A flow cytometric assay to quantify invasion of red blood cells by rodent Plasmodium parasites in vivo

Lelliott, Patrick M.; Lampkin, Shelley; McMorran, Brendan J.; Foote, Simon J.; Burgio, Gaétan

doi:10.1186/1475-2875-13-100

Cited by 39 publications

(38 citation statements)

References 41 publications

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“…Labeled-erythrocyte assays were performed as previously described (35,36), with modifications. Briefly, blood was collected from wild-type and mutant mice by cardiac puncture and suspended at 20% hematocrit in MTR (154 mM NaCl, 5.6 mM KCl, 1 mM MgCl 2 , 2.2 mM CaCl 2 , 20 mM HEPES, 10 mM glucose, 30 U/ml heparin, pH 7.4; 0.22-m filter sterilized).…”

Section: Mice and Ethicsmentioning

confidence: 99%

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Erythrocytic Iron Deficiency Enhances Susceptibility to Plasmodium chabaudi Infection in Mice Carrying a Missense Mutation in Transferrin Receptor 1

et al. 2015

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The treatment of iron deficiency in areas of high malaria transmission is complicated by evidence which suggests that iron deficiency anemia protects against malaria, while iron supplementation increases malaria risk. Iron deficiency anemia results in an array of pathologies, including reduced systemic iron bioavailability and abnormal erythrocyte physiology; however, the mechanisms by which these pathologies influence malaria infection are not well defined. In the present study, the response to malaria infection was examined in a mutant mouse line, Tfrc MRI24910 , identified during an N-ethyl-N-nitrosourea (ENU) screen. This line carries a missense mutation in the gene for transferrin receptor 1 (TFR1). Heterozygous mice exhibited reduced erythrocyte volume and density, a phenotype consistent with dietary iron deficiency anemia. However, unlike the case in dietary deficiency, the erythrocyte half-life, mean corpuscular hemoglobin concentration, and intraerythrocytic ferritin content were unchanged. Systemic iron bioavailability was also unchanged, indicating that this mutation results in erythrocytic iron deficiency without significantly altering overall iron homeostasis. When infected with the rodent malaria parasite Plasmodium chabaudi adami, mice displayed increased parasitemia and succumbed to infection more quickly than their wild-type littermates. Transfusion of fluorescently labeled erythrocytes into malaria parasite-infected mice demonstrated an erythrocyte-autonomous enhanced survival of parasites within mutant erythrocytes. Together, these results indicate that TFR1 deficiency alters erythrocyte physiology in a way that is similar to dietary iron deficiency anemia, albeit to a lesser degree, and that this promotes intraerythrocytic parasite survival and an increased susceptibility to malaria in mice. These findings may have implications for the management of iron deficiency in the context of malaria. Iron deficiency and supplementation can influence the risk and outcome of malaria infection. Epidemiology studies have indicated that iron deficiency anemia (IDA) is associated with a reduced risk of malaria in children and pregnant women (1-5), whereas clinical trials have demonstrated that iron supplementation in the absence of adequate health care increases the risk of malaria (6, 7). While the mechanisms underlying these outcomes are not well defined, it is clear that two major factors contribute: iron bioavailability and erythrocyte physiology.Iron bioavailability and homeostasis in the host during malaria infection are complex and incompletely understood (8). During infection, expression of the iron-regulatory hormone hepcidin increases significantly, leading to reduced dietary iron absorption and increased iron storage (9-12). The restriction of iron bioavailability may be a host defense mechanism designed to inhibit parasite access to iron, although the source of iron utilized by the parasite is still unclear (13,14). In liver-stage infection, reduced iron bioavailability brought on by previously ...

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Section: Mice and Ethicsmentioning

confidence: 99%

“…We employed a labeled-erythrocyte assay as previously described (35,36). The assay allows a direct comparison of parasite invasion and survival in two different erythrocyte populations within the circulation of a single host animal (Fig.…”

Section: Fig 3 Susceptibility Of Tfrcmentioning

confidence: 99%

Erythrocytic Iron Deficiency Enhances Susceptibility to Plasmodium chabaudi Infection in Mice Carrying a Missense Mutation in Transferrin Receptor 1

et al. 2015

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“…Merozoites are known to contain a single mitochondrion-like organelle which is reproduced during schizogony (23). The mitochondrial membrane potential is a key indicator of parasite viability that can be revealed using MitoTracker Red FM (MitoT; Invitrogen, Life Technologies), a dye known to stain the intact mitochondrial membrane (24)(25)(26). In some experiments, MitoT was used at a concentration of 200 nM.…”

Section: Schizont Maturation Assessment and Determination Of Ic 50 Smentioning

confidence: 99%

Ex Vivo Maturation Assay for Testing Antimalarial Sensitivity of Rodent Malaria Parasites

Chang

Malleret

Russell

et al. 2016

Antimicrob Agents Chemother

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c Ex vivo assay systems provide a powerful approach to studying human malaria parasite biology and to testing antimalarials. For rodent malaria parasites, short-term in vitro culture and ex vivo antimalarial susceptibility assays are relatively cumbersome, relying on in vivo passage for synchronization, since ring-stage parasites are an essential starting material. Here, we describe a new approach based on the enrichment of ring-stage Plasmodium berghei, P. yoelii, and P. vinckei vinckei using a single-step Percoll gradient. Importantly, we demonstrate that the enriched ring-stage parasites develop synchronously regardless of the parasite strain or species used. Using a flow cytometry assay with Hoechst and ethidium or MitoTracker dye, we show that parasite development is easily and rapidly monitored. Finally, we demonstrate that this approach can be used to screen antimalarial drugs.T he spread of artemisinin-resistant malaria parasites in Southeast Asia requires the urgent development of new antimalarial therapeutics (1). Currently, the discovery of drugs with activity against Plasmodium falciparum follows a defined pathway (2, 3). Central to this pathway is quantifying the intrinsic susceptibility of a standard laboratory strain of P. falciparum (usually strain 3D7) to the candidate antimalarial drugs, specifically by defining their half-maximal (50%) inhibitory concentration (IC 50 ) in vitro. Lead antimalarial candidates with the lowest IC 50 s (generally Յ1 M) are then tested against cultures of a range of adapted isolates (often with well-defined resistance phenotypes) or against ex vivo fresh isolates (2,(4)(5)(6). Next, to demonstrate efficacy in vivo, rodent malaria parasite models, in particular, Plasmodium berghei ANKA (7) and P. chabaudi (8) in mice, are used. Unfortunately, it is rare for the IC 50 s of the candidate drugs for rodent malaria parasites to be determined, thus preventing meaningful comparisons of the effects of the candidate drugs on human parasites and rodent malaria parasites.In vitro drug screening methods have been developed for P. berghei ANKA. However, P. berghei ANKA parasite infections are generally asynchronous, and therefore, an in vivo synchronization step is required prior to in vitro culture of the parasite (9). Additionally, rodent malaria parasites have different infection profiles, i.e., different lethalities and synchronicities, which are also influenced by the mouse genetic background and immunity (2). As the in vivo result is confounded by the choice of rodent malaria species and strain, as well as the mouse genetic background, we need a simplified ex vivo assay to accurately determine the intrinsic profile of the susceptibility of murine Plasmodium spp. to antimalarials.In the study described here, we developed a single-step protocol to enrich the ring stages of rodent malaria parasites for shortterm in vitro culture that can be used for in vitro screening and measurement of parasite sensitivities to different antimalarial compounds using flow cytometry. MATERIALS ...

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“…Parasite stocks were prepared from passage through resistant C57BL/6 and SJL/J mice, respectively, as described previously. 31 Experimental mice were infected with an IP dose of 1 3 10 4 parasitized RBCs. Blood stage parasitemia was determined by taking thin smears from tail blood (1 ml per day from days 7-14 postinfection) stained in 10% Giemsa solution.…”

Section: Infectionsmentioning

confidence: 99%

“…31 Blood from Ampd3 T689A/1 and WT mice was labeled with different fluorescent markers, mixed in equal proportions, and injected into P. chabaudi-infected WT mice while parasites were undergoing schizogony. The parasitemia of each labeled population was monitored over 21 hours to determine differences in parasite invasion and development.…”

Section: P Chabaudi Infectionmentioning

confidence: 99%

Adenosine monophosphate deaminase 3 activation shortens erythrocyte half-life and provides malaria resistance in mice

Hortle

Nijagal

Bauer

et al. 2016

Blood

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Key Points• AMPD3 activation reduces red blood cell half-life, which is associated with increased oxidative stress and phosphatidylserine exposure.• AMPD3 activation causes malaria resistance through increased RBC turnover and increased RBC production.The factors that determine red blood cell (RBC) lifespan and the rate of RBC aging have not been fully elucidated. In several genetic conditions, including sickle cell disease, thalassemia, and G6PD deficiency, erythrocyte lifespan is significantly shortened. Many of these diseases are also associated with protection from severe malaria, suggesting a role for accelerated RBC senescence and clearance in malaria resistance. Here, we report a novel, N-ethyl-N-nitrosourea-induced mutation that causes a gain of function in adenosine 59-monophosphate deaminase (AMPD3). Mice carrying the mutation exhibit rapid RBC turnover, with increased erythropoiesis, dramatically shortened RBC lifespan, and signs of increased RBC senescence/eryptosis, suggesting a key role for AMPD3 in determining RBC half-life. Mice were also found to be resistant to infection with the rodent malaria Plasmodium chabaudi. We propose that resistance to P. chabaudi is mediated by increased RBC turnover and higher rates of erythropoiesis during infection. (Blood. 2016; 128(9):1290-1301) IntroductionThe lifespan of red blood cells (RBC) is tightly regulated, lasting some 120 days in humans and 51 days in mice. 1 Senescent RBCs are cleared from the bloodstream by macrophages of the reticuloendothelial system and are replaced with new erythrocytes.2-4 This destruction is not random, and it is highly dependent on RBC age. 4 The factors determining the rate of RBC aging, and thereby RBC lifespan, have not been fully elucidated.In several genetic conditions, including pyruvate kinase deficiency, sickle cell disease, thalassemia, hereditary spherocytosis, and G6PD deficiency, 5,6 erythrocyte lifespan is significantly shortened. In most cases, cells show signs of increased senescence, including increased oxidative stress and phosphatidylserine (PS) exposure.2,7-9 Intriguingly, many of these diseases are also associated with protection from severe malaria, 10,11 which has led some authors to propose a role for RBC senescence and clearance in malaria resistance. 15,16 For example, the shortened lifespan of RBCs in pyruvate kinase-deficient individuals and associated pathology has been linked to reduced RBC ATP levels. Similarly, ATP loss contributes to pathology in sickle cell disease.17 Erythrocytic ATP levels are controlled by adenosine monophosphate deaminase (AMPD3), which converts AMP to inosine 59-monophosphate (IMP) and plays an important role in maintaining the adenylate energy charge or the ratio of ATP to AMP. Thus, ATP loss may be mediated through AMPD3, which converts AMP to IMP. In most cells, the conversion of ITP back to AMP by adenylsuccinate synthetase and adenylsuccinate lyase balances AMPD3 activity. However, in RBCs, the machinery for conversion of IMP back to AMP is absent, and to maintain AM...

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A flow cytometric assay to quantify invasion of red blood cells by rodent Plasmodium parasites in vivo

Cited by 39 publications

References 41 publications

Erythrocytic Iron Deficiency Enhances Susceptibility to Plasmodium chabaudi Infection in Mice Carrying a Missense Mutation in Transferrin Receptor 1

Erythrocytic Iron Deficiency Enhances Susceptibility to Plasmodium chabaudi Infection in Mice Carrying a Missense Mutation in Transferrin Receptor 1

Ex Vivo Maturation Assay for Testing Antimalarial Sensitivity of Rodent Malaria Parasites

Adenosine monophosphate deaminase 3 activation shortens erythrocyte half-life and provides malaria resistance in mice

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