Methotrexate and aminopterin lack in vivo antimalarial activity against murine malaria species

The antifolate anticancer drug methotrexate (MTX) has potent activity against Plasmodium falciparum in vitro. Experience of its use in the treatment of rheumatoid arthritis indicates that it could be safe and efficacious for treating malaria. We sought to establish a murine malaria model to study the mechanism of action and resistance of MTX and its analogue aminopterin (AMP). We used Plasmodium berghei, Plasmodium yoelii yoelii, Plasmodium chabaudi and Plasmodium vinckei. None of these species were susceptible to either drug. We have also tested the efficacy of pyrimethamine in combination with folic acid in P. berghei, and data indicate that folic acid does not influence pyrimethamine efficacy, which suggests that P. berghei may not transport folate. Since MTX and AMP utilise folate receptor/transport to gain access to cells, their lack of efficacy against the four tested murine malaria species may be the result of inefficiency of drug transport.     

In vivo gene silencing in Plasmodium berghei--a mouse malaria model

RNA interference (RNAi) has emerged as a specific and efficient tool to silence gene expression in a variety of organisms and cell lines. An important prospect for RNAi technology is its possible application in the treatment of diseases using short interfering RNAs (siRNAs). However, the effect of siRNAs in adult animals and their potential to treat or prevent diseases are yet to be fully investigated. The main goal of the present study is to find out whether it was possible to carry out RNAi on circulating malaria parasite in vivo. To trigger RNAi in mouse malaria parasite, we used siRNAs corresponding to cysteine protease genes of Plasmodium berghei (berghepain-1 & 2). Intravenous injections of berghepains' siRNAs in infected animal resulted in characteristic enlargement of food vacuole in circulating parasites. Protein analysis of these treated parasites showed substantial accumulation of hemoglobin, which is reminiscent of the effect observed upon treating Plasmodium falciparum with different cysteine protease inhibitors. Parasites treated with berghepain 1 & 2 siRNAs showed marked reduction in the levels of their cognate mRNAs, thereby suggesting specific inhibition of berghepains' gene expression in vivo. We also observed the generation of approximately 25 nt RNA species from berghepains' mRNAs in the treated parasites, which is a characteristic of an RNAi phenomenon. These results thus provide evidence that beyond its value for validation of gene functions, RNAi may provide a new approach for disease therapy.     

Detection of a new cerebral malaria susceptibility locus,using CBA mice

Human cerebral malaria (CM) during acute Plasmodium falciparum infection is a serious neurological complication that leads to coma and death. P. berghei ANKA infection of CBA mice is a useful experimental model of CM. To identify host susceptibility loci, we performed chromosomal mapping in crossbred populations of both CM-susceptible CBA and CM-resistant DBA/2 mice. One significant region for a CM-susceptible locus in CBA mice was mapped to H2 region on Chromosome 17, tentatively designated cmsc. cmsc was mapped to a different chromosomal region from that previously reported in the C57BL/6 mouse model of CM. It is possible that different loci contribute to CM in CBA and C57BL/6 mouse strains. Comparison of the function of CM susceptibility loci between CBA and C57BL/6 mice could have important implications for the study of the complex pathogenesis of CM in humans.     

Plasmodium berghei: parasite clearance after treatment with dihydroartemisinin in an asplenic murine malaria model

Clinical reports indicate that malaria-infected asplenic patients have a reduced capacity for parasite clearance despite intensive antimalarial therapy. The aim of this study was to evaluate the efficacy of dihydroartemisinin in an asplenic murine malaria model. Mice were inoculated with Plasmodium berghei parasitised erythrocytes and received a single dose of dihydroartemisinin 56 h later, at 2-5% parasitaemia. Haematology, liver biochemistry and histopathology of key organs were performed to evaluate organ response to malaria infection. The nadir parasitaemia occurred 20 h after dihydroartemisinin administration, falling 2.8- to 6.0-fold and 2.7- to 6.9-fold in asplenic and intact mice, respectively, (10-100 mg/kg). Histopathology indicated increased stimulation of liver function/activity during malaria infection of asplenic mice (as compared to intact mice). Overall efficacy of single-dose dihydroartemisinin treatment in asplenic mice was similar to intact mice although the rate of recrudescence in asplenic mice was significantly greater than intact mice at 30 and 100 mg/kg. The asplenic murine malaria model could be used in pre-clinical studies of splenic function and clearance of malaria parasites, pathophysiological studies or antimalarial drug efficacy in asplenia.     

Rodent and nonrodent malaria parasites differ in their phospholipid metabolic pathways

Malaria, a disease affecting humans and other animals, is caused by a protist of the genus Plasmodium. At the intraerythrocytic stage, the parasite synthesizes a high amount of phospholipids through a bewildering number of pathways. In the human Plasmodium falciparum species, a plant-like pathway that relies on serine decarboxylase and phosphoethanolamine N-methyltransferase activities diverts host serine to provide additional phosphatidylcholine and phosphatidylethanolamine to the parasite. This feature of parasitic dependence toward its host was investigated in other Plasmodium species. In silico analyses led to the identification of phosphoethanolamine N-methyltransferase gene orthologs in primate and bird parasite genomes. However, the gene was not detected in the rodent P. berghei, P. yoelii, and P. chabaudi species. Biochemical experiments with labeled choline, ethanolamine, and serine showed marked differences in biosynthetic pathways when comparing rodent P. berghei and P. vinckei, and human P. falciparum species. Notably, in both rodent parasites, ethanolamine and serine were not significantly incorporated into phosphatidylcholine, indicating the absence of phosphoethanolamine N-methyltransferase activity. To our knowledge, this is the first study to highlight a crucial difference in phospholipid metabolism between Plasmodium species. The findings should facilitate efforts to develop more rational approaches to identify and evaluate new targets for antimalarial therapy.     

Plasmodium berghei XAT: contribution of gammadelta T cells to host defense against infection with blood-stage nonlethal malaria parasite

We examined a potential role of gammadelta T cells in protective immunity to blood-stage Plasmodium berghei XAT infection. Plasmodium berghei XAT is an attenuated variant of the lethal strain P. berghei NK65 and its infection is self-resolving in immune competent mice. To determine whether gammadelta T cells are essential for the resolution of P. berghei XAT malaria, mice were depleted of gammadelta T cells with anti-TCRgammadelta antibody treatment. Although mice that had received control antibody resolved infections, mice received anti-TCRgammadelta antibody could not control their infections and eventually died. Spleen cells from infected mice produced IFN-gamma and nitric oxide (NO) within the first week of infection, however, levels of IFN-gamma and NO in gammadelta T cell-depleted mice were significantly lower than in control mice. To examine whether gammadelta T cells are involved in the antibody production, malarial-specific antibodies of the various isotypes were measured in the sera of gammadelta T cell-depleted mice and control mice. Serum levels of IgG2a, which was known to be a protective antibody in P. berghei XAT malaria, were significantly lower in gammadelta T cell-depleted mice than in control mice, whereas levels of IgG1 were comparable to those in control mice. Our results indicated that the presence of the gammadelta T cell subset was essential for resolution of blood-stage P. berghei XAT malaria and played a modulatory role in the development of Th1 response and host defense against this malarial parasites.     

Retinol supplementation in murine Plasmodium berghei malaria: effects on tissue levels, parasitaemia and lipid peroxidation

Reduced plasma retinol concentrations occur in human malaria but the benefits of supplementation remain uncertain. We assessed the in vivo efficacy of retinol administration, and its effect on lipid peroxidation, in a Plasmodium berghei murine model. Animals received vehicle (n=17) or retinol (i) before P. berghei inoculation (four doses), (ii) at parasitaemia 10-15% (three to four doses) or (iii) before and after inoculation (six to seven doses; n=15 in each group), with euthanasia on day 8 post-inoculation or when the parasitaemia exceeded 50%. Multiple-dose pre-inoculation retinol reduced endpoint parasitaemia by 24% (P=0.001 versus controls). A reduction of 18% (P=0.042) was observed when retinol was given to parasitaemic animals. Retinol was ineffective when given both before and after infection (11% reduction; P=0.47). Although retinol supplementation did not change plasma retinol concentrations, liver retinol content increased and correlated inversely with endpoint parasitaemia (r=-0.45, P=0.001). Malaria infection augmented concentrations of the free radical lipid peroxidation end-product F(2)-isoprostanes in plasma, erythrocytes and liver by 1.8-, 2.8- and 4.9-fold, respectively, but retinol supplementation had no effect on these increases. Consistent with some human malaria studies, prophylactic retinol reduces P. berghei parasitaemia. This effect relates to augmentation of tissue retinol stores rather than to retinol-associated changes in oxidant status.     

Plasmodium yoelii: novel rhoptry proteins identified within the body of merozoite rhoptries in rodent Plasmodium malaria

The biogenesis, organization and function of the rhoptries are not well understood. Antisera were prepared to synthetic peptides prepared as multiple antigenic peptides (MAPs) obtained from a Plasmodium yoelii merozoite rhoptry proteome analysis. The antisera were used in immunofluorescence and immunoelectron microscopy of schizont-infected erythrocytes. Twenty-seven novel rhoptry proteins representing proteases, metabolic enzymes, secreted proteins and hypothetical proteins, were identified in the body of the rhoptries by immunoelectron microscopy. The merozoite rhoptries contain a heterogeneous mixture of proteins that may initiate host cell invasion and establish intracellular parasite development.     

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.     

Wave expansion of CD34 progenitor cells in the spleen in rodent malaria

Defense against malaria depends upon amplification of the spleen structure and function for the clearance of parasitized red blood cells (pRBC). We studied the distribution and amount of CD34⁺ cells in the spleens of mice infected with rodent malaria. We sought to identify these cells in the spleen and determine their relationship to infection. C57BL/6J mice were infected with self-resolving, Plasmodium chabaudi CR, or one of the lethal rodent malaria strains, P. chabaudi AJ and P. berghei ANKA. We then recorded parasitemia, mortality, and the presence of CD34⁺ cells in spleen, as determined by immunohistochemistry and flow cytometry. In the non-lethal strain, the spleen structure was maintained during amplification, but disrupted in lethal models. The abundance of CD34⁺ cells increased in the red pulp on the 4th and 6th days p.i. in all models, and subsided on the 8th day p.i. Faint CD34⁺ staining on the 8th day p.i., was probably due to differentiation of committed cell lineages. In this work, increase of spleen CD34⁺ cells did not correlate with infection control.     

Murine malaria. I. Measurement of the mean rate of increase in vivo of Plasmodium berghei

Swiss mice were inoculated intraperitoneally with RBC infected with Plasmodium berghei. The moment a certain parasitemia was reached in each individual mouse was estimated by means of linear interpolation. The relationship between latent period and log inoculum was investigated by means of simple linear regression. The slopes of the latent period per log inoculum curves were significantly different using different donor mice inoculated with serial 10-fold dilution of infected RBC and exsanguinated in the same phase of the infection. It could not be demonstrated that the slopes of the regression lines depended on the stage of the disease of the donor mouse.     

Modulation of cerebral malaria by fasudil and other immune-modifying compounds

Malaria continues to cause millions of deaths annually. No specific effective treatment has yet been found for cerebral malaria, one of the most severe complications of the disease. The pathology of cerebral malaria is considered to be primarily immunological. We examined a number of compounds with known effects on the immune system, in a murine model of cerebral malaria. Of the compounds tested, only fasudil and curcumin had significant effects on the progression of the disease. Although neither drug caused a reduction in parasitemia, survival of the treated mice was significantly increased, and the development of cerebral malaria was either delayed or prevented. Our results support the hypothesis that an immunomodulator efficient in preventing CM should be administered together with anti-plasmodial drugs to prevent severe malaria disease; curcumin and fasudil should be further investigated to determine efficiency and feasibility of treatment.     

Plasmodium berghei: antiparasitic effects of orally administered hypoestoxide in mice

Hypoestoxide (HE) is a diterpene isolated from Hypoestes rosea (Acanthaceae), a plant indigenous to Nigeria. Previous studies demonstrated that HE exhibited potent anti-inflammatory and anti-cancer activities in well established animal models but weak in vitro activities in both the anti-inflammation and anti-cancer in vitro screening systems. We now report a similar observation in the in vitro and in vivo screening systems for antimalarial activity. The results indicate that while HE exhibits a relatively weak in vitro activity (IC(50) = 10 microM versus 0.11 microM for chloroquine) against different strains of cultured P. falciparum parasites, the dose of HE required to reduce parasitemia by 90% in Plasmodium berghei-infected mice, is much lower than standard antimalaria drugs (SD(90) = 250 microg/kg versus 5mg/kg for chloroquine). Furthermore, lower doses of HE were much more effective than higher doses in inhibiting parasite development. The implications of these findings are discussed.     

TNF family members and malaria: Old observations, new insights and future directions

Tumor necrosis factor (TNF) has long been recognized to promote malaria parasite killing, but also to contribute to the development of severe malaria disease. The precise molecular mechanisms that influence these different outcomes in malaria patients are not well understood, but the virulence and drug-resistance phenotype of malaria parasites and the genetic background and age of patients are likely to be important determinants. In the past few years, important roles for other TNF family members in host immune responses to malaria parasites and the induction of disease pathology have been discovered. In this review, we will summarize these more recent findings and highlight major gaps in our current knowledge. We will also discuss future research strategies that may allow us to better understand the sometimes subtle and intricate effects of TNF family molecules during malaria infection.     

Plasmodium falciparum: polymorphism in the MSP-1 gene in Indian isolates and predominance of certain alleles in cerebral malaria

Polymorphism in the block-2 region of merozoite surface protein-1 gene in 69 North Indian Plasmodium falciparum isolates was studied by PCR and RFLP using Dra-1 endonuclease. On the basis of molecular weight of the PCR products, considerable size polymorphism in target gene was seen and 69 isolates were classified into five allelic types. On RFLP, the isolates in three allelic types were further divided into two sub-allelic types each and thus eight genetic types could be identified. Interestingly, all five allelic types were identified in 47 isolates from uncomplicated (non-cerebral) malaria patients while only two allelic types (Type 2 and 3) were seen amongst 22 isolates from cerebral malaria patients. Furthermore, on RFLP, one subtype (2A) was predominantly seen in cerebral malaria patients and one subtype (3A) was exclusively found in cerebral malaria patients. These observations suggest that a few, comparatively more virulent isolates prevalent in an area may cause severe disease (cerebral malaria) which can be identified by molecular techniques like PCR-RFLP.     

Plasmodium berghei: development of an irreversible experimental malaria model in Wistar rats

A protocol to infect five-week-old Wistar rats by Plasmodium berghei which resulted in 100% mortality was developed in this work. In order to accomplish this goal, the effect of the administration of 10(7) and 10(8) parasitized erythrocytes by i.v. and i.p. route was investigated. The animals inoculated with 10(7) parasitized red blood cells by i.p. and i.v. routes showed 25 and 50% mortality, respectively. Inoculation with 10(8) parasitized erythrocytes by both routes resulted in a 100% lethal infection. The i.v. inoculation showed less scattered results and it was preferred over the i.p. route. The suitability of the protocol developed was evaluated by treating infected Wistar rats with chloroquine (30 mg/kg/day). A decreased parasitemia after the treatment was observed until the complete eradication of the parasite, around 10 days post-inoculation. Parasitemia depression after chloroquine treatment demonstrates the utility of the model developed to test new antimalarial drugs.     

Human cerebral malaria and the blood-brain barrier

Malaria represents a continuing and major global health challenge and our understanding of how the Plasmodium parasite causes severe disease and death remains poor. One serious complication of the infection is cerebral malaria, a clinically complex syndrome of coma and potentially reversible encephalopathy, associated with a high mortality rate and increasingly recognised long-term sequelae in survivors. Research into the pathophysiology of cerebral malaria, using a combination of clinical and pathological studies, animal models and in vitro cell culture work, has focussed attention on the blood-brain barrier (BBB). This represents the key interface between the brain parenchyma and the parasite, which develops within an infected red cell but remains inside the vascular space. Studies of BBB function in cerebral malaria have provided some evidence for parasite-induced changes secondary to sequestration of parasitised red blood cells and host leukocytes within the cerebral microvasculature, such as redistribution of endothelial cell intercellular junction proteins and intracellular signaling. However, the evidence for a generalised increase in BBB permeability, leading to cerebral oedema, is conflicting. As well as direct cell adhesion-dependent effects, local adhesion-independent effects may activate and damage cerebral endothelial cells and perivascular cells, such as decreased blood flow, hypoxia or the effects of parasite toxins such as pigment. Finally, a number of systemic mechanisms could influence the BBB during malaria, such as the metabolic and inflammatory complications of severe disease acting 'at a distance'. This review will summarise evidence for these mechanisms from human studies of cerebral malaria and discuss the possible role for BBB dysfunction in this complex and challenging disease.     

Murine malaria. II. Relationship between number of inoculated parasites (Plasmodium berghei) and survival time

Swiss mice were inoculated intravenously with RBC infected with Plasmodium berghei. The survival pattern appeared to be polyphasic. A close correlation existed between parasite density of the inoculum and the survival time, at least for the first two phases of the survival pattern. Significantly different numbers of mice died in the different phases, but no clear-cut relationship could be demonstrated between number of deaths and the dose. The peripheral parasitemia is closely related to the course of the infection at least in the first phase.     

Effect of artemether administered alone or in combination with praziquantel to mice infected with Plasmodium berghei or Schistosoma mansoni or both

The artemisinins have become key drugs for the treatment and control of malaria, particularly within artemisinin-based combination therapies. Since the artemisinins also exhibit antischistosomal properties, their use in areas where malaria and schistosomiasis are co-endemic may have an effect on both diseases and co-infection might alter drug efficacy. We assessed the antimalarial and antischistosomal efficacies of artemether in mice infected with Plasmodium berghei or Schistosoma mansoni or both parasites concurrently. Three oral doses of 400 mg/kg artemether at 14-day intervals reduced total and female S. mansoni worm burdens by 98.7-100%, regardless of a concurrent P. berghei infection. When four daily doses of 55 mg/kg artemether were administered, which is a standard treatment schedule to cure P. berghei-infected mice, significantly lower total and female S. mansoni worm burden reductions were observed (73.1-89.2%). Artemether, administered at both of the above-mentioned treatment schemes, showed excellent antimalarial efficacy with no indications of delayed clearance of P. berghei or recrudescence, also in mice co-infected with S. mansoni. Co-infection with P. berghei had no effect on S. mansoni worm burden reductions following artemether-praziquantel combinations. Our findings point to the need for epidemiological studies in areas where malaria and schistosomiasis co-exist and where artemisinin-based combination therapies are introduced, since artemisinin-based combination therapies as part of a malaria control package may have ancillary benefits against schistosomiasis.     

Moderate transmission but high prevalence of malaria in Madagascar

Malaria transmission remains poorly documented in areas of low transmission. A study has been carried out over two consecutive years in Analamiranga, a village located at an altitude of 885m on the western edge of the Malagasy highlands, with the aim of generating and updating malariometric indexes for both mosquitoes and schoolchildren. In this village, no vector control measures were performed during the study period nor during previous decades. Mosquitoes were collected monthly when landing on human volunteers and in various resting-places. Blood samples were taken every 3 months from schoolchildren aged 6-12 years and microscopically examined. Of 7,480 mosquitoes collected on human subjects, 5,790 were anophelines. Ten anopheline species were represented and three of these, Anopheles funestus, Anopheles arabiensis and Anopheles mascarensis, accounted for 59.2% of the collection. Of these three species 4,640 were also collected in resting places. The proportion of mosquitoes fed on bovids was high; conversely, the anthropophilic rate (mosquitoes fed on human beings) was especially low: 31%, 7% and 1%, respectively, for A. funestus, A. arabiensis and A. mascarensis. The only confirmed malaria vector was A. funestus with a low sporozoite index (of 6,830 A. funestus, five were positive for Plasmodium falciparum and four for Plasmodium vivax). The annual entomological inoculation rate (number of bites of infected anophelines per adult person) was estimated at 2.49 with low variation over the 2 years. Overall, 909 thick blood smears were tested from blood samples taken from schoolchildren with 30.3% being malaria-positive. The four Plasmodium species infecting human subjects were detected in the following proportions: P. falciparum 78.9%, P. vivax 19.4%, Plasmodium malariae 1.0% and Plasmodium ovale 0.7%. The proportions of children who were infected with any Plasmodium ranged from 10.7% in February to 51.0% in September. Parasitemic children with fever (axillary temperature >37.5 degrees C) accounted for 16.4% of the children sampled. This study demonstrates that there are substantial parasitological consequences of even a relatively low entomological transmission and also recommends including exterior resting-places of mosquitoes in future spraying campaigns in the highlands of Madagascar.