USE OF HYDROETHIDINE AND FLOW-CYTOMETRY TO ASSESS THE EFFECTS OF LEUKOCYTES ON THE MALARIAL PARASITE PLASMODIUM-FALCIPARUM

Flow cytometry was evaluated as a method of assessing in vitro the eff ects of leukocytes on blood-stage Plasmodium falciparum, Hydroethidine is converted by metabolizing cells to ethidium, a nucleic acid fluoro chrome. After incubation with hydroethidine, viable and dead leukocyte s and parasitized and uninfected erythrocytes could all be identified on the basis of fluorescence intensity and size, Leukocytes can theref ore be eliminated from further analysis; this allows assessment, at an y parasite developmental stage, of the level of parasitemia within ery throcytes in the presence of any of several types of leukocytes, Wheth er leukocytes actually kill intraerythrocytic parasites can therefore be determined and the level of cytotoxicity can be assessed, The abili ty of leukocytes to prevent merozoites from invading new erythrocytes, i,e,, inhibition of parasite invasion, can also be assessed by this m ethod, When erythrocytes containing schizont-stage parasites were cocu ltured with different leukocyte populations and the level of parasitem ia was determined after merozoite release and invasion, only cultures containing gamma delta T cells inhibited parasite invasion, The differ ent blood-stage forms of the parasite vary in nucleic acid content, wh ich allows each of the developmental stages to be distinguished by flo w cytometry; this permits assessment of changes in parasite developmen t in the presence of leukocytes, Monocyte-derived macrophages (MDMs) a ppeared to have an effect on parasite development, In this instance, w hen erythrocytes containing ring-form parasites were cocultured with M DMs and harvested 24 h later, the parasites in cultures containing MDM s were at the late schizont stage, whereas parasites in control cultur es were early trophozoites; this finding suggests that MDMs accelerate parasite development, Together, these results indicate that flow cyto metry is potentially useful for measuring the following effects mediat ed by leukocytes: (i) level of cytotoxicity, (ii) changes in parasite development, and (iii) inhibition of parasite invasion.