Elemental composition in monocytes in response to anti-malarial drugs and hemozoin.

Abstract

Every year there are approximately 300 million new cases of malaria with 2 million deaths. The majority of deaths occur in African children between the ages of 1 and 4 years and are caused by the parasite Plasmodium falciparum. Approximately R90-million is spent by the South African government each year to control malaria. Peripheral blood monocytes are the first line of defence during infection and they perform many functions, such as phagocytosis, intracellular and extracellular killing by the generation of reactive oxygen intermediates and the production of cytokines. During malaria infection some of these functions are suppressed or elevated by phagocytosis of hemozoin, fever conditions (heat shock) and the presence of anti-malarial drugs in the bloodstream of the patient. Under normal conditions phospholipase A₂ (PLA₂) is down regulated by heat shock protein 70 (HSP70) but in severe malaria PLA₂ is elevated. Two antigenic peptides were selected from the highly conserved human HSP70 and HSC70 proteins. Anti-peptide antibodies raised in chickens were affinity purified and were able to recognize the free peptide in an ELISA and the native proteins in human and canine heat shocked lymphocyte lysates on western blots. Antibodies against HSP70 detected two major proteins at 70 kDa and 33 kDa, which are most likely native HSP70 and a possible breakdown product of HSP70 respectively. The anti-HSC70 antibodies detected two proteins, an as yet unidentified 100 kDa protein and the 70 kDa HSC70. Due to the monocytes being activated during the isolation procedure, HSP70 was expressed at both 37°C and 44°C in this study. Electron-probe X-ray microanalysis enables determination of the elemental composition of any sample under the electron microscope. When the electron beam interacts with a specimen, X-rays are generated and can be used to identify and quantify the elements in the cell. Canine monocytes were analysed using this technique after incubation with therapeutically relevant concentrations of anti-malarial drugs, β-hematin and under fever conditions. The concentrations of the elements in normal canine monocytes were: Na (518.2 mmoles/kg), Mg (199.1 mmoles/kg), P (439.7 mmoles/kg), S (316.3 mmoles/kg), Cl (279.7 mmoles/kg), K (204 mmoles/kg) and Ca (81.3 mmoles/kg). All the drugs (quinine, chloroquine, primaquine, pyrimethamine, artemisinin, tetracycline, doxycycline, dapsone and suramin), phagocytosis of latex beads and β-hematin as well as heat shock, altered the elemental concentrations of canine monocytes in a unique way. Quinine, artemisinin and suramin were the most influential drugs in altering the concentrations of elements in the cells.Suramin substantially increased the concentration of Ca (356%) after 18 h and decreased K concentration (64%) after 18 h. Quinine decreased the concentrations ofNa (47%), Cl (70%), and K (67%). The concentrations of P (52%) and Ca (72%) were increased by quinine after 10 min. Artemisinin induced small increases in Mg (21 %) and K (38%) concentrations within 10 min and large increases in the concentrations of Na (291%) and Cl (389%) after 18 h. Chloroquine induced a large increase in S (212%). Quinine induced major changes after 10 min whereas artemisinin, suramin chloroquine induced huge changes after 18 h. Although artemisinin did increase the concentrations certain elements after 10 min, it was by much smaller amounts than after 18 h. Quinine, suramin and pyrimethamine altered the P/K ratios by the greatest margins whereas artemisinin had no significant effect. The P/K ratio was increased by quinine (348%) after 10 min and suramin (261%) after 18 h. Pyrimethamine decreased the P/K ratio after 18 h by 49%. The findings suggest that further investigations into the alterations in the elemental concentrations of monocytes by anti-malarial drugs, fever and hemozoin may lead to a greater understanding of the influence of these conditions in a patient during a malaria infection and its treatment.