Time course proteomic profiling of human myocardial infarction plasma samples: an approach to new biomarker discovery

BackgroundThe aim of this study was to identify novel candidate biomarker proteins differentially expressed in the plasma of patients with early stage acute myocardial infarction (AMI) using SELDI-TOF-MS as a high throughput screening technology.MethodsTen individuals with recent acute ischemic-type chest pain (< 12 h duration) and ST-segment elevation AMI (1STEMI) and after a second AMI (2STEMI) were selected. Blood samples were drawn at six times after STEMI diagnosis. The first stage (T₀) was in Emergency Unit before receiving any medication, the second was just after primary angioplasty (T₂), and the next four stages occurred at 12 h intervals after T₀. Individuals (n = 7) with similar risk factors for cardiovascular disease and normal ergometric test were selected as a control group (CG). Plasma proteomic profiling analysis was performed using the top-down (i.e. intact proteins) SELDI-TOF-MS, after processing in a Multiple Affinity Removal Spin Cartridge System (Agilent).ResultsCompared with the CG, the 1STEMI group exhibited 510 differentially expressed protein peaks in the first 48 h after the AMI (p < 0.05). The 2STEMI group, had ~ 85% fewer differently expressed protein peaks than those without previous history of AMI (76, p < 0.05). Among the 16 differentially-regulated protein peaks common to both STEMI cohorts (compared with the CG at T₀), 6 peaks were persistently down-regulated at more than one time-stage, and also were inversed correlated with serum protein markers (cTnI, CK and CKMB) during 48 h-period after IAM.ConclusionsProteomic analysis by SELDI-TOF-MS technology combined with bioinformatics tools demonstrated differential expression during a 48 h time course suggests a potential role of some of these proteins as biomarkers for the very early stages of AMI, as well as for monitoring early cardiac ischemic recovery.     

Solid-phase enzymoimmunoassay for osteocalcin in human serum or plasma, with use of a monoclonal antibody

In this solid-phase enzymoimmunoassay on microtiter plates for osteocalcin in serum or plasma, we use an osteocalcin-horseradish-peroxidase conjugate and a monoclonal antibody raised against bovine osteocalcin. We thoroughly standardized the assay for measurement of osteocalcin in both serum and plasma, demonstrating independence of sample volume, and determining the analytical recovery and within-and between-assay CVs. The detection limit was between 0.6 and 1.1 micrograms/L and the ED50 was 16 micrograms/L for a 5-microL sample volume. The intra-assay CV over the range 3 to 74 micrograms/L was less than or equal to 15%. The interassay CV over the range 3.6 to 46 micrograms/L was less than or equal to 16%. Results by this assay and by an in-house radioimmunoassay in which the same monoclonal antibody was used correlated well (r2 = 0.948). Osteocalcin concentrations in serum and plasma as measured with the present assay agreed well with published values.     

Radioimmunoassay of calcitonin in normal human plasma: problems, perspectives and prospects

Radioimmunoassay of calcitonin in normal human plasma is hindered by immunochemical heterogeneity in plasma, variable susceptibility of antisera to non-specific factors, limited assay sensitivity, and other problems. Many controversial points in the literature arise from these difficulties. Measurement of calcitonin after various extraction procedures yields greater consistency of results. Recent advances in calcitonin assay technology offer the promise of measuring calcitonin monomer with sufficient sensitivity to permit studies of normal calcitonin physiology and of the hormone's role in human disease.     

Tracking human neurologic disease status in mouse brain/plasma using reporter-tagged,EV-associated biomarkers

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Solid-phase fluoroimmunoassay of human albumin in biological fluids.

A simple fluoroimmunoassay for the determination of albumin levels in serum, urine and cerebrospinal fluid is described. It employs magnetisable particles to which antibodies to human serum albumin are covalently linked, and albumin labelled with fluorescein. Equilibrium is reached within 30 min, when separation of the bound and free fractions of the labelled albumin is performed by precipitation of the particles either with a magnet or by centrifugation. Measurement of the fluorescence in the supernatant (the free fraction) reflects the albumin concentration of the standards or samples. Correlation studies with an automated immunoprecipitation technique show good agreement.     

Multiplexed analysis of biomarkers related to obesity and the metabolic syndrome in human plasma, using the Luminex-100 system

BACKGROUND: The complex pathology of disease has sparked the development of novel protein expression profiling techniques that require validation in clinical settings. This study focuses on multiplexed analyses of adipocytokines and biomarkers linked to the metabolic syndrome, diabetes, and cardiovascular disease. METHODS: Multiplexed immunoassays using fluorescent microspheres and the Luminex-100 system were performed on plasma from 80 obese patients (40 with the metabolic syndrome) before and after 6-8 weeks of diet-induced weight loss. Leptin, insulin, C-peptide, monocyte chemoattractant protein-1 (MCP-1), eotaxin, interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha), and IL-6 concentrations measured with multiplex panels from 3 different manufacturers were compared with results from commercial ELISAs. Detection limits and between- and within-run imprecision were determined for each analyte. Bland-Altman analysis was used to determine agreement between multiplexed immunoassays and ELISAs. RESULTS: Correlation between the Luminex multiplexed assays and ELISAs was good for leptin (Linco), insulin (Linco), MCP-1 (Biosource and Upstate), and eotaxin (Biosource) with correlation coefficients of 0.711-0.895; fair for eotaxin (Upstate) and C-peptide (Linco) with correlation coefficients of 0.496-0.582; and poor for TNF-alpha, IL-8, and IL-6 (Linco, Biosource, Upstate, and R&D) with correlation coefficients of -0.107 to 0.318. Within- and between-run imprecision values for the multiplex method were generally <15%. Relative changes in plasma leptin and insulin concentrations after diet-induced weight loss were similar whether assessed by multiplex assay or ELISA. CONCLUSION: Although this technology appears useful in clinical research studies, low assay sensitivity and poor correlations with conventional ELISA methods for some analytes with very low plasma concentrations should be considered when using the Luminex platform in clinical studies.     

Solid-phase immunoenzyme analysis of alpha-fetoprotein in human blood serum]

A test system for enzyme immunoassay of alpha-fetoprotein (AFP) was developed on the basis of the diagnostic agents manufactured by the N. F. Gamaleya Research Institute of Epidemiology and Microbiology. The effect of shielding antibody active centers in sensitization of planes with excessive concentrations of antibodies was detected. The test system was used to measure AFP levels in blood sera of pregnant women. The results may be used for screening pregnant women for the prenatal diagnosis of fetal neural tube.     

Functional antibodies targeting IsaA of Staphylococcus aureus augment host immune response and open new perspectives for antibacterial therapy

Staphylococcus aureus is the most common cause of nosocomial infections. Multiple antibiotic resistance and severe clinical outcomes provide a strong rationale for development of immunoglobulin-based strategies. Traditionally, novel immunological approaches against bacterial pathogens involve antibodies directed against cell surface-exposed virulence-associated epitopes or toxins. In this study, we generated a monoclonal antibody targeting the housekeeping protein IsaA, a suggested soluble lytic transglycosylase of S. aureus, and tested its therapeutic efficacy in two experimental mouse infection models. A murine anti-IsaA antibody of the IgG1 subclass (UK-66P) showed the highest binding affinity in Biacore analysis. This antibody recognized all S. aureus strains tested, including hospital-acquired and community-acquired methicillin-resistant S. aureus strains. Therapeutic efficacy in vivo in mice was analyzed using a central venous catheter-related infection model and a sepsis survival model. In both models, anti-IsaA IgG1 conferred protection against staphylococcal infection. Ex vivo, UK-66P activates professional phagocytes and induces highly microbicidal reactive oxygen metabolites in a dose-dependent manner, resulting in bacterial killing. The study provides proof of concept that monoclonal IgG1 antibodies with high affinity to the ubiquitously expressed, single-epitope-targeting IsaA are effective in the treatment of staphylococcal infection in different mouse models. Anti-IsaA antibodies might be a useful component in an antibody-based therapeutic for prophylaxis or adjunctive treatment of human cases of S. aureus infections.     

脑卒中新的生物标志物研究进展

本综述阐述了多种用于脑卒中危险预测和诊断的新生物标志物,包括脂蛋白有关的磷脂酶A2、不对称的二甲基精氨酸、基质金属蛋白酶-9、S-100β、N-甲基-D-天门冬氨酸、神经胶质纤维酸性蛋白、二磷酸核苷酸激酶A等的临床意义,及多标志物组合的意义。     

Detection of a new heparin-dependent inhibitor of thrombin in human plasma.

The influence of the serum binding protein (DBP) for vitamin D and its metabolites on the concentration of its main ligands, 25-hydroxyvitamin D₃ (25-OHD₃) and 1,25-dihydroxyvitamin D₃ (1,25-[OH]₂D₃) was studied. The concentration of both 1,25-(OH)₂D₃ and DBP in normal female subjects (45±14 ng/liter and 333±58 mg/liter, mean±SD, respectively; n = 58) increased during the intake of estro-progestogens (69±27 ng/liter and 488±90 mg/liter, respectively; n = 29), whereas the 25-OHD₃ concentration remained unchanged. A positive correlation was found between the concentrations of 1,25-(OH)₂D₃ and DBP in these women.     

Protein oxidation biomarkers in plasma of type 2 diabetic patients

ObjectivesTo evaluate oxidative stress and the extent of oxidation of plasma proteins in type 2 diabetic patients.Design and methodsStudy was carried out on blood from 31 diabetic patients of both sexes (mean age = 58 ± 7; duration of diabetes 12 ± 5 years) and healthy age and sex matched normal subjects. Biomarkers of protein oxidation; plasma protein carbonyls (PCO), advanced oxidation protein products (AOPPs) and –SH group and free radical scavenging capacity of plasma was measured.ResultsPCO and AOPPS levels were significantly (P < 0.005) higher in diabetic patients in comparison to healthy volunteers. Reduced free radical scavenging capacity (P < 0.001) and –SH group (P < 0.05) was observed in plasma of type 2 diabetic patients.ConclusionsOur data suggest that diabetics are susceptible to protein oxidation. Oxidative modulation of proteins due to reduced radical scavenging activity of plasma patients may be one of the reasons of altered physiological processes in type 2 diabetic patients.     

Current perspectives of new agents in lung cancer

Irinotecan(CPT-11), Taxol, Taxotere, vinorelbine and gemcitabine have shown a significant activity in previously untreated non-small cell lung cancer (NSCLC). Cisplatin(CDDP) combined with vinorelbine, gemcitabine or tirapazamine was significantly superior in survival to CDDP alone in the treatment of advanced NSCLC. Patients with NSCLC treated with combination of CDDP and Taxol or vinorelbine lived longer than those treated with conventional CDDP-based chemotherapy. CPT-11, topotecan, taxol and amrubicin have demonstrated to be active against small cell lung cancer(SCLC). Combination of CPT-11 and CDDP have had a higher response rate, and better median survival(13 months) in patients with extensive disease SCLC. Clinical trials of target-based drugs including matrix metalloprotenase inhibitors, anti-angiogenesis, tyrosine kinase inhibitors, farnesyl transferase inhibitors and monoclonal antibodies have been initiated in solid tumors including lung cancer. Development of new anti-cancer agents is essential to improve outcomes of patients with lung cancer.     

Telomere targeting with a new G4 ligand enhances radiation-induced killing of human glioblastoma cells

The aim of this study was to test in vitro the efficacy of TAC, an original G-quadruplex ligand, as a potential radiosensitizing agent for glioblastoma multiforme (GBM). Two human radioresistant telomerase-positive GBM cell lines (SF763 and SF767) were analyzed, with and without TAC treatment, for telomere length, cell proliferation, apoptosis, cell-cycle distribution, gene expression, cytogenetic aberrations, clonogenic survival assay, 53BP1 immunofluorescence staining, and γH2AX phosphorylation. We found that low concentrations of TAC (0.5 and 1 μmol/L) inhibited the proliferation of GBM cells in a concentration-dependent manner after only 1 week of treatment, with minimal effects on cell cycle and apoptosis. TAC treatment had no visible effect on average telomere length but modified expression levels of telomere-related genes (hTERT, TRF1, and TRF2) and induced concentration-dependent DNA damage response and dicentric chromosomes. Survival curves analysis showed that exposure to nontoxic, subapoptotic concentrations of TAC enhanced radiation-induced killing of GBM cells. Analysis of DNA repair after irradiation revealed delayed repair kinetics in GBM cells treated with TAC. Furthermore, the combined treatment (TAC and radiation) significantly increased the frequency of chromosomal aberrations as compared with radiation alone. These findings provide the first evidence that exposure to a G4 ligand radiosensitizes human glioblastoma cells and suggest the prospect of future therapeutic applications.     

Structure-functional diversity of human L-type Ca2+ channel: perspectives for new pharmacological targets

The L-type Ca2+ channels mediate depolarization-induced influx of Ca2+ into a wide variety of cells and thus play a central role in triggering cardiac and smooth muscle contraction. Because of this role, clinically important classes of 1,4-dihydropyridine, phenylalkylamine, and benzothiazepine Ca2+ channel blockers were developed as powerful medicines to treat hypertension and angina pectoris. Molecular cloning studies revealed that the channel is subject to extensive structure-functional variability due to alternative splicing. In this review, we will focus on a potentially important role of genetically driven variability of Ca2+ channels in expression regulation and mutations, Ca2+-induced inactivation, and modulation of sensitivity to Ca2+ channel blockers with the perspective for new pharmacological targets.     

High-throughput metabolomics enables metabolite biomarkers and metabolic mechanism discovery of fish in response to alkalinity stress

High throughput mass spectrometry (MS)-based metabolomics is a popular platform for small molecule metabolites analyses that are widely used for detecting biomarkers in the research field of environmental assessment. Crucian carp (Carassius carassius, CC) is an economically and ecologically important fish in Asia. It can adapt to extremely high alkalinity, providing us with valuable material to understand the adaptation mechanism for extreme environmental stress. However, the information on the metabolite biomarkers and metabolic mechanisms of CC exposed to alkaline stress is not entirely clear. We applied high-throughput UPLC-Q-TOF/MS combined with chemometrics to identify changes in the metabolome of CC exposed to different concentrations of alkalinity for long term effects. Metabolic differences among alkalinity-treated groups were identified by multivariate statistical analysis. Further, 7 differential metabolites were found after exposure to alkaline conditions. In total, 23 metabolic pathways of these differential metabolites were significantly affected. Alkalinity exposure resulted in widespread change in metabolic profiles in the plasma with disruptions in the phenylalanine metabolism, glycine, serine and threonine metabolism, pyruvate metabolism, tyrosine metabolism, etc. The integrated pathway analysis of the associated metabolites showed that tRNA charging, l-cysteine degradation II, superpathway of methionine degradation, l-serine degradation, tyrosine biosynthesis IV, etc. appear to be the most significantly represented functional categories. Overall, this study demonstrated that metabolic changes in CC played a role in adaptation to the highly alkaline environmental stress.

High throughput mass spectrometry (MS)-based metabolomics is a popular platform for small molecule metabolites analyses that are widely used for detecting biomarkers in the research field of environmental assessment.     

Solid-phase extraction and liquid chromatography for improved assay of cyclosporine in whole blood or plasma

In this simple, precise, accurate, and specific isocratic liquid chromatographic procedure for determining cyclosporine, the cyclosporine is extracted from 1 mL of whole blood or from plasma, with 500 micrograms of cyclosporin D added per liter as internal standard, by elution from a Bond-ElutTM C18 extraction column with 300 microL of a mixture of ethanol and tetrahydrofuran. A 100-microL aliquot of the eluate, injected onto a cyano-phase analytical column, is eluted with a mixture of acetonitrile and pH 7.0 phosphate buffer at a flow rate of 1.0 mL/min and at 50 degrees C. Detection is at 210 nm. The chromatography is complete in less than 14.0 min. The method can measure less than 10.0 micrograms/L. Analytical recovery of cyclosporine added to whole blood ranged from 99 to 109% for concentrations up to 2000 micrograms/L. Between-run CVs ranged from 6.4 to 6.6%. None of numerous drugs and steroids tested interfered. Results by radioimmunoassay exceeded by 20 to 350% those measured by the present method.     

A new medium improves sampling for determination of platelet factor 4 in human plasma

The determination of alpha-granule specific platelet proteins in human plasma is a valuable indicator of in-vivo release and platelet activation provided that an artificial in-vitro release during sampling can be avoided. A new sampling medium (M) has been compared with the commercially available ones for the sampling and the subsequent determination of platelet factor 4 and beta-thromboglobulin. M allows blood withdrawal at room temperature (22 degrees C) (3.49 +/- 0.7 ng/ml) without any difference to 4 degrees C (3.47 ng/ml) up to a 30 minutes sedimentation period. However, after 40 minutes there is a significant (p less than 0.01) increase, amounting at 22 degrees C (13.30 ng/ml) and at 4 degrees C 6.68 ng/ml. The addition of prostacyclin (PGI2) in-vitro as well as blood withdrawal from volunteers infused with PGI2 at a rate of 5 ng/kg/min did not alter the values of M-preserved samples, whereas in the commercially available ones, PGI2 caused a decrease. A comparison of radioimmunoassay and enzyme immunoassay exhibited no significant difference in the absolute values. Beta-thromboglobulin was assayed as control. The findings indicate M being optimal for the sampling of platelet factor 4, but not for other alpha-granule release products. Thus, there is again evidence, that the alpha-granule products are not released to an equal extent upon a certain stimulus. Furthermore, it seems likely, that various substances inhibit their liberation from the platelet to a differing extent.