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.
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 relationship between blood CD4 + CD25 + regulatory T cells ( Treg cells) and cell immunity in patients with sepsis and its prognostic value.Methods 27 patients with sepsis admitted during August 2007 and August 2008 in ICU were enrolled, while 40 healthy volunteers served as control. According to the clinical outcome after 28 days’ treatment, the sepsis patients were assigned to a death group( n=8) and a survival group ( n =19) . Blood Treg% and CD4 /CD8 were detected by flow cytometry and total AgNOR area/nucleus area per cell ( IS%) was measured by silver nitrate staining and image processing. Results The Treg% in the patients with sepsis was significant higher than that in the normal control [ ( 5. 61 ±1. 60) % vs. ( 0. 78 ±0. 23) % , P lt; 0. 01 ] , while the level of CD4 /CD8 and IS% were significant lower[ CD4 /CD8: ( 1. 09 ±0. 30) vs. ( 1. 71 ±0. 36) , IS% : ( 5. 19 ±1. 07) % vs. ( 6. 76 ±0. 92) % , both P lt; 0. 01] . Significant correlations were found between Treg% and CD4 /CD8( r= - 0. 484, P lt;0. 01) , and between Treg% and IS% ( r = - 0. 588, P lt;0. 01) . Compared with the survival group, Treg% was significant higher [ ( 7. 09 ±1. 17) % vs. ( 5. 00 ±1. 33) % , P lt; 0. 01] , and CD4 /CD8 and IS% were significant lower[ CD4 /CD8: ( 0. 87 ±0. 22) vs. ( 1. 18 ±0. 29) , IS% : ( 3. 97 ±0. 42) % vs. ( 5. 71 ±0. 81) % , both P lt; 0. 01] in the death group. Conlusion Blood Treg% level can reflect the cell immune state of patients with sepsis and is of clinical value to assess the prognosis.
Objective To investigate the efficacy of continuous blood purification ( CBP) in the treatment of severe sepsis, and explore the related immune regulatory mechanisms. Methods Forty-eight patients with severe sepsis were randomly divided into a control group ( n =23) and a CBP group ( n =25) .CD4 + CD25 + regulatory T cells ( Treg% ) in peripheral blood and APACHEⅡ score were measured dynamically before treatment and 12, 24, 36, 48, 60, 72 hours after treatment. Meanwhile the length of ICUstay, duration of mechanical ventilation, and 28 day mortality were determined. Results Compared with the control group, the length of ICU stay, ventilator time, incidence of multiple organ failure, and mortality decreased significantly in the CBP group ( P lt; 0. 05) . And CBP also decreased Treg% and APACHEⅡ score significantly. There was a positive correlation between Treg% and APACHEⅡ score ( r =0. 804, P lt;0. 01) .Conclusion Early CBP treatment can reduce Treg%, improve cellular immunity and improve the prognosis of sepsis.
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 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 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 investigate the anti-rejection effect and the mechanism of triptolide （TPT） on islet allo- grafts in a murine model. Methods BALB/c mice were used as islet donor. C57BL/6 mice were rendered diabetic by streptozotocin （STZ） injection， and transplanted with islets under the left kidney capsule. The recipients were randomly （method of random digits table） divided into three groups （n=8）. The mice in the treatment groups were injected intrap-eritoneally with TPT at 50 μg/kg （low-dose TPT group， L-TPT group） or 100 μg/kg （high-dose TPT group， H-TPT group） daily in the first 5 days and then on alternate days until 14 days；while the mice in control group were given vehicles （1% tween 80）. Blood glucose after operation were monitored. The grafts were defined as rejection when two consecutive reading of blood glucose＞20 mmol/L. The left kidney of three recipients in each group were resected for pathological examination. The proportion of CD4＋CD25＋Foxp3＋ regulatory T cells in spleen tissues were tested by flow cytometry. Results The median survival time of islet allografts from the control group， L-TPT group， and H-TPT group were 12.6 days （9-16 days）， 21.4 days （14-27 days） ， and 27.6 days （19-34 days）， respectivly. The percentageof CD4＋CD25＋Foxp3＋regulatory T cells in spleen tissues of three groups were （5.2±0.6）%， （12.0±1.3）%， and（15.7±1.8）%， respectivly. Compared with control group， the median survival time of islet transplantation in mice exte-nded and the proportion of CD4＋CD25＋Foxp3＋ regulatory T cells in spleen tissues increased （P＜0.05）. Conclusions TPT could increase the percentage of CD4＋CD25＋Foxp3＋ regulatory T cells， reduce the rejection after islet transplanta-tion， and prolong the survival time of islet transplantation in mice. The immunosuppressive effect of TPT shows a dose-dependent.
ObjectiveTo investigate the levels of regulatory T cells (Treg) and FoxP3 gene in patients with gastric cancer before and after operation. MethodsTwenty patients with definite diagnosis of gastric cancer and 15 healthy volunteers were selected. The levels of Treg and T cell subsets in peripheral blood were determined by detecting of CD4 and CD25 with immunefluorescence stain and flow cytometry, the expressions of FoxP3 mRNA in these Treg were detected by RTPCR technique. The expression of FoxP3 protein in the gastric cancer tissue was measured by immunohistochemistry assay. ResultsThe percentage of Treg cells in total CD4+ T isolated from the patients with gastric cancer was higher than that of healthy volunteers 〔(19.39±5.58)% versus (9.91±3.23)%, Plt;0.01〕, and it markedly decreased after operation 〔（13.50±5.93）% versus (19.39±5.58)%, Plt;0.05〕. The FoxP3 mRNA expression in the patients with gastric cancer was also higher than that of healthy volunteers (0.86±0.03 versus 0.64±0.02, Plt;0.01), and decreased after operation (0.73±0.04 versus 0.86±0.03, Plt;0.05). The percentage of CD4+T cell in mononucleocytes of peripheral blood of patients with gastric cancer was significantly lower than that of healthy volunteers (Plt;0.01), but the difference was not significant between before and after operation. FoxP3 protein expressed in cytoplasm of 13 patients with gastric cancer, in which bly positive in 2 cases, middle positive in 6 cases, weakly positive in 5 cases. FoxP3 protein didn’t express in cytoplasm of 7 patients with gastric cancer. ConclusionsTreg may have a significant effect on the onset and development of gastric cancer through immunosuppressive effect. Tumor tissue is an important initiating agent on Treg proliferation.