiniMACS system.The purified CD34⁺ cells were transfected with supernatant from the retrovirus packaging cell line that expressed JAK2.Following transduction,cells were expanded into four groups: AP20187 alone,FL alone,TPO,alone,AP20187+FL+TPO,respectively.The expanded cells were monitored by GFP expression,immunophenotyping,progenitor colony assay,karyotype analysis as well as tumorigenesis in nude mice.RESULTS: The purity of selected CD34⁺ cells was over 91% and gene transfer rate was 49.32%±6.21%.Only the group of AP20187 +FL+ TPO was obtained a significant sustained outgrowth of the transduced CD34⁺ cord blood cells.The percentage of GFP+ cells consistently produced a rise to the 90% peak level by the end of 8th week of culture.Flow cytometry analysis showed that the phenotype of the expanded cells was CD33⁺,CD61⁺ and Gly-A⁺ partial positive;CD38⁺ and HLA-DR⁺ strong positive,while CD2,CD7 and CD19 were almost negative.Colony assays performed in methycelluos,which can give rise to BFU-E,CFU-GM and CFU-Mix,the CFU-GM was predominantly in all colonies.The tumor was not observed in nude mice and the karyotype analysis was normal from expanded cells.CONCLUSION: The results demonstrate that AP20187-mediated activation of JAK2 signaling is capable of stimulating expansion JAK2 transduced CB CD34⁺ cells in combination with FL and TPO.This system may have applications for studies in signaling transduction,hematopoiesis,and for gene and cell therapy.     

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

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

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.     

Comparison of Toll-like receptor expression in granulocytes from umbilical cord blood and adult peripheral blood

AIM：To compare the expression levels of Toll-like receptors (TLRs) in the granulocytes isolated from umbilical cord blood and adult peripheral blood. METHODS：The granulocytes in umbilical blood and adult peri-pheral blood were isolated by the method of density gradient centrifugation combined with red blood cell splitting. The purity of the cells was evaluated by flow cytometry. The mRNA expression levels of 10 TLRs were detected by RT-qPCR, and the protein levels of some TLRs were also tested by flow cytometry. RESULTS：The populations of CD19- CD24+ cells and CD3+ cells were （95.66±3.15）% and (4.19±1.54)% in neonatal granulocytes,respectively, and were (95.36±1.74)% and (4.30±0.96)% in adult granulocytes,respectively. The relative mRNA expression levels of TLRs in the granulocytes isolated from umbilical cord blood and adult peripheral blood were as follows: TLR1 0.141±0.091 and 0.691±0.447, TLR2 0.388±0.337 and 0.901±0.508, TLR4 0.093±0.071 and 0.254±0.147, TLR6 0.056±0.045 and 0.202±0.034, TLR7 0.001±0.001 and 0.004±0.003, and TLR8 0.046±0.040 and 0.211±0.146,and the diffe-rence had statistical significance (P<0.01). However, no difference in the expression levels of TLR3, TLR5, TLR9 and TLR10 between the neonatal and adult gra-nulocytes was observed (P>0.05). Among them, the mRNA expression of TLR3, TLR7 and TLR9 was at a low level in both neonatal and adult granulocytes. The protein level of TLR2 in adult gra-nulocytes (30.50±5.69) was higher than that in neonatal granulocytes (21.40±3.09, P<0.05). CONCLUSION：Low mRNA expression of TLR1, TLR2, TLR4 and TLR6, and low protein level of TLR2 in neonatal granulocytes indicate that the ability of recognizing bacterial pathogen by neonatal granulocytes may be defective or not yet fully mature.     

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

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

Comparative analysis of cultured endothelial progenitor cells in vitro from PBMCs and enriched CD133+ cells

AIM: To compare the methods of two currently employed isolation methods for endothelial progenitor cells (EPCs): from total peripheral blood mononuclear cells (PBMCs) and from enriched CD133+ cells, by defining the cell morphology, phenotype, reproductive activities and function in vitro, providing a reference for clinic application. METHODS: PBMCs from the healthy subjects were used for CD133+ sorting or not. The two groups of isolated cells were suspended in complete medium M199 for 7 d to 14 d. EPCs phenotype were characterized by FACS. The proliferation of differentiated EPCs was studied by MTT assay, and VEGF concentration was measured using an ELISA kit. Matrigel experiment and migration assay were imitated vascularization in vivo. RESULTS: PBMCs produced more colony-forming units (CFU) than CD133+ cells from the same volume of blood (P<0.01). From 7 d to 14 d, the two groups show decreased expression of hematopoietic stem cell markers and increased level of endothelial markers, but CD144+ cells in CD133+ group were lower than those in PBMCs groups (P<0.01). Cells in PBMCs group secreted more VEGF than that in CD133+ group on 7 d (P<0.01). Compared to CD133+ group, PBMCs group showed more potential of proliferation and vascularization in vitro. CONCLUSION: CD133+ sorted cells show a lower capacity of differentiation, secretion, proliferation and vascularization in vitro, which is unable to differentiate to mature endothelial cells, indicating that its not a preferential way to obtain EPCs for clinic therapy.     

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.