Toll-like receptor 4, Toll-like receptor 7 and Toll-like receptor 9 agonists enhance immune responses against blood-stage Plasmodium chabaudi infection in BALB/c mice.

BACKGROUND: Toll-like receptor (TLR) signals play vital roles during the blood-stage of malaria infections. However, the roles of TLR agonists in the regulation of immune responses and the development of protective immunity to malaria remain poorly understood. METHOD: BALB/c mice were pre-treated with TLR4, TLR7 and TLR9 agonists, followed by infection with Plasmodium chabaudi. After infection, splenic dendritic cells (DCs), Th1 cells and programmed death-1 (PD-1) expressed on Th1 cells, as well as regulatory T cells (Tregs) were analyzed by flow cytometry. The levels of IFN-γ, TNF-α, TGF-ß and IL-10 in splenocytes and IgG1 and IgG2a in serum were measured by ELISA. RESULT: Administration of TLR4, TLR7 and TLR9 agonists prior to infection improved disease outcomes. All TLR agonists promoted DC activation, and the proportions of Th1 cells increased. In TLR4, TLR7 and TLR9 agonist treated groups the levels of pro-inflammatory cytokines IFN-γ and TNF-α were elevated, and IgG1 and IgG2a serum levels were also significantly increased. TLR4, TLR7 and TLR9 agonists diminished the activation of Tregs and down-regulated the anti-inflammatory cytokines TGF-ß and IL-10. Finally, PD-1 expressed on Th1 cells were decreased in TLR4, TLR7 and TLR9 agonist treated groups compared with control groups. CONCLUSION: TLR4, TLR7 and TLR9 agonists activated DC-mediated innate immune responses and adaptive immune response, which against the blood-stage of Plasmodium and might be applied to malaria protection and treatment.

Plasmodium chabaudi-infected mice spleen response to synthesized silver nanoparticles from Indigofera oblongifolia extract.

Malaria is a worldwide serious-threatening infectious disease caused by Plasmodium and the parasite resistance to antimalarial drugs has confirmed a significant obstacle to novel therapeutic antimalarial drugs. In this article, we assessed the antioxidant and anti-inflammatory activity of nanoparticles prepared from Indigofera oblongifolia extract (AgNPs) against the infection with Plasmodium chabaudi caused in mice spleen. AgNPs could significantly suppress the parasitaemia caused by the parasite to approximately 98% on day 7 postinfection with P. chabaudi and could improve the histopathological induced spleen damage. Also, AgNPs were able to increase the capsule thickness of the infected mice spleen. In addition, the AgNPs functioned as an antioxidant agent that affects the change in glutathione, nitric oxide and catalase levels in the spleen. Moreover spleen IL1ß, IL-6 and TNF-α-mRNA expression was regulated by AgNPs administration to the infected mice. These results indicated the anti-oxidant and the anti-inflammatory protective role of AgNPs against P. chabaudi-induced spleen injury.

Moringa oleifera treatment increases Tbet expression in CD4+ T cells and remediates immune defects of malnutrition in Plasmodium chabaudi-infected mice.

BACKGROUND: Malaria is a worldwide problem that affects millions of people yearly. In rural areas where anti-malarial drugs are not easily accessible, many people use herbal treatments, such as Moringa oleifera, to treat a variety of diseases and ailments including malaria. While Moringa is reported to possess potent and curative anti-malarial properties, previous studies have mostly been restricted to assessment of parasitaemia. In this study, the effect of Moringa on malaria immunity in a murine model was investigated. METHODS: Using a high dose (60 mg/mouse) for a short time (7 days) or low dose Moringa (30 mg/mouse) for a longer time (3 weeks), cytokine production, and Tbet expression by effector CD4+ T cells (Teff) were determined. Mice were also treated with Moringa after infection (curatively) or before infection (prophylactically) to determine the effect of the plant extract on parasitaemia and immunity. Given that Moringa also possess many nutritional benefits, the contribution of Moringa on malnourished malaria infected mice was determined. Malnutrition was induced by limiting access to food to only 4 h a day for 4 weeks, while control mice had unlimited access to mouse laboratory chow. All data was collected by flow cytometry and analysed using one-Way ANOVA or two tailed Student's t test. RESULTS: Moringa-treated mice had increased numbers of effector CD4+ T cells accompanied by an increase in Tbet expression compared to control untreated mice. Mice that were treated with Moringa curatively also exhibited increased effector CD4+ T cell numbers, IFN-gamma and TNF secretion. Interestingly, the mice that were treated prophylactically had significantly higher Tbet expression. In the absence of adaptive immunity, high parasitaemia was observed in the RAG1 knockout mice. The food limited mice (malnourished) had reduced numbers of CD4+ T cells, TNF proportions, and significantly greater Tbet expression compared to the control group. Supplementation with Moringa in the limited group slightly restored CD4+ T cell activation, IL-2, and IL-10 production. CONCLUSIONS: Taken together, these data suggest that Moringa treatment leads to increased CD4+ T cell activation, Th1 differentiation and production of pro-inflammatory cytokines after malaria infection. Thus, Moringa may be immunologically useful in the treatment of malaria and malnutrition. Further investigations are required to identify the active components in Moringa.

Antimalarial Activity of Human Group IIA Secreted Phospholipase A2 in Relation to Enzymatic Hydrolysis of Oxidized Lipoproteins.

The level of human group IIA secreted phospholipase A2 (hGIIA sPLA2) is increased in the plasma of malaria patients, but its role is unknown. In parasite culture with normal plasma, hGIIA is inactive against Plasmodium falciparum, contrasting with hGIIF, hGV, and hGX sPLA2s, which readily hydrolyze plasma lipoproteins, release nonesterified fatty acids (NEFAs), and inhibit parasite growth. Here, we revisited the anti-Plasmodium activity of hGIIA under conditions closer to those of malaria physiopathology where lipoproteins are oxidized. In parasite culture containing oxidized lipoproteins, hGIIA sPLA2 was inhibitory, with a 50% inhibitory concentration value of 150.0 ± 40.8 nM, in accordance with its capacity to release NEFAs from oxidized particles. With oxidized lipoproteins, hGIIF, hGV, and hGX sPLA2s were also more potent, by 4.6-, 2.1-, and 1.9-fold, respectively. Using specific immunoassays, we found that hGIIA sPLA2 is increased in plasma from 41 patients with malaria over levels for healthy donors (median [interquartile range], 1.6 [0.7 to 3.4] nM versus 0.0 [0.0 to 0.1] nM, respectively; P < 0.0001). Other sPLA2s were not detected. Malaria plasma, but not normal plasma, contains oxidized lipoproteins and was inhibitory to P. falciparum when spiked with hGIIA sPLA2 Injection of recombinant hGIIA into mice infected with P. chabaudi reduced the peak of parasitemia, and this was effective only when the level of plasma peroxidation was increased during infection. In conclusion, we propose that malaria-induced oxidation of lipoproteins converts these into a preferential substrate for hGIIA sPLA2, promoting its parasite-killing effect. This mechanism may contribute to host defense against P. falciparum in malaria where high levels of hGIIA are observed.

Effect of Indigofera oblongifolia on the Hepatic Oxidative Status and Expression of Inflammatory and Apoptotic Genes during Blood-Stage Murine Malaria.

Malaria is a dangerous disease spread across several countries. Recent studies have focused on medicinal plants to discover alternative agents to the currently used drugs for malaria treatment. Here, we investigated the potential role of Indigofera oblongifolia leaf extract (IE) on hepatic inflammation in mice with Plasmodium chabaudi-infected erythrocytes. Female C57BL/6 mice were divided into three groups. The first group served as a control noninfected group, while the second and third groups were intraperitoneally injected with 106 erythrocytes parasitized by P. chabaudi. Mice from the third group were treated daily with a dose of 100 mg/kg of IE for 7 days. IE significantly reduced the number of leukocytes and apoptotic cells. The numbers of CD68-positive cells decreased in the livers of mice from the treatment group. Moreover, IE raised the hepatic antioxidant levels (glutathione and catalase) and reduced the levels of hepatic oxidative stress markers (malondialdehyde, nitric oxide, and reactive oxygen species). IE regulated some functions of the genes related to immune responses, including apoptotic genes (B-cell lymphoma-2, Bax, and caspase-3) and cytokine genes (interleukin-1ß (IL-1ß), IL-6, interferon-γ, and tumor necrosis factor-α). Therefore, IE exerts significant effects against malaria and protects the liver from injury caused by P. chabaudi via antioxidant and anti-inflammatory ways.

Antimalarial activity of vitamin D3 (VD3) does not result from VD3-induced antimicrobial agents including nitric oxide or cathelicidin.

Recent evidence suggests that 1α,25-dihydroxyvitamin D3 (VD3), the active form of vitamin D, inhibits microbial proliferation. Previously, we used in vivo murine models to investigate the antimalarial activity of VD3 and confirmed potent antimalarial activity in the acute phase. This study aimed to clarify the mechanisms underlying the antimalarial activity of VD3 in vivo, particularly extensive inhibition of parasitemia in the acute phase, focusing on nitric oxide (NO), a potent antimalarial molecule. VD3 is a good NO inducer. When most Plasmodium chabaudi AS (PcAS)-infected mice treated with VD3 survived, NO was present in blood samples obtained from VD3-treated mice at a significantly higher rate at 2 and/or 3 days post-infection than that in vehicle-treated control mice. To verify the involvement of NO in the antimalarial activity of VD3, we used aminoguanidine (AG), an inducible NO synthase (iNOS) inhibitor, to abrogate the antimalarial activity of VD3. However, despite AG-induced reductions in NO levels, parasitemia remained inhibited during the acute phase, even in the presence of AG, and the antiplasmodial faculty of VD3 was not ablated. VD3-mediated antimalarial activity irrelevant of NO compelled us to consider another candidate. In a pilot experiment, we used cathelicidin (CAMP), an antimicrobial peptide, since it is known that VD3 induces CAMP synthesis. Serum CAMP levels increased on days 4 or 5 post-infection with or without VD3 administration, but experiments using exogenous CAMP did not display curative effects in PcAS-infected mice. The present study using VD3 to target the malarial parasite thus suggests a potential novel approach to treat malarial infections.

In vivo validation of anti-malarial activity of crude extracts of Terminalia macroptera, a Malian medicinal plant.

BACKGROUND: Plasmodium falciparum malaria is still one of the most deadly pathology worldwide. Efficient treatment is jeopardized by parasite resistance to artemisinin and its derivatives, and by poor access to treatment in endemic regions. Anti-malarial traditional remedies still offer new tracks for identifying promising antiplasmodial molecules, and a way to ensure that all people have access to care. The present study aims to validate the traditional use of Terminalia macroptera, a Malian plant used in traditional medicine. METHODS: Terminalia macroptera was collected in Mali. Leaves (TML) and roots ethanolic extracts (TMR) were prepared and tested at 2000 mg/kg for in vivo acute toxicity in Albino Swiss mice. Antiplasmodial activity of the extracts was assessed against a chloroquine resistant strain P. falciparum (FcB1) in vitro. In vivo, anti-malarial efficacy was assessed by a 4-day suppressive test at 100 mg/kg in two malaria murine models of uncomplicated malaria (Plasmodium chabaudi chabaudi infection) and cerebral malaria (Plasmodium berghei strain ANKA infection). Constituents of TMR were characterized by ultra-high-performance liquid chromatography coupled to high resolution mass spectrometry. Top ranked compounds were putatively identified using plant databases and in silico fragmentation pattern. RESULTS: Lethal dose of TML and TMR were greater than 2000 mg/kg in Albino Swiss mice. According to the OECD's Globally Harmonized System of Classification, both extracts are non-toxic orally. Antiplasmodial activity of T. macroptera extracts was confirmed in vitro against P. falciparum FcB1 strain with IC50 values of 1.2 and 1.6 µg/mL for TML and TMR, respectively. In vivo, oral administration of TML and TMR induced significant reduction of parasitaemia (37.2 and 46.4% respectively) in P. chabaudi chabaudi infected mice at the 7th day of infection compared to untreated mice. In the cerebral malaria experimental model, mice treated with TMR and TML presented respectively 50 and 66.7% survival rates at day 9 post-infection when all untreated mice died. Eleven major compounds were found in TMR. Among them, several molecules already known could be responsible for the antiplasmodial activity of the roots extract of T. macroptera. CONCLUSIONS: This study confirms both safety and anti-malarial activity of T. macroptera, thus validating its traditional use.

Heterologous Expression of a Novel Drug Transporter from the Malaria Parasite Alters Resistance to Quinoline Antimalarials.

Antimalarial drug resistance hampers effective malaria treatment. Critical SNPs in a particular, putative amino acid transporter were recently linked to chloroquine (CQ) resistance in malaria parasites. Here, we show that this conserved protein (PF3D7_0629500 in Plasmodium falciparum; AAT1 in P. chabaudi) is a structural homologue of the yeast amino acid transporter Tat2p, which is known to mediate quinine uptake and toxicity. Heterologous expression of PF3D7_0629500 in yeast produced CQ hypersensitivity, coincident with increased CQ uptake. PF3D7_0629500-expressing cultures were also sensitized to related antimalarials; amodiaquine, mefloquine and particularly quinine. Drug sensitivity was reversed by introducing a SNP linked to CQ resistance in the parasite. Like Tat2p, PF3D7_0629500-dependent quinine hypersensitivity was suppressible with tryptophan, consistent with a common transport mechanism. A four-fold increase in quinine uptake by PF3D7_0629500 expressing cells was abolished by the resistance SNP. The parasite protein localised primarily to the yeast plasma membrane. Its expression varied between cells and this heterogeneity was used to show that high-expressing cell subpopulations were the most drug sensitive. The results reveal that the PF3D7_0629500 protein can determine the level of sensitivity to several major quinine-related antimalarials through an amino acid-inhibitable drug transport function. The potential clinical relevance is discussed.

Increased Plasmodium chabaudi malaria mortality in mice with nutritional iron deficiency can be reduced by short-term adjunctive iron supplementation.

BACKGROUND: Iron deficiency is the most widespread nutrient deficiency and an important cause of developmental impairment in children. However, some studies have indicated that iron deficiency can also protect against malaria, which is a leading cause of childhood morbidity and mortality in large parts of the world. This has rendered interventions against iron deficiency in malaria-endemic areas controversial. METHODS: The effect of nutritional iron deficiency on the clinical outcome of Plasmodium chabaudi AS infection in A/J mice and the impact of intravenous iron supplementation with ferric carboxymaltose were studied before and after parasite infection. Plasma levels of the iron status markers hepcidin and fibroblast growth factor 23 were measured in animals surviving and succumbing to malaria, and accompanying tissue pathology in the liver and the spleen was assessed. RESULTS: Nutritional iron deficiency was associated with increased mortality from P. chabaudi malaria. This increased mortality could be partially offset by carefully timed, short-duration adjunctive iron supplementation. Moribund animals were characterized by low levels of hepcidin and high levels of fibroblast growth factor 23. All infected mice had extramedullary splenic haematopoiesis, and iron-supplemented mice had visually detectable intracellular iron stores. CONCLUSIONS: Blood transfusions are the only currently available means to correct severe anaemia in children with malaria. The potential of carefully timed, short-duration adjunctive iron supplementation as a safe alternative should be considered.

A potent series targeting the malarial cGMP-dependent protein kinase clears infection and blocks transmission.

To combat drug resistance, new chemical entities are urgently required for use in next generation anti-malarial combinations. We report here the results of a medicinal chemistry programme focused on an imidazopyridine series targeting the Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG). The most potent compound (ML10) has an IC50 of 160 pM in a PfPKG kinase assay and inhibits P. falciparum blood stage proliferation in vitro with an EC50 of 2.1 nM. Oral dosing renders blood stage parasitaemia undetectable in vivo using a P. falciparum SCID mouse model. The series targets both merozoite egress and erythrocyte invasion, but crucially, also blocks transmission of mature P. falciparum gametocytes to Anopheles stephensi mosquitoes. A co-crystal structure of PvPKG bound to ML10, reveals intimate molecular contacts that explain the high levels of potency and selectivity we have measured. The properties of this series warrant consideration for further development to produce an antimalarial drug.Protein kinases are promising drug targets for treatment of malaria. Here, starting with a medicinal chemistry approach, Baker et al. generate an imidazopyridine that selectively targets Plasmodium falciparum PKG, inhibits blood stage parasite growth in vitro and in mice and blocks transmission to mosquitoes.

A nutrient mediates intraspecific competition between rodent malaria parasites in vivo.

Hosts are often infected with multiple strains of a single parasite species. Within-host competition between parasite strains can be intense and has implications for the evolution of traits that impact patient health, such as drug resistance and virulence. Yet the mechanistic basis of within-host competition is poorly understood. Here, we demonstrate that a parasite nutrient, para-aminobenzoic acid (pABA), mediates competition between a drug resistant and drug susceptible strain of the malaria parasite, Plasmodium chabaudi We further show that increasing pABA supply to hosts infected with the resistant strain worsens disease and changes the relationship between parasite burden and pathology. Our experiments demonstrate that, even when there is profound top-down regulation (immunity), bottom-up regulation of pathogen populations can occur and that its importance may vary during an infection. The identification of resources that can be experimentally controlled opens up the opportunity to manipulate competitive interactions between parasites and hence their evolution.

Dual antiplasmodial activity of vitamin D3 and its analog, 22-oxacalcitriol, by direct and indirect mechanisms.

Recent evidence suggests that 1α,25-dihydroxyvitamin D3 (calcitriol, VD3), the active form of vitamin D (VD), can inhibit the proliferation of microorganisms. In the present study, we conducted in vitro experiments and utilized in vivo murine models to investigate the antimalarial activity of VD3 and its analog, 22-oxacalcitriol (22-OCT), which was designed to cause less hypercalcemia than VD3. VD3 and 22-OCT treatments effectively resolved a Plasmodium chabaudi (Pc) infection in wild-type mice. Reduced parasitemia was observed during the acute phase of infection in the presence of VD3 and 22-OCT, followed by a delayed peak during the chronic stage of infection. Some anti-Pc activity was observed in VD receptor knockout (KO) mice. VD3 and 22-OCT also completely inhibited the proliferation of P. falciparum (Pf) in human red blood cells in vitro. Plasma levels of interferon (IFN)-γ in VD3-treated B10 and B6 mice were lower than those in vehicle-treated animals, and VD3 resolved a Pc infection in IFN-γ-KO mice, which greatly improved survival. These data suggest that the protective effects of VD3 are elicited through an IFN-γ-independent mechanism. Effective antiplasmodial doses of VD3 and 22-OCT resulted in a loss of body weight in mice. This loss in body weight occurred concomitantly with the development of hypercalcemia. Zoledronic acid partially attenuated VD3-induced hypercalcemia and abrogated the antiparasitic effects of VD3. This study highlights a potential therapeutic role for VD3 in the treatment of malarial infections and shows that hypercalcemia is excellent indicator of the antiplasmodial activity of VD3.

In vivo assessment of the antimalarial and spleen-protective activities of the Saudi propolis methanolic extract.

Antimalarial drug resistance is the main therapeutic challenge to the control of the disease, making the search for new compounds as alternative treatments of central importance. Propolis has a long history of medicinal use due to its antifungal, antibacterial and antiprotozoal properties. The present study therefore aimed to evaluate the antimalarial activity of the Saudi propolis methanolic extract against Plasmodium chabaudi infection in mice. To this end, albino mice were divided into five groups: the first group was the normal control; the second, third, fourth and fifth groups were infected intraperitoneally with 106 P. chabaudi-parasitized erythrocytes. The last three groups of mice were gavaged with 100 µl of propolis extract (PE) at a dose of 25, 50 and 100 mg PE/kg, respectively, once daily for 7 days. PE significantly suppressed the parasitaemia and showed significant efficacy in ameliorating anaemic conditions in P. chabaudi-infected mice in a dose-dependent manner. Histological investigation of the spleen tissue of treated and untreated mice further supports the antimalarial potential of PE. In addition, our study proved that Saudi PE reduced oxidative damage by decreasing the malondialdehyde (MDA) and increasing the catalase (CAT) activity and the glutathione (GSH) levels. Also, Saudi PE increased the level of some pro-inflammatory cytokines such as IFN-γ, TNF-α, GM-CSF and G-CSF, with the most effective dose being 100 mg PE/kg. In conclusion, PE showed antimalarial and antioxidant activities and provided protection against spleen tissue damage in P. chabaudi-infected mice.

Exploring the antimalarial potential of whole Cymbopogon citratus plant therapy.

ETHNOPHARMACOLOGICAL RELEVANCE: Cymbopogon citratus (lemon grass) has been used in traditional medicine as an herbal infusion to treat fever and malaria. Generally, whole plant extracts possess higher biological activity than purified compounds. However, the antimalarial activity of the whole C. citratus plant has not been experimentally tested. AIM OF THE STUDY: To evaluate the antimalarial activity of an herbal infusion and the whole Cymbopogon citratus plant in two experimental models of malaria. MATERIAL AND METHODS: The plant was dried for 10 days at room temperature and was then milled and passed through brass sieves to obtain a powder, which was administered to CBA/Ca mice with a patent Plasmodium chabaudi AS or P. berghei ANKA infection. We analysed the effects of two different doses (1600 and 3200mg/kg) compared with those of the herbal infusion and chloroquine, used as a positive control. We also assessed the prophylactic antimalarial activities of the whole C. citratus plant and the combination of the whole plant and chloroquine. RESULTS: The C. citratus whole plant exhibited prolonged antimalarial activity against both P. chabaudi AS and P. berghei ANKA. The low dose of the whole C. citratus plant displayed higher antimalarial activity than the high dose against P. berghei ANKA. As a prophylactic treatment, the whole plant exhibited higher antimalarial activity than either the herbal infusion or chloroquine. In addition, the combination of the whole C. citratus plant and chloroquine displayed higher activity than chloroquine alone against P. berghei ANKA patent infection. CONCLUSIONS: We demonstrated the antimalarial activity of the whole C. citratus plant in two experimental models. The whole C. citratus plant elicited higher anti-malarial activity than the herbal infusion or chloroquine when used as a prophylactic treatment. The antimalarial activity of the whole C. citratus plant supports continued efforts towards developing whole plant therapies for the management of malaria and other infectious diseases prevalent in resource-poor communities.

Cysteamine broadly improves the anti-plasmodial activity of artemisinins against murine blood stage and cerebral malaria.

BACKGROUND: The potential emergence and spread of resistance to artemisinins in the Plasmodium falciparum malaria parasite constitutes a major global health threat. Hence, improving the efficacy of artemisinins and of artemisinin-based combination therapy (ACT) represents a major short-term goal in the global fight against malaria. Mice defective in the enzyme pantetheinase (Vnn3) show increased susceptibility to blood-stage malaria (increased parasitaemia, reduced survival), and supplementation of Vnn3 mutants with the reaction product of pantetheinase, cysteamine, corrects in part the malaria-susceptibility phenotype of the mutants. Cysteamine (Cys) is a small, naturally occurring amino-thiol that has very low toxicity in vivo and is approved for clinical use in the life-long treatment of the kidney disorder nephropathic cystinosis. METHODS: The ability of Cys to improve the anti-plasmodial activity of different clinically used artemisinins was tested. The effect of different CYS/ART combinations on malarial phenotypes (parasite blood-stage replication, overall and survival from lethal infection) was assessed in a series of in vivo experiments using Plasmodium strains that induce either blood-stage (Plasmodium chabaudi AS) or cerebral disease (Plasmodium berghei ANKA). This was also evaluated in an ex vivo experimental protocol that directly assesses the effect of such drug combinations on the viability of Plasmodium parasites, as measured by the ability of tested parasites to induce a productive infection in vivo in otherwise naïve animals. RESULTS: Cys is found to potentiate the anti-plasmodial activity of artesunate, artemether, and arteether, towards the blood-stage malaria parasite P. chabaudi AS. Ex vivo experiments, indicate that potentiation of the anti-plasmodial activity of artemisinins by Cys is direct and does not require the presence of host factors. In addition, potentiation occurs at sub-optimal concentrations of artemisinins and Cys that on their own have little or no effect on parasite growth. Cys also dramatically enhances the efficacy and protective effect of artemisinins against cerebral malaria induced by infection with the P. berghei ANKA parasite. CONCLUSION: These findings indicate that inclusion of Cys in current formulations of ACT, or its use as adjunct therapy could improve the anti-plasmodial activity of artemisinin, decrease mortality in cerebral malaria patients, and prevent or delay the development and spread of artemisinin resistance.

Structure- and function-based design of Plasmodium-selective proteasome inhibitors.

The proteasome is a multi-component protease complex responsible for regulating key processes such as the cell cycle and antigen presentation. Compounds that target the proteasome are potentially valuable tools for the treatment of pathogens that depend on proteasome function for survival and replication. In particular, proteasome inhibitors have been shown to be toxic for the malaria parasite Plasmodium falciparum at all stages of its life cycle. Most compounds that have been tested against the parasite also inhibit the mammalian proteasome, resulting in toxicity that precludes their use as therapeutic agents. Therefore, better definition of the substrate specificity and structural properties of the Plasmodium proteasome could enable the development of compounds with sufficient selectivity to allow their use as anti-malarial agents. To accomplish this goal, here we use a substrate profiling method to uncover differences in the specificities of the human and P. falciparum proteasome. We design inhibitors based on amino-acid preferences specific to the parasite proteasome, and find that they preferentially inhibit the ß2-subunit. We determine the structure of the P. falciparum 20S proteasome bound to the inhibitor using cryo-electron microscopy and single-particle analysis, to a resolution of 3.6 Å. These data reveal the unusually open P. falciparum ß2 active site and provide valuable information about active-site architecture that can be used to further refine inhibitor design. Furthermore, consistent with the recent finding that the proteasome is important for stress pathways associated with resistance of artemisinin family anti-malarials, we observe growth inhibition synergism with low doses of this ß2-selective inhibitor in artemisinin-sensitive and -resistant parasites. Finally, we demonstrate that a parasite-selective inhibitor could be used to attenuate parasite growth in vivo without appreciable toxicity to the host. Thus, the Plasmodium proteasome is a chemically tractable target that could be exploited by next-generation anti-malarial agents.

Antimalarial and antioxidant activities of Indigofera oblongifolia on Plasmodium chabaudi-induced spleen tissue injury in mice.

Malaria is still one of the most common infectious diseases and leads to various public health problems worldwide. Medicinal plants are promising sources for identifying novel agents with potential antimalarial activity. This study aimed to investigate the antimalarial and the antioxidant activities of Indigofera oblongifolia on Plasmodium chabaudi-induced spleen tissue injury in mice. Mice were divided into five groups. The first group served as a vehicle control; the second, third, fourth, and fifth groups were infected with 1 × 10(6) P. chabaudi-parasitized erythrocytes. Mice of the last three groups were gavaged with 100 µl of I. oblongifolia leave extract (IOLE) at a dose of 100, 200, and 300 mg IOLE/kg, respectively, once daily for 7 days. IOLE was significantly able to lower the percentage of parasitemia. The most effective dose was the 100 mg IOLE/kg, which could reduce the parasitemia from about 38 to 12 %. The infection induced spleen injury. This was evidenced by disorganization of spleen white and red pulps, appearance of hemozoin granules and parasitized erythrocytes. These changes in spleen led to the increased histological score. Also, the infection increased the spleen oxidative damage where the levels of nitrite/nitrate, malondialdehyde, and catalase were significantly altered. All these infection-induced parameters were significantly improved during IOLE treatment. In addition, the mRNA expression of inflammatory cytokines interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha were upregulated after infection with P. chabaudi, whereas IOLE significantly reduced the expression of these genes. Our results indicate that I. oblongifolia leaves extract exhibits a significant antimalarial and antioxidant effects, and protects host spleen tissue from injuries induced by P. chabaudi.

Relevance of undetectably rare resistant malaria parasites in treatment failure: experimental evidence from Plasmodium chabaudi.

Resistant malaria parasites are frequently found in mixed infections with drug-sensitive parasites. Particularly early in the evolutionary process, the frequency of these resistant mutants can be extremely low and below the level of molecular detection. We tested whether the rarity of resistance in infections impacted the health outcomes of treatment failure and the potential for onward transmission of resistance. Mixed infections of different ratios of resistant and susceptible Plasmodium chabaudi parasites were inoculated in laboratory mice and dynamics tracked during the course of infection using highly sensitive genotype-specific quantitative polymerase chain reaction (qPCR). Frequencies of resistant parasites ranged from 10% to 0.003% at the onset of treatment. We found that the rarer the resistant parasites were, the lower the likelihood of their onward transmission, but the worse the treatment failure was in terms of parasite numbers and disease severity. Strikingly, drug resistant parasites had the biggest impact on health outcomes when they were too rare to be detected by any molecular methods currently available for field samples. Indeed, in the field, these treatment failures would not even have been attributed to resistance.

Is selection relevant in the evolutionary emergence of drug resistance?

The emergence of drug-resistant pathogens is often considered a canonical case of evolution by natural selection. Here we argue that the strength of selection can be a poor predictor of the rate of resistance emergence. It is possible for a resistant strain to be under negative selection and still emerge in an infection or spread in a population. Measuring the right parameters is a necessary first step toward the development of evidence-based resistance-management strategies. We argue that it is the absolute fitness of the resistant strains that matters most and that a primary determinant of the absolute fitness of a resistant strain is the ecological context in which it finds itself.

Blood-stage immunity to Plasmodium chabaudi malaria following chemoprophylaxis and sporozoite immunization.

Protection against malaria in humans can be achieved by repeated exposure to infected mosquito bites during prophylactic chloroquine treatment (chemoprophylaxis and sporozoites (CPS)). We established a new mouse model of CPS immunization to investigate the stage and strain-specificity of malaria immunity. Immunization with Plasmodium chabaudi by mosquito bite under chloroquine cover does not generate pre-erythrocytic immunity, which is acquired only after immunization with high sporozoite doses. Instead, CPS immunization by bite elicits long-lived protection against blood-stage parasites. Blood-stage immunity is effective against a virulent, genetically distinct strain of P. chabaudi. Importantly, if exposure to blood-stage parasitemia is extended, blood-stage parasites induce cross-stage immunity targeting pre-erythrocytic stages. We therefore show that CPS immunization can induce robust, long-lived heterologous blood-stage immunity, in addition to protection against pre-erythrocytic parasites following high dose sporozoite immunization. Cross-stage immunity elicited by blood-stage parasites may further enhance efficacy of this immunization regimen.