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IgG kappa and IgG lambda concentrations were quantified in 96 paired CSF and sera using Hevylite™ antibodies in an in-house developed sandwich ELISA method. In 56 of these samples, the results were compared with a qualitative isoelectric focusing/affinity-mediated immunoblotting assay for oligoclonal IgG kappa and IgG lambda. Normal IgG kappa/lambda ratio in the CSF was the same as in serum. In 19/33 patients with intrathecal oligoclonal IgG synthesis, skewed IgG kappa/lambda ratio was observed (increased in 16 and decreased in 3 cases). The analysis of light chain composition of intrathecally synthesised immunoglobulins could contribute to our understanding of intrathecal humoral immune response, although its diagnostic utility is limited.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina G/líquido cefalorraquidiano , Cadeias kappa de Imunoglobulina/líquido cefalorraquidiano , Cadeias lambda de Imunoglobulina/líquido cefalorraquidiano , Humanos , Imunoglobulina G/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Cadeias lambda de Imunoglobulina/imunologiaRESUMO
OBJECTIVE: The present study aimed to verify the reference intervals of plasma matrix metalloproteinases (MMPs) 2, 3, and 9 and serum asymmetric dimethylarginine (ADMA) in a healthy population with an average age corresponding to that of patients with cardiovascular diseases. METHODS: The study included 180 healthy volunteers. Plasma MMP-2, MMP-3, MMP-9, and serum ADMA levels were determined using an enzyme-linked immunosorbent assay. These levels were analyzed for association with age and gender. The Cbstat5, R software, and NCSS 2007 programs were used for statistical analysis. RESULTS: The average volunteer age was 47.4 years in the group in which MMP-3 and ADMA were analyzed, 40.3 years in the MMP-9 group, and 47.8 years for the MMP-2 group. Serum ADMA levels were determined to be independent of age and gender. Plasma MMP-2 levels were significantly correlated with age (p = 0.001), with lower levels detected in persons ≤ 49 years of age. Plasma MMP-3 was significantly associated with both age (p < 0.0001) and gender, with lower levels detected in persons of ≤ 47 years of age and among women. Plasma MMP-9 levels were not age dependent, but were associated with gender (p = 0.014), showing lower levels in women. CONCLUSIONS: Reference intervals of heparin-plasma MMP-2, MMP-3, and MMP-9 and serum ADMA levels were determined. MMP-2 and MMP-3 levels were found to be age dependent, and MMP-3 and MMP-9 levels were gender dependent.
Assuntos
Arginina/análogos & derivados , Metaloproteinase 2 da Matriz/sangue , Metaloproteinase 3 da Matriz/sangue , Metaloproteinase 9 da Matriz/sangue , Adulto , Arginina/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valores de ReferênciaRESUMO
Secretoneurin (SN) is a 33 amino-acid evolutionary conserved neuropeptide from the chromogranin peptide family. SN's main effects may be cardioprotective and are believed to be mediated through its inhibition of calmodulin-dependent kinase II (CaMKII), which influences intracellular calcium handling. SN inhibition of CaMKII suppresses calcium leakage from the sarcoplasmic reticulum through the ryanodine receptor. This action may reduce the risk of ventricular arrhythmias and calcium-dependent remodelling in heart failure. SN is also involved in reducing the intracellular reactive oxygen species concentration, modulating the immune response, and regulating the cell cycle, including apoptosis. SN can predict mortality in different disease states, beyond the classical risk factors and markers of myocardial injury. Plasma SN levels are elevated soon after an arrhythmogenic episode. In summary, SN is a novel biomarker with potential in cardiovascular medicine, and probably beyond.
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AIMS: The study aims were to verify the serum (S) and synovial fluid (SF) reference intervals (RIs) for human neutrophil defensins (HNP1-3); measure S and SF defensin concentrations in different types of SF, including non-inflammatory, inflammatory non-pyogenic, inflammatory pyogenic, and hemorrhagic; and to compare the HNP1-3 concentrations in SF and S with those of other inflammatory biomarkers. METHODS: SF and S samples were collected from 92 patients. HNP1-3 concentrations were determined using enzyme-linked immunosorbent assays; glucose, lactate, interleukin-6, and procalcitonin using an automatic analyzer; and presepsin using a Pathfast system. There were 61 non-inflammatory, 11 inflammatory non-pyogenic, 11 inflammatory pyogenic, and 9 hemorrhagic SF. Non-inflammatory SF was divided into non-inflammatory normal and non-inflammatory osteoarthritis. The former was used to estimate the HNP1-3 RI in SF and S. RESULTS: The estimated HNP1-3 RIs of SF and S were 12.47-437.42 mg/L and 5.45-44.75 µg/L, respectively. HNP1-3 differed significantly between S and SF and individual groups of SF (P<0.001 and P=0.001, respectively). There were significant relationships between SF HNP1-3 and S HNP1-3 (P<0.001), S C-reactive protein (P<0.001), and S interleukin-6 (P=0.007), and between SF HNP1-3 and SF C-reactive protein (P=0.004) and SF interleukin-6 (P<0.001). The highest kappa coefficient was between SF HNP1-3 and SF interleukin-6 (κ=0.507). CONCLUSIONS: We validated the SF HNP1-3 diagnostic kit and demonstrated that SF and S HNP1-3 are promising biomarkers for distinguishing inflammatory from non-inflammatory joint diseases.
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Ensaio de Imunoadsorção Enzimática , alfa-Defensinas , Biomarcadores , Proteína C-Reativa , Humanos , Interleucina-6 , Receptores de Lipopolissacarídeos , Fragmentos de Peptídeos , Líquido SinovialRESUMO
BACKGROUND: Total gentamicin is a sum of five congeners C1, C1a, C2, C2a and minor C2b, which differ from each other in their methylation on the purpurosamine ring. Liquid chromatography with mass detection (LC-MS/MS) and specified calibration material enables the concentration of total gentamicin and its individual congeners to be analysed. METHODS: 50 µL serum was precipitated with acetonitrile in the presence of 0.5 mol/L formic acid. A RP BEH C18 1.7 µm 2.1x50 mm column maintained at 30 °C and tobramicin as the internal standard were used. Mass detection was performed in positive electrospray. The gentamicin results were compared with fluorescence polarization immunoassay (FPIA) and chemiluminiscent microparticle immunoassay (CMIA). Passing-Bablock regression analysis and Bland-Altman analysis were used. RESULTS: Calibration curves for individual gentamicin congeners were linear with correlation coefficients between 0.997 and 0.998. Recovery was 91.6-102.0% and the coefficients of variation 1.4-8.4%. The total gentamicin concentration was compared with immunoassay FPIA (LC-MSgen = 0.9798xPFIAgen) and CMIA (LC MSgen = 0.9835xCMIAgen) both with significant correlation (p < 0.001). CONCLUSION: The LC-MS/MS method is fast and precise and can be applied to routine TDM in patients. Comparing it to immunoassays makes it possible to measure concentration of gentamicin congeners, which may be important in the case of their different pharmacokinetics.
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Gentamicinas , Espectrometria de Massas em Tandem , Cromatografia Líquida , Imunoensaio de Fluorescência por Polarização , Humanos , Imunoensaio , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Intrathecal IgM synthesis demonstrated either as cerebrospinal fluid (CSF)-restricted oligoclonal (o-) IgM bands or calculated using various formulas has been linked to more aggressive multiple sclerosis (MS) course. However, the proportion of MS patients showing intrathecal IgM synthesis varies largely between studies. We aimed to explore the relation between different formulas and results of o-IgM, and to assess the frequency of o-IgM bands in an unselected series of samples. METHODS: 432 samples were analyzed for o-IgM, o-IgG and quantitative measures of IgM and IgG synthesis. IgM index and formulas of Reiber, Auer and Öhman were compared to the result of the o-IgM test. RESULTS: At the cut-off commonly used, the non-linear formulas for intrathecal synthesis were specific (>94%) but rather insensitive (<40% even at a cut-off of 4 CSF-restricted bands) compared to o-IgM. No significant difference was noted in the performance of different formulas. At a cut-off of 4 bands, 61% of MS patients, but none of the controls were positive for o-IgM. CONCLUSIONS: Formulas for intrathecal IgM synthesis are insensitive compared to o-IgM. We propose to evaluate samples with 2 or 3 extra-CSF IgM bands as borderline and only samples with 4 or more as definitely positive.
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Esclerose Múltipla , Bandas Oligoclonais , Humanos , Imunoglobulina M , Esclerose Múltipla/diagnósticoRESUMO
OBJECTIVES: We aimed to compare various methods for free light chain (fLC) quantitation in cerebrospinal fluid (CSF) and serum and to determine whether quantitative CSF measurements could reliably predict intrathecal fLC synthesis. In addition, we wished to determine the relationship between free kappa and free lambda light chain concentrations in CSF and serum in various disease groups. METHODS: We analysed 166 paired CSF and serum samples by at least one of the following methods: turbidimetry (Freelite™, SPAPLUS), nephelometry (N Latex FLC™, BN ProSpec), and two different (commercially available and in-house developed) sandwich ELISAs. The results were compared with oligoclonal fLC detected by affinity-mediated immunoblotting after isoelectric focusing. RESULTS: Although the correlations between quantitative methods were good, both proportional and systematic differences were discerned. However, no major differences were observed in the prediction of positive oligoclonal fLC test. Surprisingly, CSF free kappa/free lambda light chain ratios were lower than those in serum in about 75% of samples with negative oligoclonal fLC test. In about a half of patients with multiple sclerosis and clinically isolated syndrome, profoundly increased free kappa/free lambda light chain ratios were found in the CSF. CONCLUSIONS: Our results show that using appropriate method-specific cut-offs, different methods of CSF fLC quantitation can be used for the prediction of intrathecal fLC synthesis. The reason for unexpectedly low free kappa/free lambda light chain ratios in normal CSFs remains to be elucidated. Whereas CSF free kappa light chain concentration is increased in most patients with multiple sclerosis and clinically isolated syndrome, CSF free lambda light chain values show large interindividual variability in these patients and should be investigated further for possible immunopathological and prognostic significance.
Assuntos
Doenças Desmielinizantes/diagnóstico , Cadeias kappa de Imunoglobulina/biossíntese , Cadeias lambda de Imunoglobulina/biossíntese , Esclerose Múltipla/diagnóstico , Estudos de Casos e Controles , Doenças Desmielinizantes/sangue , Doenças Desmielinizantes/líquido cefalorraquidiano , Ensaio de Imunoadsorção Enzimática/instrumentação , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Immunoblotting/instrumentação , Immunoblotting/métodos , Cadeias kappa de Imunoglobulina/sangue , Cadeias kappa de Imunoglobulina/líquido cefalorraquidiano , Cadeias lambda de Imunoglobulina/sangue , Cadeias lambda de Imunoglobulina/líquido cefalorraquidiano , Focalização Isoelétrica/instrumentação , Focalização Isoelétrica/métodos , Esclerose Múltipla/sangue , Esclerose Múltipla/líquido cefalorraquidiano , Nefelometria e Turbidimetria/instrumentação , Nefelometria e Turbidimetria/métodos , Variações Dependentes do Observador , Curva ROC , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Owing to the high correlation between the level of prolyl-4-hydroxyproline dipeptide in non-hydrolyzed urine and that of 4-hydroxyproline in hydrolyzed urine, we examined whether the dipeptide might function as a valuable marker of bone turnover. METHODS: Based on densitometric measurements, 68 postmenopausal women were divided into groups of non-osteopathic, osteopenic and osteoporotic subjects. The dipeptide and current urinary resorption markers were assayed in morning urine, the former using liquid chromatography, the others plus serum formation markers by means of immunoassay procedures. Together with the assay of basal levels, diet-related changes and healing effect of yearly alendronate therapy were assessed. RESULTS: Concentration levels in controls and osteoporotic subjects differed significantly; receiver operating characteristics yielded sensitivity of 0.743, specificity of 0.908, area under curve of 0.903, and cut-off of 10.2 micromol/mmol of creatinine. Spearman rank correlation showed the highest pair coefficient between the dipeptide and osteocalcin. Diet-related changes were not found. Following therapy, a significant decline occurred already within a trimester, whilst with the other resorption markers not until 6 months. CONCLUSIONS: The ease of the dipeptide assay in non-hydrolyzed urine surpasses that of hydroxyproline, and the results present the compound as a real competition to other commonly assessed markers in osteopathies.