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Objective To explore the immunopathological mechanism for the imbalance between the positive signal mediated by inducible costimulator (ICOS) and the negative signal mediated by programmed death-1 (PD-1) in patients with myasthenia gravis (MG).Methods Eighty-two patients with MG,56 healthy controls (HC) and 20 non-MG (NMG) patients,collected in the First Affiliated Hospital of Suzhou University from February 2014 to December 2016,were chosen to participate in the study.The expression of ICOS and PD-1 on peripheral blood mononuclear cells was detected by immuno-fluorescence staining and flow cytometry.The levels of soluble programmed death-1 (sPD-1),soluble programmed death ligand 1 (sPD-L1),IL-4 and other cytokines were detected by enzyme-linked immunosorbent assay.Results (1) Flow cytometry analysis:The co-expression of PD-1,ICOS on CD4 + T cells from MG group (9.64％ (8.82％)) was higher than in HC (1.81％ (2.10％),Z =-7.389,P ＜0.05) and NMG group (2.86％ (1.49％),Z =-4.636,P ＜ 0.05).The expression of ICOS on CD4 + T cells,ICOS ligand (ICOSL) on CD14+ monocytes and CD19+ B cells were increased in MG group comparing with that of the control groups.The proportion of PD-1 + CD4 + T cells (MG group 16.82％ (10.66％),HC 9.34％ (9.18％),Z =-4.345,P＜0.05;NMG group 7.07％ (3.40％),Z=-4.594,P＜0.05) and PD-1 Ligand (PD-L1) + CD14+ monocytes was higher in MG patients.All of these were detected by flow cytometry.(2) ELISA analysis:Serum sPD-1 expression significantly increased in MG group compared with that in the control groups (MG group (1.87 ± 0.64) ng/ml,NMG group (1.49 ± 0.70) ng/ml,t =2.04,P ＜ 0.05;HC (1.05 ± 0.50)ng/ml,t =2.08,P ＜ 0.05),while for serum sPD-L1,there was no significant difference between MG and control groups.(3) Serum cytokines detection:The expression of IL-4 was increased in MG patients (MG group (61.88 ±5.15) pg/ml,HC (32.03 ±1.84) pg/ml,t=2.50,P＜0.05;NMG group (42.62± 3.31) pg/ml,t =2.34,P ＜0.05),and there was a negative correlation between the expression of sPD-1 and the concentration of IL-4.Conclusions The increased expression of PD-1 + ICOS + CD4 + T cells suggested the subset involved in the pathological progress of MG.sPD-1 might disturb the ligation of PD-1 on T cells and PD-L1 on antigen presenting cells,while the ligation of ICOS and ICOSL passed positive signal,leading to over activity of the subsets and the progression of disease.
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[Objective]To investigate the relationship between clopidogrel response , early neurological deterioration and CYP2C19 gene polymorphism in sICAS patients.[Methods]116 sICAS patients were divided into deterioration group and non-deterio?ration group by whether appear early nervous function deterioration. Record included the baseline data,the genotypes of CYP2C19, platelet maximum aggregation rate after 7 days of given clopidogrel,CYP2C19 genotype and clopidogrel reaction were compared be?tween two groups.[Results]The deterioration group combination with diabetes ,stroke/TIA were significantly higher than the non-de?terioration group(P<0.05);The frequency of CYP2C19*2 AA genotype and A allele were significantly higher than those in non-dete?rioration group. The frequency of GG genotype were significantly lower than non-deterioration group (27.27% vs 2.13%,50.00% vs 14.84%,27.27% vs 72.34%)(P<0.01);The poor metabolic genotype platelet maximum aggregation rate were significantly higher than that of the fast-metabolic genotype and middle metabolic genotype(P<0.01,P<0.05),Middle metabolic genotype platelet maxi?mum aggregation rate were significantly higher than fast-metabolic genotype(P<0.01);The deterioration group fast-metabolic geno?type were significantly lower than non-deterioration group ,poor metabolic genotype were significantly higher than non-deterioration group(22.73% vs 65.96%,36.36% vs 5.32%)(P<0.01);Clopidogrel resistance rate 59.09% were significantly higher non-deteriora?tion group 28.72%(P<0.05).[Conclusion]Clopidogrel response and early neurological deterioration in sICAS patients is associatedwith CYP2C19 gene polymorphism.
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Objective To evaluate the clinical efficacy of Yang Xue Rou Gan (blood-nourishing and liver-soothing) needling method in treating post-stroke depression. Method A total of 150 patients with post-stroke depression were divided into an observation group and a control group by using the random number table, 75 cases each. In addition to the same basic treatment, the control group was intervened by Fluoxetine hydrochloride, while the observation group received Yang Xue Rou Gan needling method, twice every day, 6 d as a course at 1-day interval, for 4 weeks in total. Before and after the intervention, 24-item Hamilton Depression Scale (HAMD), Modified Edinburgh-Scandinavian Stroke Scale (MESSS), and activities of daily living [ADL, Barthel Index (BI)] were evaluated. Result After the intervention, HAMD, MESSS, and BI scores were significantly changed in both groups (P<0.05), indicating that the two groups both had improvements in depression symptoms and signs; there were significant differences between the two groups (P<0.01), suggesting that the observation group had a more significant efficacy in treating depression. Conclusion Yang Xue Rou Gan needling method can notably improve the depression and neural deficit level in post-stroke depression patients.
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Objective To investigate the role and mechanism of B7-H4, a negative costimulatory molecule, in mediating the immunomodulatory effects of mesenchymal stem cells C3H10T1/2 (C3H10) on T cell polarization. Methods The lentiviral vectors that carried the shRNA targeting mouse B7-H4 were transfected into mouse mesenchymal stem cells (C3H10-B7-H4). The cells were co-cultured with PHA-acti-vated mice spleen lymphocytes before and after the transfection. ELISA was performed to detect the concen-trations of cytokines in supernatants of cell culture in order to elucidate the effects of B7-H4 expressed by C3H10 on T cell polarization. A mouse model of experimental allergic encephalitis (EAE) was established. Fifty C57BL/6 mice were divided into five groups including control group, EAE group, C3H10 group (injec-ting EAE mice with C3H10 cells), C3H10-NC group ( injecting EAE mice with C3H10-NC cells) and C3H10-B7-H4 group (injecting EAE mice with C3H10-B7-H4 cells). ELISA was performed to detect the soluble form of IL-2, IL-17, IFN-γ and IL-4 in plasma samples. Results Knocking down the B7-H4 gene with shRNA significantly decreased the expression of B7-H4 on C3H10 cells, which weakened the inhibitory effects of C3H10 cells on the secretion of IL-2, IL-17 and IFN-γ by spleen lymphocytes. The therapeutic effects of C3H10-B7-H4 cells on mice with EAE were weakened after silencing the B7-H4 gene expression, which was manifested as higher nerve function score and earlier onset and bring forwarded peak time of EAE than those of the C3H10 group. Treating EAE mice with C3H10-B7-H4 cells was less efficient in inhibiting the expression of IL-2, IL-17 and IFN-γin plasma. However, knocking down the B7-H4 gene had no signif-icant effect on the expression of IL-4 in terms of treating EAE with C3H10 cells. Conclusion The co-inhib-itor molecule B7-H4 expressed on C3H10 cells mediated the treatment of EAE with C3H10 cells by regula-ting Th1 and Th17 effector T cells.
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To explore the physiological role and biocatalytic properties of short-chain dehydrogenases from Pseudomonas fluorescens GIM1.49, we cloned the structural gene pfd and characterized its over-expressed product. The length of gene pfd was 684 bp encoding a short-chain dehydrogenase with 227 amino acid residues and calculated molecular mass of 24.2 kDa. The recombinant plasmid pET28b-pfd was constructed and functionally expressed in Escherichia coli BL21(DE3), resulting in the over-production of recombinant short-chain dehydrogenase PFD with a size of 28 kDa. The enzyme could oxidize alcohols including 4-chloro-3-hydroxbutanoate ester and reduce 4-chloro-acetoacetate ester using either NAD(H) or NADP(H) as coenzyme. The enzyme showed the highest activity against 4-chloro-3-hydroxbutanoate ester as substrate, with Km of 186.40 mmol/L and Vmax of 89.56 U/mg. When catalying the oxidative reaction, its optimal temperature was 12 degrees C and optimal pH was 10.5, in contrast to the values of 24 degrees C and pH 8.8 in the reductive reaction. The enzyme had high solvent tolerance and its activity was improved by the addition of Ca2+ (1 mmol/L) or EDTA (5 mmol/L). These results indicated that the enzyme from Pseudomonas fluorescens GIM1.49 was a novel short-chain dehydrogenase and might play a role in oxidative degradation of halogenated secondary alcohols.
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Alcohols , Metabolism , Bacterial Proteins , Genetics , Metabolism , Butyryl-CoA Dehydrogenase , Genetics , Metabolism , Cloning, Molecular , Escherichia coli , Genetics , Metabolism , Genetic Vectors , Genetics , Oxidation-Reduction , Pseudomonas fluorescens , Genetics , Recombinant Proteins , Genetics , MetabolismABSTRACT
ObjectiveTo explore the relationship between the negative co-inhibitor programmed death-1 ( PD-1 ) and the pathogenesis of myasthenia gravis ( MG), by detecting the expression of PD-1 and programmed death ligand-1 ( PD-L1 ) on peripheral blood mononuclear cells (PBMCs) and soluble PD-1 (sPD-1) in plasma from myasthenia gravis patients. MethodsPeripheral blood samples were collected from 45 MG patients and 33 healthy persons without prednisone or other immunodepressant treatment during the half year ahead of withdrawal.The expression of PD-1 and PD-L1 on PBMCs were detected using immuno-fluorescence labeling and flow cytometry, and the concentrations of sPD-1 in plasma were measured using an ELISA kit. Results(1) The proportion of CD4+ PD-1 + T cells, as well as CD14+ PD-L1 +monocytes of the MG group was higher than that of the control group. There were no significant differences in the proportion of CD4+ PD-1 + T cells or CD14+ PD-L1 + monocytes in the MG sub-groups between different genders or MG types. While the proportion of CD4+ PD-1 + T cells of the late-onset MG (age ≥40) group was higher than that of the early-onset MG group (age ＜40). And it was higher in the MG patients with thymoma or thymus hyperplasia than that from the MG patients with normal thymus. The proportion of CD14+ PD-L1 +monocytes from the MG patients with thymoma or thymus hyperplasia group decreased obviously compared with that of the patients with normal thymus group; but no difference could be found between the late-onset group and early-onset group. (2)The concentration of sPD-1 in the plasma from the group of MG patients was(6. 92 ±0. 72) ng/ml,which was higher than that of the healthy control group ( (3.28 ±0. 42) ng/ml),even more, it was significantly higher in the early-onset MG group than that of the late-onset MG group,there was a negative correlation( r =-0. 526, P =0. 000) between the age of onset and the concentration of sPD-1. ConclusionsThe increased expressions of PD-1 on CD4+ T cells and PD-L1 on CD14+ monocytes in MG patients suggested the involvement of the couple of molecules in the pathogenesis of MG.Higher concentration of soluble PD-1 in the plasma of patients with MG suggested that it might disturb the ligation of PD-1 and PD-L1 on T cells and antigen presenting cells, which might result in the abnormal transportation of the negative modulating signal, and accelerate the pathological progress of MG.
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The receptor of the heart transplantation was a patient with terminal dilated cardiomyopathy, the donor was a patient with congenital ventricular septal defect, in situ double-chamber heart transplantation was performed, and the result of the four-year follow-up was satisfactory. At present, donor is deficient,and those donors with congenital defect can also obtain satisfactory clinical application effects after appropriate handling.
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To clone mouse phage antibodies against H-Y antigen from a phage antibody library, three cycles of affinity enrichment of the mouse phage antibody library with male spleen cells and two cycles of nonspecific absorption with female spleen cells were performed. The presence of mouse Fab on the phage surface was determined by ELISA and sequence analysis. 9 of 15 strains can bind to male spleen cells with the specific activity. Recombination rate of the phage antibody library clones is 60%. Sequence analysis of the PCR products of plasmid DNA of E5 clones show VH and Vkappa had common characteristics shared by other known variable region of antibodies. The mouse phage Fab antibody could be used for identifying H-Y antigen, and for the development of sex determination of early embryos in mammals.
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Animals , Female , Male , Mice , Base Sequence , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , H-Y Antigen , Immunoglobulin Fab Fragments , Genetics , Allergy and Immunology , Isoantibodies , Genetics , Allergy and Immunology , Molecular Sequence Data , Peptide LibraryABSTRACT
OBJECTIVE@#To detect porcine endogenous retrovirus (PERV) in Daweizi pigs and to provide basic parameters of evaluating the biological safety for xenotransplantation from pigs to humans.@*METHODS@#Ear tissues from 42 individuals were randomly collected from a Daweizi pig population. PCR and RT-PCR were performed to detect PERV proviral DNA and mRNA respectively. Finally, env-A, env-B, and env-C were amplified, sequenced, and analyzed using the BLAST software in National Center for Biotechnology Information.@*RESULTS@#PERV proviral DNA and mRNA could be detected in the 42 individuals by PCR and RT-PCR, respectively. env-A, env-B and env-C were detected in all the individuals. Compared with other pig species (AY288779, DQ011794 and AY534304), there was 1 and 8 bp differences in the sequences of env-A and env-C, while no difference in env-B.@*CONCLUSION@#PERV exists and has transcriptive activity in Daweizi pigs. The predominate subtype is PERV-ABC. Env genes are firstly cloned and sequenced in Daweizi pigs and there are polymorphism in the breed. As to the biological safety, the breed was not suitable as a donor in xenotransplantation.