Separation and Concentration of Schizonts of <i>Plasmodium falciparum</i> by Percoll Gradients1

Rivadeneira, Enid; Wasserman, Moisés; Espinal, Carlos T.

doi:10.1111/j.1550-7408.1983.tb02932.x

Cited by 133 publications

(119 citation statements)

References 8 publications

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“…For in vitro stimulation experiments, asynchronous cultures of NF54 strain parasites were harvested at a parasitemia of ϳ10 -20% and the mature asexual stages were purified by centrifugation on a 63% Percoll density gradient (16). This purification step results in preparations with 80 -90% parasitemia, consisting of Ͼ95% schizonts/mature trophozoites.…”

Section: Reagents Parasites and Mosquitoesmentioning

confidence: 99%

Plasmodium falciparum Infection Causes Proinflammatory Priming of Human TLR Responses

McCall¹,

Netea

Hermsen³

et al. 2007

The Journal of Immunology

106

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TLRs are a major group of pattern recognition receptors that are crucial in initiating innate immune responses and are capable of recognizing Plasmodium ligands. We have investigated TLR responses during acute experimental P. falciparum (P.f.) infection in 15 malaria-naive volunteers. TLR-4 responses in whole blood ex vivo stimulations were characterized by significantly (p < 0.01) up-regulated proinflammatory cytokine production during infection compared with baseline, whereas TLR-2/TLR-1 responses demonstrated increases in both proinflammatory and anti-inflammatory cytokine production. Responses through other TLRs were less obviously modified by malaria infection. The degree to which proinflammatory TLR responses were boosted early in infection was partially prognostic of clinical inflammatory parameters during the subsequent clinical course. Although simultaneous costimulation of human PBMC with P.f. lysate and specific TLR stimuli in vitro did not induce synergistic effects on cytokine synthesis, PBMC started to respond to subsequent TLR-4 and TLR-2 stimulation with significantly (p < 0.05) increased TNF-α and reduced IL-10 production following increasing periods of preincubation with P.f. Ag. In contrast, preincubation with preparations derived from other parasitic, bacterial, and fungal pathogens strongly suppressed subsequent TLR responses. Taken together, P.f. primes human TLR responses toward a more proinflammatory cytokine profile both in vitro and in vivo, a characteristic exceptional among microorganisms.

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Section: Reagents Parasites and Mosquitoesmentioning

confidence: 99%

Plasmodium falciparum Infection Causes Proinflammatory Priming of Human TLR Responses

McCall¹,

Netea

Hermsen³

et al. 2007

The Journal of Immunology

106

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“…Free parasites were isolated by treating parasitized erythrocytes with 0.15% saponin. The parasitized culture was enriched for mature stages (trophozoites and schizonts) by centrifugation in a 70% Percoll (GE Healthcare) solution (15).…”

mentioning

confidence: 99%

Metabolic Fate of Fumarate, a Side Product of the Purine Salvage Pathway in the Intraerythrocytic Stages of Plasmodium falciparum

Bulusu¹,

Jayaraman²,

Balaram

2011

Journal of Biological Chemistry

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In aerobic respiration, the tricarboxylic acid cycle is pivotal to the complete oxidation of carbohydrates, proteins, and lipids to carbon dioxide and water. Plasmodium falciparum, the causative agent of human malaria, lacks a conventional tricarboxylic acid cycle and depends exclusively on glycolysis for ATP production. However, all of the constituent enzymes of the tricarboxylic acid cycle are annotated in the genome of P. Plasmodium falciparum is a parasitic protozoan, which causes the most severe form of malaria in humans. Because of the emergence in the parasite of widespread drug resistance to most of the first-line antimalarials, the development of new drugs that target the parasite metabolism is needed. Over several years, malarial parasites have been studied to understand various novel biochemical features, which not only gives opportunities for chemotherapeutic interventions but also stimulates broader scientific interests.During the intraerythrocytic stages of P. falciparum, the tricarboxylic acid cycle does not seem to function like a conventional tricarboxylic acid cycle for the following reasons. (a) The bulk of the glucose is metabolized to lactate by anaerobic glycolysis, which is then secreted by the parasite as a metabolic waste (1) (2). As a result, unlike aerobic cells, in P. falciparum there is minimal carbon flow from the cytoplasm to the mitochondrial tricarboxylic acid cycle. (b) The multi-enzyme complex pyruvate dehydrogenase, which channels pyruvate into the tricarboxylic acid cycle through acetyl-CoA, has been found to localize on the apicoplast membrane in P. falciparum (3), unlike mammalian cells, where the enzyme is present on the inner mitochondrial membrane (4). (c) The enzyme isocitrate dehydrogenase generates NADPH rather than NADH (5), thereby implying that its main role is probably to act as a redox sensor rather than donating reducing equivalents to the electron transport chain. In addition, P. falciparum synthesizes ATP mainly by substrate level phosphorylation through glycolysis and not through oxidative phosphorylation (6), suggesting that the primary function of the tricarboxylic acid cycle in generating reducing equivalents for the electron transport chain might be dispensable for the parasite. However, genes for all of the enzymes of the tricarboxylic acid cycle are present and are expressed in P. falciparum (7,8) during the intraerythrocytic stages, thereby implying that the pathway probably has important, yet unidentified biosynthetic functions.Unlike its human host, P. falciparum lacks the de novo purine biosynthetic pathway and is hence completely dependent on the purine salvage pathway to meet its purine nucleotide requirements (9). In the purine salvage pathway, hypoxanthine salvaged from the host is phosphoribosylated to IMP, which then branches out to form AMP and GMP (Fig. 1). IMP is converted to AMP in two steps, catalyzed by two distinct enzymes: (a) adenylosuccinate synthetase, which adds a molecule of aspartate to the 6-oxo position of IMP with a concomitan...

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“…Cells were usually harvested or subcultured when the parasitemia reached 15-20%, as determined microscopically by thin blood smears stained with Giemsa. Trophozoite and early schizont stages were routinely isolated by the gelatin flotation method (21) or the Percoll-alanine method as described elsewhere (20,22 Effect on parasite growth. Upon reaching the trophozoite stage (10-20% parasitemia), the cells were washed with growth medium, resuspended to 2% hematocrit and 1-2% parasitemia, and distributed into wells of a 24-well microtiter plate (0.6 ml per well).…”

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confidence: 99%

The antimalarial action of desferal involves a direct access route to erythrocytic (Plasmodium falciparum) parasites.

Loyevsky

Lytton²,

Mester³

et al. 1993

J. Clin. Invest.

103

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We designed the N-methylanthranilic-desferrioxamine (MA-DFO) as a fluorescent iron(III) chelator with improved membrane permeation properties. Upon binding of iron(III), MA-DFO fluorescence is quenched, thus allowing traceability of drug-iron(III) interactions. MA-DFO is well tolerated by mammalian cells in culture. Its antimalarial activity is pronounced: IC50 values on in vitro (24-h) growth of Plasmodium fakciparum were 3±1 MM for MA-DFO compared with 30±8 for DFO. The onset of growth inhibition of rings or trophozoites occurs 2-4 h after exposure to 13 gM MA-DFO. This effect is commensurate with MA-DFO permeation into infected cells. In a 24-h exposure to MA-DFO or DFO, trophozoites take up either compound to -10% of the external concentration, rings to 5%, and noninfected cells to < 1%. Red cells encapsulated with millimolar concentrations of DFO or MA-DFO fully support parasite invasion and growth. We conclude that extracellular MA-DFO and DFO gain selective access into parasites by bypassing the host. The rate-limiting step is permeation through the parasite membrane, which MA-DVO accomplishes faster than DFO, in accordance with its iigher hydrophobicity. These views are consistent with the proposed duct, which apparently provides parasitized cells with a window to the external medium. (J. Clin. Invest. 1993. 91:218-224.)

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Separation and Concentration of Schizonts of Plasmodium falciparum by Percoll Gradients1

Cited by 133 publications

References 8 publications

Plasmodium falciparum Infection Causes Proinflammatory Priming of Human TLR Responses

Plasmodium falciparum Infection Causes Proinflammatory Priming of Human TLR Responses

Metabolic Fate of Fumarate, a Side Product of the Purine Salvage Pathway in the Intraerythrocytic Stages of Plasmodium falciparum

The antimalarial action of desferal involves a direct access route to erythrocytic (Plasmodium falciparum) parasites.

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