Transcriptional profiling of TLR-4/7/8-stimulated guinea pig splenocytes and whole blood by bDNA assay
Transcriptional profiling of TLR-4/7/8-stimulated guinea pig splenocytes and whole blood by bDNA assay
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DOI：
10.1016/j.jim.2011.07.021
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发表时间：
2011-10-28
影响因子：
2.2
通讯作者：
Baldwin, Susan L.
中科院分区：
文献类型：
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作者：
Ching, Lance K.;Mompoint, Farah;Baldwin, Susan L.
Toll样受体（TLR）激动剂目前正在研究作为疫苗的佐剂，目前有几种主要候选药物已进入许可产品或处于后期临床开发阶段。豚鼠被广泛用于药物和疫苗的临床前试验;然而，在这种模型中评价TLR激动剂受到免疫学工具和试剂有限的阻碍。在这项研究中，我们验证了使用的支链DNA（bDNA）测定被称为QuantiGene 2.0试剂系统测量先天性细胞因子和趋化因子mRNA水平后，豚鼠细胞TLR刺激。在用TLR-4或TLR-7/8激动剂离体刺激豚鼠脾细胞和全血后，T辅助细胞-1（Th 1）极化细胞因子（TNF-α、IL-1 β、IL-12）和趋化因子（CXCL 1、CCL 2）的基因表达上调。这些数据证实了QuantiGene系统作为RT-PCR的替代方法用于测量转录水平和作为高通量筛选工具用于解剖豚鼠对TLR刺激的免疫应答的实用性。总体而言，QuantiGene平台是可靠的、可重复的和灵敏的。这些激动剂具有用作针对各种病原体的疫苗中的佐剂组分的潜力。(C)2011爱思唯尔有限公司版权所有。
Toll-like receptor (TLR) agonists are currently being examined as adjuvants for vaccines, with several lead candidates now in licensed products or in late-stage clinical development Guinea pigs are widely used for preclinical testing of drugs and vaccines; however, evaluation of TLR agonists in this model is hindered by the limited availability of immunological tools and reagents. In this study, we validated the use of a branched-chain DNA (bDNA) assay known as the QuantiGene Plex 2.0 Reagent System for measuring innate cytokine and chemokine mRNA levels following TLR stimulation of guinea pig cells. Gene expression for T-helper-1 (Th1) polarizing cytokines (TNF-alpha, IL-1 beta, IL-12) and chemokines (CXCL1, CCL2) was upregulated following ex vivo stimulation of guinea pig splenocytes and whole blood with TLR-4 or TLR-7/8 agonists. These data confirm the utility of the QuantiGene system both as an alternative to RT-PCR for measuring transcript levels and as a high-throughput screening tool for dissecting the immunological response to TLR stimulation in guinea pigs. Overall, the QuantiGene platform is reliable, reproducible, and sensitive. These agonists have the potential to be used as adjuvant components in vaccines against various pathogens. (C) 2011 Elsevier B.V. All rights reserved.