Sequential Plasmodium chabaudi and Plasmodium berghei infections provide a novel model of severe malarial anemia.
ABSTRACT: Lack of an adequate animal model of Plasmodium falciparum severe malarial anemia (SMA) has hampered the understanding of this highly lethal condition. We developed a model of SMA by infecting C57BL/6 mice with P. chabaudi followed after recovery by P. berghei infection. P. chabaudi/P. berghei-infected mice had an initial 9- to 10-day phase of relatively low parasitemia and severe anemia, followed by a second phase of hyperparasitemia, more profound anemia, reticulocytosis, and death 14 to 21 days after infection. P. chabaudi/P. berghei-infected animals had more intense splenic hematopoiesis, higher interleukin-10 (IL-10)/tumor necrosis factor alpha and IL-12/gamma interferon (IFN-?) ratios, and higher antibody levels against P. berghei and P. chabaudi antigens than P. berghei-infected or P. chabaudi-recovered animals. Early treatment with chloroquine or artesunate did not prevent the anemia, suggesting that the bulk of red cell destruction was not due to the parasite. Red cells from P. chabaudi/P. berghei-infected animals had increased surface IgG and C3 by flow cytometry. However, C3(-/-) mice still developed anemia. Tracking of red cells labeled ex vivo and in vivo and analysis of frozen tissue sections by immunofluorescence microscopy showed that red cells from P. chabaudi/P. berghei-infected animals were removed at an accelerated rate in the liver by erythrophagocytosis. This model is practical and reproducible, and its similarities with P. falciparum SMA in humans makes it an appealing system with which to study the pathogenesis of this condition and explore potential immunomodulatory interventions.
Project description:Parasite cytoadherence within the microvasculature of tissues and organs of infected individuals is implicated in the pathogenesis of several malaria syndromes. Multiple host receptors may mediate sequestration. The identity of the host receptor(s), or the parasite ligand(s) responsible for sequestration of Plasmodium species other than Plasmodium falciparum is largely unknown. The rodent malaria parasites may be useful to model interactions of parasite species, which lack the var genes with their respective hosts, as other multigene families are shared between the species. The role of the endothelial receptors ICAM-1 and CD36 in cytoadherence and in the development of pathology was investigated in a Plasmodium chabaudi infection in C57BL/6 mice lacking these receptors. The schizont membrane-associated cytoadherence (SMAC) protein of Plasmodium berghei has been shown to exhibit reduced CD36-associated cytoadherence in P. berghei ANKA-infected mice.Parasite tissue sequestration and the development of acute stage pathology in P. chabaudi infections of mice lacking CD36 or ICAM-1, their respective wild type controls, and in infections with mutant P. chabaudi parasites lacking the smac gene were compared. Peripheral blood parasitaemia, red blood cell numbers and weight change were monitored throughout the courses of infection. Imaging of bioluminescent parasites in isolated tissues (spleen, lungs, liver, kidney and gut) was used to measure tissue parasite load.This study shows that neither the lack of CD36 nor the deletion of the smac gene from P. chabaudi significantly impacted on acute-stage pathology or parasite sequestration. By contrast, in the absence of ICAM-1, infected animals experience less anaemia and weight loss, reduced parasite accumulation in both spleen and liver and higher peripheral blood parasitaemia during acute stage malaria. The reduction in parasite tissue sequestration in infections of ICAM-1 null mice is maintained after mosquito transmission.These results indicate that ICAM-1-mediated cytoadherence is important in the P. chabaudi model of malaria and suggest that for rodent malarias, as for P. falciparum, there may be multiple host and parasite molecules involved in sequestration.
Project description:The purpose of this research is to identify and evaluate the global gene expression of the rodent malaria parasites Plasmodium yoelii, Plasmodium berghei and Plasmodium chabaudi blood-stage parasites and specifically compare the blood stage gene expression profiles of samples derived from previous studies on Plasmodium falciparum, Plasmodium vivax and Plasmodium knowlesi Overall design: Mice were infected by intraperitoneal injections of P. berghei ANKA, P. chabaudi AS or P. yoelii 17x parasitized erythrocytes and parasitaemia and parasite stages were monitored by thin blood smears stained with Giemsa. Mice infected with P. chabaudi were highly synchronized and terminal bled every 2 hours under anesthesia over the course of 24 hr. For P. berghei and P. yoelii infection, mice were terminal bleed and the stage-specific parasitized erythrocytes were separated via Nycodenz density gradient. The ring stage interface was isolated, washed and subjected to ex vivo culture, which was then collected every 2 hr over the course of 24 hr over a complete IDC life-cycle. Samples labeled with Cy5 were hybridized against a reference RNA pool labeled with Cy3, consisting of equal amounts of P. yoelii or P. berghei or P. chabaudi RNA from each time point.
Project description:Malaria is a global disease that clinically affects more than two hundred million people annually. Despite the availability of effective antimalarials, mortality rates associated with severe complications are high. Hepatopathy is frequently observed in patients with severe malarial disease and its pathogenesis is poorly understood. Previously, we observed high amounts of hemozoin or malaria pigment in livers from infected mice. In this study, we investigated whether hemozoin is associated with liver injury in different mouse malaria models. C57BL/6J mice infected with the rodent parasites Plasmodium berghei ANKA, P. berghei NK65 or P. chabaudi AS had elevated serum liver enzymes without severe histological changes in the liver, in line with the observations in most patients. Furthermore, liver enzymes were significantly higher in serum of P. chabaudi AS-infected mice compared to mice infected with the P. berghei parasite strains and a strong positive correlation was found between hepatic hemozoin levels, hepatocyte damage and inflammation in the liver with P. chabaudi AS. The observed liver injury was only marginally influenced by the genetic background of the host, since similar serum liver enzyme levels were measured in infected C57BL/6J and BALB/c mice. Intravenous injection of P. falciparum-derived hemozoin in malaria-free C57BL/6J mice induced inflammatory gene transcription in the liver, suggesting that hemozoin may be involved in the pathogenesis of malaria hepatopathy by inducing inflammation.
Project description:Membrane electrochemical potential is a feature of the molecular profile of the cell membrane and the two-dimensional arrangement of its charge-bearing molecules. Plasmodium species, the causative agents of malaria, are intracellular parasites that remodel host erythrocytes by expressing their own proteins on erythrocyte membranes. Although various aspects of the modifications made to the host erythrocyte membrane have been extensively studied in some human Plasmodium species (such as Plasmodium falciparum), details of the structural and molecular biological modifications made to host erythrocytes by nonhuman Plasmodium parasites have not been studied. We employed zeta potential analysis of erythrocytes parasitized by P. chabaudi, a nonhuman Plasmodium parasite. From these measurements, we found that the surface potential shift was more negative for P. chabaudi-infected erythrocytes than for P. falciparum-infected erythrocytes. However, electron microscopic analysis of the surface of P. chabaudi-infected erythrocytes did not reveal any modifications as compared with nonparasitized erythrocytes. These results suggest that differences in the membrane modifications found herein represent unique attributes related to the pathogenesis profiles of the two different malaria parasite species in different host animals and that these features have been acquired through parasite adaptations acquired over long evolutionary time periods.
Project description:Malaria is a parasitic disease that causes severe hemolytic anemia in Plasmodium-infected hosts, which results in the release and accumulation of oxidized heme (hemin). Although hemin impairs the establishment of Plasmodium immunity in vitro and in vivo, mice preconditioned with hemin develop lower parasitemia when challenged with Plasmodium chabaudi adami blood stage parasites. In order to understand the mechanism accounting for this resistance as well as the impact of hemin on eryptosis and plasma levels of scavenging hemopexin, red blood cells were labeled with biotin prior to hemin treatment and P. c. adami infection. This strategy allowed discriminating hemin-treated from de novo generated red blood cells and to follow the infection within these two populations of cells. Fluorescence microscopy analysis of biotinylated-red blood cells revealed increased P. c. adami red blood cells selectivity and a decreased permissibility of hemin-conditioned red blood cells for parasite invasion. These effects were also apparent in in vitro P. falciparum cultures using hemin-preconditioned human red blood cells. Interestingly, hemin did not alter the turnover of red blood cells nor their replenishment during in vivo infection. Our results assign a function for hemin as a protective agent against high parasitemia, and suggest that the hemolytic nature of blood stage human malaria may be beneficial for the infected host.
Project description:AbstractInbred mice are commonly used to test candidate malaria vaccines, but have been unreliable for predicting efficacy in humans. To establish a more rigorous animal model, we acquired African woodland thicket rats of the genus Grammomys, the natural hosts for Plasmodium berghei. Thicket rats were acquired and identified as Grammomys surdaster by skull and teeth measurements and mitochondrial DNA genotyping. Herein, we demonstrate that thicket rats are highly susceptible to infection by P. berghei, and moderately susceptible to Plasmodium yoelii and Plasmodium chabaudi: 1-2 infected mosquito bites or 25-100 sporozoites administered by intravenous injection consistently resulted in patent parasitemia with P. berghei, and resulted in patent parasitemia with P. yoelii and P. chabaudi strains for at least 50% of animals. We then assessed efficacy of whole-organism vaccines to induce sterile immunity, and compared the thicket rat model to conventional mouse models. Using P. berghei ANKA radiation-attenuated sporozoites, and P. berghei ANKA and P. yoelii chemoprophylaxis vaccination approaches, we found that standard doses of vaccine sufficient to protect laboratory mice for a long duration against malaria challenge, are insufficient to protect thicket rats, which require higher doses of vaccine to achieve even short-term sterile immunity. Thicket rats may offer a more stringent and pertinent model for evaluating whole-organism vaccines.
Project description:Rodent malaria species Plasmodium yoelii and P. chabaudi have been widely used to validate vaccine approaches targeting blood-stage merozoite antigens. However, increasing data suggest the P. berghei rodent malaria may be able to circumvent vaccine-induced anti-merozoite responses. Here we confirm a failure to protect against P. berghei, despite successful antibody induction against leading merozoite antigens using protein-in-adjuvant or viral vectored vaccine delivery. No subunit vaccine approach showed efficacy in mice following immunization and challenge with the wild-type P. berghei strains ANKA or NK65, or against a chimeric parasite line encoding a merozoite antigen from P. falciparum. Protection was not improved in knockout mice lacking the inhibitory Fc receptor CD32b, nor against a ?smac P. berghei parasite line with a non-sequestering phenotype. An improved understanding of the mechanisms responsible for protection, or failure of protection, against P. berghei merozoites could guide the development of an efficacious vaccine against P. falciparum.
Project description:BACKGROUND: The emerging resistance of Plasmodium species to currently available anti-malarials remains a public health concern, hence the need for new effective, safe and affordable drugs. Natural products remain a reliable source of drugs. Nefang is a polyherbal anti-malarial of the Cameroonian folklore medicine with demonstrated in vitro antiplasmodial and antioxidant activities. It is composed of Mangifera indica (bark and leaf), Psidium guajava, Carica papaya, Cymbopogon citratus, Citrus sinensis, Ocimum gratissimum (leaves). This study aimed at investigating the suppressive, prophylactic and curative activities of Nefang in Plasmodium infected rodent models. METHODS: Systemic acute oral toxicity of Nefang aqueous and ethanol extracts was assessed in mice up to a dose of 5,000 mgkg(-1) body weight. BALB/c mice and Wistar rats were inoculated with Plasmodium chabaudi chabaudi and Plasmodium berghei, respectively, and treated with Nefang, the Mangifera indica bark/Psidium guajava combination and a Psidium guajava leaf aqueous extracts (75, 150, 300 and 600 mgkg(-1) bwt). Their schizonticidal activity was then evaluated using the Peter's 4-day suppressive test). The prophylactic and curative (Rane's Test) activity of Nefang was also evaluated by determining the parasitaemia, survival time, body weight and temperature in pre-treated rodents. RESULTS: Acute oral toxicity of the extract did not cause any observed adverse effects. Percent suppressions of parasitaemia at 600 mgkg(-1) bwt were as follows (P. berghei/P. chabaudi): Nefang - 82.9/86.3, Mangifera indica bark/Psidium guajava leaf combination extract - 79.5/81.2 and Psidium guajava leaf - 58.9/67.4. Nefang exhibited a prophylactic activity of 79.5% and its chemotherapeutic effects ranged from 61.2 - 86.1% with maximum effect observed at the highest experimental dose. CONCLUSION: These results indicate that Nefang has excellent in vivo anti-malarial activities against P. berghei and P. chabaudi, upholding earlier in vitro antiplasmodial activities against multi-drug resistant P. falciparum parasites as well as its traditional use. Hence, Nefang represents a promising source of new anti-malarial agents.
Project description:Cerebral malaria (CM) is a severe and rapidly progressing complication of infection by Plasmodium parasites that is associated with high rates of mortality and morbidity. Treatment options are currently few, and intervention with artemisinin (Art) has limited efficacy, a problem that is compounded by the emergence of resistance to Art in Plasmodium parasites. Rocaglates are a class of natural products derived from plants of the Aglaia genus that have been shown to interfere with eukaryotic initiation factor 4A (eIF4A), ultimately blocking initiation of protein synthesis. Here, we show that the rocaglate CR-1-31B perturbs association of Plasmodium falciparum eIF4A (PfeIF4A) with RNA. CR-1-31B shows potent prophylactic and therapeutic antiplasmodial activity in vivo in mouse models of infection with Plasmodium berghei (CM) and Plasmodium chabaudi (blood-stage malaria), and can also block replication of different clinical isolates of P. falciparum in human erythrocytes infected ex vivo, including drug-resistant P. falciparum isolates. In vivo, a single dosing of CR-1-31B in P. berghei-infected animals is sufficient to provide protection against lethality. CR-1-31B is shown to dampen expression of the early proinflammatory response in myeloid cells in vitro and dampens the inflammatory response in vivo in P. berghei-infected mice. The dual activity of CR-1-31B as an antiplasmodial and as an inhibitor of the inflammatory response in myeloid cells should prove extremely valuable for therapeutic intervention in human cases of CM.
Project description:BACKGROUND: Vascular endothelial growth factor (VEGF) is taken up by parasitized red blood cells during malaria and stimulates intra-erythrocytic growth of Plasmodium falciparum in vitro. The cause and consequence of this uptake is not understood. METHODS: Plasmodium falciparum was cultured in vitro. Parasite growth and intracellular VEGF levels were assessed using flow cytometry. Intracellular VEGF was visualized by fluorescence immunocytochemistry. Phosphorylated tyrosine was measured by western blotting. In vivo assessment of intra-erythrocytic VEGF was performed in Plasmodium berghei ANKA-infected C57BL/6 mice. RESULTS: VEGF accumulated intracellularly in infected red blood cells, particularly in schizonts. In vitro growth of P. falciparum was unchanged when co-cultured with the anti-VEGF antibody bevacizumab or with an anti-VEGF receptor-1 peptide. In contrast, the VEGF receptor-2 inhibitor, SU5416, dose-dependently inhibited growth. None of the treatments reduced intracellular VEGF levels. Thus, the anti-parasitic effect of SU5416 seemed independent of VEGF uptake. SU5416 reduced phosphorylated tyrosine in parasitized red blood cells. Similarly, the broad-spectrum tyrosine kinase inhibitor genistein dose-dependently inhibited P. falciparum growth and reduced tyrosine phosphorylation. Neither bevacizumab nor anti-VEGF receptor-1 peptide affected tyrosine kinase activity. Finally, in vivo uptake of VEGF in P. berghei ANKA was demonstrated, analogous to the in vitro uptake in P. falciparum, making it a possible model for the effects of VEGF signalling in vivo during malaria. CONCLUSIONS: Inhibition of VEGFR-2 signalling reduces intra-erythrocytic growth of P. falciparum, likely due to tyrosine kinase inhibition. Internalisation of VEGF in P. falciparum-infected red blood cells does not rely on VEGF receptors. The function of in vivo uptake of VEGF can be studied in rodent malaria models.