【 Abstract】 Objective To construct a lentiviral expression vector carrying Nogo extra cellular peptide residues 1-40(NEP1-40) and to obtain NEP1-40 efficient and stable expression in mammalian cells. Methods The DNA fragment ofNEP1-40 coding sequence was ampl ified by PCR with designed primer from the cDNA l ibrary including NEP1-40 gene, and then subcloned into pGC-FU vector with in-fusion technique to generate the lentiviral expression vector, pGC-FU-NEP1-40. The positive clones were screened by PCR and the correct NEP1-40 was confirmed by sequencing. Recombinant lentiviruses were produced in 293T cells after the cotransfection of pGC-FU-NEP1-40， and packaging plasmids of pHelper 1.0 and pHelper 2.0. Green fluorescent protein (GFP) expression of infected 293T cells was observed to evaluate gene del ivery efficiency. NEP1-40 protein expression in 293T cells was detected by Western blot. Results The lentiviral expression vector carrying NEP1-40 was successfully constructed by GFP observation， and NEP1-40 protein expression was detected in 293T cells by Western blot. Conclusion The recombinant lentivirus pGC-FU-NEP1-40 is successfully constructed and it lays a foundation for further molecular function study of NEP1-40.
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 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 expression of the histone deacetylases 1( HDAC1) and the level of whole histone acetylation and methylation in lung T cells of asthmatic rats, and investigate their role in the pathogenesis of asthma.Methods Sixteen wistar rats were randomly divided into a control group and an asthma group( n =8 in each group) . The rats was sensitized with ovalbumin( OVA) and challenged with aerosol OVA to establish asthma model. The asthmatic ratmodel was confirmed by measurement of pulmonary function, histochemical staining, HE staining, and the levels of interleukin-4 ( IL-4 ) , interferon-gamma ( IFN-γ) and immunoglobulin E( IgE) in serum and bronchoalveolar lavage fluid ( BALF) . T cells were isolated fromrat lungs and the purity was identified. The expression of the HDAC1, the level of whole histone H3 and H4 acetylation, and whole H3K9 dimethylation were analyzed by Western blot in lung T cells. Results Compared with the control group, the protein expression of HDAC1 was significantly lower( 0. 465±0. 087 vs 0. 790 ±0. 076, P lt;0. 05) in lung T cells of the asthma group. No significant differences werefound in regard to the level of whole histone H3 and H4 acetylation and whole H3K9 dimethylation betweenthe two groups. Conclusions HDAC1 in lung T cells may be involved in the pathogenesis of asthma.Histone modification by HDAC1 may be a specific eventwith gene transcription which may not be associated with asthma.
ObjectivesTo systematically review the clinical response rate of CD19 chimeric antigen receptor modified-T cells (CD19CART) in the treatment of B cell hematological malignancies.MethodsPubMed, EMbase, CNKI, WanFang Data and VIP databases were searched to collect cohort studies about CD19CART in the treatment of B cell hematological malignancies from 2000 to 2016. Two reviewers independently screened literature, extracted data and assessed the risk of bias of included studies. Then, a single rate meta-analysis was performed by R software and SPSS 16.0 software.ResultsA total of 13 prospective cohort studies were included. The results of single group rate meta-analysis showed that the overall pooled response rate of CD19 CART was 68% (95%CI 0.51 to 0.82). The 6 months and 1-year PFS after CD19 CART infused by Kaplan-Meier were 46% (95%CI 0.35 to 0.56) and 24% (95%CI 0.16 to 0.34), respectively. The median duration was 180 days (95%CI 138 to 222). The COX regression model showed lymphodepletion to be the only influence factor of PFS.ConclusionsCD19 CART has a good clinical response rate in the treatment of B cell hematological malignancies. Lymphodepletion is the only important impact on the response rate and PFS. Due to limited quality and quantity of included studies, more high quality studies are required to verify the above conclusions.
Objective To summarize research progress of the mechanism of natural killer cells (NK cells) acted in regulating the T cell immunity in chronic infectious disease. Method Literatures about recent studies concerning how NK cells act as a regulator for T cells in chronic infectious disease were reviewed according to the results obtained from PubMed, Embase, CNKI, CBM, and Wanfang databases. Results NK cells that acted as regulators of T cell immunity could affect T cell immune responses through influencing antigen presentation, secreting cytokine, and presenting lytic activities, thus playing an important role in the immunological therapy of chronic infectious diseases. Conclusion NK cells are critical for T cell immune regulation, which could provide noval strategies for immunological therapy of chronic infectious disease, transplantation-related immune rejection, and autoimmune disease.
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 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 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.
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.