F. Miltgen

Malaria sporozoite penetration: a new approach by double staining

To determine, whether a sporozoite is outside the hepatocyte membrane or internalized, a double staining test was carried out using, successively, antibody labeled with peroxidase and fluorescein. This test permits the quantification of sporozoite entry and outline sporozoite-hepatocyte interactions.

Inhibitory activity of IL‐6 on malaria hepatic stages

Summary Addition of recombinant inlcrleukin‐6 (IL‐6) to Plasmodium yoelii hepatic cultures resulted in a specific dose‐dependent inhibition of parasite development. Time course experiments showed that, without any direct effect on free sporozoites, IL‐6 exerts its action during both the early phase of infection and during the subsequent maturation of the schizonts. Elicitation of the oxidative burst appears lo be one mechanism by which IL‐6 interferes with the development of hepatic phase. Catalase and superoxide dismutase, two scavengers of hydrogen peroxide and superoxide anions, reversed the IL‐6 mediated parasiticidal activity.

A method for the quantitative assessment of malaria parasite development in organs of the mammalian host

A non-radioactive PCR method was developed to quantify the development of malaria parasites in the infected host. This was achieved by using Plasmodium genus-specific primers corresponding to the parasites small subunit ribosomal RNA genes. The quantification of the PCR product was performed by high performance liquid chromatography, and calibration curves were obtained by amplification from defined quantities of purified Plasmodium genomic DNA. Using this method, it was possible to quantify development of P. berghei and P. yoelii blood-stage parasites from blood and brain samples of infected mice, and of hepatic stage parasites, from liver samples of mice infected with different numbers of sporozoites.

Hepatic phase of malaria is the target of cellular mechanisms induced by the previous and the subsequent stages. A crucial role for liver nonparenchymal cells.

Both the sporozoites and the erythrocytic stages can modulate the hepatic phase by cytokines, notably IFN-gamma, TNF and IL-6, either directly or as a result of a cascade of events, and by MHC-restricted and antibody-dependent cell-mediated cytotoxicity. The role played by CD8+ T cells in inducing protective immunity against pre-erythrocytic stages is clearly established. The potential interest of triggering peptide-primed CD4+ T cells has to be considered regarding protection. Indeed, CD4+ T cells induced by the non-repetitive part of the CS protein of Plasmodium yoelii are protective, by eliminating malaria from hepatocytes. The crucial role of the liver NPC has to be emphasized, their participation in TNF schizonticidal effect and in ADCC mechanisms being strongly supported by our data.

Plasmodium ovale: In vitro development of hepatic stages

Primary cultures of human hepatocytes, a culture-derived clone from the human hepatoma Hep G2 line, and cultured rat hepatocytes were inoculated in vitro with Plasmodium ovale sporozoites extracted from Anopheles stephensi, An. gambiae, and An. dirus mosquitoes. Penetration and differentiation of P. ovale sporozoites into trophozoite stage parasites occurred in all three cell types, but with a lower transformation rate in the Hep G2 cell line than in the primary cultured hepatocytes. Further maturation was obtained only in the human hepatocytes, in which the parasites were uninucleate until the third day after infection, before development to 60 micron in length by the eighth day. Additionally, this culture system was used to assess the ability of an anti-P. ovale sporozoite monoclonal antibody to inhibit penetration of sporozoites into hepatocytes and to detect sporozoite determinants in the maturing liver stage parasites.

Inflammatory status and preerythrocytic stages of malaria: Role of the C-reactive protein

In the acquisition of protection against malaria, the role played by nonspecific factors, some being part of the cascade effect of cytokines, has to be considered. The C-reactive protein, a major acute phase reactant secreted by interleukin-1 stimulated hepatocytes, has an effect on the hepatic development of Plasmodia, both by preventing penetration of the sporozoite into the hepatocyte and by blocking parasite division through an antibody-like effect. This latter effect confirms the potential interest of targeting the uninuclear form of the parasite. Nevertheless, C-reactive Protein alone does not account for all the effects of the inflammatory response, other reactants from both serum and hepatocytes are also involved.

The gametocytes of Plasmodium vinckei petteri, their morphological stages, periodicity and infectivity.

Gametocyte production by P. vinckei petteri was cyclic, occurring at each schizogony every 24 h. They matured in 27 h from merozoite to type 0 microgametocyte, in 3 h from type 0 to type I, 6 h from type I to type II and 3 h from type II to type III. Transmission experiments showed that the time of maximum infectivity was midday when mice were inoculated at midnight, and midnight when mice were inoculated at midday. In all instances, maximum infectivity coincided with a peak in intensity by type II microgametocytes, a relationship confirmed by multiple correspondence analysis. The proportion of type II microgametocytes was higher in the mosquitoes blood meal than in smears of tail blood of mice, suggesting a sequestration phenomenon with this stage.

Protective immunity against malaria: cellular changes in the liver vary according to the method of immunization.

Characterization of cells present in the extravascular compartment of murine liver was performed after different immunization procedures against the malaria parasite Plasmodium yoelii. Mice were immunized with live or irradiated sporozoites or with parasitized erythrocytes. Whatever the immunization protocol used, the mice were protected against a sporozoite challenge but each immunization procedure induced a specific profile of cell types. Immunization with irradiated sporozoite induce a significant increase in CD8+ lymphocytes, parasitized erythrocytes stimulates production of monocytes/macrophages and CD8+ lymphocytes while, after live sporozoites immunization, polymorphonuclear cells, macrophages/ monocytes, B cells and a range of T cell subsets were increased in number.

Activity of dihydrofolate reductase inhibitors on the hepatic stages of Plasmodium yoelii yoelii in vitro

The effects of the dihydrofolate reductase inhibitors proguanil and chlorproguanil, their active metabolites cycloguanil and chlorcycloguanil, and pyrimethamine, against the hepatic stages of Plasmodium yoelii yoellii were investigated in cultured BALB/c mouse hepatocytes. Proguanil was inactive at concentrations of 10(-8) M, whereas the other compounds were fully active at this and lower concentrations. Chlorcycloguanil was the most active compound and almost completely inhibited schizont development in concentrations as low as 10(-12) M.

The chemotherapy of rodent malaria XLI: Causal prophylaxis Part V. Effect of mefloquine on exoerythrocytic schizogony in Plasmodium yoelii yoelii

Previous studies using mefloquine in rodents have suggested that this compound has no effect on pre-erythrocytic schizogony. In the present study, direct observations on 46-hour-old pre-erythrocytic schizonts of P. yoelii in the liver of rats have shown that mefloquine does induce recognizable changes in the peripheral, enzyme-containing vesicles but that these are insufficient to hinder the maturation of the parasites, thus confirming that this compound has no causal prophylactic value.