Stage independent chloroquine resistance and chloroquine toxicity revealed via spinning disk confocal microscopy
Stage independent chloroquine resistance and chloroquine toxicity revealed via spinning disk confocal microscopy
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DOI：
10.1016/j.molbiopara.2007.12.014
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发表时间：
2008-05-01
影响因子：
1.5
通讯作者：
Roepe, Paul D.
中科院分区：
文献类型：
--
作者：
Gligorijevic, Bojana;Purdy, Kyle;Roepe, Paul D.
我们之前定制了Nipkow旋转圆盘共聚焦显微镜（SDCM）以获取活的红细胞内疟疾寄生虫的4D数据[Gligorijevic B，麦卡利斯特R，Urbach JS，Roepe，PD.活的红细胞内疟疾寄生虫的旋转圆盘共聚焦显微镜。1.疟原虫色素在药物敏感与耐药疟疾中的定量发展。Biochemistry 2006;45：12400-10]。我们报道了氯喹（CQ）治疗似乎并没有影响细胞周期的进展，并建议毒性可能表现为后生殖。我们现在使用SDCM、同步细胞培养和连续与推注药物给药来详细研究阶段特异性CQ效应。我们开发了一种新的，非常快速的方法，用于在3D中计数质子核。然后，我们量化了细胞周期不同阶段用CQ脉冲的活寄生虫培养物的裂殖体核和疟原虫色素（Hz）的产生，并发现环的团注处理影响核分裂的多样性。我们量化了CQ暴露后后续周期中的寄生虫血症和裂殖子发育，并发现一部分CQ毒性表现为“延迟死亡”。使用这些方法和其他方法，我们比较了CQ敏感（CQS）与抗性（CQR）菌株以及通过引入突变PfCRT而成为CQR的转染子。令人惊讶的是，我们发现PfCRT赋予对在发育的非常早期的环阶段施用的CQ的抗性，其中消化空泡尚未形成，以及在前体阶段，其中Hz产生被认为是平稳的。总之，这些数据迫使重新思考CQ药理学和CQR的机制。(C)2007 Elsevier B. V.保留所有权利。
We previously customized a Nipkow spinning disk confocal microscope (SDCM) to acquire 4D data for live, intraerythrocytic malarial parasites [Gligorijevic B, McAllister R, Urbach JS, Roepe, PD. Spinning disk confocal microscopy of live, intraerythrocytic malarial parasites. 1. Quantification of hemozoin development for drug sensitive versus resistant malaria. Biochemistry 2006;45:12400-10]. We reported that chloroquine (CQ) treatment did not appear to affect progress through the cell cycle, and suggested that toxicity may be manifested post-schizogony. We now use SDCM, synchronized cell culture and continuous vs. bolus drug dosing to investigate stage specific CQ effects in detail. We develop a novel, extremely rapid method for counting schizont nuclei in 3D. We then quantify schizont nuclei and hemozoin (Hz) production for live parasite cultures pulsed with CQ at different stages in the cell cycle and find that bolus treatment of rings affects the multiplicity of nuclear division. We quantify parasitemia and merozoite development in subsequent cycles following bolus CQ exposure and find that a portion of CQ toxicity is manifested post-schizogony as "delayed death". Using these methods and others we compare CQ sensitive (CQS) vs. resistant (CQR) strains as well as transfectants that are CQR via introduction of mutant PfCRT. Surprisingly, we find that PfCRT confers resistance to CQ administered at the very early ring stage of development, wherein a digestive vacuole is not yet formed, as well as at the schizont stage, wherein Hz production is thought to plateau. Taken together, these data force a rethinking of CQ pharmacology and the mechanism of CQR. (C) 2007 Elsevier B.V. All rights reserved.