van der Heyde H C, Elloso M M, vande Waa J, Schell K, Weidanz W P
Department of Medical Microbiology and Immunology, University of Wisconsin, Madison 53706, USA.
Clin Diagn Lab Immunol. 1995 Jul;2(4):417-25. doi: 10.1128/cdli.2.4.417-425.1995.
Flow cytometry was evaluated as a method of assessing in vitro the effects of leukocytes on blood-stage Plasmodium falciparum. Hydroethidine is converted by metabolizing cells to ethidium, a nucleic acid fluorochrome. After incubation with hydroethidine, viable and dead leukocytes and parasitized and uninfected erthrocytes could all be identified on the basis of fluorescence intensity and size. Leukocytes can therefore be eliminated from further analysis; this allows assessment, at any parasite developmental stage, of the level of parasitemia within erythrocytes in the presence of any of several types of leukocytes. Whether leukocytes actually kill intraerythrocytic parasites can therefore be determined and the level of cytotoxicity can be assessed. The ability of leukocytes to prevent merozoites from invading new erythrocytes, i.e., inhibition of parasite invasion, can also be assessed by this method. When erythrocytes containing schizont-stage parasites were cocultured with different leukocyte populations and the level of parasitemia was determined after merozoite release and invasion, only cultures containing gamma delta T cells inhibited parasite invasion. The different blood-stage forms of the parasite vary in nucleic acid content, which allows each of the developmental stages to be distinguished by flow cytometry; this permits assessment of changes in parasite development in the presence of leukocytes. Monocyte-derived macrophages (MDMs) appeared to have an effect on parasite development. In this instance, when erythrocytes containing ring-form parasites were cocultured with MDMs and harvested 24 h later, the parasites in cultures containing MDMs were at the late schizont stage, whereas parasites in control cultures were early trophozoites; this finding suggests that MDMs accelerate parasite development. Together, these results indicate that flow cytometry is potentially useful for measuring the following effects mediated by leukocytes: (i) level of cytotoxicity, (ii) changes in parasite development, and (iii) inhibition of parasite invasion.
流式细胞术被评估为一种在体外评估白细胞对恶性疟原虫血液阶段影响的方法。氢化乙锭被代谢细胞转化为核酸荧光染料乙锭。与氢化乙锭孵育后，可根据荧光强度和大小识别存活和死亡的白细胞以及被寄生和未感染的红细胞。因此，可以将白细胞从进一步分析中排除；这使得在任何寄生虫发育阶段，都能够在存在几种类型白细胞的情况下评估红细胞内的疟原虫血症水平。因此，可以确定白细胞是否真的杀死红细胞内的寄生虫，并评估细胞毒性水平。白细胞阻止裂殖子侵入新红细胞的能力，即抑制寄生虫入侵，也可以通过这种方法进行评估。当含有裂殖体阶段寄生虫的红细胞与不同白细胞群体共培养，并在裂殖子释放和入侵后测定疟原虫血症水平时，只有含有γδT细胞的培养物抑制了寄生虫入侵。寄生虫的不同血液阶段形式在核酸含量上有所不同，这使得每个发育阶段都可以通过流式细胞术区分开来；这允许评估在存在白细胞的情况下寄生虫发育的变化。单核细胞衍生的巨噬细胞（MDM）似乎对寄生虫发育有影响。在这种情况下，当含有环状体阶段寄生虫的红细胞与MDM共培养并在24小时后收获时，含有MDM的培养物中的寄生虫处于晚期裂殖体阶段，而对照培养物中的寄生虫是早期滋养体；这一发现表明MDM加速了寄生虫的发育。总之，这些结果表明流式细胞术可能有助于测量白细胞介导的以下影响：（i）细胞毒性水平，（ii）寄生虫发育的变化，以及（iii）对寄生虫入侵的抑制。
