To counter the global threat caused by Plasmodium falciparum malaria, new drugs and vaccines are urgently needed. However, there are no practical animal models because P. falciparum infects human erythrocytes almost exclusively. Here we describe a reliable falciparum murine model of malaria by generating strains of P. falciparum in vivo that can infect immunodeficient mice engrafted with human erythrocytes. We infected NODscid/β2m−/− mice engrafted with human erythrocytes with P. falciparum obtained from in vitro cultures. After apparent clearance, we obtained isolates of P. falciparum able to grow in peripheral blood of engrafted NODscid/β2m−/− mice. Of the isolates obtained, we expanded in vivo and established the isolate Pf3D70087/N9 as a reference strain for model development. Pf3D70087/N9 caused productive persistent infections in 100% of engrafted mice infected intravenously. The infection caused a relative anemia due to selective elimination of human erythrocytes by a mechanism dependent on parasite density in peripheral blood. Using this model, we implemented and validated a reproducible assay of antimalarial activity useful for drug discovery. Thus, our results demonstrate that P. falciparum contains clones able to grow reproducibly in mice engrafted with human erythrocytes without the use of myeloablative methods.
Murine models of Plasmodium falciparum malaria may become crucial tools in drug discovery. Here we show that non-myelodepleted NOD-scid IL2R␥ null mice engrafted with human erythrocytes support an infectious burden up to tenfold higher than that supported by engrafted NOD-scid 2microglobulin null mice. The new model was validated for drug discovery and was used to assess the therapeutic efficacy of 4-pyridones, selective inhibitors of P. falciparum cytochrome bc 1 .Malaria is caused by the erythrocytic stages of protozoan parasites of the genus Plasmodium. Among the species pathogenic for humans, Plasmodium falciparum is responsible for 300 to 500 million cases of malaria and over a million deaths annually, particularly in developing countries. The development of new antimalarial medicines and vaccines is a key part of the global strategy for malaria eradication (6).P. falciparum almost exclusively infects human erythrocytes (hE). As a result, candidate drugs and vaccines in early stages of preclinical development are usually tested in vivo by measuring their therapeutic efficacy against rodent-adapted plasmodial species and by assessing the antiparasitic response of non-human immune systems, respectively (11). To overcome the host specificity issue, two conceptually different murine models of erythrocytic stages of P. falciparum malaria have been developed. The first one requires chemical in vivo depletion of phagocytic cells from immunodeficient mice engrafted with hE in order to allow the growth of P. falciparum after intraperitoneal (i.p.) infection (2, 8). However, its variable kinetics of parasitemia and, particularly, the use of toxic reagents, which might affect the efficacy of antimalarials or effector cells, have limited its use in drug discovery (5). Recently, a new P. falciparum murine model that does not require in vivo myeloablative treatment of mice and is suitable for drug discovery was described (1). In this new model, NOD-scid mice genetically deficient in beta-2 microglobulin (2 m tm1Unc , abbreviated as 2 m null ) engrafted with hE (HM-2 m null ) are infected intravenously with P. falciparum strains selected in vivo for their competence to grow reproducibly in hE-engrafted immunodeficient mice (1).The NOD-scid 2 m null mouse strain retains residual NK cell activity as well as other innate immune functions and shows a high incidence of early thymic lymphomas, which dramatically diminish their life span (4). These characteristics may be a serious problem for addressing long-term pharmacokinetic/ pharmacodynamic (PK/PD) studies because of the relatively low total parasite burden per mouse achievable (1) and the short life span of NOD-scid 2 m null mice (4). Interestingly, NOD-scid strains carrying a null mutation of the interleukin 2 (IL-2) receptor ␥ chain (IL2R␥ tm1Wjll , abbreviated as IL2R␥ null ) have been developed (10). These murine strains lack fully mature NK cells and show additional defects in their innate immune system that explain their greater ability to support the engraftme...
Background: Microscopic analysis of blood smears is currently the most frequently used method to measure parasitemias in experiments of drug efficacy in murine models of malaria. However, it is subjective and labour intensive, which preclude its utilization in large-scale evaluation programs. Flow cytometry is an alternative method, but due to the limited specificity achieved with the currently available techniques, it has not been widely used in murine models of malaria during preclinical evaluation. We describe a new flow cytometric method based on the differences of autofluorescence and DNA content measured after staining with YOYO-1 that are observed in infected erythrocytes compared with noninfected erythrocytes. Methods: Samples of blood from Plasmodium yoeliiinfected animals were fixed with glutaraldehyde, incubated with RNAase, and stained with YOYO-1 in 96-well plate format. After acquisition, erythrocytes gated in loga-
Flow cytometry is a powerful tool for measuring parasitemias in murine malaria models used to test new antimalarials. Measurement of the emission of the nonpermeable nucleic acid dye YOYO-1 (at 530 and 585 nm after excitation at 488 nm) allowed the unambiguous detection of low parasitemias (!0.01%) but required prolonged fixation and permeabilization of the sample. Thus, we tested whether this issue could be overcome by use of the cell-permeant dye SYTO-16 with this same bidimensional method. Blood samples from CD1 mice infected with Plasmodium yoelii, Plasmodium vinckei, or Plasmodium chabaudi or from NOD scidb2m-/-engrafted with human erythrocytes and infected with P. falciparum were stained with SYTO-16 in the presence or absence of TER-119 mAb (for engrafted mice) in 96-well plate format and acquired in Trucount TM tubes. Bidimensional analysis with SYTO-16 was quantitatively equivalent to YOYO-1. Moreover, by combining SYTO-16 with the use of TER-119-PE antimouse erythrocyte mAb and Trucount tubes, the measurement of the concentration of P. falciparuminfected erythrocytes over a range of five orders of magnitude was achieved. Bidimensional analysis using SYTO-16 can be used to accurately measure the concentration of Plasmodium spp.-infected erythrocytes in mice without complex sample preparation. MALARIA is caused by the erythrocytic stages of protozoa of the genus Plasmodium, which colonize and destroy host's erythrocytes (1). To counter this disease, murine models of malaria are essential tools for research (2), particularly for drug discovery (3). In addition to the standard rodent experimental systems, different murine models of P. falciparum malaria are currently available (4-6). These are of special interest for drug discovery because, with the exception of human subjects, these are the only experimental systems available that allow the evaluation in vivo of the real human pathogen growing inside human erythrocytes (hE) previously engrafted into immunodeficient mice. Not surprisingly, the peripheral blood of these chimeric mice [humanized mice (HM)] is a complex mixture of murine erythrocytes (mE) and hE, in which the hematological effects of massive transfusions of hE and their elimination from peripheral blood may have important effects. Hence, the specific and quantitative measurement of different erythrocytic subpopulations is crucial in HM models, particularly when these models are used to establish the relationship between the amount of an antimalarial drug in blood and the effect on parasitemia through experimental pharmacokinetic and pharmacodynamic studies (PK/PD). In this kind of
The emergence of resistance to available antimalarials requires the urgent development of new medicines. The recent disclosure of several thousand compounds active in vitro against the erythrocyte stage of Plasmodium falciparum has been a major breakthrough, though converting these hits into new medicines challenges current strategies. A new in vivo screening concept was evaluated as a strategy to increase the speed and efficiency of drug discovery projects in malaria. The new in vivo screening concept was developed based on human disease parameters, i.e. parasitemia in the peripheral blood of patients on hospital admission and parasite reduction ratio (PRR), which were allometrically down-scaled into P. berghei-infected mice. Mice with an initial parasitemia (P0) of 1.5% were treated orally for two consecutive days and parasitemia measured 24 h after the second dose. The assay was optimized for detection of compounds able to stop parasite replication (PRR = 1) or induce parasite clearance (PRR >1) with statistical power >99% using only two mice per experimental group. In the P. berghei in vivo screening assay, the PRR of a set of eleven antimalarials with different mechanisms of action correlated with human-equivalent data. Subsequently, 590 compounds from the Tres Cantos Antimalarial Set with activity in vitro against P. falciparum were tested at 50 mg/kg (orally) in an assay format that allowed the evaluation of hundreds of compounds per month. The rate of compounds with detectable efficacy was 11.2% and about one third of active compounds showed in vivo efficacy comparable with the most potent antimalarials used clinically. High-throughput, high-content in vivo screening could rapidly select new compounds, dramatically speeding up the discovery of new antimalarial medicines. A global multilateral collaborative project aimed at screening the significant chemical diversity within the antimalarial in vitro hits described in the literature is a feasible task.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.