ABSTRACT
Objective: To investigate the expression and prognosis effect of methylation-regulated SLIT3 and SPRCL1 genes in smoking-related lung adenocarcinoma. Methods: The expression levels of SLIT3 and SPARCL1 in cigarette smoke-induced malignant transformed cell (S30) and lung adenocarcinoma (LUAD) cell lines were measured by real-time fluorescence quantitative PCR (qPCR). Datasets of mRNA expression, DNA methylation and patient information data were obtained from The Cancer Genome Altas (TCGA) database. The mRNA expression levels of SLIT3 and SPARCL1 were validated in LUAD tissues. The 10-year survival curve of LUAD patients with different smoking history was plotted, and the correlation between mRNA expression level and DNA methylation level of LUAD patients was further analyzed. S30 cells were treated with 5-azacytidine (5-aza), an inhibitor of DNA methyltransferase, to analyze the methylation regulatory mechanism of SLIT3 and SPRCL1. Results: The qPCR results showed the significant down-regulation of SLIT3 and SPARCL1 in S30 cell and four LUAD cell lines (SLIT3: 0.493±0.134 and 0.041±0.014, 0.161±0.023, 0.277±0.055, 0.035±0.005; SPARCL1: 0.507±0.131 and 0.453±0.045, 0.420±0.040, 0.153±0.035, 0.430±0.050; all P<0.01). Bioinformatics analysis showed that SLIT3 and SPARCL1 were low expressed in LUAD tissue (8.12±1.58 vs 10.84±0.69 and 11.46±1.06 vs 13.57±0.67; both P<0.001) compared with adjacent peritumoral tissues, and expression levels of SLIT3 and SPARCL1 were significantly correlated with smoking history (both P<0.001). Non-smoker with high expression of SLIT3 and SPARCL1 was associated with better prognosis among LUAD patients. There was a significant negative correlation between promoter methylation and mRNA expression level of the two genes (r=-0.208, -0.574; both P<0.001). 5-aza treatment significantly up-regulated the expression levels of SLIT3 and SPARCL1 genes in S30 cells (2.137±0.281, 3.657±0.882; both P<0.01). Conclusion: SLIT3 and SPARCL1 can be regulated by DNA methylation and down-regulated in LUAD tissue, which has important prognostic significance on the smoking-induced LUAD patients.
Subject(s)
Adenocarcinoma of Lung , Lung Neoplasms , Calcium-Binding Proteins , Extracellular Matrix Proteins , Humans , Membrane Proteins , Prognosis , SmokingABSTRACT
The purpose of this investigation was to describe the effect of antibacterial stewardship and evaluate the trends and correlation of antibacterial resistance and usage from 2009 to 2013 in a tertiary-care teaching hospital in northwest China. Antibacterial usage was expressed as defined daily doses per 100 patients per day (DDDs/100 PDs). Hospital-wide population-level data and time series analysis were used to evaluate the trends and determine associations between antibacterial exposure and acquisition of resistance. Yearly consumption of overall antibacterials significantly decreased from 66.54 to 28.08 DDDs/100 PDs (ß = -10.504, p < 0.01). The resistant rates of the five most frequently isolated species (including Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae) significantly decreased or remained stable, and none of them showed a statistically significant upward trend. The medical quality indicators got better or remained stable. Autoregressive integrated moving average (ARIMA) models demonstrated that the monthly resistance rate of P. aeruginosa to imipenem was strongly correlated with antipseudomonal carbapenems usage (ß = 34.94, p < 0.001), as did the correlation of P. aeruginosa to meropenem with antipseudomonal third-generation cephalosporins usage (ß = 32.76, p < 0.01) and K. pneumoniae to amikacin with aminoglycosides usage (ß = 22.01, p < 0.001). The decreased antibacterial use paralleled the improved bacterial resistance without deteriorating medical quality indicators during antimicrobial stewardship. It also suggests that optimum antibiotic use is necessary to alleviate the threat posed by resistant microorganisms at the hospital level.
Subject(s)
Anti-Bacterial Agents/therapeutic use , Bacteria/drug effects , Bacterial Infections/drug therapy , Bacterial Infections/microbiology , Drug Resistance, Bacterial , Drug Therapy/standards , Drug Utilization/standards , Bacteria/isolation & purification , Bacterial Infections/epidemiology , China , Drug Prescriptions/standards , Drug Therapy/methods , Health Policy , Hospitals, Teaching , Humans , Microbial Sensitivity Tests , Organizational Policy , Prevalence , Tertiary Care Centers , Time FactorsABSTRACT
OBJECTIVE: To elucidate the potential of monoclonal antibodies (mAbs) of culture filtrate protein 10 (CFP-10) and early secretory antigenic target 6 (ESAT-6) in tuberculosis (TB) diagnosis. DESIGN: We generated and characterised monoclonal and polyclonal antibodies against Mycobacterium tuberculosis-specific antigens ESAT-6 and CFP-10 by immunising BALB/c mice with an ESAT-6/CFP-10 fusion protein. Stable hybridoma cell lines were established and mAbs were specifically identified by immunoblotting and immunoprecipitation. The mouse mAbs were used to coat plates, and biotin-labelled polyclonal antibodies were used to detect the antigens. One hundred and seventy-three samples of sputum culture supernatants and pleural effusion aspirates have been tested. RESULTS: The ESAT-6 enzyme-linked immunosorbent assay (ELISA) detected the culture supernatants and pleural effusion specimens that were positive for M. tuberculosis, but failed to identify M. tuberculosis-positive specimens in the non-M. tuberculosis culture supernatants or control specimens. This yielded a sensitivity of 95.4% and a specificity of 100% for the ESAT-6-specific ELISA. The CFP-10 ELISA presented less satisfactory sensitivity and specificity, of respectively 81.6% and 92.2%. Results showed positive detection rates of ESAT-6 and CFP-10 of 86.8% (33/38) and 76.3% (29/38) for the diagnosis of tuberculous pleural effusion in patients bacteriologically negative for M. tuberculosis culture. CONCLUSION: The ESAT-6 and CFP-10 ELISAs incorporating mAbs generated in this study serve as potential tools in the laboratory diagnosis of TB.
Subject(s)
Antibodies, Monoclonal , Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Enzyme-Linked Immunosorbent Assay/methods , Mycobacterium tuberculosis/immunology , Tuberculosis, Pleural/diagnosis , Animals , DNA Primers , Databases, Nucleic Acid , Disease Models, Animal , Humans , Immunoblotting , Immunoprecipitation , Mice , Mice, Inbred BALB C , Mycobacterium tuberculosis/isolation & purification , Sensitivity and Specificity , Sputum/microbiology , Tuberculin/immunologyABSTRACT
OBJECTIVE: To validate a high performance liquid chromatography (HPLC) method for serum teicoplanin measurement and use the method for clinical monitoring of teicoplanin levels to analyze the clinical application of teicoplanin. METHODS: 55 patient profiles were collected and analyzed for the clinical teicoplanin application. 10 critically ill patients of the 55 cases were monitored for teicoplanin trough concentration using the HPLC method. RESULTS: The modified HPLC method exhibited excellent linearity, with correlation coefficient r = 0.9995. The intra-day and inter-day coefficients of variation were less than 10%. The lower limit of detection of teicoplanin was 5.63 mg/l. The recovery of teicoplanin was above 90%. Of the 55 patients in this study, there were 42 patients without load-dosing. There were only 29 patients treated with teicoplanin documented Gram-positive infections by etiological diagnoses. In the 10 patients with teicoplanin serum trough concentration monitoring, all cases received a loading dose of 400 mg every 12 h for 3 doses, and the mean trough concentration of teicoplanin was 10.82 ± 4.51 mg/l. The mean trough levels were 13.04 ± 6.23 mg/l in 4 patients with microbiological eradication and improvement of symptoms of diseases and 9.34 ± 2.61 mg/l in 6 patients with persistence of previous clinical infectious symptoms, respectively. CONCLUSION: The modified HPLC method is robust, highly reproducible and suited to monitor the concentration of teicoplanin. In critically ill Chinese patients, we should consider more appropriate loading doses and evaluate the relationship between teicoplanin trough concentration and the efficacy using microbiological and clinical parameters.
Subject(s)
Anti-Bacterial Agents/blood , Chromatography, High Pressure Liquid/methods , Drug Monitoring , Teicoplanin/blood , Adult , Aged , Female , Hospitals, Teaching , Humans , Male , Middle Aged , Sensitivity and SpecificityABSTRACT
OBJECTIVE: To determine the effects of triptolide (TP) on the expression of interleukin-18 (IL-18) and its receptor in phorbol 12-myristate 13-acetate (PMA)-stimulated rheumatoid arthritis synovial fibroblasts (RASF). MATERIALS AND METHODS: RASF were obtained from the synovial tissue of patients with RA. RASF were pretreated with TP (0~100 ng/ml) for 2 h before stimulation with PMA (50 ng/ml). The bioactivity of IL-18 in the supernatant was detected based on IFN-gamma secretion from IL-18-responding human myelomonocytic KG-1 cells. IL-18 level was analyzed by ELISA. In situ expression of IL-18Ralpha was determined by immunofluorescence assay. To estimate the protein and mRNA expression of IL-18 and IL-18Ralpha in RASF, western blot and quantitative RT-PCR were performed. Nuclear factor-kappaB (NF-kappaB) activity in the whole-cell extract of treated RASF was also measured using an ELISA-based method. RESULTS: TP effectively inhibited the bioactivity of IL-18 in PMA-stimulated RASF. The expression of IL-18 and IL-18R at protein and gene levels was reduced by TP. NF-kappaB activity in PMA-stimulated RASF was profoundly suppressed by TP. These effects showed a high correlation with TP concentration (0~100 ng/ml). CONCLUSION: TP effectively inhibited the expression of IL-18 and its receptor in PMA-stimulated RASF. These results suggest a mechanism of TP in RA therapy.
Subject(s)
Arthritis, Rheumatoid/immunology , Diterpenes/metabolism , Fibroblasts/metabolism , Immunosuppressive Agents/metabolism , Interleukin-18/immunology , Phenanthrenes/metabolism , Receptors, Interleukin-18/immunology , Synovial Membrane/cytology , Animals , Arthritis, Rheumatoid/pathology , Cells, Cultured , Diterpenes/pharmacology , Epoxy Compounds/metabolism , Epoxy Compounds/pharmacology , Fibroblasts/cytology , Fibroblasts/drug effects , Humans , Immunosuppressive Agents/pharmacology , Interleukin-18/genetics , Phenanthrenes/pharmacology , Receptors, Interleukin-18/genetics , Tetradecanoylphorbol Acetate/metabolism , Transcription Factor RelA/metabolismABSTRACT
The aim of this study was to develop a tumour vaccine with the ability to induce and expand higher affinity cytotoxic T lymphocytes and stimulate an effective antitumour immune response. The hypothesis tested was that G422 glioblastoma cells modified with B7-1 and interferon (IFN)-gamma genes could serve as a tumour vaccine. It was found that therapeutic subcutaneous immunizations with this tumour vaccine significantly induced a cytotoxic T-cell response and prolonged the survival of female Kuming mice with intracerebral G422 tumour isografts. The data collectively suggested that G422 glioblastoma cells genetically modified with B7-1 and IFN-gamma genes could serve as a tumour vaccine.
Subject(s)
Brain Neoplasms/prevention & control , Cancer Vaccines/administration & dosage , Disease Models, Animal , Glioblastoma/prevention & control , Immunotherapy, Adoptive , Animals , B7-1 Antigen/biosynthesis , B7-1 Antigen/genetics , Brain Neoplasms/genetics , Brain Neoplasms/immunology , Cancer Vaccines/genetics , Cancer Vaccines/immunology , Cell Line, Tumor , Female , Glioblastoma/genetics , Glioblastoma/immunology , Immunotherapy, Adoptive/methods , Injections, Subcutaneous , Interferon-gamma/genetics , Mice , Mice, Inbred Strains , T-Lymphocytes, Cytotoxic/immunologyABSTRACT
Successful adoptive immunotherapy for leukemia depends on the generation of T cells that can specifically react with malignant cells. Dendritic cells (DCs) are important antigen-presenting cells in the development of antileukemia T-cell responses. Mononuclear cells (MNC) were isolated from peripheral blood or bone marrow of patients with chronic myelogenous leukemia (CML), and acute myelogenous leukemia (AML). After incubation with granulocyte-macrophage colony-stimulating factor, interleukin (IL)-4, and tumor necrosis factor-alpha, MNC developed morphological characteristics of DCs in vitro, which were confirmed by phenotypic assay. Fluorescence in situ hybridization demonstrated the presence of fusion gene in the nuclei of representative CML or AML-M3 samples, indicating that the cells were leukemic in origin. IL-12 levels were significantly higher in AML-DCs and CML-DCs prestimulated with phorbol 12-myristate 13-acetate than in the corresponding leukemic cells, but were lower than that of healthy donors. These cells were potent stimulators of lymphocyte proliferation in specific in vitro assays for DC function. However, the stimulatory abilities of allogeneic T cells in a mixed lymphocyte reaction were impaired compared with those of mature DCs derived from healthy donors, although T-cell stimulatory effects were significantly increased in these differentiated leukemia-DCs. These results suggest that functional DCs may be derived from leukemic (AML, CML) blasts in a significant number of patients and may be capable of inducing leukemia-specific immune responses with potentially clinically beneficial effects.
Subject(s)
Cell Differentiation/immunology , Dendritic Cells/immunology , Leukemia, Myeloid/immunology , Cells, Cultured , Flow Cytometry , Humans , In Vitro Techniques , Leukemia, Myeloid/pathology , Lymphocyte Culture Test, MixedABSTRACT
AIM: Recombiant human telomerase reverse transcriptase (hTERT)-human IL-18(hIL18) was constructed to investigate its expression and biological function in eukaryotic cells. DCs transfected with hTERT-IL18 acquired stronger telomerase activity and were able to elicit an hTERT-specific cytotoxic T lymphocyte CTL response in vitro. METHODS: hIL-18 gene fragment was amplified by polymerase chain reaction (PCR) and TA cloned. The hIL-18 gene was then subcloned into eukaryotic expression vector pcDNA3.1(+) containing human TERT via a linker. The sequence of gene fusion was confirmed using both restrictive enzyme digestion and DNA sequencer. The expression vector with gene fusion was transfected into 3T3 cell line with Lipofectamine 2000. ELISA, Western blot, immunofluorescence stain were performed to determine the expression properties of hTERT-hIL18 in 3T3 cells. Its biological effect on the anti-apoptosis was measured by Flow cytometry and its effect on INF-gamma expression was determined using ELISA. After preparation of dendritic cells, hTERT-hIL18, hTERT, hIL-18 expression vectors were transfected into DCs respectively by electroporation to generate hTERT-specific DCs lines. The peripheral blood mononuclear cells PBMCs were stimulated with different DCs lines to create specific CTL. The response of target cell (leukemia cell line-K562 cell) to hTERT-specific CTL was evaluated by LDH release assay. RESULTS: The human IL-18 gene fragment was amplified from the human mononuclear cells and was inserted into pcDNA3.1(+)/hTERT vector successfully. The correct sequence was proved by both restrictive enzyme digestion and sequencing. The correct open reading frame was also verified. Fusion protein of hTERT-hIL18 was effectively expressed in eukaryotic cells, which was detected by both Western-blotting and immunofluorescence stain. The expressed recombinant fusion protein induced similar levels of INF-gamma to that of native IL-18 protein. FCM assay showed that the transfected fusion protein inhibited the apoptosis, which was consistent with the effects of hTERT as a universal tumor associated antigen. CTL assay shows that hTERT- hIL18 and hTERT gene-transfected DCs stimulated T-cell responses that recognized and lysed tumor target cells of high hTERT expression, whereas DCs transfected with hIL-18 gene didn't induced the response of tumor targets lyses. CONCLUSION: The Recombinant hTERT- hIL18 fusion protein had both biological activity of hTERT and hIL-18, indicating that this rationally designed protein can be further developed as novel tumor therapeutics. DCs transfected with hTERT-IL18 gene were capable of eliciting a stronger hTERT-specific CTL response in vitro.
Subject(s)
Genetic Vectors/genetics , Interleukin-18/genetics , T-Lymphocytes, Cytotoxic/immunology , Telomerase/genetics , 3T3 Cells , Animals , Apoptosis/drug effects , Apoptosis/genetics , Cancer Vaccines/genetics , Cancer Vaccines/immunology , Cell Line, Tumor , Coculture Techniques , Culture Media, Conditioned/pharmacology , Cytotoxicity Tests, Immunologic , Dendritic Cells/cytology , Dendritic Cells/immunology , Dendritic Cells/metabolism , Humans , Interferon-gamma/metabolism , Interleukin-18/metabolism , K562 Cells , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Methotrexate/pharmacology , Mice , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , T-Lymphocytes, Cytotoxic/cytology , T-Lymphocytes, Cytotoxic/metabolism , Telomerase/immunology , Telomerase/metabolism , TransfectionABSTRACT
Aberrant expression of the RON receptor tyrosine kinase has been implicated in the pathogenesis of epithelial tumours. The aim of this study was to determine RON expression in various normal epithelial cells and their corresponding tumours by immunohistochemistry. The role of RON in regulating tumourigenic phenotypes was also studied using thyroid cancer cells as a model. RON was almost exclusively expressed at variable levels in normal epithelial cells from the digestive track, lung, kidney, pancreas, liver, breast, bladder, skin, and others. Among 15 types of cancer studied, RON was overexpressed in significant numbers in cancers derived from breast (56%), colon (51%), lung (48), thyroid (42%), skin (37%), bladder (36%), and pancreas (33%). In contrast, limited RON overexpression was observed in cancers from stomach, kidney, brain, liver, ovary, and prostate. Detailed analysis of thyroid tissues showed that RON was hardly detected in normal thyroid cells, moderately expressed in adenoma samples, but overexpressed in about half of papillary and follicular cancer specimens. Overexpression correlated with advanced clinical stage and was associated with lymph node metastasis. In cultured thyroid cancer cells, RON was highly expressed, with constitutive phosphorylation. Activation of RON increased cell growth and migration via the MAP kinase and AKT pathways. Silencing RON expression significantly prevented cell growth and increased cell apoptotic death. These findings show that RON overexpression occurs in a particular group of epithelial cancers. The requirement for RON in sustaining tumourigenic phenotypes suggests that it is a potential target for therapeutic intervention.
Subject(s)
Neoplasm Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Thyroid Neoplasms/metabolism , Antibodies, Monoclonal/immunology , Apoptosis , Cell Division , Cell Transformation, Neoplastic , Disease Progression , Epithelial Cells/metabolism , Female , Gene Expression Regulation, Neoplastic , Gene Silencing , Humans , Lymphatic Metastasis , Male , Neoplasm Proteins/genetics , Neoplasm Proteins/immunology , Neoplasm Staging , Neoplasms/metabolism , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/immunology , Thyroid Neoplasms/pathology , Tissue Array Analysis/methods , Tumor Cells, CulturedABSTRACT
Hepatic fibrosis is a common outcome of chronic liver diseases. In schistosomiasis, chronic parasite egg-induced granuloma formation can lead to fibrosis, which is immunologically characterized by the dominant Th2 response. Recently, it has been shown that gene therapy is an attractive approach for the treatment of hepatic fibrosis. To investigate the antifibrotic effects of IL-18 gene transfer, a normal murine liver cell line BNL.CL2 was transfected with recombinant adenovirus encoding mouse IL-18, and then intrasplenically transplanted into mice infected with Schistosoma japonicum (S. japonicum). Our data show that IL-18 gene-modified hepatocytes intrasplenically transplanted into mice can effectively express IL-18 in the liver and in peripheral blood. Intrasplenic transplantation of IL-18 gene-modified hepatocytes into S. japonicum-infected mice could result in a significantly increased IFN-gamma and IL-2 but decreased IL-4 and IL-10 concentration both in the liver and in the serum, suggesting that the dominant Th2 response in mice with schistosomiasis could be reversed by this intervention. Consistent with the changes in Th1 and Th2 cytokine production, mice intrasplenically transplanted with IL-18 gene-modified hepatocytes developed much less hepatic fibrosis at 20 weeks after infection, which was evaluated by liver content of hydroxyproline, collagens, and hepatic mRNA expression of procollagens. These data indicate that intrasplenic transplantation of IL-18 gene-modified hepatocytes can be a candidate for therapeutic intervention in hepatic fibrosis through induction of a dominant Th1 response.
Subject(s)
Genetic Therapy/methods , Hepatocytes/transplantation , Interleukin-18/genetics , Liver Diseases, Parasitic/therapy , Schistosomiasis/therapy , Th1 Cells/immunology , Adenoviridae/genetics , Animals , Gene Expression , Genetic Vectors , Hepatocytes/immunology , Liver Cirrhosis/therapy , Liver Diseases, Parasitic/immunology , Male , Mice , Mice, Inbred BALB C , Schistosomiasis/immunology , SpleenABSTRACT
OBJECTIVE: To investigate the immunoregulatory effect of immunostimulatory DNA sequence (ISS) on the production of Th1 and Th2 cytokines induced by mite allergen in PBMC of the patients with mite allergic asthma in vitro. METHODS: PBMC from the patients with allergic asthma and normal controls were isolated and cultured in vitro stimulated by ISS and Dermatophagoides farinae allergen (Df). IL-12, IFN-gamma and IL-5 in the cell supernatants were detected by ELISA. Df specific IgE in sera of patients were assayed by fluorescent enzyme immunoassay. RESULTS: PBMC from both the patients and normal controls stimulated by ISS plus Df produced a significant increase in the level of both IFN-gamma and IL-12 compared with non-ISS and Df stimulations, whereas IL-5 was decreased. Moreover, the levels of IFN-gamma, IL-12 produced were significantly higher in normal controls than in the patients, on the contrary, IL-5 was down regulated. It was also shown that the level of IL-12 produced by PBMC of the patients with ISS plus Df stimulation correlated positively with that of IFN-gamma. CONCLUSION: ISS not only promotes the expressions of Th1 cytokines but also downregulates the production of Th2 cytokines induced by Df in both allergic and non-allergic individuals, indicating its potential application in the immunotherapy of mite allergy.
Subject(s)
Adjuvants, Immunologic/pharmacology , Antigens, Dermatophagoides/pharmacology , Asthma/immunology , Th1 Cells/immunology , Th2 Cells/immunology , Adult , Animals , Asthma/metabolism , Base Sequence , Cells, Cultured , Female , Humans , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/immunology , Male , Middle Aged , Mites/immunology , Th1 Cells/metabolism , Th2 Cells/metabolismABSTRACT
OBJECTIVE: To study the inhibitory effect of Kangxian Recipe (KXR) on TGF-beta 1 induced hepatocyte apoptosis. METHODS: The in vitro model of hepatocyte apoptosis was established by cell biologic methods, utilizing the characteristics of TGF-beta 1 to observe the inhibitory effect of KXR on hepatocyte apoptosis. RESULTS: TGF-beta 1 induced apoptosis of hepatocyte in a dose-dependent manner. The apoptosis rate of 2.2.15 cells was 63% when 500 ng/L TGF-beta 1 was used, while for HepG2 cell, it was merely 44%. After treatment of 20 ng/L KXR, the apoptosis rate of the two kinds of cell lines lowered to 33% and 24% respectively. The inhibition rate of both groups was about 50%. CONCLUSION: KXR had strong inhibitory effect on hepatocyte apoptosis induced by TGF-beta 1.