An in vitro model was developed to study the hepatic phase of Plasmodium falciparum, the only malaria parasite lethal to man. Primary cultures of human hepatocytes were inoculated with sporozoites of Brazilian and African strains of P. falciparum. On days 1 through 7 after inoculation examination of fluorescence-labeled and Giemsa-stained preparations demonstrated the presence of many intracellular parasites. In three separate sets of experiments all cultures were found to be infected with as many as 650 liver schizonts measuring up to 40 micrometers. After the addition of red blood cells, intraerythrocytic forms of P. falciparum were detected on days 12 and 13 by an immunofluorescence assay, indicating that the hepatic cycle had been completed in vitro.

There is growing evidence that cytokines (interleukin [IL] 1, IL 6, interferon-gamma, tumor necrosis factor [TNF]) directly or indirectly interfere with the intrahepatic development of malaria parasites. Recent work in our laboratory clearly showed that TNF can affect the hepatic development of parasites via IL 6 secreted by liver nonparenchymal cells. The possible participation of an L-arginine-dependent effector mechanism has been studied to explain the TNF/IL 6-induced inhibition. We thus investigated if NGmonomethyl-L-arginine and N omega-nitro-L-arginine, two specific inhibitors of inorganic nitrogen oxide synthesis from L-arginine, were able to affect the inhibitory effect of TNF and/or IL 6 in co-cultures. At 0.1 and 0.5 mM both L-arginine analogues reversed the inhibitory effect of these cytokines. An interesting observation is that L-arginine analogues enhance schizont development in the absence of prior cytokine contact. This result indicates an hepatic basal L-arginine-dependent anti-parasitic activity which might explain the existence of self-degenerating hepatic forms as previously reported.

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Cultured hepatic stages of Plasmodium falciparum and P. yoelii and with a monoclonal antibody recognizing a C-terminal fragment of the P. falciparum heat-shock-like protein (Pfhsp70) revealed that synthesis of this antigen first occurs during intrahepatic development of the parasite, at the two nuclei stage. Using a variety of techniques, including scanning electron microscopy, we observed that this antigenic determinant was expressed on the infected hepatocyte membrane. Its participation in antibody-dependent cell-mediated cytotoxicity was investigated. While no effect was obtained with peripheral blood cells, we found that 25% of the schizonts were specifically lysed when using spleen cells at a killer/target ratio of 30/1. More interestingly, with nonparenchymal liver cells, up to 50% of the hepatic parasites disappeared with a killer/target ratio of 10/1.

In previous work, a T-helper epitope was mapped within the circumsporozoite protein of the murine malaria parasite Plasmodium yoeli. A 21-mer synthetic peptide corresponding to this epitope (amino acid positions 59-79; referred to as Pyl) induced a specific T-cell proliferation in BALB/c and C57BL/6 mice and provided help for the production of antibodies to peptides from the repetitive region,