Plasmodium yoelii: blood oxygen and brain function in the infected mouse

The carriage of oxygen by the blood and the in vivo response of the brain were investigated in mice infected with a lethal strain of Plasmodium yoelii. All mice with parasitaemia exceeding 70% were severely anaemic (Hb 3.5 +/- 1.8 g/dl; mean +/- 1 SD), acidotic (blood pH 7.04 +/- 0.06) and hypoglycaemic (blood glucose 0.6 +/- 0.76 mumol/ml). The oxyhaemoglobin dissociation curve (ODC) of blood from heavily infected mice was shifted right as compared to controls, but the increase in p50 was less than expected from the accompanying acidosis. The reduced shift right was due to a decrease in the 2,3-DPG/Hb ratio in infected animals (0.72 +/- 0.12, n = 17 vs 1.10 +/- 0.09, n = 12 in controls). Despite the severity of terminal infection, the cerebral pH and the relative steady-state concentrations of PCr, ATP and Pi measured in vivo by nuclear magnetic resonance (31P NMR) were normal. Alterations in brain energy status and pH cannot account for cerebral signs or death in this proposed mouse model of cerebral malaria.     

Plasmodium berghei: diet and drug dosage regimens influencing selection of drug-resistant parasites in mice

Two different diets for the host and three drug dosage regimens were used to select lines resistant to sulfadoxine and pyrimethamine from the parent strain of the rodent malaria parasite Plasmodium berghei [the N (K173) strain]. A higher yield of resistance was obtained when a high parasitemia was present at the beginning of the drug pressure schedule. The development of resistance to the association of sulfadoxine plus pyrimethamine was accelerated by a relatively high para-aminobenzoic acid (PABA) content diet. Reproducibility was satisfactory when one of the dosage regimens was applied independently by two different technicians at different times.     

Plasmodium berghei: Architectural analysis by freeze-fracturing of the intraoocyst sporozoite's pellicular system

The technique of freeze-fracturing has been used to study the architecture of the pellicular complex of the intraoocyst sporozoite of Plasmodium berghei. The sporozoite is surrounded by three plasma membranes and a layer of subpellicular microtubules. During freeze-fracturing, each of the three membranes can split along its hydrophobic interior to yield a total of six fracture faces. The most obvious feature of each fracture face is the presence of globular intramembranous particles on the surface. The six fracture faces differ from one another in arrangement, size, and density of these intramembranous particles. Two of the fracture faces exhibit a unique arrangement of particles in well-organized parallel rows along the long axis of the sporozoite. This arrangement has not been reported in either the erythrocytic or the exoerythrocytic forms of Plasmodium spp. Another unique feature in the sporozoite revealed through freeze-fracturing is a single suture line that traverses the long axis of the inner two membranes of the parasite.     

Plasmodium yoelii: the thymus-dependent lymphocyte in mice immunodepressed by malaria

Euthymic mice, athymic nude mice, and mice treated with antithymocyte serum were infected with Plasmodium yoelii and immunized 10 days postinfection with pneumococcal polysaccharide (SSSIII). As a control, uninfected mice were also immunized with SSSIII. Splenic plaque-forming cells as well as serum antibody titers to SSSIII were measured 5 days after immunization. In infected euthymic mice, both plaque-forming cells (PFC) and serum antibody were severely depressed. In contrast, plaque-forming cells and serum antibody were approximately normal in infected nude mice and in infected mice treated with antithymocyte serum. Splenic adherent cells from infected euthymic mice failed to function as accessory cells in the in vitro antibody response to a second antigen, the sheep erythrocyte. Moreover, they lacked suppressor activity when cultured with spleen cells from uninfected mice. In contrast, adherent spleen cells from infected mice treated with antithymocyte serum displayed accessory cell function.     

Plasmodium yoelii: Antibody and the maintenance of immunity in BALB/c mice

Subpatent persistence of parasitemia was detected for up to 7 weeks after infection of BALB/c mice with Plasmodium yoelii. Serum taken from recovered mice maintained parasitemias in recipient mice at a subpatent level when transferred repeatedly at 2-day intervals. Single doses of serum from convalescent donors delayed the course of infection in recipients. Small doses of transferred hyperimmune serum had the same effect, whereas large doses (>0.5 ml) totally suppressed parasitemia. Only a single secondary challenge of recovered mice was required in order to produce a maximally protective hyperimmune serum. Mice completely protected from a primary challenge with P. yoelii by transfer of hyperimmune serum were not at all resistant to a second challenge given some weeks later. After transfer of hyperimmune serum into mice with established P. yoelii infection, parasitemia fell to subpatent levels within 48 hr. During the first 21 hr after serum transfer, a progressive reduction in the proportion of ring forms present in blood smears was observed.     

Plasmodium berghei: Uptake and distribution of chloroquine in infected mouse erythrocytes

The capacity of mouse erythrocytes infected with Plasmodium berghei to accumulate chloroquine is developed with maturation of the parasites. This is shown by direct comparison of the early and mature stages, which are separated by density difference. After drug accumulation, infected cells were fractionated by saponin lysis or nitrogen decompression to study the drug distribution. Effectiveness of isolating intact parasites and host components was checked by SDS-polyacrylamide gel electrophoresis and by low leakage of parasite-specific lactate dehydrogenase used as a marker enzyme. At low external drug concentration (~10^?7M), chloroquine is principally accumulated in the parasites. However, at higher drug concentrations (~10^?5and ~10^?3M), the proportion of the drug found in the host cytosol fraction is increased. A small but significant proportion of the drug (<20%) is associated with the host cell membrane. The pellet fraction of the freed parasites, further fractionated by freeze-thaw lysis, contains a major proportion of the drug at low external concentrations. However, the pellet fraction obtained from prolonged sonication of the parasites, which contains the bulk of hemozoin pigment, carries only a small proportion of the drug. This indicates that parasite membrane components may bind most of the drug. As external chloroquine concentration is increased, the proportion of drug in the parasite supernatant increases, some or most of which is probably bound by soluble hemecontaining compounds. However, the presence of chloroquine in the parasite does not affect the partition of heme in particulate and soluble forms.     

Plasmodium chabaudi: relationship between the occurrence of recrudescent parasitaemias in mice and the effective levels of acquired immunity

In NIH inbred mice infected with the $\text{A}\text{S}$ strain of Plasmodium chabaudi the erythrocytic infection shows an acute primary parasitaemia which becomes subpatent after about 2 weeks. A period (7–10 days) of subpatency follows before a short-lasting patent recrudescence appears. The appearance of the recrudescent parasitaemia was examined in relation to (1) the level of antiplasmodial antibody detected by the indirect fluorescent antibody (IFA) test, (2) the antiparasite activity of the serum measured by the passive protection test, and (3) the ability of the mice to control and eliminate a large intravenous challenge infection. In the period between the primary parasitaemia becoming subpatent and the onset of the recrudescence there was a slight drop in the IFA levels, and a steep decline in passive protective levels and in the ability of the mice to control a large intravenous challenge infection. It is suggested that a decline in the effector arm in the immune response contributes to emergence of the recrudescent parasitaemias.     

Plasmodium yoelii: Genetic analysis of crosses between two rodent malaria subspecies

New electrophoretic variants of the enzyme adenosine deaminase (ADA) are described from two malaria subspecies. Differences were found in the mobility of ADA enzymes from Plasmodium yoelii yoelii (isolate 17X) and P. y. nigeriensis (isolate N67). Five attempts to cross these two subspecies were made, each of which demonstrated successful hybridization. The four genetic markers differentiating the parent lines appeared to segregate independently of each other. There was no marked preference for the gametes of one subspecies to self-fertilize rather than cross-fertilize with the other subspecies.     

Plasmodium: experimental animals for human malaria and research needs. A review.

The literature on experimental animals for human malaria has been selectively reviewed. Criteria for the selection of host animals and the experimental approaches for establishing malarial infections in them are discussed and analyzed on the bases of previous work. It is suggested that the research experiments of other countries, where feasible and applicable, should be adopted in attempts to establish animal models for human malaria indigenous to the People's Republic of China.     

Plasmodium cynomolgi: folic acid antagonist combinations for treatment of malaria in rhesus monkeys

The dihydrofolate reductase inhibitor 2,4-diamino-6-(2-naphthyl-sulfonyl)-quinazoline and its tetrahydro analog 2,4-diamino-6-(2-naphthyl-sulfonyl)-5,6,7,8-tetrahydroquinazoline were evaluated for possible synergism in treating trophozoite-induced Plasmodium cynomolgi var. bastianellii malaria of rhesus monkeys. The degree of synergism, if it exists, was found to be not nearly as great as observed previously with this combination of drugs against Plasmodium berghei infections of mice.     

Plasmodium berghei: modification of sialic acid on red cells from infected mouse blood

The surface membrane glycoproteins of normal mouse erythrocytes can be labeled by oxidation with either periodate or galactose oxidase in the presence of neuraminidase, followed by reduction with NaB³H₄. Without neuraminidase there is little galactose oxidase-catalyzed labeling of protein. Analysis of labeled proteins by SDS-polyacrylamide gel electrophoresis showed that both methods labeled the same set of glycoproteins. Plasmodium berghei infection dramatically reduced the sialoglycoprotein labeling of red blood cells from infected blood using the periodate/NaB³H₄ method. Provided neuraminidase was present, labeling by the galactose oxidase method gave identical results to normal erythrocytes. We conclude that the glycoprotein sialic acid of uninfected as well as infected red cells is modified during infection such that it is refractory to periodate oxidation. Acylation of the exocyclic hydroxyls of sialic acid is suggested to account for this. Lectin binding and cell agglutination experiments using Limulin, soybean and wheatgerm lectins, and concanavalin A confirmed and extended these observations. The possible implications of these results with regard to anemia induced by malaria are briefly discussed.     

Plasmodium lophurae: composition and properties of hemozoin, the malarial pigment.

Plasmodium lophurae hemozoin (malarial pigment) is composed of proteinaceous macromolecules bonded to iron III protoporphyrin IX by coordination bonding, van der Waals forces, and hydrophobic interactions but not by covalent bonding. Hemozoin is not composed of partially degraded globin peptides coordinated to heme, since fragments of molecular size less than that of globin monomers were not observed by SDS-PAGE. Two major polypeptides constituted the macromolecular portion of hemozoin; these had molecular weights of 21,000 and 15,000. The 21,000-molecular-weight protein is probably of parasite origin. The 15,000-molecular-weight polypeptide is believed to consist of globin monomers, and indicates the presence of irreversibly denatured hemoglobin (hemiglobin), as a constituent of hemozoin. The formation of hemozoin is hypothesized to play the following roles: protection of the parasite against molecular oxygen and compartmentation of the iron porphyrin which is a product of hemoglobin digestion by the plasmodium.     

Plasmodium falciparum: in vitro induction of resistance to aminopterin

Plasmodium falciparum parasites were grown on microplates in the presence of aminopterin. The FCR-8 strain was more sensitive to aminopterin than a Richards strain and died within 1 week of treatment. A few parasites of the Richards strain survived treatment and developed normal parasitemias. This strain was resistant to aminopterin at concentrations not higher than those used for its selection. Removal of aminopterin did not affect the growth of the resistant variant, showing that it was not aminopterin dependent. Aminopterin affected the sensitive parasites by interfering with nucleic acid synthesis, whereas protein synthesis was not impaired. Gametocytogenesis was unaffected by aminopterin.     

Plasmodium berghei: a mouse model for the "sudden death" and "malarial lung" syndromes

A mouse model for the "sudden death" and "malarial lung" syndromes is described. Mice of the C3H/z strain succumb suddenly approximately 7 days after an infection with Plasmodium berghei becomes patent, at a time when parasitemia is still moderate (6 to 8%). Death could be shown to be due to anaphylactoid shock, probably induced by soluble immune complexes. Increased vascular permeability caused transudation and leakage of serum proteins into the interstitium and the alveoli. The lungs were found to be edematous, with a fine granular precipitate in the alveoli and adherent to the vascular walls. The precipitates reacted with antiglobulins G and M, and could be shown to also contain malaria antigens and C3/4. A dramatic drop in hematocrit was recorded several hours before death, indicating the sudden release of malaria antigens. The myocardium of animals that had died very suddenly showed a patchy loss of phosphorylase activity. This loss of activity was much more extensive, and sometimes almost total, when there had been an agonal period of several (1 to 3) hours before death. In these cases the irreversibility of the myocardial damage was also indicated by the loss of activity of the dehydrogenases, as well as by typical inflammatory reactions of granulocytic and histiocytic infiltrations. The hearts thus presented a typical picture of the acute and peracute shock syndromes. In acute shock cardiac insufficiency develops so suddenly that death ensues before irreversible damage has occurred, and cardiac insufficiency can only be demonstrated by the most sensitive of enzyme histochemical means. In the present case shock was induced by the anaphylactoid activity of immune complexes with the lung as target organ. The described syndrome appears analogous to human "malarial lung."     

Plasmodium berghei: uptake of clindamycin and its metabolites by mouse erythrocytes with clindamycin-sensitive and clindamycin-resistant parasites.

Tritiated Clindamycin was used to compare the uptake of Clindamycin in plasma and red cells of mice infected with clindamycin-sensitive or clindamycin-resistant Plasmodium berghei and in uninfected mice. Red cells infected with either sensitive or resistant parasites have a higher concentration of [³H]clindamycin and its active metabolites 1 hr after drug administration than uninfected red blood cells. There was no significant difference in uptake of Clindamycin by red blood cells parasitized by sensitive or resistant parasites. Levels of Clindamycin and its metabolites were consistently higher in red cells than in plasma, both in infected and uninfected mice, but the drug was readily removed by washing red cells with phosphate buffered saline in either case. It is concluded that resistance to Clindamycin is not due to an impaired uptake of the drug by the parasitized red cell as has been shown for chloroquine resistance in P. falciparum and P. berghei.     

Plasmodium falciparum: effect of time in continuous culture on binding to human endothelial cells and amelanotic melanoma cells

An in vitro correlate of the binding in vivo of Plasmodium falciparum-infected erythrocytes to capillary and venular endothelium, using cultured human endothelial cells and amelanotic melanoma cells, was previously developed. The effects of different times in continuous culture on binding of erythrocytes infected with nine different isolates of P. falciparum is now reported. Four isolates, which bound at the time they were first tested, rapidly lost the ability to bind after 26-43 days in culture. One of these, the Cameroun isolate, tested 12 h after the blood was obtained from the patient, had the highest rate of binding of all isolates (680 infected erythrocytes per 100 melanoma cells). After 37 days in culture, only 18 infected erythrocytes per 100 melanoma cells bound. Three isolates first tested after 30-62 days in culture bound poorly. In contrast, two others, the Vietnam (VI) and Brazil (It), continued to bind during the period of study. The Brazil (It) isolate studied after 43 days in culture bound 505 infected erythrocytes per 100 melanoma cells; its clone ItG2G1 continued to bind equally well after 400 days in culture. The ultrastructural morphology of knobs on the binding and nonbinding infected erythrocytes were indistinguishable. Since evidence from other studies indicates that knobs are necessary for binding to endothelium, it is proposed that some parasites in continuous culture may not express the molecules responsible for binding, although the morphologic knobs are still present.     

Plasmodium falciparum: one-step growth in a semi-defined medium and the stimulatory effect of human seric lipoproteins and liposomes

The ring stages of Plasmodium falciparum within red blood cells cultured with complete medium stop growing when transferred to a basic medium containing RPMI plus fatty acid-free bovine serum albumin and dialyzable factors from human serum. Growth and multiplication can be partially restored by the addition of lipoprotein fractions prepared from human serum. No specificity was observed with subclasses of lipoproteins. Synthetic liposomes containing lecithin, oleic acid, and cholesterol mimic the effect of lipoproteins.     

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 berghei: influence on granulopoiesis and macrophage production in BALB/c mice

Granulocyte and macrophage progenitor cells forming colonies in vitro (GM-CFC) from bone marrow, spleen, and peripheral blood of BALB/c mice infected with Plasmodium berghei were cultured at various times postinfection in a viscous, 0.8% methylcellulose system. The numbers of GM-CFCs from bone marrow increased gradually during the first week of infection, reaching a maximum around the tenth day of the disease. Subsequently, a rise of GM-CFCs in cultures of nucleated cells from the peripheral blood was observed and, with some delay, in spleen cell cultures also, with a maximum around the end of the second week. After the tenth day of malaria infection a fall of colony frequency in bone marrow-derived cells took place, leading to subnormal values of GM-CFCs during the third week of infection. Subsequently, a decrease in the spleen cell cultures followed, but colony numbers did not fall to normal values. The general increase in GM-CFCs in the different organs was preceded by a rise in serum levels of colony-stimulating activity (CSA), attaining a maximum 1 week after P. berghei inoculation. During the following period the CSA levels fell and reached normal values around the seventeenth day of the disease. Chemotherapy with chloroquine started on the fifteenth day of infection, when GM-CFCs in the bone marrow have dropped to normal values, stopped their further decrease. In the spleen a gradual normalization took more than 2 weeks. A challenge infection evoked an elevation of GM-CFC numbers in the bone marrow and in the spleen during the first 10 days in only about 50% of immune mice. The reaction was immediate in some animals, but generally lower and of shorter duration than during primary infection. The results have indicated that a lethal P. berghei infection in mice caused a transient increase in production of CSA followed by a general recruitment of GM-CFCs in all hemopoietic organs.