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1.
Sci Transl Med ; 11(515)2019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31645455

RESUMO

Improved tuberculosis (TB) prevention and control depend critically on the development of a simple, readily accessible rapid triage test to stratify TB risk. We hypothesized that a blood protein-based host response signature for active TB (ATB) could distinguish it from other TB-like disease (OTD) in adult patients with persistent cough, thereby providing a foundation for a point-of-care (POC) triage test for ATB. Three adult cohorts consisting of ATB suspects were recruited. A bead-based immunoassay and machine learning algorithms identified a panel of four host blood proteins, interleukin-6 (IL-6), IL-8, IL-18, and vascular endothelial growth factor (VEGF), that distinguished ATB from OTD. An ultrasensitive POC-amenable single-molecule array (Simoa) panel was configured, and the ATB diagnostic algorithm underwent blind validation in an independent, multinational cohort in which ATB was distinguished from OTD with receiver operator characteristic-area under the curve (ROC-AUC) of 0.80 [95% confidence interval (CI), 0.75 to 0.85], 80% sensitivity (95% CI, 73 to 85%), and 65% specificity (95% CI, 57 to 71%). When host antibodies against TB antigen Ag85B were added to the panel, performance improved to 86% sensitivity and 69% specificity. A blood-based host response panel consisting of four proteins and antibodies to one TB antigen can help to differentiate ATB from other causes of persistent cough in patients with and without HIV infection from Africa, Asia, and South America. Performance characteristics approach World Health Organization (WHO) target product profile accuracy requirements and may provide the foundation for an urgently needed blood-based POC TB triage test.


Assuntos
Tosse/diagnóstico , Triagem/métodos , Tuberculose Pulmonar/diagnóstico , Anticorpos Antibacterianos/análise , Tosse/microbiologia , Tosse/patologia , Humanos , Aprendizado de Máquina , Sistemas Automatizados de Assistência Junto ao Leito , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologia
2.
Biotechniques ; 35(3): 624-9, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-14513568

RESUMO

Here we describe a new approach to study apoptosis pathways using multiplex suspension arrays. Apoptosis was induced in Jurkat T cells using the protein synthesis inhibitor, anisomycin. Cells grown in 96-well plates were treated with anisomycin for up to 7 h, washed, and lysed in their respective wells. Samples of each lysate were analyzed using Beadlyte suspension arrays consisting of total Akt/PKB, phosphorylated Akt/PKB, active caspase-3, and single-stranded DNA (ssDNA)-specific Beadmate microspheres and quantified with the X-MAP system. We found that phosphorylated Akt levels dropped dramatically with 2 h or more of anisomycin treatment, whereas active caspase-3 levels rose sharply with 2 h of treatment, signifying the onset of apoptosis. Longer incubation with anisomycin showed increases in ssDNA, which is consistent with the characteristic degradation of DNA that occurs in late-stage apoptosis. This approach demonstrates how apoptosis pathways can be studied from a small amount of sample without the use of more lengthy techniques such as immunoprecipitation or Western blotting. The suspension array is being expanded to measure many other intracellular proteins including posttranslational modifications and should prove to be extremely useful for studying apoptosis and other important cellular pathways.


Assuntos
Apoptose/fisiologia , Biomarcadores/análise , Caspases/metabolismo , Contagem de Células/métodos , Técnicas de Cultura de Células/métodos , Imunoensaio de Fluorescência por Polarização/métodos , Mapeamento de Interação de Proteínas/métodos , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Anisomicina/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3 , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , DNA/metabolismo , Relação Dose-Resposta a Droga , Humanos , Células Jurkat/citologia , Células Jurkat/efeitos dos fármacos , Células Jurkat/fisiologia , Microesferas , Proteínas Proto-Oncogênicas c-akt , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
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