RESUMO
In microarray studies alterations in gene expression in circulating leukocytes have shown utility for ischemic stroke diagnosis. We studied forty candidate markers identified in three gene expression profiles to (1) quantitate individual transcript expression, (2) identify transcript clusters and (3) assess the clinical diagnostic utility of the clusters identified for ischemic stroke detection. Using high throughput next generation qPCR 16 of the 40 transcripts were significantly up-regulated in stroke patients relative to control subjects (p<0.05). Six clusters of between 5 and 7 transcripts were identified that discriminated between stroke and control (p values between 1.01e-9 and 0.03). A 7 transcript cluster containing PLBD1, PYGL, BST1, DUSP1, FOS, VCAN and FCGR1A showed high accuracy for stroke classification (AUC=0.854). These results validate and improve upon the diagnostic value of transcripts identified in microarray studies for ischemic stroke. The clusters identified show promise for acute ischemic stroke detection.
Assuntos
Isquemia Encefálica/genética , Família Multigênica , Acidente Vascular Cerebral/genética , Transcriptoma , ADP-Ribosil Ciclase/genética , ADP-Ribosil Ciclase/metabolismo , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/genética , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Isquemia Encefálica/metabolismo , Estudos de Casos e Controles , Fosfatase 1 de Especificidade Dupla/genética , Fosfatase 1 de Especificidade Dupla/metabolismo , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Perfilação da Expressão Gênica , Glicogênio Fosforilase Hepática/genética , Glicogênio Fosforilase Hepática/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Fosfolipase D/genética , Fosfolipase D/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Acidente Vascular Cerebral/metabolismo , Versicanas/genética , Versicanas/metabolismoRESUMO
T lymphocytes may play an important role in the evolution of ischemic stroke. Depletion of γδT cells has been found to abrogate ischemia reperfusion injury in murine stroke. However, the role of γδT cells in human ischemic stroke is unknown. We aimed to determine γδT cell counts and γδT cell interleukin 17A (IL-17A) production in the clinical setting of ischemic stroke. We also aimed to determine the associations of γδT cell counts with ischemic lesion volume, measures of clinical severity and with major stroke risk factors. Peripheral blood samples from 43 acute ischemic stroke patients and 26 control subjects matched on race and gender were used for flow cytometry and complete blood count analyses. Subsequently, cytokine levels and gene expression were measured in γδT cells. The number of circulating γδT cells was decreased by almost 50% (pâ=â0.005) in the stroke patients. γδT cell counts did not correlate with lesion volume on magnetic resonance diffusion-weighted imaging or with clinical severity in the stroke patients, but γδT cells showed elevated levels of IL-17A (pâ=â0.048). Decreased γδT cell counts were also associated with older age (pâ=â0.004), pre-existing hypertension (pâ=â0.0005) and prevalent coronary artery disease (pâ=â0.03), with pre-existing hypertension being the most significant predictor of γδT cell counts in a multivariable analysis. γδT cells in human ischemic stroke are reduced in number and show elevated levels of IL-17A. A major reduction in γδT lymphocytes also occurs in hypertension and may contribute to the development of hypertension-mediated stroke and vascular disease.
Assuntos
Hipertensão/imunologia , Interleucina-17/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Acidente Vascular Cerebral/imunologia , Linfócitos T/imunologia , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Contagem de Células Sanguíneas , Citocinas/sangue , Feminino , Citometria de Fluxo , Humanos , Hipertensão/complicações , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Estatísticas não Paramétricas , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/patologia , Linfócitos T/metabolismoRESUMO
Clinical studies of gene expression are increasingly using the whole blood, peripheral blood mononuclear cells, and leukocyte subsets involved in the innate and adaptive immune responses. However, the small amount of RNA available in the clinical setting is a limitation for commonly used methods such as quantitative polymerase chain reactions (qPCR) and microarrays. Our aim was to design 96 gene assays to simultaneously measure gene expression in the whole blood and seven leukocyte subsets using a new-generation qPCR method--high-throughput nanofluidic reverse transcription qPCR (HT RT-qPCR). The leukocyte subset purity was 94% to 98% for seven subsets and was less for the γδ T-cell receptor subset (80%). The HT RT-qPCR replicate sample measurements were highly reproducible (r = 0.997, p < 2.2 × 10(-16)), and the ΔΔCt values from HT RT-qPCR correlated significantly with those from qPCR. The control genes were differentially expressed across the eight leukocyte subsets in the control subjects (p = 1.3 × 10(-5), analysis of variance). Two analytical methods, absolute and relative, gave concordant results and were significantly correlated (p = 1.9 × 10(-9)). HT RT-qPCR permits the rapid, reproducible, and quantitative measurement of multiple transcripts using minimal sample amounts. The protocol described yielded leukocyte subsets of high purity and identified two analytic methods for use.