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Objective To construct an electrochemical immunosensor for ultrasensitive determination of myeloperoxidase (MPO) .Methods The electrochemical immunosensor for M PO was prepared by modifying the electrode using Au-graphitized me-soporous carbon nanoparticles(AuNPs@ GMCs) hybrid and immobilizing MPO antibodies onto the glass carbon electrode surface . The effect of experimental parameters on the immunosensor and results comparison with ELISA were investigated .Results The immunosensor was sensitive to M PO with a linear relationship between 2 .000 and 300 .000 ng/mL and a correlation coefficient of 0 .999 ;the detection limit was 0 .5 ng/mL .The correlation coefficient of two methods was 0 .983 .Conclusion The immunosensor can be used for ultrasensitive detection of MPO .
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Objective To prepare a novel monoclonal antibody (mAb) that specifically against Mycobacterium tuberculosis culture filtrate protein 10 (CFP-10).Methods The BALB/c mice were immunized by a peptide with 14 amino acids (aa residues 53 to 66) of CFP-10,and then the splenocytes of mice were fused with myeloma cell line SP2/0.The resultant fused cells were subjected to screening culture,enzyme linked immunosorbent assay (ELISA) assay and subcloning by limited dilution to establish hybridoma cell lines of stable secreting anti-the peptide of CFP-10 antibody.The antibody was purified,and its isotypes were analyzed.Then,the antibody was further evaluated by Western blotting,immunoprecipitation and ELISA in 38 culture supernatant samples of Mycobacterium tuberculosis,20 culture supernatant samples of non-Mycobacterium tuberculosis,32 samples of tuberculous pleural effusion,24 samples of non-tuberculous pleural effusion,and 20 serum samples from healthy controls.Results The isotype of the mAb against the specific peptide of CFP-10 was an IgG1 with κ chain,and it was applicable for Western blotting and immunoprecipitation analysis.ELISA quantitative test showed that the sensitivity and specificity for diagnosis of Mycobacterium tuberculosis were 78.6％ (55/70) and 92.2％ (59/64),respectively.Conclusion The mAb generated against the specific peptide of CFP-10 is high in sensitivity and specificity,and it might be used in the early diagnosis of tuberculosis.
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ObjectiveTo determine the protein and mRNA expression levels of interleukin-17 (IL-17) and proportions of Th17 cells in peripheral blood of patients with atopic dermatitis (AD),and to analyze their clinical significance.MethodsVenous blood samples were obtained from 63 patients with AD and 30 normal human controls.Enzyme linked immunosorbent assay was performed to measure the plasma level of IL-17 protein,real time fluorescence reverse transcription (RT)-PCR to detect the mRNA expression level of IL-17 in peripheral blood,flow cytometry to determine the percentage of Th17 cells in peripheral blood mononuclear cells(PBMCs).Spearman's rank correlation analysis was performed to assess the relationship of peripheral blood Th17 cell percentage and IL-17 levels with disease severity in patients with AD.Results The percentage of Th17 cells in PBMCs was 1.83％ ± 0.47％ in patients with acute AD,significantly higher than that in normal human controls (0.85％ ± 0.45％,t =4.128,P ＜ 0.01 ) and in patients with chronic AD ( 1.12％ ± 0.69％,t =2.439,P ＜ 0.05).There was a significant increase in the plasma level (98.37 vs.63.75 ng/L,U =168,P ＜ 0.05) and mRNA expression level of IL-17 in peripheral blood in patients with AD compared with normal controls.Both the Th17 cell percentage and plasma IL-17 level were positively correlated with eczema area and severity index(EASI) score in patients with AD (r =0.681,P ＜ 0.01;r =0.427,P ＜ 0.05).ConclusionsThere is an elevated proportion of Th17 cells and an abnormal expression of IL-17 protein and mRNA in peripheral blood from patients with acute AD,which are positively correlated with disease severity,implicating the possible roles of IL-17 and Th17 cells in the pathogenesis of AD.
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Objective Atherosclerosis is widely accepted as a chronic inflammatory disease. Serum biomarkers for vulnerable plaques not only serve as diagnostic tools for the identification of patients with acute coro-nary syndrome, but also assist the identification of high-risk patients. However, the existing data are limited and conflicting. In the present study, we determined whether the plasma levels of interleukin-1β (IL-1β) are correlated with adverse cardiac outcomes in patients with ST-evaluate acute myocardial infarction (STEAMI) undergoing pri-mary percutaneous coronary intervention (PCI). Effect of the plasma intedeukin-1β level on prognosis of patients with ST-segment elevation acute myocardial infarction. Method This prospective single-center study included 96 patients with SIEAMI with onset < 12 h who underwent primary PCI, 271 patients with stable angina pectoris (SAP) and 148 control subjects without coronary artery disease who were consecutively admitted to hospital be-tween Mar, 2006 and Mar, 2008. Plasma IL-1β levels were measured by enzyme-linked immunosorbent assay in all subjects. The patients with STEAMI were then followed prospectively for the occurrence of major adverse car-diac events (MACE) (including cardiovascular death, non-fatal myocardial infarction, heart failure, and cardio-genie shock) during hospitalization. We determined the association between IL-1β levels with the risk of MACE using multivariate logistic regression. Results Compared with the SAP patients and control subjects, patients with STEAMI had higher levels of IL-1β (P < 0.05). During hospitalization, 32 patients (33.3%) experienced MACE [23 males, 9 females; age: (75.44±13.45) years]. In the STEAMI patients, IL-1β was elevated in patients with MACE compared with patients without MACE (median [range]: 26.52 [12.010 to 155.244] pg/mL vs 2.157 [0.433 to 83.021] pg/mL; P < 0.01) by non-parameter analysis. Significant and positive correlations be-tween IL-1β and cardiac troponin-I (cTnI) (r = 0.353, P =0.004) were observed by Spearman's correlations analysis. Multivariate logistic regression analysis revealed that IL-1β levels ≥20 pg/mL were significantly and in-dependently associated with MACE during hospitalization (odds ratio: 32.05; 95% confidence interval: 4.28 to 240.151; P =0.001). Conclusions The present study revealed that patients with STEAMI had elevated IL-1β levels on admission. The plasma IL- 1β level is an independent inflammatory predictor for in-hospital MACE in pa-tients with STEAMI undergoing percutaneous coronary intervention.
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Objective A recombinant adenoviral vector containing mIL-18BP and mIL-4 fusion gene(AdmIL-18BP/mIL-4) was constructed and used to investigate the role of mIL-18BP and mIL-4 in medula-ring the expressions of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthetase (iNOS) and their inducing products PGE2, NO in murine collagen-induced arthritis. Methods Male DBA-1/BOM mice were used in this study. Mice with CIA were intra-articularly injected with 107 pfu/6μl of AdmIL-18BP/mIL-4.Intra-articular injections of AdLacZ or PBS were used as controls. The mRNA expression of COX-2, iNOS in synovial tissue was analyzed by semi-quantitative RT-PCR. Expression of COX-2 and iNOS protein was estimated by Western blot method. The production of PGE2 and NO in synovia was detected by competitive ELISA and enzyme reduction of nitrate. Results The expression of COX-2, iNOS mRNA in routine synovial tissue of AdmIL-18BP/mIL-4 treatment group was significantly lower than that of AdLacZ group (0.15 vs 0.42,P<0.01 ; 0.05 vs 0.77, P<0.01) and PBS group (0.15 vs 0.65, P<0.01; 0.05 vs 0.64, P<0.01 ). And the protein expression of COX-2, iNOS from AdmIL-18BP/mIL-4 treatment group was also obviously lower than that of AdLacZ group (0.08 vs 0.92, P<0.01; 0.11 vs 1.00, P<0.01) and PBS group (0.08 vs 0.77, P<0.01; 0.11 vs 0.84, P<0.01 ). The PGE2 and NO production in synovia of AdmIL-18BP/mIL-4 treatment group was significantly lower than that of AdLacZ group [(0.68x0.06) vs (2.58±0.21)ng/mL, P<0.01; (23.4+2.5) vs (60.0±11.3)μmol/L, P<0.01 ] and PBS group [(0.68±0.06) vs (2.57±0.20)ng/mL, P<0.01; (23.4+2.5) vs (60.3±13.4)μmol/L, P<0.01]. Conclusion These data indicat that local over-expre-ssion of mIL-18BP and mIL-4 can down-regulate COX-2, iNOS and their induced product PGE2, NO in CIA mice. The combination treatment with mIL-18BP and mIL-4 is a promising therapeutic target for RA.
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<p><b>OBJECTIVE</b>To investigate the antitumor effects of intrasplenically transplanted interleukin-18 (IL-18) gene-modified hepatocytes on murine implanted liver carcinoma.</p><p><b>METHODS</b>Embryonic murine hepatocyte cell line (BNL-CL2) was transfected with a recombinant adenovirus encoding IL-18 and used as delivery cells for IL-18 gene transfer. Two cell lines, BNL-LacZ and BNL-CL2, were used as controls. One week after intrasplenic injection of C26 cells (colon carcinoma line), tumor-bearing syngeneic mice underwent the intrasplenic transplantation of IL-18 gene-modified hepatocyte cell line and were divided into treatment group (BNL IL-18) and control groups (BNL-LacZ and BNL-CL2). Two weeks later, the serum levels of IL-18, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in the implanted liver carcinoma-bearing mice were assayed, the cytotoxicity of murine splenic cytotoxic T-lymphocytes (CTLs) was measured, and the morphology of the hepatic tumors was studied to evaluate the antitumor effects of the approach.</p><p><b>RESULTS</b>In the treatment group, the serum levels of IL-18, IFN-gamma, TNF-alpha and NO increased significantly. The splenic CTL activity increased markedly (P < 0.01), accompanied by a substantial decrease in tumor volume and the percentage of tumor area and prolonged survival of liver carcinomo-being mice.</p><p><b>CONCLUSIONS</b>In vivo IL-18 expression by ex vivo manipulated cells with IL-18 recombinant adenovirus is able to exert potent antitumor effects by inducing a predominantly T-cell-helper type 1 (Th1) immune response. Intrasplenic transplantation of adenovirus-mediated IL-18 gene-modified hepatocytes could be used as a targeting treatment for implanted liver carcinoma.</p>
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Animais , Masculino , Camundongos , Adenoviridae , Linhagem Celular , Técnicas de Transferência de Genes , Terapia Genética , Métodos , Vetores Genéticos , Hepatócitos , Interleucina-18 , Genética , Neoplasias Hepáticas Experimentais , Terapêutica , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Linfócitos T Auxiliares-Indutores , Alergia e Imunologia , TransfecçãoRESUMO
Objective To investigate the therapeutic effects of intrasplenically transplanted interferon ? (IFN ?) gene modified hepatocytes on murine implanted liver carcinoma. Methods Murine fetal hepatocytes (BNL?CL2) were transfected with recombinant adenovirus expressing IFN ?. Two cell lines BNL?Lac Z and BNL?CL2 were taken as control. One week after intrasplenically injected C26 cells(colon carcinoma line), sixty tumor bearing syngeneic mice were intrasplenically transplanted IFN ? gene modified hepatocytes and were divided into treated group (BNL?IFN ?) and two control groups (BNL?Lac Z and BNL?CL2), two weeks later, levels of IFN ?, tumor necrosis factor alpha(TNF ?) and nitric oxide(NO) in the serum of liver implanted carcinoma bearing mice were assayed, the cytotoxicity of murine splenic cytotoxic T lymphocyte (CTL) was measured. Mophology of hepatic tumors were studied. The therapeutic effects on the mice with the implanted liver carcinoma were also evaluated. Results In treated group (compared with control groups), the levels of IFN ?, TNF ? and NO in the serum increased significantly ( P