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 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 approach the role of CD4+CD25+ regulatory T cells in the maintenance of immunotolerance in mouse liver allograft. MethodsThe mouse orthotopic liver transplantation was performed. After the liver transplantation immunotolerance induction, antiCD25 monoclonal antibody (PC61) was injected into the recipients with a delayed timing to remove the CD4+CD25+ T cells. The percentage of CD4+CD25+ T cells and the expression of forkhead/winged helix transcription factor (Foxp3) in the recipients were examined. Furthermore, the survival time of the recipient was observed. ResultsC3H/HeJ recipients receiving DBA/2 hepatic allografts survived over 70 d as in the syngeneic liver transplantation (C3H/HeJ recipients receiving C3H/HeJ hepatic grafts). With various protocols of the delayed PC61 treatment, the CD4+CD25+ T cell was completely disappeared as observed. However, the removal of CD4+CD25+ regulatory T cells after the induction of transplantation immunotolerance did not affect the survival of hepatic allografts. ConclusionCD4+CD25+ regulatory T cells are not essential for the maintenance of spontaneous mouse liver transplantation immunotolerance.
Objective To investigate the influence of T helpers 17 (Th17) cells, regulatory T (Treg) cells and their related cytokines on postoperative atrial fibrillation (POAF) after coronary artery bypass graft (CABG). Methods A total of 132 consecutive patients undergoing CABG between May 2013 and July 2016 were recruited. There were 82 males and 50 females with the age ranging from 39-76 years. Venous blood samples were collected within 2 hours after surgery. The expression of Th17 cells, Treg cells and their related cytokines in the peripheral blood was determined. Results POAF occurred in 35 patients (a POAF group) and 97 patients did not develop POAF (a No POAF group). Compared to the No POAF group, the proportion of Th17 cells and Th17/Treg ratio in the peripheral blood significantly increased in the POAF group (P>0.05) while proportion of Treg cells remained no significant change (P>0.05). The expression of Th17-related cytokines (IL-6, IL-8 and IL-17) all obviously increased in the POAF group (P>0.05). However, no significant difference was found in the expression of Treg-related cytokines (IL-10 and TGF-β) between the two groups (P>0.05). Conclusion Th17/Treg is unbanlanced in POAF patients and regulation of this imbalance may decrease the occurrence of POAF.
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.
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.
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.
ObjectiveThrough the analysis of quantitative and functional changes in peripheral blood CD4+ CD25+FOXP3+ regulatory T cells (Treg) of early HCC patients before and after operation, to discuss the operation effect on the immune function from the aspect of immune suppression. MethodsExtracted the lymphocytes of peripheral blood in HCC patients before and after operation (case group, n=15) and normal people (control group, n=5 cases), and analyze the number and function of Treg by flow cytometer after extracellular (CD4, CD25) and intracellular (FOXP3) staining. ResultsCD4+CD25+ T cells and CD25+FOXP3+ T cells in preoperative peripheral blood in case group were significantly higher than those in control group (12.43±2.57)% vs. (5.56±1.02)%, (5.14±1.4)% vs. (2.18±0.83)%, Plt;0.05). These two cells decreased at 1 week after operation. 〔(10.56±2.13)%, (4.28±1.08)%〕, but there was not statistically significant (Pgt;0.05), they decreased significantly at 2 weeks after operation 〔(7.30±0.89)%, (3.43±0.83)%， Plt;0.05〕. CD8+ T cells and CD4+CD25- T cells in preoperative peripheral blood in case group were significantly lower than those in control group 〔（23.42±1.80）% vs. （29.22±2.26）%， （36.14±1.12）% vs. （43.69±2.78）%， Plt;0.05〕， These two cells decreased significantly at 2 weeks after operation 〔（27.15±1.71）%， （40.30±2.00）%〕. The analysis on the Treg and AFP correlation found that they have low correlation (r=048, Plt;0.05 ). ConclusionsThe hepatectomy can improve the immune response of HCC patient. Treg may have a certain auxiliary significance in the diagnosis, treatment and prognosis of patients with hepatocellular carcinoma.
【Abstract】ObjectiveTo find a stable and efficient method for ex vivo concentration of CD4+CD25+ regulatory T cells in rats. MethodsCD4+CD25+ regulatory T cells were separated from the rat splenic cells in two steps by magnetic cell sorting (MACS) system. CD4+ T cells were first negatively sorted by cocktail antibodies and antiIgG magnetic microbeads. And then CD4+CD 25+T cells were positively sorted by antiCD25 PE and antiPE magnetic microbeads. The purity and the cell survival rate of the sorted cells were measured by flow cytometry and trypan blue dyeing respectively, and the immunosuppressive action of CD4+CD25+ T cells on the proliferation of CD4+CD25- T cells was also assessed by in vitro cell proliferation assay. ResultsThe purity of negatively sorted CD4+ T cells were (83.6±2.5)% (79%~87%), and the purity of positively sorted CD4+CD25+ T cells was (90.2±1.8)% (86%~93%) with the survival rate of (92.8±3.4)% (92%~95%). These concentrated cells significantly suppressed the proliferation of CD4+CD25- T cells in mixed lymphocyte culture （CD25+/CD25- versus CD25-, P＜0.01）. ConclusionWe created a twostep procedure of magnetic cell sorting system for CD4+CD25+ regulatory T cells sorting, which insures the cells to be satisfactorily purified and well functioned.