Daniel C. Martin

Electron microscopy of Plasmodium falciparum 1. The structure of trophozoites in erythrocytes of human volunteers

Abstract Electron microphotographs of early trophozoites of Plasmodium falciparum from human volunteers were compared with those of Plasmodium berghei in the rat. Human and rodent trophozoites exhibited certain general similarities: (1) both lacked a nucleolus, (2) pigment and smooth membrane vesicles were similar, (3) both forms exhibited double limiting membranes, (4) both lacked typical mitochondial forms, (5) both forms exhibited micropyle-type structures which are apparently specialized sites of absorption. Although multilaminated-membraned bodies (whorled-patterns) were present in both forms, P. falciparum differed in that these bodies appeared to be derived from the nuclear membranes; the origin of the body in P. berghei appeared to be more related to the external limiting membranes. The erythrocytic forms of P. berghei showed a great predilection for reticulocytes or younger red cells, but P. falciparum was found only in mature red cells.

Human Subjects in Clinical Research — A Report of Three Studies

THE search for new drugs and technics of medical care requires that man be the final experimental animal. The safeguards for volunteers have thus far assumed two forms. The first has been formal re...

The effect of parasite populations on the curative action of pyrimethamine

Abstract A non-resistant strain of P. falciparum was used to demonstrate a mechanism of acquired drug resistance. The dose of pyrimethamine required to cure a non-resistant P. falciparum infection depends directly on the population size of the parasitaemia. The surviving parasites demonstrate maximum resistance. This may explain widely different findings about the ease of resistance of P. falciparum to various drugs. This must also be considered as a mechanism for potentiation between two drugs effective on asexual forms of the parasite.

Studies on an East African strain of Plasmodium falciparum

Abstract An East African strain of P. falciparum was obtained from a child in Kampala, Uganda. It proved to be very responsive to all antimalarials tested. Chloroquine 0·45 g. base and quinine 2·5 g. base given over 5 days, each produced radical cure. A review of responsiveness of strains of P. falciparum used for infection of volunteers is presented. The data on the response of P. falciparum strains from different world areas indicate that sensitivity to chloroquine and quinine are closely related.

Electron microscopy of plasmodium berghei: I. on the migration of trophozoites from infected erythrocytes in the rat

Abstract Electron microphotographs are presented of trophozoites of Plasmodium berghei migrating from parasitized rat erythrocytes. The structure of the merozoite (young trophozoite) is compared with that of the maturing trophozoite containing pigment and forming rings. The significance of trophozoite migration, regarding the extracellular viability and infectivity of the trophozoite form, remains to be investigated.

Chemical agents effective in mediating control of growth and division synchrony of Plasmodium berghei in pinealectomized mice.

Pinealectomy abolishes the growth and division synchrony of Plasmodium berghei which is normally augmented by the photoperiodic rhythm. The augmented synchrony typical of the intact animal may be restored by ubiquinone, members of the vitamin K series, or partly by alpha-tocopherol. This restoration of growth and division synchrony by the 1,4-quinones or the 6-chromanols requires the continued exposure of the pinealectomized animal to the appropriate photoperiodic rhythm. Diets deficient in vitamin K or vitamin E will have the same effect on growth and division synchrony as does pinealectomy. The vascular release of mature schizonts, which is also under the control of the pineal, is only partially restored by the 1,4-quinones, or 6-chromanols. Pinealectomy can be shown to abolish the effects of photoperiodism in augmenting growth and division synchrony of Plasmodium berghei in the mouse (Arnold et al., 1969b). In addition, pinealectomy changes the pattern of mature schizont sequestration of the same infection in mice. The mechanism by which the pineal is activated is not yet known, nor is the mechanism known by which the pineal carries out its part in regulating growth and division synchrony. Similar uncertainties apply to the means by which the pineal regulates vascular capture and release of mature schizonts. It seems likely that the pineal is the junction point for a neural pathway and a chemical pathway. In studying the nature of the pineal as a chemical transducer, we have explored the role of several known chemical agents

Augmentation of growth and division synchrony and of vascular sequestration of Plasmodium berghei by the photoperiodic rhythm.

Growth and division pattern of Plasmodium berghei in mice is either absent or hard to detect under most circumstances. An augmented pattern of growth and division synchrony occurs under the influence of certain light rhythms. These light rhythms require specific time patterns, specific light quality, and specific light intensities. Generation times of the parasite can be calculated from syn- chronous growth patterns and correspond to the length of the solar day. The final merozoite number does not seem to be modified by the entrainment mechanism. Diet exerts a permissive effect on the augmentation of growth and division synchrony by light, but neither light nor diet seem to play a role in the operation of the master clock. Sequestration of mature schizonts in blood vessels is also depend- ent on photoperiodism. One light rhythm will produce synchronous growth and division with most of the cell division occurring in the peripheral blood, while other light rhythms will induce growth and division synchrony with most of the cell division occuring in cells sequestered in the blood vessels. In addition, vascular capture and release of late growth forms appear also to be under the control of the photoperiodic rhythm.

Role of the Endocrine System in Controlling Growth and Division Synchrony of Plasmodium berghei in Mice

The malaria parasite of mice may be induced by certain photoperiodic rhythms to show an augmented growth and division synchrony. This process is mediated by the testes and pineal but not by other endocrine organs. Certain steroids will compensate for the missing testes. Four hydroxy-17alpha-methyl testosterone and 4-chlorotestosterone produced an effect nearly equal to that seen in the intact animal. Estrogen, progesterone, and testosterone used alone and in combination were not able to replace the testes. In addition to the partial control of growth and division synchrony, the testes also influence the vascular capture and release of mature parasites along the walls of the blood vessels. Hypophysectomy had no effect on growth and division synchrony or on parasite sequestration even after partial gonadal atrophy had occurred. Neither cyclic peaks in concentration of replacement androgens nor the photoperiodic rhythm are the time clue to which the growth and division synchrony of the parasite is entrained. The nature of the time clue is still unknown. Malaria infections have a striking dependence on the solar day. This is apparent from the fact that the generation time of most species of parasite is an integral multiple of 24 hr. In addition, many species of malaria are so closely tied to a diurnal rhythm that all parasites of the species are found in growth and division synchrony in the same host individual. A species of rodent malaria, Plasmodium berghei, offers certain insights into the mechanism of parasite entrainment to a host rhythm. In this system of mouse-with-parasite, the entrainment only occurs when the host mouse is exposed to sunlight or a nearly equivalent light for at least 16 hr a day. Parasite division occurs between 12 midnight and 6 AM during the time of entrainment (Arnold et al., 1969a). The timing of cell division is not by the timing of the light rhythm. The on-off cycle of the light can be moved in any fashion around the clock as long as the duration of light is at least 16 hr. The parasites still divide from 12 midnight to 6 AM solar time. In some respects, P. berghei is a convenient indicator of the host rhythm. The parasite has a 24-hr generation time. There are no problems with multiple broods of parasites. There are convenient morphologic indicators of the Received for publication 1 April 1969. * This study supported by U. S. Army Contract No. DA-49-193-MD-2545 from the U. S. Army Research and Development Command, Office of the Surgeon General. This paper is contribution No. 561 from the Army Research Program on Malaria. age of the parasite. On the other hand, there are problems with sequestration (the vascular capture and release of mature parasites). Vascular sequestration is another curious feature of the host-parasite relationship. In many species of malaria, including P. berghei, the older and dividing forms of the parasite, together with the host red cell, stick to the blood vessel surfaces, until such time as cell division is complete (Garnham, 1966). On completion of cell division, the young forms usually circulate freely in the blood until they reach a degree of maturity appropriate for sequestration. The cycle is then repeated. In preliminary work from this laboratory, we have found that the pineal body is part of the mediating system for both growth and division synchrony and for vascular sequestration (Arnold et al., 1969b). The effect of the pineal body on growth and division synchrony can be replaced in part by vitamin K, vitamin E, and ubiquinone (Arnold et al., 1969c). Although the presence of an intact pineal or its chemical alternates, vitamin K, vitamin E, or ubiquinone, is needed for growth and division synchrony of P. berghei, none of these factors appears to act as the master clock. The diurnal growth rhythm of the parasites remains on the same time schedule regardless of the time of administration of the ubiquinone or of the phase relationship to the photoperiodic rhythm. In addition, the photop2riodic rhythm must be maintained if chemical replacement is to work in the pinealectomized animal. For this reason, a continued search for other medi-

The drug response of a normal and a multi-resistant strain of P. falciparum to sulphalene

The intermediate-acting sulphonamide, sulphalene, has been tested as an antimalarial agent in non-immune human volunteers. It is very effective against a chloroquine-pyrimethamine-resistant P. falciparum. It cured 7 of 7 cases when 1 gramme was given as a single dose. Against a strain of P. falciparum sensitive to all common antimalarial drugs, sulphalene was not as satisfactory; it cured only 2 of 5 patients receiving 1 gramme and 3 of 6 receiving 2·5 grammes. In both P. falciparum strains the speed of action was slow. The clearance of parasites was slower than with quinine. When sulphalene is combined with the new inhibitor of dihydrofolate reductase trimethoprim, the combination is extremely effective and rapid against normal and resistant P. falciparum.

Enhanced sensitivity of P. falciparum to sulphalene as a consequence of resistance to pyrimethamine

Abstract Experiments were conducted with P. falciparum in volunteers concerning the increased sensitivity of the malaria to a sulphonamide (sulphalene) induced by the parasites resistance to pyrimethamine (a dihydrofolate inductase inhibitor). The “normal” P. falciparum strain was relatively resistant to treatment with sulphalene; only 5 of 11 patients were cured by 2·5 grammes. After resistance to pyrimethamine was induced, the strain was much more sensitive to sulphalene: 9 of 11 patients were cured by 1 gramme. Evidence is presented for the postulate that the sulphonamide sensitivity of a strain depends on whether it does or does not synthesize its own folic acid. It is further postulated that P. falciparum is capable of either using exogenous folic acid or of synthesizing it. The parasites probably do not do both at the same time.