RESUMO
The recent introduction of fluorescent bead-based technology, allowing the measurement of multiples analytes in a single 25-50 microl sample has revolutionized the study of cytokine responses. However, such multiplex approaches may compromise the ability of these assays to accurately measure actual cytokine levels. This study evaluates the performance of three commercially available multiplex cytokine fluorescent bead-based immunoassays (Bio-Rad's Cytokine 17-plex kit; LINCO Inc's 29-plex kit; and RnD System's Fluorokine-Multi Analyte Profiling (MAP) base kit A and B). The LINCO Inc kit was found to be the most sensitive assay for measuring concentrations of multiple recombinant cytokines in samples that had been spiked with serial dilutions of the standard provided by the manufacturer, followed respectively by the RnD Fluorokine-(MAP) and Bio-Rad 17-plex kits. A positive correlation was found in the levels of IFN-gamma measured in antigen stimulated whole blood culture supernatants by the LINCO Inc 29-plex, RnD Fluorokine-(MAP) and RnD system IFN-gamma Quantikine ELISA kits across a panel of controls and stimulated samples. Researchers should take the limitation of such multiplexed assays into account when planning experiments and the most appropriate use for these tests may currently be as screening tools for the selection of promising markers for analysis by more sensitive techniques.
Assuntos
Citocinas/sangue , Imunofluorescência , Humanos , Indicadores e Reagentes/química , Kit de Reagentes para Diagnóstico , Sensibilidade e EspecificidadeRESUMO
This study investigated interleukin-4 (IL-4), IL-4 delta 2, transforming growth factor beta (TGF-beta), TGF-beta RII, Foxp3, GATA-3, T-bet, and gamma interferon (IFN-gamma) transcription in peripheral blood samples of adult pulmonary tuberculosis patients prior to and after 1 week of therapy. Twenty patients with positive results for sputum culture for Mycobacterium tuberculosis were enrolled and treated with directly observed short-course antituberculosis chemotherapy. Early treatment response was assessed. At the end of the intensive phase of treatment (month 2), 12 patients remained sputum culture positive (slow responders) and 8 converted to a negative culture (fast responders). Only the expression levels of IL-4 (4-fold decrease) and IL-4 delta 2 (32-fold increase) changed significantly during the first week of therapy in the 20 patients. No baseline differences were present between the responder groups, but fast responders had significantly higher IL-4 transcripts than slow responders at week 1. Fast responders showed a 19-fold upregulation and slow responders a 47-fold upregulation of IL-4 delta 2 at week 1. Only slow responders also showed a significant decrease in IL-4 expression at week 1. There were no significant differences in expression of TGF-beta, TGF-beta RII, Foxp3, IFN-gamma, and GATA-3 between the groups. These data show that differential IL-4-related gene expression in the early stage of antituberculosis treatment accompanies differential treatment responses and may hold promise as a marker for treatment effect.
Assuntos
Antituberculosos/uso terapêutico , Regulação da Expressão Gênica , Interleucina-4/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , RNA Mensageiro/metabolismo , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/imunologia , Adolescente , Adulto , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-4/genética , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificação , RNA Mensageiro/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Escarro/microbiologia , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Resultado do Tratamento , Tuberculose Pulmonar/microbiologiaRESUMO
This study investigates how the extent of pre-treatment radiological disease and early anti-tuberculous treatment response affect levels of selected circulating host immune markers. Twenty HIV-uninfected tuberculosis patients with BACTEC culture positivity for Mycobacterium tuberculosis at diagnosis and treated with directly observed short course anti-tuberculosis chemotherapy and 13 healthy community controls were enrolled. Serum samples were collected throughout treatment. After the intensive phase of treatment, 12 patients remained sputum culture-positive (slow responders) and eight patients were culture negative (fast responders). C-reactive protein (CRP), soluble intercellular adhesion molecule-1 (sICAM-1), soluble urokinase plasminogen activator receptor (suPAR), soluble lymphocyte activation gene-3 (sLAG-3), granzyme B, soluble tumour necrosis factor receptor one and two (sTNFR I and sTNFR II) and soluble death receptor 5 (sDR5) concentrations were measured. High levels of CRP at diagnosis were found to be associated (p
