Plasmodium berghei: inhibitors of ornithine decarboxylase block exoerythrocytic schizogony

DL-alpha-difluoromethylornithine and DL-alpha-monofluoromethyldehydroornithine methyl ester, inhibitors of ornithine decarboxylase, blocked exoerythrocytic schizogony of Plasmodium berghei in mice and in cultured human hepatoma cells. These effects were reversed by exogenous administration of the polyamine, spermidine. The antimalarial drug, primaquine, the side chain of which is structurally analogous to a natural polyamine, did not enhance the activity of alpha-difluoromethylornithine or alpha-monofluoromethyldehydroornithine methyl ester. These results extend previous observations that polyamines influence the malaria parasite's schizogony outside the red blood cell but not within it.     

Plasmodium falciparum and Plasmodium berghei: effects of ornithine decarboxylase inhibitors on erythrocytic schizogony

Five ornithine decarboxylase inhibitors: alpha-difluoromethylornithine (DFMO) (eflornithine); alpha-monofluoromethyl-3,4-dehydroornithine; alpha-monofluoromethyl-3,4-dehydroornithine methyl ester; alpha-monofluoromethyl-3,4-dehydroornithine ethyl ester; and (2R,5R)-delta-methyl-alpha-acetylenic putrescine were shown to inhibit erythrocytic schizogony of Plasmodium falciparum in vitro and reduced spermidine levels in infected erthrocytes. Only DFMO was effective at limiting erythrocytic schizogony of P. berghei in vivo. Administration of DFMO as a 2% solution in the drinking water for 4 days reduced parasitemia in mice by 50% in a 4-day suppression test but did not increase survival time of infected mice. This is the first demonstration of an effect of DFMO on plasmodial erythrocytic schizogony in vivo and suggests that interference with polyamine biosynthesis may, in fact, be a viable chemotherapeutic target in erythrocytic malaria.     

Observation on complete schizogony of Plasmodium vivax in vitro

Attempts to grow Plasmodium vivax in vitro were made on 43 isolates in three different culture media. Complete schizogony occurred in the new medium SCMI 612 in which 34 out of 43 isolates produced merozoites. The RPMI 1640 and Waymouth media suitable for the cultivation of P. falciparum were also used with markedly less success. Results of the experiments indicate differences in nutritional requirements between the two species of Plasmodium.     

Long-term in vitro cultivation of Plasmodium berghei

Long-term in vitro culture of Plasmodium berghei was established using the Petri dish candle jar method of Trager and Jensen (1976). Cultures were established at 22, 27 and 37°C. As optimal growth was observed at 27°C, subsequent cultivation was carried out at this temperature. RPMI 1640 medium was modified by incorporating additional glucose (1 mg ml⁻¹) and bactopeptone (1 mg ml⁻¹) in the medium. This medium was found suitable for maintenance of mouse erythrocytes in vitro. P. berghei cultures were maintained using candle jars and this modified RPMI 1640 medium for 45 weeks.     

Motility of Plasmodium berghei ookinetes in vitro

Motility of Plasmodium berghei ookinetes, which developed in primary and established cell line cultures obtained from Anopheles stephensi mosquitoes, was studied by using still photomicrographs and normal speed cinephotomicrography. At 18–72 hr after inoculation of P. berghei infected blood from hamsters or mice, motile ookinetes were seen in both mosquito cell cultures; the most active specimens were observed at 24–30 hr. Ookinetes underwent a sporadic forward gliding movement, during which a variable degree of rotation of the body upon its longitudinal axis usually occurred. Some specimens rotated repeatedly upon their axes without any forward progression. The direction of the gliding movement always coincided with the curvature of the ookinete body. In those specimens in which no rotation of the body occurred, a circular course resulted. Ookinetes covered a distance of as much as 50 μm during a single gliding movement. A few ookinetes undergoing locomotion appeared to leave a path or trail on the substrate. Occasionally, an ookinete penetrated a red cell with its slender anterior projection, resulting in lysis of the cell. After red cells had been penetrated by ookinetes, the parasites already within these cells fused with each other to form larger spheroidal bodies. Penetration of cultured cells was not observed.     

Changes in schizogony and drug response in two lines of rodent Plasmodium, P. berghei NK 65 and P. berghei ANKA

White mice were infected with two strains, ANKA and NK 65, of Plasmodium berghei. The parasites were subjected to chloroquine pressure (60 mg/kg at each passage) during 20 passages. We then compared the behaviour of the strains as they acquired chemoresistance. The drug resistance was estimated by the 2% delay time test (D2%), and the schizogonic rhythm by the synchronicity index (SI). Before drug pressure, the ANKA strain had a D2% of 4.34, and a SI of 0.2. This strain became highly drug resistant, but synchronicity increased: the D2% was 2.93, and the SI was 0.36 at the 20th passage. The NK 65 strain had an initial D2% of 4.12, and an SI of 0.2. The chemoresistance acquired during 20 passages was very irregular for this strain: after drug pressure, the D2% was 2.03 and the SI was 0.28. Drug pressure was then removed (for both strains), for 10 passages (no chloroquine). Resistance and synchronicity returned to their initial values. The two strains behaved very differently, in terms of their affinity for reticulocytes, and with chloroquine activity which favours an increase in SI because only merozoites are preserved.     

Plasmodium berghei: in vitro and in vivo activity of dequalinium

Bisquinoline compounds have exhibited remarkable activity in vitro and in vivo against Plasmodium parasites by inhibition of heme detoxification. We have tested the ability of dequalinium 1,1'-(1,10-decanediyl)bis(4-amino-2-methylquinoline), a known antimicrobial agent, to inhibit beta-hematin synthesis using a non-emzymatic colorimetric assay and globin proteolysis by electrophoretic analysis (SDS-PAGE-15%). Dequalinium was able to inhibit both processes in vitro with close correlation to a murine malaria model, reducing parasitemia levels, prolonging the survival time post-infection and curing 40% of infected mice using a combination therapy with a loading dose of chloroquine. These results confirm that dequalinium is a promising lead for antimalarial drug development.     

Interactions of Plasmodium berghei sporozoites and murine Kupffer cells in vitro

Malaria sporozoites must leave the bloodstream and cross a layer of sinusoidal lining cells in order to infect hepatocytes and undergo exoerythrocytic schizogony. To determine whether Kupffer cells (KC) derived from this layer interact with sporozoites, murine KC were isolated from perfused livers of BALB/cJ mice and incubated in vitro with Plasmodium berghei sporozoites. Isolated KC had characteristic macrophage surface Ag and were phagocytic, ingesting both latex particles and Leishmania major amastigotes. In the absence of immune serum, sporozoites associated with fewer than 10% of these KC. By 30 min, 10% of the cell-associated sporozoites were completely ingested, 30% were in the process of being ingested, and 60% were attached to the surface of the cells. Opsonization of sporozoites with monoclonal or polyclonal antibodies directed against P. berghei circumsporozoite protein markedly enhanced sporozoite association with KC. Up to 40% of cells exposed to opsonized sporozoites had parasites inside or attached to their surfaces. Sporozoites attached to or ingested by KC were uniformly destroyed within 240 min in all cultures; there was no evidence of conversion of sporozoites to the exoerythrocytic stage within KC by light microscopy, and there was no evidence of residual sporozoites, either inside or outside of cells, by either light or electron microscopy. These data suggest that under nonimmune conditions, KC play a minor role in resistance to infection by malaria sporozoites. However, when sporozoites are opsonized by circumsporozoite antibodies, phagocytosis by KC may be an important immune mechanism that prevents parasitization of hepatocytes.     

A protective monoclonal antibody with dual specificity for Plasmodium falciparum and Plasmodium berghei circumsporozoite proteins.

An IgM monoclonal antibody (Mab 36) which reacts with the circumsporozoite (CS) proteins of both P. falciparum and P. berghei was isolated from Plasmodium falciparum sporozoite-immunized mice. In assays of biological activity, Mab 36 induces the CS precipitation reaction with live sporozoites and blocks the invasion of hepatoma cells by sporozoites in vitro at concentrations much lower than those observed for previously reported CS protein-specific monoclonal antibodies. Mab 36 also provided complete protection against P. berghei sporozoite challenge in mice at low doses. Linear epitope mapping revealed that the epitope specificities recognized by Mab 36 are completely encompassed by other monoclonals previously shown to be associated in vivo with protection against P. falciparum or P. berghei sporozoite infection. These results suggest that the ability to make high-affinity IgM antibody to specific CS protein repeat epitopes may be important for eliciting protection against malarial infection.     

Long-term in vitro cultures of Plasmodium berghei and preliminary observations on gametocytogenesis

Several long-term in vitro cultures of the rodent malaria parasite Plasmodium berghei were established. In these cultures, ranging over 17–90 days, peak parasitaemias of over 20% and multiplication rates of up to 7·7 were observed. A previously described culture method was used. The method for medium refreshment was changed and rat erythrocytes were used as host cells. The long-term cultivation of Plasmodium berghei enables us to study the process of gametocytogenesis since male and female gametocytes were produced in all cultures and reached full maturity, demonstrated by exflagellation and fertilization in vitro.     

Development of Plasmodium berghei ookinetes to young oocysts in vitro.

The mosquito stage of Plasmodium berghei was cultivated in vitro, with special attention to ookinete transformation into early oocyst. The ookinetes were obtained by in vitro culture of gametocytes taken from infected mice, purified by density gradient of metrizoic acid or a lymphocyte separation medium, and incubated either in acellular culture or in co-cultivations with mosquito cells. In acellular culture, the ookinetes were found to aggregate with each other and transformed from banana to round shapes. Their inner pellicular membranes and subpellicular microtubules partially disappeared, indicating that development to early oocyst had occurred. Co-cultivation wtih Aedes albopictus cells (C6/36 clone) revealed that ookinetes transformed into early oocyst in the medium, or invaded the cells and then transformed to early oocysts within the cell cytoplasm as well. However all of these transformed cells failed to develop further, i.e., neither deposition of the oocyst capsule nor nuclear division was observed. Many ookinetes which failed to penetrate the Aedes cells were phagocytized within three days of culture. A significant difference between invaded and transformed oocysts and phagocytized ookinetes was seen in that the former lacked vacuole membrane. Co-cultivation with Toxorhynchites amboinensis cells (TRA-284-SFG clone) permitted transformation of ookinetes into early oocysts in the medium as in the acellular culture, but no ookinete invasion nor phagocytosis by the cell was observed.