Neutrophils mediate T lymphocyte function in septic mice via the CD80/cytotoxic T lymphocyte antigen-4 signaling pathway / 中华危重病急救医学

Objective:To investigate the effect of neutrophils on T lymphocyte function in septic mice and the role of CD80/cytotoxic T lymphocyte antigen-4 (CTLA-4) signaling pathway in this modulated effects.Methods:① In vivo experiment: 6-8 weeks old male C57BL/6 mice were divided into sham operation group (Sham group, n = 20), Sham+CTLA-4 antibody treatment group (Sham+aCTLA-4 group, n = 20), cecal ligation and perforation (CLP) induced sepsis model group (CLP group, n = 30) and CLP+CTLA-4 antibody treatment group (CLP+aCTLA-4 group, n = 30) according to the random number table. CLP was used to reproduce mouse sepsis model. The mice in the Sham group were treated identically but their cecums were neither punctured nor ligated. In CTLA-4 antibody treatment groups, 50 μg CTLA-4 antibody was injected intraperitoneally 6 hours and 24 hours after the operation. Forty-eight hours after operation, 6 mice in Sham group and Sham+aCTLA-4 group, 14 mice in CLP group and CLP+aCTLA-4 group were randomly selected to detect the expression of CD69 in spleen. At the same time, spleen, bone marrow and peripheral blood were collected, and the expression of CD80 on neutrophils was detected by flow cytometry. The expression of CTLA-4 on the surface of T lymphocytes in spleen was detected by immunofluorescence and flow cytometry. The remaining mice in each group were used to observe the 96-hour survival after operation.② In vitro experiment 1: neutrophils were extracted from bone marrow of healthy mice and stimulated with LPS (1 mg/L) for 4, 8 and 12 hours respectively. The control group was added with the same amount of phosphate buffered saline (PBS) at each time point, and the expression of CD80 was detected at each time point.③ In vitro experiment 2: splenic T lymphocytes of healthy mice were extracted and divided into PBS control group, LPS group (final concentration of LPS 1 mg/L), neutrophil group and neutrophil+LPS group. In the latter two groups, the co-culture model of neutrophils and T lymphocytes was established, and then the corresponding treatment was given to detect the expression of CTLA-4 on the surface of T lymphocytes. With the above four groups as controls, CTLA-4 antibody treatment groups (final concentration of CTLA-4 antibody 50 mg/L) were set up respectively. After 48 hours, the level of interleukin-2 (IL-2) in the cell supernatant was detected by enzyme linked immunosorbent assay (ELISA). Results:① Results of in vivo experiment: compared with Sham group, the expression of CD80 on neutrophils in spleen, bone marrow and peripheral blood was significantly up-regulated, while the expression of CTLA-4 on the surface of T lymphocytes was significantly increased [(9.98±0.84)% vs. (3.48±0.64)%, P < 0.05]. It suggested that neutrophils may affect T lymphocytes function through CD80/CTLA-4 pathway in sepsis. Compared with CLP group, CTLA-4 antibody could significantly improve the 96-hour cumulative survival rate of CLP mice (56.25% vs. 18.75%, P < 0.05), and increase the expression of CD69 on the surface of T lymphocytes. It suggested that CTLA-4 antibodies might increase T lymphocytes activation in sepsis and improve survival. ② Results of in vitro experiment: with the prolongation of LPS stimulation, the expression of CD80 on neutrophils gradually increased in time-dependent manner as compared with PBS control group [4 hours: (6.35±0.40)% vs. (3.41±0.40)%, 8 hours: (8.57±0.64)% vs. (3.09±0.27)%, 12 hours: (19.83±1.06)% vs. (5.16±0.36)%, all P < 0.05]. Compared with PBS control group, the expression of CTLA-4 on CD4 +/CD8 + T lymphocytes was not significantly affected by LPS stimulation alone, but CTLA-4 was increased after co-culture with neutrophils [CD4 +: (4.92±0.30)% vs. (3.33±0.25)%, CD8 +: (4.26±0.21)% vs. (2.53±0.66)%, both P < 0.05], and the increased trend of CTLA-4 was more obvious after co-culture with LPS-stimulated neutrophils [CD4 +: (6.34±0.50)% vs. (3.33±0.25)%, CD8 +: (6.21±0.41)% vs. (2.53±0.66)%, both P < 0.05]. In the PBS control group and LPS group, CTLA-4 antibody had no significant effect on IL-2 secretion of T lymphocytes. Compared with PBS control group, co-culture with neutrophils could inhibit the secretion of IL-2 by T lymphocytes (ng/L: 1 938.00±68.45 vs. 2 547.00±218.00, P < 0.05), and the inhibitory effect of neutrophils stimulated by LPS was more obvious (ng/L: 1 073.00±34.39 vs. 2 547.00±218.00, P < 0.05). CTLA-4 antibodies could partially restore IL-2 secretion. In conclusion, after promoting the expression of CTLA-4 on the surface of T lymphocytes, neutrophils might mediate the inhibition of T lymphocytes function by reducing the production of IL-2. Conclusions:Neutrophils mediate T lymphocytes dysfunction in sepsis, and the CD80/CTLA-4 pathway plays an important role. The CTLA-4 antibody improves survival and T lymphocytes function in sepsis mice, which may be a new method of immunotherapy for sepsis.

Delivery strategies for macromolecular drugs in cancer therapy

With the development of biotherapy, biomacromolecular drugs have gained tremendous attention recently, especially in drug development field due to the sophisticated functions . Over the past few years, a motley variety of drug delivery strategies have been developed for biomacromolecular drugs to overcome the difficulties in the druggability, ., the instability and easily restricted by physiologic barriers. The application of novel delivery systems to deliver biomacromolecular drugs can usually prolong the half-life, increase the bioavailability, or improve patient compliance, which greatly improves the efficacy and potentiality for clinical use of biomacromolecular drugs. In this review, recent studies regarding the drug delivery strategies for macromolecular drugs in cancer therapy are summarized, mainly drawing on the development over the last five years.

Research progress on the application of LAG-3 and its inhibitors in cancer immunotherapy / 中国肿瘤生物治疗杂志

@# Lymphocyte-activation gene 3 (LAG-3), also known as CD223, is a 498-amino-acid type I transmembrane protein encoded by LAG-3 gene, which consists of extracellular, transmembrane and intracellular regions.LAG-3 negatively regulates T lymphocyte by binding extracellular domain to ligand, thus avoiding autoimmunitycaused by T cell over-activation. Like programmed cell death 1 (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA-4), LAG-3 is an important immune checkpoint in vivo and plays a balanced regulatory role in human immune system.Tumor cells escape the surveillance of the immune system by over-expressing LAG-3 ligand. With the development in research of immune checkpoints, LAG-3 has become a new generation of immunotherapy targets after PD-1 and CTLA-4. This article reviews the structure and function of LAG-3 and the application of its inhibitors in tumor immunotherapy, in order to provide reference for the further study of LAG-3.

Immunostimulatory monoclonal antibodies for hepatocellular carcinoma therapy: Trends and perspectives

Hepatocellular carcinoma (HCC) is the second cause of cancer-related death in the world and is the main cause of death in cirrhotic patients. Unfortunately, the incidence of HCC has grown significantly in the last decade. Curative treatments such as surgery, liver transplantation or percutaneous ablation can only be applied in less than 30% of cases. The multikinase inhibitor sorafenib is the first line therapy for advanced HCC. Regorafenib is the standard of care for second-line patients. However, novel and more specific potent therapeutic approaches for advanced HCC are still needed. The liver constitutes a unique immunological microenvironment, although anti-tumor immunity seems to be feasible with the use of checkpoint inhibitors such as nivolumab. Efficacy may be further increased by combining checkpoint inhibitors or by applying loco-regional treatments. The success of immune checkpoint blockade has renewed interest in immunotherapy in HCC.

El hepatocarcinoma (HCC) es la segunda causa de muerte relacionada con el cáncer en el mundo y es la principal causa de muerte en pacientes cirróticos. Desafortunadamente, la incidencia de HCC ha crecido significativamente en la última década. Los tratamientos curativos como la cirugía, el trasplante de hígado o la ablación solo pueden aplicarse en menos del 30% de los casos. El sorafenib es el tratamiento de primera línea para el HCC avanzado, mientras que el regorafenib se reserva como segunda línea. Sin embargo, todavía son necesarios nuevos enfoques terapéuticos potentes y más específicos para el HCC avanzado. El hígado constituye un microambiente inmunológico único, aunque la inmunidad antitumoral parece ser factible mediante el uso de inhibidores de punto de control como nivolumab. La eficacia puede aumentarse adicionalmente combinando inhibidores de puntos de control inmunitario o aplicando tratamientos loco-regionales. En este sentido, el éxito del uso de anticuerpos monoclonales, que bloquean el control inmunitario, ha renovado el interés en la inmunoterapia para el HCC.

Association between CTLA-4 gene + 49A/G polymorphism and the risk of periodontitis: A Meta-analysis / 实用口腔医学杂志

Objective:To study the relationship between cytotoxic T lymphocyte antigen 4 (CTLA-4) gene + 49A/G polymorphism and the risk of periodontitis.Methods:A comprehensive literature research was electronically performed to retrieve currently published studies regarding the association of CTLA-4 gene +49A/G polymorphism with periodontitis susceptibility.The individual OR with 95％ CI was pooled to calculate the strength of the association using RevMan 5.2 software.Results:4 out of 18 seached studies satisfied the standard for Meta-analysis.A total of 702 cases and 926 controls were finally included in the Meta-analysis.Overall,no significant association of CTLA-4 gene + 49A/G polymorphism with the risk of periodontitis was observed (P ＞ 0.05).In the subgroup analysis by ethnicity,the results showed a significant association of CTLA-4 gene + 49A/G polymorphism with increased risk of periodontitis in Asian population(P ＜ 0.05) but not in Caucasian population(all P ＞ 0.05).The stratification analysis by subtypes of periodontitis revealed no significant association of the polymorphism with chronic and aggressive periodontitis respectively (all P ＞ 0.05).Conclusion:The present studies suggest that CTLA-4 gene + 49A/G polymorphism may be not associated with the risk of periodontitis in the overall population,but correlated with an increased risk of periodontitis in Asian population.

The expression of FOXP3 and CTLA-4 in peripheral blood mononuclear cells in children with newly onset type 1 diabetes mellitus / 中国小儿急救医学

Objective To investigate the expression levels of forkhead transcription factor 3 (FOXP3) and cytotoxic T lymphocyte-associated antigen-4(CTLA-4) in the peripheral blood of children with newly diagnosed type 1 diabetes mellitus(T1DM),and explore their roles in the pathogenesis of T1DM. Methods Fifty children with newly diagnosed T1DM and 30 healthy children( control group) were select-ed. FOXP3 and CTLA-4 mRNA expressions were detected using real-time PCR. Soluble FOXP3 ( sFOXP3 ) and CTLA-4(sCTLA-4) protein levels in the serum were assayed by ELISA,and the levels of diabetes anti-bodies,HbA1C and C-peptide were determined using Western blotting,high performance liquid ion-exchange chromatography,and electrochemiluminescence, respectively. Results The expressions of FOXP3 mRNA and protein in T1DM patients were lower than those in the control group[0. 95 ± 0. 48 vs. 2. 11 ± 0. 79,(6. 27 ± 1. 49) ng/ml vs. (9. 02 ± 2. 37) ng/ml,respectively,both P <0. 01],while the expressions of CTLA-4 mRNA and protein were higher than those in the control group[2. 43 ± 0. 83 vs. 1. 94 ± 0. 84,(77. 88 ± 22. 34) ng/ml vs. (65. 97 ± 12. 11) ng/ml,respectively,both P<0. 01]. In T1DM patients,the expressions of FOXP3 and CTLA-4 genes presented positive correlation with the protein expression( r=0. 758,0. 396, P<0. 05), and FOXP3 protein expression presented correlation with CTLA-4 protein expression ( r =-0. 624,P<0. 05). Conclusion There are abnormal expressions of FOXP3 and CTLA-4 genes as well as their proteins in the peripheral blood of the children with newly diagnosed T1DM,and FOXP3 regulated the expression of CTLA-4 in T regulatory cells,indicating the involvement of immune mechanisms in the devel-opment of T1DM.

Analysis on correlation between cytotoxic T lymphocyte antigen 4 gene polymorphisms and susceptibility to hepatocellular carcinoma / 重庆医学

Objective To analyse the correlation between cytotoxic T-lymphocyte antigen-4(CTLA-4)gene polymorphism-318 T/C ,CT60 G/A and the susceptibility of hepatocellular carcinoma (HCC) .Methods Totally 277 cases of HCC and 306 healthy controls in our hospital from September 2012 to September 2014 were selected as the research subjects ,all subjects were Han nationality .The peripheral blood (5 mL) in each case was collected for separating serum ,the AFP level was detected by chemilunescent method ,the serum IL-2 ,IL-4 and TGF-β levels were detected by ELISA .Genomic DNA was extracted ,after PCR amplification ,the CTLA-4-318 T/C ,CT60 G/A gene polymorphism distributions were detected by the direct sequencing method . Results The CTLA-4-318 CC and CT60 AA genotypes all conformed to the Hardy-Weinberg equilibrium(P> 0 .05) .CTLA-4-318 CC and CT60 AA were related with the HCC risk decrease ,different genotypes of CTLA-4 CT60 G/A had obvious relation with serum AFP level(χ2 =12 .779 ,P=0 .012) .The serum IL-2 and IL-4 levels in the HCC patients carrying CTLA-4-318 T and CT60 G allele were significantly decreased ,while the TGF-βlevel was significantly increased ,moreover CTLA-4-318 had obvious relation with the HCC grading .Conclusion CTLA-4-318 TT could promote the occurrence and progression of HCC ,which might be related with the down-regulation of Th1/Th2 type cytokines ,and up-regulation of Th3 type cytokine .

Radiotherapy and immune checkpoint blockades: a snapshot in 2016

Immune checkpoint blockades including monoclonal antibodies (mAbs) of cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed death-1 (PD-1), and programmed death-ligand 1 (PD-L1) have been emerged as a promising anticancer therapy. Several immune checkpoint blockades have been approved by US Food and Drug Administration (FDA), and have shown notable success in clinical trials for patients with advanced melanoma and non-small cell lung cancer. Radiotherapy is a promising combination partner of immune checkpoint blockades due to its potent pro-immune effect. This review will cover the current issue and the future perspectives for combined with radiotherapy and immune checkpoint blockades based upon the available preclinical and clinical data.

Association of genetic polymorphisms of STAT4, the cytotoxic T-lymphocyte antigen-4 and chromosome 9p21.3 with susceptibility to rheumatoid arthritis in Minnan population / 中华风湿病学杂志

Objective A very high prevalence of rheumatoid arthritis (RA) is observed in Minnan population in China.We aimed to explore the genetic characteristics of RA in Minnan population and genetic mechanisms of RA by studying the associations of three single nucleotide polymorphisms (SNPs) of signal transducer and activator of transcription 4 (STAT-4) (rs7574865),the cytotoxic T-lymphocyte antigen-4 (CTLA)-4 (rs3087243) and chromosome 9p21.3(rs1333049) with RA in Minnan population.Methods A case-control study of 119 RA patients and 125 normal controls from Quanzhou were enrolled.SNPs (rs7574865,rs3087243,rs1333049) were genotyped by allele-specific polymerase chain reaction (PCR) and analyzed by SPSS 18.0.x2-test was applied to compare allele and genotype frequeneies betweeen cases and controlsLogistic regression models were used to analyze the SNPs.Results The results showed the genotype distributions of STAT4 genes were significantly different between case and control groups (P＜0.01).Compared with the GT heterozygous genotype,TT and GG homozygosity carriers had a lower risk (OR=0498 and 0.300,P=0.018 and P=0.002 respectively).There was not statistical difference in genotypes and allele in CTLA-4 (rs3087243) between RA patients and healthy controls (x2=4.083,P=0.130),but compared with the AG genotyoe,GG homozygosity carriers had a lower risk on basis of statistics (OR=0.580,P=0.04).There was not statistical difference in genotypes and allele in the chromosome 9p21.3 (rs1333049) (P＞0.05),but compared with the GG genotype carriers,CC and GC genotypes carriers had a lower risk on basis of statistics (OR=0.565,P=0.0495).Conclusion Chromosome 9p21.3 (rs1333049) and CTLA-4 rs3087243 G/A may not be associated with susceptibility to RA in Minnan popula-tions.This replication study confirmes that STAT4 rs7574865 G/T polymorphism is associated with susceptibility to RA in Minnan population.

Association between CTLA-4 gene polymorphisms and the susceptibility to Graves' disease / 中华内分泌代谢杂志

Objective To determine whether the single nucleotide polymorphisms (SNPs) rs231775 and rs3087243 of the cytotoxic T lymphocyte antigen 4 (CTLA-4) gene are associated with susceptibility to Graves' disease (GD) in Chinese Han population.Methods Patients were enrolled from outpatient department and wards.Blood samples from each subject were collected to extract DNA,and the genotypes were determined by TagMan-MGB probe.Results The frequencies of allele G (OR =1.244,95％ CI 1.124-1.377,P＜0.01) and genotype GG (55.3 ％ vs 49.1％,OR =1.279,95 ％ CI 1.126-1.454,P＜0.01) of rs231775 in GD group were higher than those in control group.The frequencies of allele G (OR =1.303,95％ CI 1.166-1.457,P＜0.01) and genotype GG (76.8％ vs 71.8％,OR=1.302,95％ CI 1.143-1.484,P＜0.01) of rs3087243 in GD group were also higher.Conclusion GG genotypes in rs231775 and rs3087243 of CTLA-4 gene are related to the high risk of GD.

A novel evaluation system for T lymphocytes immune function based on the expression of costimulatory molecules in chronic HBV-infected patients / 中华临床感染病杂志

Objective To investigate the expression and significance of CD28,cytotoxic T-lymphocyte antigen-4 (CTLA-4),programmed death-1 (PD-1) and T cell immunoglobulin mucin-3 (Tim3) on T lymphocytes in chronic HBV-infected patients.Methods A total of 102 chronic HBV-infected patients,including 42 patients with chronic hepatitis B (CHB),30 patients with hepatitis B-induced liver cirrhosis (LC),and 30 patients with hepatocellular carcinoma (HCC),were enrolled from the First Affiliated Hospital to Soochow University during October 2012 and June 2013.Thirty healthy individuals were also enrolled as controls.Expression of CD28,CTLA-4,PD-1,Tim-3 on T lymphocytes in peripheral blood were determined by flow cytometry,and the differences among groups were analyzed using one-way ANOVA and LSD-t test.Spearman correlation test was performed to analyze the correlations of the expression of CD28,CTLA-4,PD-1,Tim-3 on T lymphocytes with HBV DNA loads,HBeAg and ALT.Results The expression of CD4 + CD28 +,CD8 + CD28 +,CD4 + CTLA-4 + in chronic HBV-infected patients were lower than those in healthy controls.CD4 + CD28 + expression in HCC group was lower than that in CHB group (t =2.373,P ＜ 0.05) ; CD8 + CD28 + expression in LC and HCC group was lower than that in CHB group (t =4.324 and 4.088,P ＜ 0.01) ; CD8 + PD-1 +,CD4 + Tim-3 + and CD8 + Tim-3 + expressions in CHB group were higher than those in LC,HCC group and healthy controls (t =3.051,3.130,3.121,3.254 and 3.723,P ＜0.01).CD8 + PD-1 + expression was positively correlated with ALT levels and HBV DNA loads (r =0.516 and 0.582,P ＜ 0.01) ; CD8 + Tim-3 + expression was also positively correlated with ALT levels andHBV DNA loads (r =0.578 and 0.556,P ＜0.01); PD-1 and Tim-3 expressions on CD8 T lymphocytes were positively correlated with each other (r =0.578,P ＜ 0.01).Conclusion The abnormal expression of the molecules on T lymphocytes in chronic HBV-infected patients is closely correlated with immune function disorder and the progression of the disease.

The diseases associated with costimulatory molecules B7 and cytotoxic T lymphocyte antigen-4 / 国际儿科学杂志

Tcells need two signals to promote their activation.Only two signals existing at the same time,T cells can effectively activate,proliferate and secrete cell factors or play the toxic effects of cells.One signal is that T cell receptor recognize antigen and CD3 will transduce it to cells.The other is the combination of costimulatory molecules from antigen-presenting cell and costimulatory molecules receptor on the T cell surface.B7 on antigen-presenting cell and CD28/cytotoxic T lymphocyte-antigen-4 expressed on T cells are the most important costimulatory molecules.

High levels of serum soluble cytotoxic T lymphocyte associated antigen 4 and its clinical significance in patients with Crohn's disease / 中华消化杂志

Objective To investigate the expression of serum soluble cytotoxic T lymphocyte associated antigen 4 (sCTLA4), the association of sCTLA4 level with erythrocyte sedimentation rate (ESR) and C reactive protein (CRP), as well as its role in patients with Crohn's disease (CD). The relationship-1661A/G and -1722T/C polymorphisms of CTLA4 gene and between disease susceptibility and phenotype of CD was analyzed. Methods A total of 126 CD patients and 300 healthy controls were enrolled in the study. Serum sCTLA4 level was determined by enzyme-linked immunosorbent assay. The concentrations of ESR and CRP were analyzed by automatic ESR Analyzer SRS 100/Ⅱ and rate nephelometry, respectively. The polymorphisms of CTLA4-1661A/G and -1722 T/C were genotyped by DNA sequencing. Results Serum sCTLA4 level was higher in CD patients than in healthy controls [(18. 70±3. 72) ng/ml vs (1.72±0. 32) ng/ml, P＜0. 01)]. Among CD patients, sCTLA4 level was higher in patients with active disease when compared to those with inactive disease [(19.83±4.35) ng/ml vs (18. 02±3.14) ng/ml, P=0. 015)]. sCTLA4 level was positively correlated with ESR and CRP levels (r=0. 267, P=0. 003; r=0. 524 P ＜0.01, respectively). In CD patients, serum sCTLA4 level was significantly higher in those with stricturing disease behavior than that in those without stricturing and penetrating or with penetrating disease behavior (P= 0.021; P=0. 015, respectively). Detection of CTLA4 -1661A/G and -1722T/C polymorphisms showed no significant difference between CD patients and healthy controls. Conclusions The high level of serum sCTLA4 in CD patients is correlated with disease activity, CRP levels and disease behavior. It suggests that sCTLA4 may play an important role in pathogenesis of CD.

Association of Cytotoxic T Lymphocyte Antigen-4 Gene Polymorphisms and HLA Class II Alleles with the Development of Type 1 Diabetes in Korean Children and Adolescents

We studied the association of cytotoxic T lymphocyte antigen-4 gene (CTLA4) polymorphisms with the development of type 1 diabetes (T1D) in Korean children and adolescents. A total of 176 Korean subjects (92 females and 84 males) with childhood-onset T1D were studied. The A/G polymorphism at position 49 in CTLA4 exon 1 and the C/T polymorphism at position -318 in the CTLA4 promoter were analyzed by PCR-RFLP methods. The genotype and allele frequencies of the CTLA4 polymorphisms in the T1D patients were not different from those in the controls. These polymorphisms were not associated with the clinical characteristics or the development of autoimmune thyroid disease in the T1D patients. The frequency of the A allele was significantly higher in the patients that did not have two out of the three susceptible HLA-DRB1 alleles, which were DRB1*0301, *0405 and *09012, compared to the controls (P<0.05). These results suggest that CTLA4 polymorphisms do not directly confer any susceptibility to T1D. However, a CTLA4-mediated susceptibility effect on the development of T1D might be significant in children and adolescents that do not have susceptible HLA class II alleles.

Preventing acute rejection after liver transplantation via blockading B7/CD28 costimulatory pathway in rats / 中国现代普通外科进展

Objective:To observe the effect of CTLA-4Ig on preventing acute rejection and the role of costimulatory molecule B7-1 and B7-2 via the model of orthotopic liver transplantation(ROLT)in rats.Methods:Build the model of ROLT(DA-Lewis).All rats were divided into 2 groups randomly.Group A was control group.Group B was injected with CTLA4-Ig into abdominal cavity after 48 hours of operation.Animals were sacrificed on the day 3,5,7,10 after ROLT to detect the changes of serum biochemistry(ALT,TBIL and DBIL).Liver grafts were collected for measuring the expression of B7-1 and B7-2 mRNA by RT-PCR.Results:1)Compared with the control group,the expression of B7-1 and B7-2 mRNA increased significantly(P

CTLA-4 Polymorphism in Myasthenia Gravis

BACKGROUND: Cytotoxic T lymphocyte associated antigen 4 (CTLA-4) plays a role in down-regulating both the cellular and the humoral responses by suppressing the ongoing responses of activated T-cells. There are evidences to suggest the genetic contribution of the CTLA-4 locus to a number of autoimmune diseases, such as insulin dependent diabetes mellitus, multiple sclerosis and systemic lupus erythematosus. The aim of the present study is to analyze CTLA-4 gene polymorphism in patients with myasthenia gravis (MG) compared to healthy controls. METHODS: Thirty healthy controls and 31 patients with MG were genotyped into G/G, A/G and A/A of CTLA-4 gene polymorphism at position 49 and the relationship with the clinical feature was analysed. RESULTS: In the patients with MG, the genotype frequencies of G/G, A/G and A/A were 61.3%, 35.5% and 3.2%, respectively. In healthy controls, the frequencies of each genotype were 50%, 43% and 7%, respectively. There was no significant difference in the genotype frequencies of CTLA-4 gene between patients with MG and the control group. There were also no significant differences in the genotype frequencies of CTLA-4 gene between ocular and generalized MG. CONCLUSIONS: These data suggest that the CTLA-4 polymorphism at position 49 do not affect the development of MG. However, further study is needed to clarify the possible role of the CTLA-4 polymorphism in the susceptibility to MG.

Application of Cytotoxic T Lymphocyte Antigen 4 Immunoglobulin G in Prevention of Rejection and the Induction of Immune Tolerance of Orthotopic Liver Transplantation in Rats / 中国普外基础与临床杂志

Objective This study was conducted to build experimental model of orthotopic liver transplantation in rat(ROLT) with the character of acute rejection;and to study the effect of cytotoxic T lymphocyte antigen 4 immunoglobulin G(CTLA4-Ig) on prevention of rejection and the induction of immune tolerance of ROLT.(Methods Build model) of Wistar→SD ROLT(performed by the two cuff method) with character of acute rejection.Recipients were injected with CTLA4-Ig 75 ?g per ROLT into abdominal cavity after 2 days of operation.Contrast was made with no treatment group,the clinical characters,the liver function,the transplantated liver pathologic character and the concentrations of TNF-? in serum were observed and measured on postoperative day 7.In treatment group,all above observation were done on postoperative month 4.Above all,determination of the effect of CTLA4-Ig on preventing acute rejection and inducing tolerance in ROLT was observed.Results ①Recipients(no treatment group) died one by one within 6th～14th days;pathologic character of rejection in transplantation liver could be found;② In treatment group,on postoperative day 7 and month 4,no clinical rejection character and no pathologic character of rejection in transplantation liver were found and serum concentration of cytokins related to TNF-? found lower than that of contrast group(P

Cytotoxic T Lymphocyte Antigen-4 (CTla-4) Polymorphism in Korean Autoimmune Thyroid Disease / 대한내분비학회지

BACKGROUND: The cause of autoimmune thyroid diseases (AITD), including Graves disease and Hashimotos thyroiditis, is largely unknown. To identify the genes responsible, most attention has been focussed on the HLA regions in the early studies. However, these studies have repeatedly shown a weak association between AITD and the HLA-DR3 in Caucasians. To understand and find out the mechanisms underlying the development of AITD, a search for non-HLA linked susceptibility genes is important. A recent study from American population have indicated an association between a polymorphism of CILA-4 gene and Graves disease. To clarify the relationship of the CTLA-4 polymorphism and AITD, the allele frequency of CTLA-4 gene from the patients with Graves disease and with Hashimotos thyroiditis in Korean papulation were analysed. METHODS: The CTLA-4 exon 1 polymorphism (49, A/G) was analysed by PCR-based, RFLP (Restriction Fragment Length Polymorphism) from 92 women and 37 men with Graves disease and 50 women and 9 men with Hashimotos thyroiditis diagnosed. Also, 287 healthy controls including 155 women and 132 men with no clinical evidence or family history of thyroid disease were enrolled. RESULTS: 1) In the group of Graves disease, there was significantly more patients with alanine homozygote (GG) than in control group (P<0.0005, RR=1.40). However, there was not significant with threonine homozygote (AA) between two groups (P=0.052). In the group of Hashimotos thyroiditis, no significant differences were found between all homozygotes and heterozygote. 2) In the group of Graves disease, there were significantly more patients with alanine homozygote (GG) (P<0.0001, RR=1.85) and significantly fewer patients with threonine homozygote (AA) than in the group of Hashimoto's thyroiditis (P<0.005, RR 0.25). CONCLUSION: Regardless of sex difference, alanine homozygote (GG) at exon 1 (codon 17) of CTLA-4 is associated with Graves disease in Korean population, which suggests genetic susceptibility is some role in the pathogenesis of Graves disease.

Expression of adenovirus-mediated CTLA4Ig-IRES-CTLA4 gene in bone marrow mesenchymal stem cells / 解放军医学杂志

Objective To construct adenovirus vector harboring CTLA4Ig-IRES-CTLA4 gene, and to express the gene in bone marrow mesenchymal stem cells (BMMSCs). Methods The cDNA fragments of CTLA4, and the extracellular domains of CTLA4 and IgGFc were cloned to pT-Easy vectors by RT-PCR from the activated splenocytes of Wistar rats. Recombinant adenovirus vector harboring CTLA4Ig-IRES-CTLA4 was produced from AdeasyTM Adenoviral Vector System by homologous recombination between the Adtrack vector (pAdtrack-GFP-CTLA4Ig-IRES-CTLA4) and the plasmid pAdEasy-1 containing most of the human adenovirus serotype 5 (Ad5) genome in E. coli BJ5183 strain, and then packaged and propagated in 293 cells. BMMSCs were infected with the recombinant adenovirus, and the co-expression of both CTLA4Ig and CTLA4 was then detected by RT-PCR and Western blotting. The immunosuppression function of the transfected BMMSCs was investigated by mixed lymphocyte response (MLR). Results Fusion gene CTLA4Ig was constructed successfully, and the recombinant CTLA4Ig-IRES-CTLA4 adenovirus was then generated by homologous recombination and packaged in 293 cells. The transduction efficiency of recombinant adenovirus in BMMSCs was elevated up to 98.67% determined by flow cytometry using a GFP as marker. CTLA4Ig and CTLA4 were expressed at the same time in BMMSCs detected by RT-PCR and Western blot, and the transfected stem cells showed stronger immunosuppression function than that of BMMSCs or BMMSCs transduced by Ad-CTLA4Ig. Conclusion The BMMSCs transfected by the recombinant adenovirus can co-express CTLA4Ig and CTLA4, which improved the immunosuppression function of BMMSCs in the way of costimulatory pathway blockade.