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1.
Cell ; 136(3): 551-64, 2009 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-19185337

RESUMO

The generation of cortical projection neurons relies on the coordination of radial migration with branching. Here, we report that the multisubunit histone acetyltransferase Elongator complex, which contributes to transcript elongation, also regulates the maturation of projection neurons. Indeed, silencing of its scaffold (Elp1) or catalytic subunit (Elp3) cell-autonomously delays the migration and impairs the branching of projection neurons. Strikingly, neurons defective in Elongator show reduced levels of acetylated alpha-tubulin. Reduction of alpha-tubulin acetylation via expression of a nonacetylatable alpha-tubulin mutant leads to comparable defects in cortical neurons and suggests that alpha-tubulin is a target of Elp3. This is further supported by the demonstration that Elp3 promotes acetylation and counteracts HDAC6-mediated deacetylation of this substrate in vitro. Our results uncover alpha-tubulin as a target of the Elongator complex and suggest that a tight regulation of its acetylation underlies the maturation of cortical projection neurons.


Assuntos
Movimento Celular , Córtex Cerebral/citologia , Histona Acetiltransferases/metabolismo , Neurônios/citologia , Tubulina (Proteína)/metabolismo , Acetilação , Animais , Linhagem Celular , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Humanos , Camundongos , Complexos Multienzimáticos/metabolismo , Neurogênese
2.
J Biol Chem ; 287(39): 32535-45, 2012 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-22854966

RESUMO

The Elongator complex is composed of 6 subunits (Elp1-Elp6) and promotes RNAPII transcript elongation through histone acetylation in the nucleus as well as tRNA modification in the cytoplasm. This acetyltransferase complex directly or indirectly regulates numerous biological processes ranging from exocytosis and resistance to heat shock in yeast to cell migration and neuronal differentiation in higher eukaryotes. The identity of human ELP1 through ELP4 has been reported but human ELP5 and ELP6 have remained uncharacterized. Here, we report that DERP6 (ELP5) and C3ORF75 (ELP6) encode these subunits of human Elongator. We further investigated the importance and function of these two subunits by a combination of biochemical analysis and cellular assays. Our results show that DERP6/ELP5 is required for the integrity of Elongator and directly connects ELP3 to ELP4. Importantly, the migration and tumorigenicity of melanoma-derived cells are significantly decreased upon Elongator depletion through ELP1 or ELP3. Strikingly, DERP6/ELP5 and C3ORF75/ELP6-depleted melanoma cells have similar defects, further supporting the idea that DERP6/ELP5 and C3ORF75/ELP6 are essential for Elongator function. Together, our data identify DERP6/ELP5 and C3ORF75/ELP6 as key players for migration, invasion and tumorigenicity of melanoma cells, as integral subunits of Elongator.


Assuntos
Proteínas de Transporte/metabolismo , Movimento Celular , Melanoma/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas/metabolismo , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Deleção de Genes , Células HEK293 , Histona Acetiltransferases , Humanos , Melanoma/genética , Melanoma/patologia , Complexos Multiproteicos/genética , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Proteínas/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo
3.
Trends Biochem Sci ; 33(4): 171-80, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18353649

RESUMO

The IkappaB kinases (IKKs) IKK-alpha and IKK-beta, and the IKK-related kinases TBK1 and IKK-epsilon, have essential roles in innate immunity through signal-induced activation of NF-kappaB, IRF3 and IRF7, respectively. Although the signaling events within these pathways have been extensively studied, the mechanisms of IKK and IKK-related complex assembly and activation remain poorly defined. Recent data provide insight into the requirement for scaffold proteins in complex assembly; NF-kappaB essential modulator coordinates some IKK complexes, whereas TANK, NF-kappaB-activating kinase-associated protein 1 (NAP1) or similar to NAP1 TBK1 adaptor (SINTBAD) assemble TBK1 and IKK-epsilon complexes. The different scaffold proteins undergo similar post-translational modifications, including phosphorylation and non-degradative polyubiquitylation. Moreover, increasing evidence indicates that distinct scaffold proteins assemble IKK, and potentially TBK1 and IKK-epsilon subcomplexes, in a stimulus-specific manner, which might be a mechanism to achieve specificity.


Assuntos
Regulação Enzimológica da Expressão Gênica , Quinase I-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Humanos , Quinase I-kappa B/fisiologia , Modelos Biológicos , Modelos Genéticos , NF-kappa B/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de Sinais , Ubiquitina/metabolismo
4.
Crit Care Med ; 40(8): 2304-9, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22809906

RESUMO

OBJECTIVES: To test the usefulness of procalcitonin serum level for the reduction of antibiotic consumption in intensive care unit patients. DESIGN: Single-center, prospective, randomized controlled study. SETTING: Five intensive care units from a tertiary teaching hospital. PATIENTS: All consecutive adult patients hospitalized for >48 hrs in the intensive care unit during a 9-month period. INTERVENTIONS: Procalcitonin serum level was obtained for all consecutive patients suspected of developing infection either on admission or during intensive care unit stay. The use of antibiotics was more or less strongly discouraged or recommended according to the Muller classification. Patients were randomized into two groups: one using the procalcitonin results (procalcitonin group) and one being blinded to the procalcitonin results (control group). The primary end point was the reduction of antibiotic use expressed as a proportion of treatment days and of daily defined dose per 100 intensive care unit days using a procalcitonin-guided approach. Secondary end points included: a posteriori assessment of the accuracy of the infectious diagnosis when using procalcitonin in the intensive care unit and of the diagnostic concordance between the intensive care unit physician and the infectious-disease specialist. MEASUREMENTS AND MAIN RESULTS: There were 258 patients in the procalcitonin group and 251 patients in the control group. A significantly higher amount of withheld treatment was observed in the procalcitonin group of patients classified by the intensive care unit clinicians as having possible infection. This, however, did not result in a reduction of antibiotic consumption. The treatment days represented 62.6±34.4% and 57.7±34.4% of the intensive care unit stays in the procalcitonin and control groups, respectively (p=.11). According to the infectious-disease specialist, 33.8% of the cases in which no infection was confirmed, had a procalcitonin value>1µg/L and 14.9% of the cases with confirmed infection had procalcitonin levels<0.25 µg/L. The ability of procalcitonin to differentiate between certain or probable infection and possible or no infection, upon initiation of antibiotic treatment was low, as confirmed by the receiving operating curve analysis (area under the curve=0.69). Finally, procalcitonin did not help improve concordance between the diagnostic confidence of the infectious-disease specialist and the ICU physician. CONCLUSIONS: Procalcitonin measuring for the initiation of antimicrobials did not appear to be helpful in a strategy aiming at decreasing the antibiotic consumption in intensive care unit patients.


Assuntos
Antibacterianos/uso terapêutico , Calcitonina/sangue , Infecção Hospitalar/tratamento farmacológico , Unidades de Terapia Intensiva , Precursores de Proteínas/sangue , Idoso , Antibacterianos/administração & dosagem , Peptídeo Relacionado com Gene de Calcitonina , Infecção Hospitalar/sangue , Infecção Hospitalar/diagnóstico , Feminino , Humanos , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Método Simples-Cego
5.
Nephrol Dial Transplant ; 27(5): 1950-6, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-21940481

RESUMO

BACKGROUND: The recommended target range for serum parathyroid hormone (PTH) in dialysis patients has changed from 150 to 300 pg/mL in the KDOQI guidelines to two to nine times the upper normal limit in the KDIGO ones. Although inclusion/exclusion criteria for the reference population are highly important, they are usually not mentioned in the commercial kits. In this study, we used the same reference population of vitamin D-replete normal subjects to establish reference values for 10 commercial PTH kits. We evaluated whether this may improve the classification of dialysis patients according to the KDIGO compared to the use of reference values proposed by the manufacturers. METHODS: We measured serum PTH with 10 different kits in 149 haemodialysis patients, and 240 25-OH-vitamin D-replete (>75 nmol/L) individuals with an estimated glomerular filtration rate >60 mL/min/1.73 m(2). RESULTS: For the 10 kits, our upper normal limit was lower than those of the manufacturers. The difference was, however, variable from one kit to another. The two kits that yielded the lowest and the highest absolute concentrations classified differently 84/149 patients (56.4%) according to the KDOQI and 53/149 (36.2%) according to the KDIGO using the manufacturers' normal values. Using our normal values significantly decreased the discrepancies with 24/149 patients (16.1%) being still classified differently. Taking the measurement uncertainty into consideration, 8% of the patients only remained differently classified by these two kits. CONCLUSIONS: Using the same vitamin-D-replete population to establish the reference range for 10 commercial PTH kits significantly improved the classification of haemodialysis patients according to the KDIGO target range.


Assuntos
Nefropatias/sangue , Nefropatias/terapia , Hormônio Paratireóideo/sangue , Guias de Prática Clínica como Assunto/normas , Kit de Reagentes para Diagnóstico , Diálise Renal , Idoso , Idoso de 80 Anos ou mais , Densidade Óssea/fisiologia , Doenças Ósseas Metabólicas/sangue , Doenças Ósseas Metabólicas/diagnóstico , Doenças Ósseas Metabólicas/etiologia , Doença Crônica , Feminino , Taxa de Filtração Glomerular/fisiologia , Humanos , Nefropatias/complicações , Masculino , Pessoa de Meia-Idade , Valores de Referência , Vitamina D/sangue
6.
Clin Lab ; 58(5-6): 515-25, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22783583

RESUMO

BACKGROUND: In the second generation of the point-of-care (POC) assay Roche CARDIAC proBNP, the upper limit of the measuring range was extended from 3000 to 9000 ng/L. METHODS: A thirteen-site multicentre evaluation was carried out to assess the analytical performance of the POC NT-proBNP assay and to compare it with a laboratory N-terminal pro-brain natriuretic peptide (NT-proBNP) assay. RESULTS: In method comparisons of six lots of POC NT-proBNP with the lab reference method (Elecsys proBNP) mean bias ranged from -10 to +17%. In lot-to-lot comparisons all six investigated lots of POC NT-proBNP showed excellent agreement, with mean bias between -7% and +2%. The majority of all coefficients of variation obtained from ten-fold measurements using 56 native blood samples were below 8%. No interference was observed with hemolytic blood (hemoglobin concentrations up to 0.12 mmol/L), lipemic blood (triglyceride concentrations up to 14.0 mmol/L) nor icteric blood (bilirubin concentrations up to 63 micromol/L). Hematocrit values between 24% and 51% had no influence on the assay result. High NT-proBNP concentrations above the measuring range of POC NT-proBNP did not lead to false low results due to potential high-dose hook effect. Results with POC NT-proBNP were not influenced by different ambient temperatures (18 degrees C to 32 degrees C), the sample material used, nor by over- or underdosing by 15 microL compared to the regular sample volume of 150 microL. CONCLUSIONS: The POC NT-proBNP assay showed an excellent analytical performance including a good agreement with the laboratory method. The assay is therefore suitable for its intended use in point-of-care settings.


Assuntos
Fator Natriurético Atrial/sangue , Técnicas de Diagnóstico Cardiovascular/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito , Precursores de Proteínas/sangue , Técnicas de Diagnóstico Cardiovascular/normas , Humanos , Sistemas Automatizados de Assistência Junto ao Leito/normas , Controle de Qualidade , Reprodutibilidade dos Testes , Temperatura
7.
J Biol Chem ; 285(33): 25831-40, 2010 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-20558726

RESUMO

The oncogenic protein BCL-3 activates or represses gene transcription through binding with the NF-kappaB proteins p50 and p52 and is degraded through a phospho- and GSK3-dependent pathway. However, the mechanisms underlying its degradation remain poorly understood. Yeast two-hybrid analysis led to the identification of the proteasome subunit PSMB1 as a BCL-3-associated protein. The binding of BCL-3 to PSMB1 is required for its degradation through the proteasome. Indeed, PSMB1-depleted cells are defective in degrading polyubiquitinated BCL-3. The N-terminal part of BCL-3 includes lysines 13 and 26 required for the Lys(48)-linked polyubiquitination of BCL-3. Moreover, the E3 ligase FBW7, known to polyubiquitinate a variety of substrates phosphorylated by GSK3, is dispensable for BCL-3 degradation. Thus, our data defined a unique motif of BCL-3 that is needed for its recruitment to the proteasome and identified PSMB1 as a key protein required for the proteasome-mediated degradation of a nuclear and oncogenic IkappaB protein.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína 3 do Linfoma de Células B , Proteínas de Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Proteínas F-Box/genética , Proteína 7 com Repetições F-Box-WD , Imunofluorescência , Células HeLa , Humanos , Imunoprecipitação , Lisina/metabolismo , Subunidade p50 de NF-kappa B/genética , Subunidade p50 de NF-kappa B/metabolismo , Subunidade p52 de NF-kappa B/genética , Subunidade p52 de NF-kappa B/metabolismo , Fosforilação/genética , Fosforilação/fisiologia , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica/genética , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética , Ubiquitinação/fisiologia
8.
Clin Chem Lab Med ; 49(2): 277-80, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21143018

RESUMO

BACKGROUND: The ImmunoCAP© ISAC allows for the determination of specific immunoglobulin E (IgE) against 103 recombinant or purified allergen components in a single analytical step. The aim of our study was to perform a comparison of the specific IgE results measured with the microarray method to those obtained using the traditional method of ImmunoCAP©. METHODS: We selected 86 clinically relevant patients on the basis of their specific IgE for recombinant allergens (ImmunoCAP© 250, Phadia). Also, we selected two patients with a high total IgE to evaluate the non-specific binding of IgE. All samples were screened with the ImmunoCAP© ISAC. Then, we compared the 555 specific IgE antibodies results provided by ImmunoCAP© ISAC with the specific IgE levels obtained with ImmunoCAP© 250. RESULTS: We observed that 82 of the 384 results found to be positive with ImmunoCAP© were negative with ISAC© (concordance 78.65%). Of 171 negative results obtained with ImmunoCAP©, 11 were positive with ISAC© (concordance 93.57%). No non-specific binding was observed. CONCLUSIONS: Our results show that the ImmunoCAP© ISAC has good analytical performance when compared with the ImmunoCAP© 250 method. We did not observe any non-specific binding. However, better sensitivity for some clinically relevant allergen components, such as rPru p 3 is needed.


Assuntos
Hipersensibilidade/diagnóstico , Hipersensibilidade/imunologia , Análise Serial de Proteínas/métodos , Adulto , Animais , Especificidade de Anticorpos , Feminino , Humanos , Imunoglobulina E/análise , Imunoglobulina E/imunologia , Masculino
9.
Clin Chem Lab Med ; 49(2): 271-5, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21083442

RESUMO

BACKGROUND: We validated the DiaSorin Liaison Calcitonin_II-Gen, an improved method for calcitonin (CT) measurements, compared this method with the Cisbio_h-CT kit and established the reference range of CT in a normal adult population. METHODS: We determined the precision, functional sensitivity, traceability to the 2nd IS 89/620, linearity and measurement uncertainty, accuracy profile and ß-expectation limits. We evaluated the specificity, the susceptibility to human anti-animal antibodies (HAMA), hook-effect and carry over. To establish a reference range, we selected 267 adults without renal insufficiency presenting with normal thyroid stimulating hormone (TSH), free thyroxin (T4) and calcium concentrations and without anti-thyroglobulin antibodies as our "reference" healthy population. We compared the method with Cisbio on 250 consecutive and 45 samples from a post-pentagastrin stimulation test. RESULTS: Precision (expressed as CV) was < 10% for the measurement range, functional sensitivity: 5.3 ng/L and the method was found linear until to a 1/10 dilution. Uncertainty ranged from 25% to 7.2%, and the risk that one result falls out of the ± 20% acceptance limits was < 5% between 2.9 and 1513 ng/L. The Bland and Altman plot showed no systematic bias between the two methods. The test is still prone to HAMA influence, does not present any hook-effect, although carry over was observed. Ninety-five percent of our adult reference population showed CT concentrations < 7.4 ng/L, with an important gender difference: 95% of the men showed CT values < 9.8 ng/L, whereas 95% of women were < 4.0 ng/L. CONCLUSIONS: The Liaison Calcitonin_II-Gen is an analytically robust method. The important difference in gender observed in our population might lead to re-evaluation of the generally used "10 ng/L" cut-off in a multicentre prospective study.


Assuntos
Análise Química do Sangue/métodos , Calcitonina/sangue , Adulto , Análise Química do Sangue/normas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valores de Referência
10.
Cell Mol Life Sci ; 67(8): 1255-64, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20082207

RESUMO

Lysine acetylation is a post-translational modification that critically regulates gene transcription by targeting histones as well as a variety of transcription factors in the nucleus. More recent reports have also demonstrated that numerous proteins located outside the nucleus are also acetylated and that this modification has profound consequences on their functions. This review describes the latest findings on the substrates acetylated outside the nucleus and on the acetylases and deacetylates that catalyse these modifications. Protein acetylation is emerging as a major mechanism by which key proteins are regulated in many physiological processes such as migration, metabolism and aging as well as in pathological circumstances such as cancer and neurodegenerative disorders.


Assuntos
Lisina/metabolismo , Proteínas/metabolismo , Acetilação , Animais , Humanos , Processamento de Proteína Pós-Traducional
11.
J Biomed Biotechnol ; 2010: 906082, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20029632

RESUMO

Protein profiling using SELDI-TOF-MS has gained over the past few years an increasing interest in the field of biomarker discovery. The technology presents great potential if some parameters, such as sample handling, SELDI settings, and data analysis, are strictly controlled. Practical considerations to set up a robust and sensitive strategy for biomarker discovery are presented. This paper also reviews biological fluids generally available including a description of their peculiar properties and the preanalytical challenges inherent to sample collection and storage. Finally, some new insights for biomarker identification and validation challenges are provided.


Assuntos
Biomarcadores/análise , Líquidos Corporais/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Humanos
12.
Clin Chem Lab Med ; 48(1): 67-72, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-19943811

RESUMO

BACKGROUND: The goal of this study was to validate the DiaSorin Liaison BAP OSTASE, a new method for measurement of bone alkaline phosphatase (BAP), and to compare this method with the Beckman-Coulter Access Ostase. We also wanted to establish the reference range for BAP in adults and children. METHODS: We determined the precision, functional sensitivity, recovery, linearity and measurement uncertainty, accuracy profile and beta-expectation limits. We defined an adult reference interval using individuals with 25-OH vitamin D >80 nmol/L, parathormone <58 ng/L, and normal calcium, phosphorous and estimated glomerular filtration rate. Each adult subclass (men/non-menopausal women/menopause women) contained 120 individuals. We also determined the 2.5th and 97.5th percentiles from a population of 450 children, stratified according to age and gender. RESULTS: The results of the validation showed: precision <6%, functional sensitivity <0.74 microg/L, mean recovery 98.8+/-4.2% and good linearity. Relative uncertainty ranged from 9.0% to 12.9%, and the risk of one result falling out of the +/-15% acceptance limits was <5% for concentrations between 7 and 94 microg/L. The Bland-Altman plot showed no systematic bias between the two methods. In adults, we did not find any statistical difference between the different subclasses. The upper limit of normality observed in the entire population (n=360) was 21.3 microg/L (90% CI: 18.3-24.2 microg/L). CONCLUSIONS: The Liaison BAP OSTASE is a robust method, and is completely validated between 7 and 93 microg/L: in this range, 95% of the values obtained will be within +/-15% of the true value.


Assuntos
Fosfatase Alcalina/sangue , Imunoensaio/métodos , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Valores de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Incerteza
13.
Clin Chem Lab Med ; 48(3): 365-71, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20020820

RESUMO

BACKGROUND: We report a Dutch-Belgian multicentre evaluation of the Tosoh HLC-723G8 glycohaemoglobin analyser, an ion-exchange HPLC instrument for the separation and quantification of haemoglobin A1c (HbA1c) in whole blood. METHODS: We evaluated the analytical performances of the Tosoh G8 analyser and compared the results for blood samples with its predecessor, the Tosoh G7, and with two other widely used analysers, the Bio-Rad Variant II and Adams Arkray HA-8160. RESULTS: Within- and between-batch imprecision [coefficient of variation (CVs)] was <0.5% and 2%, respectively, and compared favourably with the G7. The excellent performances in terms of speed (1.6 min/analysis) did not result in increased variability of the results or carry-over between samples. The method shows no interference from carbamylated haemoglobin, and recognises the presence of haemoglobinopathies, which triggers the correction of the HbA1c result. Comparison with established methods showed good correlation, not only with the G7 but also with the Variant II and HA-8160 systems. CONCLUSIONS: With respect to reproducibility, chromatographic resolution, speed of analysis and identification of Hb variants, the Tosoh G8 analyser can be considered to be state of the art.


Assuntos
Cromatografia Líquida de Alta Pressão , Hemoglobinas Glicadas/análise , Análise Química do Sangue/instrumentação , Análise Química do Sangue/métodos , Análise Química do Sangue/normas , Calibragem , Humanos , Fenótipo , Isoformas de Proteínas/análise , Reprodutibilidade dos Testes
14.
Clin Lab ; 56(1-2): 37-49, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20380358

RESUMO

BACKGROUND: The cobas h 232 point-of-care analyzer by Roche is the instrument successor of the Cardiac reader allowing the quantitative determination of troponin T, creatine kinase MB, myoglobin, NT-proBNP and D-dimer. METHODS: In this study 1329 patients with acute coronary syndromes, heart failure, thromboembolic or other diseases and 945 healthy donors were assessed. Comparisons versus central laboratory methods were carried out with 2379 samples from these individuals; out of these, 1591 samples gave quantitative results within the measuring range and were included in the evaluation. RESULTS: The point-of-care assays for creatine kinase MB, myoglobin, NT-proBNP and D-dimer were within a relative bias range of -5.9 to +6.9% compared to the laboratory assay. The troponin T assay showed a bias of -11.0% and after change of the calibration procedure of +1.9%. None of the five point-of-care assays had a relative difference between the new system and the precursor device that was higher than +5.0%. Within-series coefficients of variation of patient samples were found in a range from 4.8 to 14.8%. No significant interference was observed with lipemic, hemolytic and icteric blood or at different hematocrit values. CONCLUSIONS: Due to its good analytical agreement with the laboratory methods and with its precursor device, the cobas h 232 system can be reliably used to support on-site decision making for cardiovascular patients in acute and non-acute settings.


Assuntos
Cardiopatias/diagnóstico , Sistemas Automatizados de Assistência Junto ao Leito/normas , Tromboembolia/diagnóstico , Síndrome Coronariana Aguda/sangue , Síndrome Coronariana Aguda/diagnóstico , Calibragem , Creatina Quinase Forma MB/sangue , Desenho de Equipamento , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Cardiopatias/sangue , Insuficiência Cardíaca/sangue , Insuficiência Cardíaca/diagnóstico , Humanos , Masculino , Mioglobina/sangue , Peptídeo Natriurético Encefálico/sangue , Variações Dependentes do Observador , Fragmentos de Peptídeos/sangue , Valores de Referência , Reprodutibilidade dos Testes , Tromboembolia/sangue , Troponina T/sangue
15.
Ann Biol Clin (Paris) ; 68(2): 149-56, 2010.
Artigo em Francês | MEDLINE | ID: mdl-20348047

RESUMO

Recently, anti-deamidated gliadin antibodies were proposed for the serological diagnosis of celiac disease. We evaluate the specificity of different anti-deamidated gliadin antibodies ELISA in comparison with conventional anti-native gliadin kits. Serum samples from 46 non celiac patients were analyzed by five different quantitative ELISA for anti-native gliadin, anti-deamidated gliadin and anti-transglutaminase neo-epitope antibodies together with a screening ELISA. Twenty-four percent of the patients demonstrated anti-native gliadin IgA and 63% IgG antibodies. Using anti-deamidated gliadin antibodies, the number of false positive IgA and, particularly, IgG results, markedly decreased in the non celiac patients: 21 and 24% respectively with anti-Gliadin (GAF-3X) Euroimmun kit, 7 and 26% with Bindazyme Human Anti-Gliadin (MGP) The Binding Site kit and 0 and 41% with Celiac G+ Immco kit. The new assay which makes use of the physiological complex of tissue transglutaminase cross-linked with deamidated gliadin peptides, called neo-epitope, did not improve the differential diagnosis of celiac disease with 30% of false positive results in IgG (2% in IgA). Using the Inova screening kit, a positive result for IgA and/or IgG anti-deamidated gliadin and/or anti-tissue transglutaminase antibodies was obtained in 24% of the non celiac patients. In conclusion, our study assessed the superiority, in terms of specificity, of anti-deamidated gliadin antibodies, over the conventional anti-gliadin antibodies for the differential diagnosis of celiac disease.


Assuntos
Anticorpos/sangue , Doença Celíaca/sangue , Gliadina/imunologia , Autoanticorpos/sangue , Doença Celíaca/diagnóstico , Doença Celíaca/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Reações Falso-Positivas , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Sensibilidade e Especificidade , Transglutaminases/imunologia
16.
J Proteome Res ; 8(10): 4810-22, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19705920

RESUMO

Ceramides are central molecules in sphingolipid metabolism. They are involved in the regulation of cancer-cell growth, differentiation, senescence and apoptosis. To better understand how these secondary messengers induce their biological effects, adenocarcinoma cells (HCT116) were treated with exogenous long-chain ceramides (C16-ceramide) in order to mimic endogenous sphingolipids. This treatment induced a decrease of cell viability partly due to apoptosis as shown by PARP cleavage and a decrease of pro-caspase 3. Two-dimensional differential in-gel electrophoresis (2D-DIGE) revealed the differential expression of 51 proteins in response to C16-ceramide. These proteins are notably involved in cell proliferation, apoptosis, protein transport and transcriptional regulation. Among them, the cell death-promoting factor Btf was found to be implicated in the apoptotic signal triggered by ceramide. In adenocarcinoma cells, Btf regulates apoptosis related proteins such as Mdm2, p53, BAX and pBcl-2 and thus plays an important role in the ceramide mediated cell death. These findings bring new insight into the proapoptotic ceramide-dependent signaling pathway.


Assuntos
Adenocarcinoma/metabolismo , Ceramidas/farmacologia , Neoplasias do Colo/metabolismo , Proteômica/métodos , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Apoptose/efeitos dos fármacos , Sobrevivência Celular , Eletroforese em Gel Bidimensional , Células HCT116 , Humanos , Proteoma/efeitos dos fármacos , Proteoma/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais , Espectrometria de Massas em Tandem , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismo
17.
Heart Vessels ; 24(4): 267-70, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19626398

RESUMO

High-sensitivity C-reactive protein predicts future cardiovascular events in both healthy individuals and patients with unstable and stable coronary syndromes. Few data are available about the incidence and the relation to inflammation of troponin elevation following percutaneous coronary intervention (PCI), a potential predictor of longterm outcome. We sought to confirm the impact of embolization on long-term outcome and evaluate the ability of baseline inflammation to predict troponin elevation induced by PCI. We prospectively analyzed 200 patients treated by PCI for stable or Braunwald IIA class unstable angina. The patients were recruited between January 1997 and May 1999, and the population was followed during a mean follow-up of 32 months. Major adverse cardiac events (MACEs) were defined as the occurrence of death, myocardial infarction or recurrent angina requiring repeat PCI, or coronary artery bypass grafting. During the follow-up period, 58 MACEs were observed. By multivariate analysis, independent predictors for the occurrence of MACEs were unstable angina and troponin I level after PCI (P < 0.0001 for both). No correlation was found between baseline inflammation and significant troponin I elevation post PCI and by multivariate analysis, no biological variable was a predictor of troponin I elevation post PCI. Baseline inflammation cannot predict onset of minor myonecrosis damage (expressed by troponin elevation) induced by PCI, a significant predictor of long-term outcome in this setting.


Assuntos
Angioplastia Coronária com Balão/efeitos adversos , Proteína C-Reativa/metabolismo , Estenose Coronária/terapia , Inflamação/complicações , Infarto do Miocárdio/etiologia , Miocárdio/metabolismo , Troponina I/sangue , Idoso , Angina Instável/sangue , Angina Instável/etiologia , Angioplastia Coronária com Balão/mortalidade , Biomarcadores/sangue , Estenose Coronária/sangue , Estenose Coronária/complicações , Estenose Coronária/mortalidade , Feminino , Humanos , Inflamação/sangue , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/sangue , Infarto do Miocárdio/mortalidade , Miocárdio/patologia , Necrose , Razão de Chances , Modelos de Riscos Proporcionais , Recidiva , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Resultado do Tratamento
18.
Clin Chim Acta ; 387(1-2): 150-2, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17904113

RESUMO

BACKGROUND: As other immunoassays, PTH determination is not free from interferences. Indeed, natural antibodies like heterophile antibodies (HAMA) and rheumatoid factor (RF) can induce falsely elevated results, leading to misdiagnosis and expensive unnecessary explorations. However, in routine practice, these interferences are not always obvious to detect. METHODS: On 2084 PTH samples, we applied a validation strategy in four steps to screen for HAMA and rheumatoid factor interferences. RESULTS: 36% of our samples presented an elevated PTH. We found a clinically plausible reason for 91% of them. The remaining 63 suspicious samples were treated with HBT and 40% of them were found to be HAMA positive. RF determination was performed on the HAMA-negative samples and RF was positive in 21 of them. They were then treated with RF-Absorbent. Nine of these 21 samples presented RF interference. CONCLUSION: Applying this strategy in our routine validation, we managed to avoid spuriously elevated PTH results, which could have caused medical errors as well as unnecessary cost-effective extra-investigations.


Assuntos
Hormônio Paratireóideo/sangue , Artefatos , Reações Falso-Positivas , Humanos
19.
Clin Chim Acta ; 398(1-2): 118-24, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18805407

RESUMO

BACKGROUND: Cystatin C-based equations are used to estimate GFR. However, three cystatin C immunoassays are on the market. Difference in cystatin C assays could have strong consequences on the accuracy and precision of cystatin C-based equations. We have performed an analytical study of these three assays and studied potential differences between assays on the precision of cystatin C-based equations. METHODS: We have studied imprecision, recovery, linearity and interferences of the three immunoassays (nephelometric assay from Siemens and turbidimetric assays from Dako and Gentian). The impact of differences in cystatin C assays has been studied for the equations published by Levey (Siemens assay) and Grubb (Dako assay). RESULTS: Analytical performance of the Dako assay is slightly less high. For cystatin C values below 2.5 mg/L, no statistical difference is found between results given by the Dako and the Gentian assays. So, both assays can be used in the Grubb equation. Cystatin C results are different with the Siemens assay. The Levey equation, built with the Siemens assay, can only be used with cystatin C values measured with this assay. Using the Dako or Gentian assay results in the Levey equation can lead to differences in estimating GFR up to 6 mL/min/1.73 m2. Differences can reach 9.5 mL/min/1.73 m2 if the Siemens assay is used in the Grubb equation. CONCLUSION: The Siemens and Gentian assays seem analytically more valid than the Dako assay for cystatin C determination. Differences in cystatin C assays can lead to significant differences in cystatin C-based equations. However, these differences seem less important than the differences observed with creatinine and creatinine-based equations.


Assuntos
Cistatina C/análise , Taxa de Filtração Glomerular/fisiologia , Algoritmos , Calibragem , Creatina/sangue , Hemoglobinas/análise , Humanos , Imunoensaio , Nefelometria e Turbidimetria , Valor Preditivo dos Testes , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes
20.
Clin Chim Acta ; 396(1-2): 80-5, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18687322

RESUMO

BACKGROUND: Determination of glomerular filtration rate plays an important role in nephrological practice. Iohexol is a reference marker for glomerular filtration rate determination. It is available and safe. The aim of this study was to develop a simple, efficient and easy to use analytical method for the quantification of iohexol in serum and urine by high performance liquid chromatography and to thoroughly validate this method. METHODS: The HPLC method was inspired from the method published by Krutzen. The e.noval software V2.0 (Arlenda, Liège, Belgium) was used to compute all validation results. RESULTS: The validation results indicate that the method will give accurate and reliable results for serum values ranging from 12.95 to 1295 microg/ml and for urine values ranging from 86.0 to 4144 microg/ml. In routine practice, iohexol concentrations found in plasma after injection range from 40 to 600 microg/ml. The expected urinary values are much wider. One should not hesitate to dilute urine samples to fit with the validated range over 5000 microg/ml. CONCLUSION: This is the first time that a reference method for the determination of GFR is validated with such a rigorous and thorough protocol. Contrary to other GFR markers, iohexol is now strongly validated from an analytical point of view.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Iohexol/análise , Iohexol/metabolismo , Incerteza , Medição de Risco , Soro/química , Urina/química
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