Plasmodium berghei: acid-insensitive phosphofructokinase in infected mouse erythrocytes

The rate of glucose utilization by red blood cells infected with Plasmodium berghei was not inhibited by an acidic pH which completely inhibited normal red cell glucose consumption. This insensitivity to acid conditions by P. berghei-parasitized red cells was associated with an electrophoretically separable and kinetically distinct form of the enzyme phosphofructokinase (EC 2.7.1.11) which exhibited a pH response similar to that of whole-cell glucose consumption.     

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 berghei: Glycolytic enzymes of the infected mouse erythrocyte

This paper documents the maximal activities of the glycolytic enzymes in the red blood cells of normal mice and mice infected with Plasmodium berghei. There appears to be sufficient parasite-related activity of each glycolytic enzyme to support the increased glycolytic rate, i.e., increased glucose consumption, of the parasite-infected red blood cell. The relative proportions of glycolytic enzyme activities in parasite-infected red cells are different from the proportions in either normal or reticulocyte-rich blood, indicating that the increased enzyme activities associated with infected cells are not due to contaminating host red cells or reticulocytes. A comparison of maximal enzyme activities to the rate of whole cell glucose consumption indicates that different glycolytic control mechanisms are operating in the infected RBC from those in the uninfected cells.     

Changes in rodent-erythrocyte methemoglobin reductase system produced by two malaria parasites, viz. Plasmodium yoelii nigeriensis and Plasmodium berghei

The methemoglobin reductase system plays a vital role in maintaining the equilibrium between hemoglobin and methemoglobin in blood. Exposure of red blood cells to oxidative stress (pathological/physiological) may cause impairment to this equilibrium. We studied the status of erythrocytic methemoglobin and the related reductase system during Plasmodium yoelii nigeriensis infection in mice and P. berghei infection in mastomys. Malaria infection was induced by intraperitoneal inoculation with 10⁶ infected erythrocytes. The present investigation revealed a significant decrease in the activity of methemoglobin reductase, with a concomitant rise in methemoglobin content during P. yoelii nigeriensis infection in mice erythrocytes. This was accompanied with a significant increase in reduced glutathione and ascorbate levels. The activity of lactate dehydrogenase, glucose 6-phosphate dehydrogenase and glutathione reductase increased with a progressive rise in parasitemia. However, no methemoglobin or associated reductase activity was detected in normal and P. berghei-infected mastomys. P. berghei infection in mastomys resulted in an increase in the level of reduced glutathione and ascorbate in erythrocytes, and also in the activity of lactate dehydrogenase, glucose 6-phosphate dehydrogenase and glutathione reductase. These results suggest that antioxidants/antioxidant enzymes may prevent or reduce the formation of methemoglobin in the host and thereby protect the host from methemoglobinemia.     

Cultivation of the exoerythrocytic stage ofPlasmodium berghei in primary cultures of mouse hepatocytes and continuous mouse cell lines

Summary Plasmodium berghei exoerythrocytic (EE) stages have been cultured in vitro in human continuous cell lines and primary cultures of both human and rat hepatocytes. Although the predominant experimental model of irradiated sporozoite-induced protective immunity is the mouse,P. berghei has not been cultivated in primary mouse hepatocytes or in continuous mouse lines. Because of this, target cells are not available for determining if these immunized mice produce cytotoxic T lymphocytes (CTLs) that recognizeP. berghei antigens expressed on hepatocytes in the context of class I major histocompatability (MHC) antigens. We report the development of methods for cultivating the (EE) stage ofP. berghei in murine hepatocytes and in two cell lines derived from the livers of BALB/c mice; one line produced from a primary hepatocyte culture and the other produced by fusion of mouse hepatocytes with a continuous rat liver line. Mature parasites were detected by microscopy and by DNA probe in both cell lines, each of which supported complete development ofP. berghei liver stages and production of infectious merozoites. Since class I MHC antigens are present on the surface of primary hepatocytes and the mouse X rat hybrid line, these cells can be used to detect cytotoxic T cells against liver stage parasites. This work was supported by the Naval medical Research and Development Command, Bethesda, MD, work unit no. 3M161102B510AK111, ONR contract N00014-83-C-0355, and by contract DPE-0453-C-00-3051-00 of the U.S. Agency for International Development, Washington, D.C. The opinions and assertions herein are not to be construed as official or as reflecting the views of the Navy Department or the naval service at large. The experiments reported herein were conducted according to the principles set forth in the current edition of the “Guide for the Care and Use of Laboratory Animals”, Institute of Laboratory Animal Resources, National Research Council, DHHS, Pub. no. (NIH)85-23     

Circulating immune complexes in rodent and simian malaria

The circulating immune complexes have been detected in the sera of albino rats infected withPlasmodium berghei and rhesus monkeys infected with P.knowlesi by (i) quantitative cryoprecipitation assay and (ii) polyethylene glycol assay. In the rodent model, the levels of circulating immune complexes increased during infection and decreased considerably in the post-infection period. In the simian system, high levels were detected during peak parasitaemia. Polyethylene glycol precipitate obtained from the sera during acuteP. knowlesi infection when analysed by Immunoelectrophoresis was found to contain (i) monkey IgG, (ii) four other components of monkey plasma, (iii) two components of normal monkey erythrocytes and (iv) antigen(s) ofP. knowlesi.     

Impairment of T-Helper Function by a Plasmodium berghei-Derived Immunosuppressive Factor1

Mice injected with an immunosuppressive factor (ISF) extracted from Plasmodium berghei-infected rat erythrocytes have a reduced antibody response to unrelated antigens. T-cells from ISF-treated mice failed to provide adequate help to naive, syngeneic B-cells in the primary IgM response in vitro to sheep red blood cells and to dinitrophenylated keyhole limpet hemocyanin. The same T-cells, however, were able to cooperate with memory B-cells in the secondary IgG response. No other cellular deficit was delected in ISF-treated mice; B-cells and macrophages behaved normally, and there was no detectable excess of suppressor cells. The T-cell impairment was not reflected in decreased production of interleukin 2, but was also shown by the diminished delayed type hyperaensitivity reaction to sheep red blood cells of ISF-treated mice.     

Decreased level of 2,3-diphosphoglycerate and alteration of structural integrity in erythrocytes infected with Plasmodium falciparum in vitro

2,3-Diphosphoglycerate (2,3-DPG), an intracellular metabolite of glycolytic pathway is known to affect the oxygen binding capacity of haemoglobin and mechanical properties of the red blood cells. 2,3-DPG levels have been reported to be elevated during anaemic conditions including visceral leishmaniasis. 2,3-DPG activity in P. falciparum infected red blood cells, particularly in cells infected with different stages of the parasite and its relationship with structural integrity of the cells is not known. Chloroquine sensitive and resistant strains of P. falciparum were cultured in vitro and synchronized cultures of ring, trophozoite and schizont stage rich cells along with the uninfected control erythrocytes were assayed for 2,3-DPG activity and osmotic fragility. It was observed that in both the strains, in infected erythrocytes the 2,3-DPG activity gradually decreased and osmotic fragility gradually increased as the parasite matured from ring to schizont stage. The decrease in 2,3-DPG may probably be due to increased pyruvate kinase activity of parasite origin, which has been shown in erythrocytes infected with several species of Plasmodium. The absence of compensatory increase in 2,3-DPG in P. falciparum infected erythrocytes may aggravate hypoxia due to anaemia in malaria and probably may contribute to hypoxia in cerebral malaria. As 2,3-DPG was not found to be increased in erythrocytes parasitized with P. falciparum, the increased osmotic fragility observed in these cells is not due to increased 2,3-DPG as has been suggested in visceral leishmaniasis.     

Influence of Reticulocytosis on the Course of Infection of Plasmodium chabaudi and P. berghei*

SYNOPSIS. Reticulocytosis, stimulated by the destruction of red blood cells by phenylhydrazine, altered the course of infection of both Plasmodium chabaudi and P. berghei in the mouse. P. chabaudi, lacking a preference for reticulocytes, was adversely affected when young cells were present in abundance. Parasitemias diminished and most of the animals survived the otherwise fatal infection. P. berghei preferentially invaded reticulocytes to the extent that the parasitemia became contained largely in the reticulocyte population. This was accompanied by a delay in time to death.     

Scanning electron microscope observations of Plasmodium berghei ookinetes in primary mosquito cell cultures

Plasmodium berghei-infected blood from mice was inoculated into primary cell cultures (PCC) obtained from the mosquito Anopheles stephensi. Immature and mature ookinetes of Plasmodium berghei, which developed in these cultures were studied with the scanning electron microscope. Immature ookinetes had a bulbous-like structure at the posterior end and a slightly wrinkled surface. Mature ookinetes were smoother in appearance and somewhat longer than immature forms. Shallow spiraling waves were observed on the surface of some ookinetes, especially in the anterior half of the body. Such waves may be involved in ookinete locomotion. Penetration of cultured cells by ookinetes was not observed. Infected red cells, which were present in the inoculum, had small depressions on the red cell surface, whereas some uninfected red cells had accentuated concavities. Mouse blood cells adhered closely to PCC cells; some attached red cells were irregular in shape.     

Plasmodium berghei: vaccination of mice against malaria with heat inactivated parasitized blood

Heat inactivated Plasmodium berghei-infected blood acted as a vaccine against P. berghei infection in mice. The heat inactivated blood was noninfective. Intact or splenectomized vaccine-treated mice, as well as P. berghei susceptible mice inoculated with whole blood or homogenized spleens from vaccine-treated animals, did not become infected. A/J, DDS and Carworth CF₁ mice were all protected against P. berghei challenge after vaccination. A/J and DDS mice developed good immunity after a single vaccination injection. Similar levels of immunity were obtained in CF₁ mice after at least two vaccine injections. Immunized mice responded to P. berghei challenge with mild anemias and low level parasitemias. Resolution of infection occurred between the first and third weeks after challenge. Nonvaccinated mice developed progressive anemia and parasitemia during the same time period. The immunity appears to be caused by P. berghei antigens; it could not be induced by homologous or heterologous noninfected red blood cells, P. gallinaceum-infected blood or Freund's Complete Adjuvant.     

Dynamics of telomere turnover inPlasmodium berghei

Non-uniform composition in telomeric repeats at the extremities ofPlasmodium chromosomes was exploited in order to obtain data on intraclonal diversification of telomeric sequences, relevant for the study of telomere regeneration dynamics. Families of sibling telomeric clones were obtained from several chromosomal ends ofPlasmodium berghei, and analysed so as to determine the exact points from which individual clones start to diverge. As much as 90% of the telomeric tract appears to be subject to events causing abrupt changes in the sequence of telomeric repeats. The results are compatible with the hypothesis that breakpoint probability is a continuously increasing function over the entire telomeric tract.     

Chloroquine resistant malaria: Association with enhanced plasmodial protease activity

Chloroquine resistant Plasmodium berghei has several unusual features including (i) lack of malaria “pigment”, (ii) more efficient host catabolism of heme from infected erythrocytes, and (iii) relatively inefficient uptake of external chloroquine by infected red cells. The malaria pigment produced by chloroquine sensitive P. berghei is probably incompletely catabolized hemoglobin, the heme group of which is unavailable for subsequent catabolism by the host's reticuloendothelial system. This pigment has been suggested by others as the site of high affinity chloroquine binding. We hypothesized that all three characteristics of chloroquine resistant infections might be explained by enhanced proteolytic digestion of host cell hemoglobin. In confirmation, we report that chloroquine resistant P. berghei has 700–800% greater protease activity than the chloroquine sensitive form. This greatly elevated protease activity may explain the aforementioned characteristics of chloroquine resistant P. berghei and may help elucidate the basis of chloroquine resistance in human P. falciparum.     

Plasmodium yoelii inhibitor of cysteine proteases is exported to exomembrane structures and interacts with yoelipain‐2 during asexual blood‐stage development

Plasmodium falciparum (Pf) blood stages express falstatin, an inhibitor of cysteine proteases (ICP), which is implicated in regulating proteolysis during red blood cell infection. Recent data using the Plasmodium berghei rodent malaria model suggested an additional role for ICP in the infection of hepatocytes by sporozoites and during liver‐stage development. Here we further characterize the role of ICP in vivo during infection with Plasmodium yoelii (Py) and Pf. We found that Py‐ICP was refractory to targeted gene deletion indicating an essential function during asexual blood‐stage replication, but significant downregulation of ICP using a regulated system did not impact blood‐stage growth. Py‐ICP localized to vesicles within the asexual blood‐stage parasite cytoplasm, as well as the parasitophorous vacuole, and was exported to dynamic exomembrane structures in the infected RBC. In sporozoites, expression was observed in rhoptries, in addition to intracellular vesicles distinct from TRAP containing micronemes. During liver‐stage development, Py‐ICP was confined to the parasite compartment until the final phase of liver‐stage development when, after parasitophorous vacuolemembrane breakdown, it was released into the infected hepatocyte. Finally, we identified the cysteine protease yoelipain‐2 as a binding partner of Py‐ICP during blood‐stage infection. These data show that ICP may be important in regulating proteolytic processes during blood‐stage development, and is likely playing a role in liver stage‐hepatocyte interactions at the time of exoerythrocytic merozoite release.     

Phosphorylation of membrane proteins from Plasmodium berghei-infected red cells.

Membrane from Plasmodium berghei-infected mouse red cells has a different pattern of phosphorylation by (γ-³²P)ATP from normal membrane. A phosphorylated membrane protein of apparent molecular weight 42,000, absent in membrane from normal cells, can be detected in membrane from infected cells. The new phosphorylated protein can be extracted by 0.1 mM EDTA but not by triton X-100, indicating that it may be red cell actin.     

Plasmodium falciparum: Invasion and development in highly parasitized cultures

Summary We report that synchronized cultures ofPlasmodium falciparum with up to 40% parasitized cells can be obtained with the use of low, red blood cell suspensions and only daily replacement of culture medium. These cultures contained not only a reduced proportion of uninfected red cells but also of population of cells with brief and equal time of exposure to culture conditions. Such high parasitemias are desirable for studies of ring-staged parasites (for which enrichment techniques are not available) and late-staged parasites when the manipulations for enrichment are inappropriate or unsuccessful. This work was supported by Grant HL-21016 from the National Institutes of Health, Bethesda, MD Preliminary observations on this report were presented at the Poster session at the Red Cell 6th Ann Arbor Conference, Michigan, October, 1983.     

In vivo noninvasive visualization of retinal perfusion dysfunction in murine cerebral malaria by camera‐phone laser speckle imaging

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection associated with impaired cerebral blood flow. Visualization of the eye vasculature, which is embryologically derived from that of the brain, is used clinically to diagnose the syndrome. Here, we introduce camera‐phone laser speckle imaging as a new tool for in vivo, noncontact two‐dimensional mapping of blood flow dynamics in the experimental cerebral malaria (ECM) murine model of Plasmodium berghei ANKA. In a longitudinal study, we show that the camera‐phone imager can detect an overall decrease in the retinal blood‐flow‐speed (BFS) as ECM develops in P. berghei ANKA infected mice, with no similar change observed in uninfected control mice or mice infected with a non‐ECM inducing strain (P. berghei NK65). Furthermore, by analyzing relative alterations in the BFS of individual retinal vessels during the progression of ECM, we illustrate the strength of our imager in identifying different BFS‐change heterogeneities in the retinas of ECM and uninfected mice. The technique creates new possibilities for objective investigations into the diagnosis and pathogenesis of CM noninvasively through the eye. The camera‐phone laser speckle imager along with measured spatial blood perfusion maps of the retina of a mouse infected with P. berghei ANKA—a fatal ECM model—on different days during the progression of the infection (top, day 3 after infection; middle, day 5 after infection; and bottom, day 7 after infection).      

Aryl piperazine and pyrrolidine as antimalarial agents. Synthesis and investigation of structure-activity relationships

Piperazine and pyrrolidine derivatives were synthesised and evaluated for their capacity to inhibit the growth of Plasmodium falciparum chloroquine-resistant (FCR-3) strain in culture. The combined presence of a hydroxyl group, a propane chain and a fluor were shown to be crucial for the antiplasmodial activity. Five compounds of the aryl-alcohol series inhibited 50% of parasite growth at doses ?10 μM. The most active compound 1-(4-fluoronaphthyl)-3-[4-(4-nitro-2-trifluoromethylphenyl)piperazin-1-yl] propan-1-ol was almost 20–40 times more active on P. falciparum (IC₅₀: 0.5 μM) than on tumorogenic and non-tumorogenic cells. In vivo it has a very weak effect; inhibiting 35% of parasite growth only, at 10 mg/kg/day against Plasmodium berghei infected mice without any impact on survival time. In silico molecular docking study and molecular electrostatic potential calculation revealed that this compound bound to the active site of Plasmodium plasmepsin II enzyme.     

GLUT1‐mediated glucose uptake plays a crucial role during Plasmodium hepatic infection

Intracellular pathogens have evolved mechanisms to ensure their survival and development inside their host cells. Here, we show that glucose is a pivotal modulator of hepatic infection by the rodent malaria parasite Plasmodium berghei and that glucose uptake via the GLUT1 transporter is specifically enhanced in P. berghei‐infected cells. We further show that ATP levels of cells containing developing parasites are decreased, which is known to enhance membrane GLUT1 activity. In addition, GLUT1 molecules are translocated to the membrane of the hepatic cell, increasing glucose uptake at later stages of infection. Chemical inhibition of GLUT1 activity leads to a decrease in glucose uptake and the consequent impairment of hepatic infection, both in vitro and in vivo. Our results reveal that changes in GLUT1 conformation and cellular localization seem to be part of an adaptive host response to maintain adequate cellular nutrition and energy levels, ensuring host cell survival and supporting P. berghei hepatic development.     

Reduced activity of the epithelial sodium channel in malaria-induced pulmonary oedema in mice

Lung complications during malaria infection can range from coughs and impairments in gas transfer to the development of acute respiratory distress syndrome (ARDS). Infecting C57BL/6 mice with Plasmodium berghei K173 strain (PbK) resulted in pulmonary oedema, capillaries congested with leukocytes and infected red blood cells (iRBCs), and leukocyte infiltration into the lungs. This new model of malaria-associated lung pathology, without any accompanying cerebral complications, allows the investigation of mechanisms leading to the lung disease. The activity of the amiloride-sensitive epithelial sodium channel (ENaC) in alveolar epithelial cells is decreased by several respiratory tract pathogens and this is suggested to contribute to pulmonary oedema. We show that PbK, a pathogen that remains in the circulation, also decreased the activity and expression of ENaC, suggesting that infectious agents can have indirect effects on ENaC activity in lung epithelial cells. The reduced ENaC activity may contribute to the pulmonary oedema induced by PbK malaria.