Curriculum in Toxicology, University of North Carolina-Chapel Hill, CB# 7270, Chapel Hill, NC 27599-7270, USA.
Toxicol Appl Pharmacol. 2010 Apr 15;244(2):144-55. doi: 10.1016/j.taap.2009.12.027. Epub 2010 Jan 4.
Effective hazard screening will require the development of high-throughput or in vitro assays for the identification of potential sensitizers. The goal of this preliminary study was to identify potential biomarkers that differentiate the response to allergens vs non-allergens following an acute exposure in naïve individuals. Female BALB/c mice received a single intratracheal aspiration exposure to Metarhizium anisopliae crude antigen (MACA) or bovine serum albumin (BSA) in Hank's Balanced Salt Solution (HBSS) or HBSS alone. Mice were terminated after 1, 3, 6, 12, 18 and 24 h. Bronchoalveolar lavage fluid (BALF) was evaluated to determine total and differential cellularity, total protein concentration and LDH activity. RNA was isolated from lung tissue for microarray analysis and qRT-PCR. MACA administration induced a rapid increase in BALF neutrophils, lymphocytes, eosinophils and total protein compared to BSA or HBSS. Microarray analysis demonstrated differential expression of genes involved in cytokine production, signaling, inflammatory cell recruitment, adhesion and activation in 3 and 12 h MACA-treated samples compared to BSA or HBSS. Further analyses allowed identification of approximately 100 candidate biomarker genes. Eleven genes were selected for further assessment by qRT-PCR. Of these, 6 demonstrated persistently increased expression (Ccl17, Ccl22, Ccl7, Cxcl10, Cxcl2, Saa1), while C3ar1 increased from 6-24 h. In conclusion, a single respiratory exposure of mice to an allergenic mold extract induces an inflammatory response which is distinct in phenotype and gene transcription from the response to a control protein. Further validation of these biomarkers with additional allergens and irritants is needed. These biomarkers may facilitate improvements in screening methods.
有效的危害筛选将需要开发高通量或体外测定法，以鉴定潜在的敏化剂。本初步研究的目的是鉴定潜在的生物标志物，以区分在无经验个体中急性暴露后对过敏原和非过敏原的反应。雌性 BALB/c 小鼠接受单次气管内吸入异孢小菇粗抗原（MACA）或牛血清白蛋白（BSA）在 Hank's 平衡盐溶液（HBSS）或 HBSS 中的暴露。在 1、3、6、12、18 和 24 小时后处死小鼠。评估支气管肺泡灌洗液（BALF）以确定总细胞和分类细胞、总蛋白浓度和 LDH 活性。从肺组织中分离 RNA 进行微阵列分析和 qRT-PCR。与 BSA 或 HBSS 相比，MACA 给药导致 BALF 中性粒细胞、淋巴细胞、嗜酸性粒细胞和总蛋白迅速增加。微阵列分析表明，与 BSA 或 HBSS 相比，3 和 12 小时 MACA 处理的样品中涉及细胞因子产生、信号转导、炎症细胞募集、黏附和激活的基因表达差异。进一步的分析允许鉴定出大约 100 个候选生物标志物基因。选择 11 个基因通过 qRT-PCR 进行进一步评估。其中，6 个基因表现出持续增加的表达（Ccl17、Ccl22、Ccl7、Cxcl10、Cxcl2、Saa1），而 C3ar1 则在 6-24 小时内增加。总之，小鼠单次呼吸暴露于过敏原性霉菌提取物会引起炎症反应，其表型和基因转录与对照蛋白的反应明显不同。需要用其他过敏原和刺激物进一步验证这些生物标志物。这些生物标志物可能有助于改善筛选方法。
