William Trager

Bio: William Trager is an academic researcher from Rockefeller University. The author has contributed to research in topics: Plasmodium falciparum & Plasmodium lophurae. The author has an hindex of 52, co-authored 184 publications receiving 15452 citations.

Human malaria parasites in continuous culture

Abstract: Plasmodium falciparum can now be maintained in continuous culture in human erythrocytes incubated at 38 degrees C in RPMI 1640 medium with human serum under an atmosphere with 7 percent carbon dioxide and low oxygen (1 or 5 percent). The original parasite material, derived from an infected Aotus trivirgatus monkey, was diluted more than 100 million times by the addition of human erythrocytes at 3- or 4-day intervals. The parasites continued to reproduce in their normal asexual cycle of approximately 48 hours but were no longer highly synchronous. The have remained infective to Aotus.

Acquired Immunity to Ticks.

Abstract: It is well known that the American dog tick, Dermacentor variabilis Say, can be reared through its entire life cycle on guinea pigs. In the course of permitting successive batches of larvae of this tick to engorge on the same guinea pig, it was repeatedly observed that, while the first batch always gave a large number of engorged larvae, the following batches gave few or none. This suggested that guinea pigs, after one infestation with larvae of D. variabilis, acquire an effective immunity against these ectoparasites. The experiments reported in this paper show that such an immunity is indeed developed, and that it may be solid enough to prevent larvae from engorging and to reduce the amount of blood taken by nymphs and adults.

Plasmodium falciparum in culture: use of outdated erthrocytes and description of the candle jar method.

Abstract: Plasmodium falciparum has been cultured continuously in a simplified technique using plastic petri dishes in a candle jar. This method has the advantage of being readily adaptable to many experiments, especially those requiring numerous replicates. Experiments using this method have allowed examination of some factors responsible for variations in parasite multiplication rates. Generally, fetal calf serum is not comparable to human serum, modifications to the RPMI 1640 medium are usually deleterious, and erythrocytes aged in citrated plasma are better for malarial cultures than freshly obtained cells; thus, erythrocytes outdated by blood-bank standards could provide a readily available supply of cells for large-scale production of malarial antigens. Continuous culture of Plasmodium falciparum in vitro was first obtained by a method providing for a slow flow of medium over a thin settled layer of human erythrocytes (Trager and Jensen, 1976; Trager, 1976). This method lends itself to partial automation and provides for continuous production of parasite material. It is not convenient, however, for the study of the many different factors that might affect growth of the parasites. For this purpose we developed a simplified method using plastic petri dishes in a candle jar. The essentials of this petri dish method have already been briefly described (Trager and Jensen, 1976). Here we present full details of the method and its application in demonstrating the feasibility of using outdated erythrocytes and for testing certain other modifications of the culture conditions. MATERIALS AND METHODS

Characterization of the in vitro biotransformation of the HIV-1 reverse transcriptase inhibitor nevirapine by human hepatic cytochromes P-450.

Abstract: Nevirapine (NVP), a non-nucleoside inhibitor of HIV-1 reverse transcriptase, is concomitantly administered to patients with a variety of medications. To assess the potential for its involvement in drug interactions, cytochrome P-450 (CYP) reaction phenotyping of NVP to its four oxidative metabolites, 2-, 3-, 8-, and 12-hydroxyNVP, was performed. The NVP metabolite formation rates by characterized human hepatic microsomes were best correlated with probe activities for either CYP3A4 (2- and 12-hydroxyNVP) or CYP2B6 (3-and 8-hydroxyNVP). In studies with cDNA-expressed human hepatic CYPs, 2- and 3-hydroxyNVP were exclusively formed by CYP3A and CYP2B6, respectively. Multiple cDNA-expressed CYPs produced 8- and 12-hydroxyNVP, although they were produced predominantly by CYP2D6 and CYP3A4, respectively. Antibody to CYP3A4 inhibited the rates of 2-, 8-, and 12-hydroxyNVP formation by human hepatic microsomes, whereas antibody to CYP2B6 inhibited the formation of 3- and 8-hydroxyNVP. Studies using the CYP3A4 inhibitors ketoconazole, troleandomycin, and erythromycin suggested a role for CYP3A4 in the formation of 2-, 8-, and 12-hydroxyNVP. These inhibitors were less effective or ineffective against the biotransformation of NVP to 3-hydroxyNVP. Quinidine very weakly inhibited only 8-hydroxyNVP formation. NVP itself was an inhibitor of only CYP3A4 at concentrations that were well above those of therapeutic relevance (K(i) = 270 microM). Collectively, these data indicate that NVP is principally metabolized by CYP3A4 and CYP2B6 and that it has little potential to be involved in inhibitory drug interactions.

Fine structure of human malaria in vitro.

Abstract: SYNOPSIS. The erythrocytic cycle of the human malaria parasite, Plasmodium falciparum, was examined by electron microscopy. Three strains of parasites maintained in continuous culture in human erythrocytes were compared with in vivo infections in Aotus monkeys. The ultrastructure of P. falciparum is not altered by continuous cultivation in vitro. mitochondria contain DNA-like filaments and some cristae at all stages of the erythrocytic life cycle. The Golgi apparatus is prominent at the schizont stage and may be involved in the formation of rhoptries. In culture, knob-like protrusions first appear on the surface of trophozoite-infected erythrocytes. The time of appearance of knobs on cells in vitro correlates with the life cycle stage of parasites which are sequestered from the peripheral circulation in vivo. Knob material of older parasites coalesces and forms extensions from the erythrocyte surface. Some of this material is sloughed from the host cell surface. The parasitophorous vacuole membrane breaks down in erythrocytes containing mature merozoites both in vitro and in vivo. Merozoite structure is similar to that of P. knowlesi. The immature gametocytes in culture have no knobs.

Human malaria parasites in continuous culture

Abstract: Plasmodium falciparum can now be maintained in continuous culture in human erythrocytes incubated at 38 degrees C in RPMI 1640 medium with human serum under an atmosphere with 7 percent carbon dioxide and low oxygen (1 or 5 percent). The original parasite material, derived from an infected Aotus trivirgatus monkey, was diluted more than 100 million times by the addition of human erythrocytes at 3- or 4-day intervals. The parasites continued to reproduce in their normal asexual cycle of approximately 48 hours but were no longer highly synchronous. The have remained infective to Aotus.

Synchronization of Plasmodium falciparum erythrocytic stages in culture.

Abstract: Synchronous development of the erythrocytic stages of a human malaria parasite, Plasmodium falciparum, in culture was accomplished by suspending cultured parasites in 5% D-sorbitol and subsequent reintroduction into culture. Immediately after sorbitol treatment, cultures consisted mainly of single and multiple ring-form infections. At the same time, varying degrees of lysis of erythrocytes infected with the more mature stages of the parasite was evident. Approximately 95% of the parasites were in the ring stage of development at 48 and 96 hr after sorbitol treatment-likewise, a high percentage of trophozoite and schizont stages was observed at 24, 72, and 120 hr. D-Mannitol produced similar, selective, lytic effects.

Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique.

Abstract: A rapid, semiautomated microdilution method was developed for measuring the activity of potential antimalarial drugs against cultured intraerythrocytic asexual forms of the human malaria parasite Plasmodium falciparum. Microtitration plates were used to prepare serial dilutions of the compounds to be tested. Parasites, obtained from continuous stock cultures, were subcultured in these plates for 42 h. Inhibition of uptake of a radiolabeled nucleic acid precursor by the parasites served as the indicator of antimalarial activity. Results of repeated measurements of activity with chloroquine, quinine, and the investigational new drug mefloquine demonstrated that the method is sensitive and precise. Several additional antimalarial drugs and compounds of interest were tested in vitro, and the results were consistent with available in vivo data. The use of P. falciparum isolates with known susceptibility to antimalarial drugs also permitted evaluation of the cross-resistance potential of each compound tested. The applications and expectations of this new test system within a drug development program are discussed.