Mixed culture of human iPSCs with rabbit corneal endothelial cells induces differentiation of iPSCs

AIM:To investigate the morphology and protein expression of human induced pluripotent stem cells(iPSCs) in the mixed-culture environment with rabbit corneal endothelial cells(CECs) and to provide the experimental basis and the mechanism of iPSC differentiation into CECs. METHODS:Primary rabbit CECs were isolated with trypsin and subcultured. The human iPSCs were cultured and amplified by a feeder-free method and their characteristics were evaluated by Western blotting. iPSCs labeled with quantum dots of appropriate concentration were used to establish mixed-culture model with rabbit CECs. The morphology of iPSCs was evaluated by atomic force microscopy(AFM) and inverted microscopy. The protein expression of CD31, CD34, CD133 and aquaporin 1(AQP1) in iPSCs was tested by the method of immunofluorescence. RESULTS:The rabbit CECs were hexagonal and showed a typical cobblestone appearance. iPSCs grew in the cloning form, and 3 pluripotent proteins Oct4, Nanog and Sox2 were expressed positively. 1/4 suspension of iPSCs labeled with 10 nmol/L quantum dots and 60% confluence of rabbit CECs made best mixed culture for each other. Under AFM and inverted microscope, the volume of iPSC became bigger and the nuclear-cytoplasm ratio was decreased after 7 days of mixed culture. Some granular protrusions of the membrane were observed and the surface roughness of the cell membrane increased. The protein expression of CD31, CD34, CD133 and AQP1 in iPSCs was negative, while AQP1 was detected after mixed culture for 2 weeks. CONCLUSION:iPSCs morphologically change to endothelial-like cells after mixed culture with rabbit CECs and express the marker protein AQP1 of CECs at the same time.     

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.     

5-FU upregulates stem cell marker CD133 expression in colon cancer cells

AIM: To investigate the effect of 5-fluorouracil(5-FU)on the expression of the stem cell marker CD133 on colon cancer stem cells. METHODS: CD133 expression on several colon cancer cell lines was detected by flow cytometry. The CD133 positive cells from DLD1 cells were separated by the method of magnetic activated cell separation. Colony assay was used to measure self-renew ability and MTS assay was used to detect the sensitivity to 5-FU after separation. After 5-FU treatment, the change of CD133 mRNA level was measured by qPCR. RESULTS: CD133 expression on the surface of colon cacner cell lines DLD1, HT29, SW480, HCT116, Lovo, RKO was 30.20%, 82.00%, 0.34%, 91.80%, 85.30%, 0.28% respectively. DLD1 cells had two obvious populations according to CD133 expression. CD133 positive cells were separated from DLD1 cells, the positive purity was 87.21%±5.33% and the negative purity was 84.30%±4.65%. CD133 positive cells formed more colonies with limited dilution colony assay(46.33%±4.44% vs 31.00%±2.00%, P<0.05). CD133 positive cells were less sensitive to 5-FU compared to CD133 negative cells(20% less, P<0.01). 5-FU at concentration of 1 mg/L upregulated CD133 mRNA expression in both DLD1 and HT29 cells, the relative quantity was increased from 1 to 1.684±0.012(P<0.01)and 30.702±0.280 to 49.379±0.460(P<0.01)in HT29 and DLD1, respectively. CONCLUSION: Compared to CD133 negative cells, CD133 positive cells show more ability to form colonies in vitro, and are less sensitive to 5-FU. 5-FU upregulats the mRNA expression of CD133, resulting in the CD133 colon cancer stem cells enrichment during 5-FU treatment.     

Transwell contact co-culture promotes growth and differentiation of single-dissociated iPSCs

AIM：To investigate the promoting role of Transwell contact co-culture system in the growth and differentiation of single-dissociated induced pluripotent stem cells (iPSCs). METHODS：Bovine corneal endothelial cells (CECs) at passage 1~2 (P1~2) were seeded on the underside of Transwell inserts placed into culture plates and were cultured in 37 ℃ and 5% CO2 for 8 h. Accutase digestion and 40 μm filter process disaggregated colony-aggregated iPSCs into single-dissociated iPSCs, and the cells were seeded on the inside of Transwell inserts with CECs in medium of mTeSR1 for 3 d and then in low-glucose DMEM supplemented with 10% FBS for 2 weeks. The characteristics and differentiation markers were evaluated by real-time fluorescence quantitative polymerase chain reaction (qPCR), immunofluorescence staining, live & dead cell staining and alkaline phosphatase (ALP) staining. The group of iPSCs cultured in conventional medium was used as control group 1. The group of single-dissociated iPSCs co-cultured with CECs was set as experimental group, while single-dissociated iPSCs without co-culture were as control group 2. RESULTS：The bovine CECs showed typical hexagonal cobblestone shape. iPSCs showed colony-like growth, while became single-dissociated cells after Transwell contact co-culture with bovine CECs for 3 d. The single-dissociated iPSCs positively expressed the undifferentiated markers, Nanog and Oct4. The mRNA expression levels of Nanog, Oct4 and Sox2 between experimental group and control group 1 were both positive and had no statistical significance difference (P>0.05). The dead cells in experimental group decreased significantly, and there was statistically significant difference compared to control group 2 (P<0.01). After 14 d of induced differentiation co-culture, the single-dissociated iPSCs showed rather uniform polygonal morphology, increased dimension and no obvious colony existence. Negative ALP staining, positive immunofluorescence staining for ZO-1, AQP1 and CD31, and negative for CD34 and CD133 were also observed. The results of qPCR showed that the mRNA expression of Oct4, Nanog and Sox2 significantly decreased, and had statistically significant difference compared with control group 1 (P<0.01). CONCLUSION：When co-cultured with bovine CECs, iPSCs morphologically changed to endothelial-like cells and expressed some markers of CECs. Transwell contact co-culture system not only enhances the growth of single-dissociated iPSCs, but also promotes their differentiation.     

Effects of statin reloading before percutaneous coronary intervention on circulatory endothelial progenitor cells and inflammatory cytokines

AIM:To investigate the effects of atorvastatin reloading in pre-percutaneous coronary intervention (PCI) period on endothelial progenitor cell (EPC) count and inflammatory cytokine expression in the stable angina pectoris patients who had previously received long-term statin treatment. METHODS:The patients with stable angina pectoris that had received long-term statin therapy and planned to accept PCI were randomized into 3 groups: 80 mg atorvastatin 12 h and 40 mg 2 h before coronary angioplasty (80 mg reloading), pre-operatively with 40 mg/d atorvastatin for 7 d (40 mg reloading), and without atorvastatin reloading (no reloading). CD45-/CD133+/CD34+, CD45-/CD34+/KDR+ and CD45-/CD144+/KDR+ EPCs in 100 μL peripheral blood were determined by flow cytometry 1 h prior to PCI and 1 h, 6 h and 24 h after PCI. The serum concentrations of soluble intercellular adhesion molecule 1 (sICAM-1), C-reactive protein (CRP) and troponin I (TnI) were analyzed immediately prior to and 24 h after PCI. RESULTS: (1) In 80 mg reloading group, the numbers of circulating CD45-/CD133+/CD34+ and CD45-/CD34+/ KDR+ early differentiation stage EPCs 1 h and 6 h after coronary angioplasty was significantly elevated compared with those before PCI (P<0.05). (2) In control group, the serum concentrations of sICAM-1 and CRP 24 h after PCI were significantly elevated (P<0.05) compared with preoperative values. (3) The rise in serum TnI concentration from pre- to post-operation in 80 mg reloading group was lower than that in control group. CONCLUSION: The method of atorvastatin reload before PCI affects the number of EPCs in peri-operative period. High dose of atorvastatin application before PCI triggers early EPC circulation. The serum levels of post-operative inflammatory cytokine sICAM-1 as well as CRP are reduced by atorvastatin reloading before PCI.     

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.     

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.     

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.     

Effect of 7-hydroxyisoflavone on HCT116 cells proliferation by Id1

AIM:To investigated the effect of 7-hydroxyisoflavone (7-HIF) on the proliferation, apoptosis and stem-like cell feature of colorectal cancer cells. METHODS:The effect of 7-HIF on the proliferation of HCT116 cells was detected by WST-1 assay and colony formation assay. The effects of 7-HIF on the cell cycle distribution and apoptosis in the HCT116 cells were analyzed by flow cytometry. The expression of cell-cycle related proteins and the stemness related proteins was determined by Western blot. RESULTS:After treated with 7-HIF (200 μmol/L), the viability of HCT116 cells was inhibited, and the size and number of the colony were decreased as compared with control group (P<0.05). The G₀/G₁ phase of the cell cycle was increased. The proportion of S phase was decreased and the cells were mainly arrested in G₀/G₁ phase. The apoptotic rate of HCT116 cells was 21.4%, which was significantly higher than that in the control group (1.1%). The results of Western blot revealed that the expression of inhibitor of differentiation 1(Id1) was significantly decreased (P<0.05). The expression of cell cycle markers cyclin D1 and cyclin E, the proliferative markers survivin and PCNA, and stem cell markers CD133, ALCAM and EpCAM were all down-regulated by 7-HIF treatment (P<0.05). CONCLUSION:7-HIF inhibits the proliferation and induces the apoptosis of colorectal cancer cells, and inhibits the stem-like cell feature, which may be related to Id1 inhibition.     

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.     

The influence of anesthesia and means of postoperative pain control on T lymphocyte subtypes of blood in patients with rectal cancer

AIM: To evaluate the influence of anesthesia and different means of postoperative pain control on the T-lymphocyte during the perioperative period in patients with rectal cancer.METHODS: 40 adult patients, aged 65 or older, of American Society of Anesthesiologists (ASA) class 2-3 were divided into two groups according to the type and means of postoperative pain managements. Group Ⅰ (n=20) received intravenous anesthesia and patient controlled analgesia(PCA), fentanyl (13 μg/kg) for post pain; group Ⅱ (n=20) received intravenous anesthesia plus lumber epidural anesthesia and epidural PCA of morphine 5 mg plus ropivacaine 100 mg for post operative pain. Blood samples from internal jugular vein were obtained before surgery, at the completion of surgery and 24, 48, and 120 h post surgery for detecting CD3⁺, CD4⁺, CD4/CD8 counts of peripheral T-lymphocytes. In addition, blood cortisol level and pain intensity were assessed by visual analogue score (VAS)at each time point. RESULTS: Baseline(before anesthesia) values of CD3⁺,CD4⁺, CD4/CD8 in patients were messured and there was a significant decrease of all these values from completion of surgery to 48 h after surgery in both groups (P<0.01). However, group Ⅱ showed a higher CD4⁺ at 48 h, higher CD3⁺,CD4⁺, CD4/CD8 at 120 h post surgery than group Ⅰ (P<0.05). Patients in both groups obtained good pain relief post surgery,but VAS in group Ⅱ were significantly lower than those in group Ⅰ at 24 and 48 h post surgery (P<0.01). Compared with baseline, blood cortisol levels in both groups increased markedly at completion of surgery, and at 24, 48 h after surgery (P<0.01),while the increased cortisol level in group Ⅱ at completion of surgery and 24 h after surgery was less than that in group Ⅰ (P<0.05).CONCLUSION: Combined intravenous anesthesia with lumber epidural anesthesia appears to reduce the perioperative stress response and exerts less negative effects on the T-lymphocytes, suggesting that such a means of anesthesia might be more suitable to the elderly patients with rectal cancer.     

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.     

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.     

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.     

Expression of EPCAM,CD44 and CD24 in gastric cancer tissues and its clinical significance

AIM: To evaluate the relationship between epithelial cell adhesion molecule (EPCAM)/cluster of differentiation 44 (CD44)/cluster of differentiation 24 (CD24) expression and the clinicopathological characteristics/prognosis in 95 gastric cancer patients. METHODS: The expression levels of EPCAM, CD44 and CD24 were detected using the two-step method of immunohistochemistry in 95 gastric cancer patients who underwent surgical excision and were pathologically diagnosed as gastric cancer. RESULTS: There were 56 EPCAM-positive patients (58.95%), 41 CD44-positive patients (43.16%) and 56 CD24-positive patients (58.95%). Thirty patients were both EPCAM and CD44 positive (31.58%), 45 patients were both EPCAM and CD24 positive (47.37%), 32 patients were both CD44 and CD24 positive (33.68%), and 25 patients were EPCAM, CD44 and CD24 positive (26.32%). EPCAM expression was correlated with age, depth of tumor infiltration and WHO histological classification. CD44 expression was correlated with BORRMANN and WHO histological classification as well as CEA value. CD24 expression was correlated with the depth of infiltration, location of the tumor, WHO histological classification and viscera invasion. All positive expression of EPCAM, CD44 and CD24 was correlated with the depth of infiltration, location of the tumor and WHO histological classification (P<0.05). The difference of survival rate between EPCAM positive group and negative group was observed, and the CD44 positive group and negative group had the same result (P<0.05 and P<0.01, respectively). The difference of survival rate between EPCAM⁺CD44⁺CD24⁺ group and EPCAM^-CD44^-CD24^- group was statistically significant (P<0.05). The difference of survival rate between EPCAM^-CD44⁺CD24⁺ group and EPCAM^-CD44^-CD24^- group was also significant (P<0.05). CONCLUSION: The positive rates of EPCAM, CD44 and CD24 expression are high in gastric cancer tissues and these 3 proteins can be used as primary screening targets.     

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.     

Relationship between invasion of tumor-associated macrophages and phenotype and immune efficacy of tumor-infiltrating lymphocytes in advanced ovarian carcinoma

AIM:To explore the relationship between the invasion of tumor-associated macrophages(TAM) and the phenotype and immune efficacy of tumor-infiltrating lymphocytes(TIL) in advanced ovarian carcinoma. METHODS:Immunohistochemical analysis of TAM density in 175 cases of poorly-differentiated ovarian cancer tissue biopsy was performed. The cases were divided into TAM high-density(TAM^High) group and TAM low-density(TAM^Low) group according to the median of TAM density. The control group included 32 cases of benign ovarian lesions. The changes of CD8⁺ and CD25⁺ phenotypes of TIL were detected by flow cytometry analysis. TIL in the 2 groups were cultured in vitro and the conditioned-medium was collected for detecting the expression of IL-2, IL-10, TGF-β and IFN-γ by ELISA. RESULTS:The average TAM infiltration density was 62.8/high-power field(HP, ×400) in 175 cases of poorly-differentiated ovarian carcinoma, and the median was 53.3/HP. TAM^High group was 87 cases and TAM^Low group was 88 cases. A significant difference between malignant ovarian carcinoma group and control group(10.5/HP) was observed. The mean expression of CD8⁺ TIL in TAM^High group was 24%, and CD8⁺ TIL in TAM^Low group was 52%(P<0.05). The mean expression of CD25⁺ TIL in TAM^High was 48%, and CD25⁺ TIL in TAM^Low was 25%(P<0.05). The average infiltration density of CD8⁺ and CD25⁺ TIL in control group was 7%. The average infiltration density of CD8⁺ and CD25⁺ TIL in TAM^High and TAM^Low groups was significantly higher than that in control group(P<0.05). Compared with TAM^Low group, TIL destruction cytokines IL-2 and IFN-γ were significantly decreased in TAM^High group(P<0.05), while the inhibitory cytokines IL-10 and TGF-β were significantly increased(P<0.05). CONCLUSION:In high-density TAM infiltration of ovarian cancer tissues, CD25⁺ TIL type and inhibitory cytokines IL-10 and TGF-β increase, while CD8⁺ TIL type and destruction cytokines IL-2/IFN-γ decrease, suggesting that the high-density TAM has relationship with the phenotype and immune efficacy of TIL.     

Influence of histone deacetylase inhibitor romidepsin on phenotype and function of T lymphocytes in vitro

AIM: To explore the effects of romidepsin (FK228), a novel histone deacetylase inhibitor, on the effector and regulatory T cells in vitro.METHODS: As the reactive cells, lymphocytes, CD4⁺ T cells and CD8⁺ T cells were labelled with CFSE, and stimulated with anti-CD3 and anti-CD28 mAbs in the presence and absence of different levels of romidepsin (experimental group and positive control group), or PBS (placebo group).After 72 h, the proliferation of the cells was detected in different groups. The lymphocytes were stimulated with anti-CD3 and anti-CD28 mAbs in the presence and absence of different levels of romidepsin (experimental group and positive control group),or PBS (placebo group). After 72 h, the percentage of CD4⁺ Foxp3⁺ T cells and the levels of related cytokines were detected in different groups. RESULTS: The proliferation of CFSE-labelled lymphocytes, CD4⁺ T cells and CD8⁺ T cells triggered by anti-CD3 and anti-CD28 mAbs all were inhibited when cultured with romidepsin at concentrations of 1 μmol/L, 3 μmol/L and 5 μmol/L in a dose-dependent manner (P<0.05). Compared with placebo group, in the presence of anti-CD3 and anti-CD28 mAbs, 1 μmol/L romidepsin did not increase the percentage of CD4⁺ Foxp3⁺ T cells (P>0.05). When cultured with romidepsin at concentrations of 3 μmol/L and 5 μmol/L, the percentage of CD4⁺ Foxp3⁺ T cells was enhanced markedly (P<0.05). The levels of IL-10 and TNF-α in the supernatant were markedly increased in positive control group and 3 experimental groups (P<0.05), and the levels of cytokines in different experimental groups were gradually decreased with the elevation of FK228 concentration (P<0.05). The level of TGF-β was slightly increased in positive control group with no significant difference compared with placebo group (P>0.05). With the increase in the concentration of FK228 in different experimental groups, the TGF-β level was increased in a dose-dependent manner and there were significant differences in the 3 experimental groups. Meanwhile, significant differences existed between experimental groups and placebo group and between experimental groups and positive control group (P<0.05). CONCLUSION: Romidepsin inhibits the proliferation of CD4⁺ and CD8⁺ effector T cells and increases the percentage of CD4⁺ Foxp3⁺ regulatory T cells. It may be related to the increased level of TGF-β, but independent of IL-10.     

Effects of bortezomib on Treg cells in multiple myeloma and its relationship with tumor burden and chemoresponse

AIM: To investigate the changes of regulatory T (Treg) cells in peripheral blood from multiple myeloma (MM) patients with bortezomib-based treatment, and to analyze the effects of bortezomib on Treg cells in MM and the relationship with tumor burden and chemoresponse. METHODS: Treg cells from 30 newly-diagnosed MM patients were detected by flow cytometry before and after bortezomib-based treatment. Meanwhile, 12 cases with conventional treatment were enrolled as controls. The correlation of Treg cell count with the levels of M protein and serum β₂-microglobulin (β₂-MG) was then statistically analyzed. RESULTS: The patients in early disease stage had lower Treg cell count. Decreased Treg cell count was found in 31 cases with chemoresponse (P<0.01), which did not exist in the 11 cases without chemoresponse. The patients achieved >50% serum β₂-MG reduction showed more obvious changes of Treg cell count than those achieved ≤50% serum β₂-MG reduction (P<0.05). The patients with bortezomib-based treatment showed more apparent decline in Treg cell count than the patients with conventional treatment, which were related to chemoresponse and serum β₂-MG reduction (P<0.05). CONCLUSION: Treg cell count is a reliable index of tumor budern. Both bortezomib-related treatment and conventional treatment can reduce Treg cell count, but the reduction is more obvious in bortezomib-related treatment. Such reduction is related to chemoresponse and tumor burden, suggesting the mechanism of bortezomib curing MM may be by the way of Treg suppression.     

Detection and clinical significance of myeloid-derived suppressor cells in peripheral blood of patients with Parkinson disease

AIM: To detect the myeloid-derived suppressor cells (MDSCs) in peripheral blood from the patients with Parkinson disease (PD) and its clinical significance. METHODS: The patients (n=80) diagnosed PD from January 2016 to March 2017 in our hospital and 20 healthy volunteers were selected as the subjects. According to the Hoehn-Yahr staging, 80 PD patients were staged, of whom 22 were I, 24 were Ⅱ, 20 were Ⅲ, 14 were IV, and 0 was V. Peripheral blood (5 mL) samples from the patients with PD and the healthy volunteers were collected and the mononuclear cells were isolated. The levels of CD14⁺CD11b⁺ cells and CD14^-CD11b⁺ cells in the peripheral blood were detected by flow cytometry. The two populations of the cells were sorted by magnetic beads. The mRNA levels of arginase 1 (ARG1), interleukin-10 (IL-10) and cyclooxygenase 2 (COX-2) were detected by qPCR. The expression of surface membrane proteins CD14 and CD11b, and immunosuppressive factors ARG1, IL-10 and COX-2 was determined by Western blot and ELISA. RESULTS: No significant change of CD14⁺CD11b⁺ cells between the patients with PD and normal controls was observed, but the cells with CD14^-CD11b⁺ increased significantly in the patients with PD compared with the control people (P<0.05). The CD14^-CD11b⁺ cells in peripheral blood of the patients were related to the stage of Hoehn-Yahr. The CD14^-CD11b⁺ and CD14⁺CD11b⁺ cells showed high levels of IL-10 and COX-2, and the high level of ARG1 was only expressed in the CD14^-CD11b⁺ cells. The expression of ARG1 in the CD14^-CD11b⁺ population from PD patients was significantly different from that of CD14⁺CD11b⁺ population and normal subjects (P<0.05). CONCLUSION: The CD14^-CD11b⁺ cells and ARG1 expression level in peripheral blood of the PD patients can be used to evaluate the pathogenesis and staging. Immunosuppression may play an important role in the pathogenesis and development of PD.