Objective To investigate the changes of CD4 + CD25 + Foxp3 + regulatory T cells( Treg) in peripheral blood of patients with acute exacerbation of COPD( AECOPD) , and analyze the relationship of CD4 + CD25 + Foxp3 + Treg with insulin resistance. Methods A total of 79 patients with AECOPD were divided into four groups according to disease severity( 11 cases in stage Ⅰ,31 cases in stage Ⅱ,28 cases in stage Ⅲ, an 9 cases in stage Ⅳ) .42 healthy volunteers were recruited as control. Fast blood glucose( FBG) and fast insulin( FINS) were measured for calculating the insulin resistance index. The CD4 + CD25 + Foxp3 + Treg were detected by flow cytometry. The relationship between the proportion and number of CD4 + CD25 + Foxp3 + Treg with insulin resistance was statistically analyzed. Results Compared with the healthy control group, the levels of FBG, FINS, and insulin resistance index in the AECOPD patients were significantly higher ( P lt; 0. 01, P lt; 0. 05) . The proportion and number of CD4 + CD25 + Foxp3 + Treg in peripheral blood decreased significantly( P lt; 0. 01, P lt; 0. 05) . The insulin resistance index increased with the severity of AECOPD while the proportion and number of CD4 + CD25 + Foxp3 + Treg in peripheral blood decreased. The insulin resistance index in the AECOPD patients of stage Ⅲ and Ⅳ were higher than those of stage Ⅰ and Ⅱ. The proportion and number of CD4 + CD25 + Foxp3 + Treg in the AECOPD patients of stage Ⅲ and Ⅳ were significantly lower than those of stage Ⅰ and Ⅱ. Both the proportion and number of CD4 + CD25 + Foxp3 + Treg were negatively correlated with insulin resistance ( r = - 0. 633, - 0. 871, P lt; 0. 01) . Conclusions CD4 + CD25 + Foxp3 + Treg cells might may play important role in modulating insulin resistance in AECOPD. The more serious the disease, the lower the CD4 + CD25 + Foxp3 + Treg and the worse insulin resistance.
Objective To assess the effects of different immunosuppressive drugs on proliferation and function of regulatory T cells (Tregs). Methods We searched MEDLINE (1966 to November 2009), EMbase (from inception to September 2009), and The Cochrane Library (Issue 4, 2009) for clinical and basic research about the effects of various immunosuppressive drugs on Tregs. Data were extracted and methodological quality was assessed by two independent reviewers. Outcome measures for clinical research included blood Tregs levels, acute rejection episodes, and graft function. Outcome measures for basic research included percentage of Tregs proliferation, function, Tregs phenotype, and evidence for possible mechanisms. We analyzed data qualitatively. Results Forty-two studies, including 19 clinical trials and 23 basic studies, were included. The immunosuppressive drugs studied were calcineurin inhibitors (CNIs), Rapa, anti-metabolism drugs, IL-2 receptor-blocking antibodies, T-cell depleting antibodies, and co-stimulation blockade antibodies. Most of the studies were on Rapa and CNIs. Eight basic studies on Rapa and CNIs showed that Rapa could promote the proliferation and function of Tregs, while CNIs could not. Five clinical trials involving a total of 158 patients showed that patients taking Rapa had higher blood concentration of Tregs than those taking CNIs, but no differences were found in graft function (6-42-month follow-up). Conclusion There is substantial evidence that Rapa favors Tregs survival and function. However, the larger number of the blood Tregs in the patients treated with Rapa does not show any correlation with better graft function. Large-sample and high-quality clinical studies with longer follow-up are needed to thoroughly assess the efficacy of immunosuppressive drugs on Tregs and to reveal whether a relationship exists between Tregs and graft function.
Objective To establish a method to isolate the CD4+CD25+ regulatory T cells (Tregs) and to identify the purity and function of these cells. Methods The peripheral blood (8 mL) were collected from the great saphenous vein of 10 rhesus monkeys (4 females and 6 males, aged 4-5 years, and weighing 5-8 kg). The mononuclear cells were isolated with density gradient centrifugation. CD4+ T cells were separated by the Magnetic cell sorting (MACS) negative selection and MACS positive selection. The cell yield rate, the cell viability, and the cell purity were compared between MACS negative selection and MACS positive selection. In CD4+ MACS negative selection, the anti-biotin MicroBeads and biotin-antibody cocktai in CD4+CD25+ Tregs isolation kit non-human primate were used, and in MACS positive selection, the anti-APC MicroBeads in CD4+CD25+ Tregs isolation kit non-human primate and CD4-APC were used. The CD4+ T cells separated by positive selection were selected to obtain CD4+CD25 Tregs with CD25 MicroBeads. The purity, activity, the FoxP3 level, and the suppressive function to concanavalin A (ConA) activated autologous CD4+CD24- effective T cells (Teffs) of CD4+CD25+ Tregs were detected by flow cytometry. Results After CD4+ T cells were separated by MACS negative selection and MACS positive selection, the cell viabilities were all up to 95%, showing no significant difference (P gt; 0.05). The cell yield rate and purity of CD4+ T cells by positive selection were significantly higher than those of CD4+ T cells by negative selection (P lt; 0.05). CD4+CD25+ Tregs can be successfully isolated by MACS double positive selection. The classifying purity was 76.2% ± 8.6%; the cell activity was 93.3% ± 4.7%; and the level of FoxP3 was 74.2% ± 6.9%. The CD4+CD25+ Tregs had suppressive effect on ConA activated autologous CD4+CD25- Teffs. Conclusion MACS double positive selection can be used to isolate high-purity CD4+CD25+ Tregs from the peripheral blood of rhesus monkeys and the process does not influence the activity of CD4+CD25+ Tregs.
Objective To investigate the effects of ulinastatin on Treg/Th17 and immune status in patients with severe sepsis.Methods A total of 80 patients with severe sepsis, who were hospitalized in ICU during October 2011 to July 2012, were randomly divided into a routine group and a ulinastatin group. The patients in the ulinastatin group were intravenously administered 30mg ulinastatin three times per day for 5 days in addition to routine bundle treatment. The expression of Treg, Th17 and HLA-DR were detected on the first day in ICU and 5 days after treatment. 20 healthy individuals served as controls. Results Compared with the control group, the severe sepsis group had overexpression of Treg and Th17 ( P lt;0. 01) , higher ratio of Treg/Th17( P lt;0. 01) , and decreased HLA-DR expression of CD14 monocyte ( P lt; 0. 01) . In the severe sepsis patients, ulinastatin injection reduced the abnormal expression of Treg and Th17 ( P lt; 0. 01) , decreased the ratio of Treg/Th17( P lt; 0. 01) , and improved the expression of HLA-DR ( P lt; 0. 01) more effectively compared with the routine treatment. Ulinastatin also lowered 28-day mortality of the patients with sepsis, but the difference between the ulinastatin group and the routine group was not significant. Conclusions In severe sepsis patients, there were abnormal overexpression of Treg and Th17, imbalance of Treg/Th17, and underexpression of HLA-DR which imply an immune suppression. Ulinastatin can decrease the expression of Treg and Th17, inverses the ratio of Treg/Th17, and improve the expression of HLA-DR, so as to improve the prognosis of severe sepsis patients.
Objective To examine the expression of promoter CpG island methylation of Notch1 gene and explore the variable sites for DNA methylation in lung CD4 + T cells of asthmatic rat models.Methods An ovalbumin ( OVA) sensitized- challenged asthmatic rat model was established. Total T cells were isolated and CD4 + T lymphocytes were purified using magnetic beads. Twenty Wistar rats were randomly divided into a control group and an asthma group ( n = 10 in each group) . CD4 + T cells were isolated by immunomagnetic beads and identified by flow cytometry ( FCM) . Realtime PCR was employed to examine the mRNA expression of Notch1 gene in lung CD4 + T cells and the methylation level of Notch1 gene was examined by methylation-specific PCR. Results The mRNA expression of Notch1 in lung CD4 + T cells of the asthma group was 2. 254 ±0. 403 times as much as that of the control group. The total methylation level of asthma group was lower than that of the control group ( 0. 150 ±0. 108 vs. 0. 300 ±0. 667, P lt;0. 01) . Conclusion Promoter demethylation is one of the major mechanisms of Notch1 gene up-regulation in pathogenesis of asthma.
Objective To compare the difference in the expressions of forkhead box protein 3 (FoxP3) and adenosine 2a receptor (A2aR) in gastric cancer tissues and its adjacent tissues, and to investigate the relationship between the elevated expression of FoxP3/A2aR and clinicopathological features in gastric cancer. Methods Gastric cancer tissues and their adjacent tissues from 52 patients with gastric cancer were collected, who underwent surgery in the Affiliated Hospital of Xuzhou Medical University from July 2015 to November 2016, immunohistochemical staining was used to detect the expressions of FoxP3 and A2aR. Results ① The high-expression rate of FoxP3 in gastric cancer tissues was 69.2% (36/52), which was higher than that of adjacent tissues (11.5%, 6/52), P<0.001. The high-expression rate of A2aR in gastric cancer tissues was 69.2% (36/52), which was higher than that of adjacent tissues (25.0%, 13/52),P<0.001. ② The expression of FoxP3 was positively correlated with the expression of A2aR in gastric cancer tissues (r=0.76, P<0.05). ③ In gastric cancer tissues, high-expressions of FoxP3 and A2aR were not related to gender, age, diameter of tumor, tumor location, degree of differentiation, gross type, and histological type (P>0.05), but both associated with TNM stage, T stage, number of lymph node metastasis, and distant metastasis (P<0.05), the high-expression rates of FoxP3 and A2aR in patients with stage Ⅲ+Ⅳ were higher than those of patients with stage Ⅰ+Ⅱ, the high-expression rates of FoxP3 and A2aR in patients with stage T3+T4 were higher than those of patients with stage T1+T2, the high-expression rates of FoxP3 and A2aR in patients with distant metastasis were higher than those of patients without distant metastasis, and the high-expression rates of FoxP3 and A2aR increased gradually with the increase in the number of lymph node metastasis. Conclusion There are high expressions of FoxP3 and A2aR in gastric cancer tissues, and both of them may play important role in promoting the occurrence and development of gastric cancer.
Objective To investigate the percentage of CD4+CD25+ Treg in peripheral blood of patients with severe multiple trauma and systemic inflammatory response syndrome（SIRS） and its effects on cellular immunity and secondary infection.Metheds Peripheral blood of 23 patients with severe multiple trauma was collected in 24 h after SIRS was diagnosed，and flow cytometry was used to determine the percentage of CD4+CD25+ Treg and CD4/CD8 ratio.Simultaneously，in order to explore the cell proliferation，silver staining was used to determine Ag-NORs of leukomonocyte in peripheral blood represented by IS%.In order to investigate the infection in patients，sputum and secretion sample were collected for bacteriological examination on 1 and 5 day after SIRS was established.Forty healthy volunteers were enrolled as control.Results Compared with the control，the percentage of CD4+CD25+ Treg was significant higher[（14.21±3.43）% vs（9.53±3.22），Plt;0.01] and the ratio of CD4/CD8 and IS% were significant lower in patients with severe multiple trauma[（5.94±0.66）% vs（6.74±0.95）%，（1.22±0.25）% vs（1.72±0.36）%，respectively，both Plt;0.01].In those patients（n=14） who developed secondary infection，Treg% was significant higher [（18.69±4.21）% vs（12.58±2.49）%，Plt;0.01]，while IS% and CD4/CD8 were significant lower [（5.79±0.68）% vs（6.15±0.57）%，（1.15±0.25）% vs（1.39±0.25）%，both Plt;0.01].compared to the patients without secondary infection Conlusion CD4+CD25+ Treg is valuable to estimate the cellular immunity and predict secondary infection in patients with severe multiple trauma.
Objective To investigate the role of T helper 17 ( Th17) cells and CD4 + CD25 + Foxp3+regulatory T cells ( Treg) in the pathogenesis of asthma in a mouse model. Methods Twenty-four BALB/ c mice were randomly divided into an asthma group and a normal control group, with 12 mice in each group.Asthma model was established by ovalbumin sensitization and aerosol challenge in the asthma group. Airway reactivity was measured by plethysmography. The total and differential cell counts in bronchoalveolar lavage fluid ( BALF) were measured. The ratio of Th17 and Treg cells to mononuclear cells in the spleens of mice were detected by flow cytometry. The levels of IL-17 and IL-10 in BALF and lung homogenates were measured by ELISA. Results The bronchial provocation test showed that the average lung resistance increased remarkably in the asthma group. In spleens of the asthmatic mice, the percentage of Th17 cells was significantly higher [ ( 5.68 ±1. 99)% vs ( 2.80 ±0. 82) %, P lt; 0. 01] , and the percentage of Treg cells was significantly lower [ (2.88 ±0. 46) % vs ( 6.10 ±2.44) % , Plt; 0. 01] , with the ratio of Th17 to Treg significantly increased( 1. 93 ±0. 41 vs 0. 50 ±0. 15,P lt;0. 01) . In BALF and lung homogenates of the asthma group, the level of IL-17 was significantly higher[ ( 22. 37 ±3. 00) pg/mL vs ( 11. 42 ±2. 15) pg/mL, ( 52. 93 ±5. 39) pg/mL vs ( 19. 38 ±2. 65) pg/mL, both Plt; 0. 01] , and the level of IL-10 was significantly lower[ ( 6. 05 ±1. 25) pg/mL vs ( 14. 23 ±2. 94) pg/mL, ( 9. 33 ±1. 79) pg/mL vs ( 21. 40 ±2. 44) pg/mL, both P lt; 0. 01] compared with the control group.Conclusion The imbalance of Th17/ Treg plays an important role in the pathogenesis of asthma.
Objective To investigate the suppression effect and mechanism of Astilbin on lung allograft rejection in rats, in order to know the function of Astilbin on rats’ lung acute rejection. Methods The model of rat left lung transplantation was set up. Sixty lung transplanted rats were divided into two groups randomly, control group: rats were fed with normal saline 1ml per day, experimental group: rats were fed with Astilbin 1mg/kg per day. Survival time, transforming rate of T cells in spleen, activity of interleukin 2 (IL-2) in spleen lymph cells and apoptosis of T cells were observed. Changes in ultrastructure of pulmonary arteries were observed by electron microscope. Results The survival time in experimental group was prolonged than that in control group (25.4±2.1 d vs. 13.4±1.2 d;t=2.042, Plt;0.05）. Transforming rate of T cells of spleen in experimental group was significant lower than that in control group (23 465.8±8 783.4 cpm vs. 74 567.3±12 874.6 cpm; t=2.284，Plt;0.05）.Activity of IL-2 of spleen lymph cells in experimental group was significant lower than that in control group (425±2.65U/ml vs. 23.46±1.82U/ml; t=3.165, Plt;0.01).Effectively derive apoptosis of activated T cells in acute rejection were observed in experimental group, the ultrastructure of pulmonary arteries showed attenuated injury in experimental group. Conclusion Astilbin decreased the IL-2 concentration in plasma and induced the apoptosis in activated T cells, then suppressed the acute rejection of lung allograft and prolonged the survival period of lung transplantation rats.
ObjectiveTo investigate the proportion of peripheral blood CD4+CD25+ regulatory T cells (Tregs) in patients with pancreatic head carcinoma, the dynamic changes of these cells before and after pancreatoduodenectomy were also analyzed. MethodsThe proportions of peripheral blood CD4+CD25+ Tregs in patients with pancreatic head carcinoma and normal individuals were examined by using flow cytometric analysis. The CD4+/CD8+ ratio was also studied before and after operation. ResultsThe patients with pancreatic head carcinoma showed higher ratio of CD4+CD25+ and CD4+CD25high Tregs compared with normal control before operation (Plt;0.05). However, the percentage of these T cells reduced significantly after pancreatoduodenectomy, which was most obviously on the 3rd day after operation (Plt;0.01, Plt;0.05). After operation, CA199 level began to decrease, which was obvious on the fourteen day after operation. This tendency of CD4+CD25high Tregs changes was similar to that of CA199. The patients showed an decreased ratios of CD4+/CD8+ compared with normal controls, which further declined after operation, and reached the lowest point on the seventh day after operation (Plt;0.05). ConclusionsPancreatoduodenectomy may be helpful for the recovery of antitumor immunity. The perioperative period of patients with pancreatic head carcinoma may be a beneficial windowphase for immune intervention and Tregs may be served as target cells.