Transcription profiling of mouse with experimental cerebral malaria following P. berghei ANKA infection

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection, predominantly experienced by children and non-immune adults, which results in great mortality and long-term sequelae. Recent reports based on histology of post-mortem brain tissue suggest that CM may be the common end point for a range of syndromes. Here, we have analysed the gene expression profiles in brain tissue taken from experimental CM (ECM)-susceptible, Plasmodium berghei ANKA (PbA)-infected C57BL/6 (B6) and CBA/CaH (CBA) mice with ECM. Gene expression profiles were largely heterogeneous between the two ECM-susceptible strains. These results, combined with experimental data, support the existence of distinct pathogenic pathways in CM. Experiment Overall Design: C57BL/6 and CBA/CaH mice were infected with 10e5 Plasmodium berghei ANKA-infected RBCs and monitored for ECM development. At onset of ECM symptoms, infected mice and naive controls were culled, perfused (in order to remove non-adherent circulating cells), and brains were removed. Total RNA was extracted from these brains and pooled (n=6 mice/ group). Pooled RNA samples were converted to cDNA and antisense cRNA, labelled and hybridized to GeneChip Mouse Genome 430 2.0 Arrays (Affymetrix, Surrey Hills, Australia). Arrays were scanned using the GeneChip Scanner 3000 (Affymetrix) and GeneChip Operating Software v1.1.1 (Affymetrix). Normalisation and initial analyses were carried out in GeneSpring v7 (Agilent Technologies). Values below 0.01 were set to 0.01. Each measurement was divided by the 50th percentile of all measurements in that sample. The data was filtered for genes flagged as present, which had at least an expression level of 50. Following this, a threshold of 2.5 fold up-regulation or down-regulation of genes differentially expressed during ECM was set.脑型疟疾（Cerebral malaria, CM）是恶性疟原虫（Plasmodium falciparum）感染引发的严重并发症，主要累及儿童与非免疫成人，可导致极高的死亡率及长期后遗症。近期基于尸检脑组织组织学的研究提示，脑型疟疾可能是多种综合征的共同终点。本研究对罹患实验性脑型疟疾（experimental CM, ECM）的两种易感品系小鼠——伯氏疟原虫ANKA株（Plasmodium berghei ANKA, PbA）感染的C57BL/6（B6）及CBA/CaH（CBA）小鼠的脑组织基因表达谱进行了分析。两种ECM易感品系的基因表达谱存在显著异质性。上述结果结合实验数据，支持脑型疟疾存在不同的致病通路。 实验整体设计：将C57BL/6及CBA/CaH小鼠感染10^5个伯氏疟原虫ANKA株感染的红细胞，持续监测其ECM发病情况。在ECM症状发作时，处死感染小鼠及未感染对照小鼠，通过心脏灌注去除非黏附循环血细胞，随后取出脑组织。从脑组织中提取总RNA并进行混合（每组n=6只小鼠）。将混合后的RNA样本反转录为cDNA并合成反义cRNA，经标记后与GeneChip小鼠基因组430 2.0阵列（Affymetrix，澳大利亚萨里山）进行杂交。使用GeneChip扫描仪3000（Affymetrix）及GeneChip操作软件v1.1.1（Affymetrix）扫描芯片阵列。数据标准化及初步分析在GeneSpring v7（安捷伦科技）中完成。将低于0.01的表达值统一设为0.01；将每个测量值除以该样本中所有测量值的第50百分位数。随后对标记为“存在”且表达水平至少为50的基因进行数据过滤。在此基础上，设置2.5倍上调或下调的阈值，以筛选ECM进程中差异表达的基因。