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The quest for the development of a novel antimalarial drug informed the decision to subject phytol to in vivo trials following a demonstration of therapeutic potential against chloroquine sensitive strain of Plasmodium falciparum under in vitro condition. On this basis, the in vivo anti-Plasmodium berghei activity of phytol including the ameliorative effects of the compound on P. berghei-associated anaemia and organ damage were investigated. Mice were infected with chloroquine-sensitive strain of P. berghei and were treated with phytol at a dose of 10 and 20 mg/kg body weight (BW) for four days. The levels of parasitemia, packed cell volume and redox sensitive biomarkers of liver, brain and spleen tissues were determined. Our result revealed that phytol significantly (p < 0.05) suppressed the multiplication of P. berghei in a dose-dependent manner. Additionally, the phytol significantly (p < 0.05) ameliorated the P. berghei-induced anaemia and brain damage. Data from the present study demonstrated that phytol has suppressive effect on P. berghei and could ameliorate some P. berghei-induced pathological changes., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A hallmark of mortality and morbidity, malaria is affecting nearly half of the world's population. Emergence of drug-resistant strains of malarial parasite prompts identification and evaluation of medicinal plants and their constituents that may hold the key to a new and effective anti-malarial drug. In this context, nineteen methanolic extracts from seventeen medicinal plants were evaluated for anti-plasmodial potential against Plasmodium falciparum strain 3D7 (Chloroquine (CQ) sensitive) and INDO (CQ resistant) using fluorescence based SYBR-Green assay and for cytotoxic effects against mammalian cell lines. Leaf extract of two plants showed promising in vitro anti-malarial activity (Pf3D7 IC 50  ≤ 10 μg/ml); one plant extract showed good activity (Pf3D7 IC 50  = 10.1-20 μg/ml); seven were moderately active (IC 50  = 20.1-50 μg/ml), four plant extracts showed poor activity (PfD7 IC 50  = 50.1-100 μg/ml) and five extracts showed no activity up to IC 50  = 100 μg/ml. Further, six extracts were found equipotent to PfINDO (resistance index ranging 0.4-2) and relatively nontoxic to mammalian cell lines HEK293 (cytotoxicity index ranging 1.4-12.5). Based on good resistance and selectivity indices, three extracts were evaluated for in vivo activity in Plasmodium berghei ANKA infected mice at a dose of 500 mg/kg and they showed significant suppression of P. berghei parasitemia. Further, these active plant extracts were fractionated using silica-gel chromatography and their fractions were evaluated for anti-plasmodial action. Obtained fractions showed enrichment in antimalarial activity. Active fractions were analyzed by gas chromatography and mass-spectrometery. Results suggests that the three active plant extracts could serve as potent source of anti-malarial agent and therefore require further analysis., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The development and spread of multidrug-resistant strains of malarial parasites have led to an overwhelming increase in the resistance to current antimalarial drugs. The urgent need for alternative antimalarial drugs has directed some of the current studies toward folkloric medicine approaches. Interestingly, the Zizyphus spina Cristi leaf extract (ZLE) has been found to exhibit antiplasmodial activity. This study evaluated the protective effect of ZLE against Plasmodium berghei-induced cerebral tissue injuries in mice. Male C57Bl/6 mice received an injection of P. berghei-infected red blood cells. Mice were divided into three groups (control, infected, and ZLE-treated), and were subjected to histological, biochemical, and molecular analyses. Murine malaria infections induced significant weight loss; however, upon ZLE treatment, the weight of mice was markedly restored. Additionally, infected mice showed brain histopathological changes and induction of oxidative damage. Significantly, ZLE treatment restored the levels of oxidative markers and antioxidant enzyme to the normal ranges. The mRNA expression of several genes in the brain of mice including Cacnb4, Adam23, Glrb, Vdac3, and Cabp1 was significantly upregulated during P. berghei infection. In contrast, ZLE markedly reduced the mRNA expression of these genes. To conclude, the results indicate that ZLE could play an important role in reducing the destructive effect of P. berghei-induced cerebral malaria owing to its antiplasmodial and antioxidant activities., (Copyright © 2017. Published by Elsevier Ltd.)

Theileria equi and Babesia caballi are the causative agents of equine piroplasmosis (EP), which affects equine production in various parts of the world. However, a safe and effective drug is not currently available for treatment of EP. Dihydroorotate dehydrogenase (DHODH) is the fourth enzyme in the de novo pyrimidine synthesis pathway and has been known as a novel drug target for several apicomplexan protozoan parasites. In this study, we evaluated four DHODH inhibitors; atovaquone (ATV), leflunomide (LFN), brequinar (Breq), and 7-hydroxy-5-[1,2,4] triazolo [1,5,a] pyrimidine (TAZ) on the growth of T. equi and B. caballi in vitro and compared them to diminacene aceturate (Di) as the control drug. The growth of T. equi and B. caballi was significantly hindered by all inhibitors except TAZ. The half maximal inhibitory concentration (IC 50 ) of ATV, LFN, Breq and Di against T. equi was approximately 0.028, 109, 11 and 40 μM, respectively, whereas the IC 50 of ATV, LFN, Breq and Di against B. caballi was approximately 0.128, 193, 5.2 and 16.2 μM, respectively. Using bioinformatics and Western blot analysis, we showed that TeDHODH was similar to other Babesia parasite DHODHs, and confirmed that targeting DHODHs could be useful for the development of novel chemotherapeutics for treatment of EP., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Phenylhydrazine (PHZ) treatment is generally used to enhance parasitemia in infected mice models. Transient reticulocytosis is commonly observed in iron-deficient anemic hosts after treatment with iron supplementation, and is also associated with short-term hemolysis caused by PHZ treatment. In this study, we investigated the relationship between reticulocytosis and cerebral malaria (CM) in a murine model induced by PHZ administration before Plasmodium berghei ANKA (PbA) infection. Mortality and parasitemia were checked daily. Pro-inflammatory cytokines and IL-10 were quantified by ELISA. The expression of CXCL9, CXCL10, CCL5, and CXCR3 mRNAs was determined by real-time PCR. Brain sequestration of CD4(+) and CD8(+) T cells and populations of splenic Th1 CD4(+) T cells, dendritic cells (DCs), CD11b(+) Gr1(+) cells, and regulatory T cells (Tregs) were assessed by FACS. PHZ administration dramatically increased parasitemia from day 3 to day 5 post infection (p.i.) compared with the untreated control infected mice group; also, CM developed at day 5 p.i., compared with day 7 p.i. in untreated control infected mice, as well as significantly decreased blood-brain barrier function (P < 0.001). PHZ administration during PbA infection significantly increased the expression of CXCL9 (P <0.05) and VCAM-1 (P <0.001) in the brain, increased the expression of CXCL10, CCL5 and CXCR3, and significantly increased the recruitment of CD4(+) and CD8(+) T cells (P <0.001 and P <0.01, respectively) as well as CD11b(+) Gr1(+) cells to the brain. In addition, PHZ administration significantly increased the numbers of IL-12-secreting DCs at days 3 and 5 p.i. compared to those of untreated control infected mice (P <0.001 and P <0.01, respectively). Consequently, the activation of CD4(+) T cells, especially the expansion of the Th1 subset (P <0.05), was significantly and dramatically enhanced and was accompanied by marked increases in the production of protein and/or mRNA of the Th1-type pro-inflammatory mediators, IFN-γ and TNF-α (P <0.01 for both for protein; P <0.05 for TNF-α mRNA). Our results suggest that, compared to healthy individuals, people suffering from reticulocytosis may be more susceptible to severe malaria infection in malaria endemic areas. This has implications for the most appropriate selection of treatment, which may also cause reticulocytosis in patients living in such areas., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Plasmodium parasites degrade hemoglobin producing reactive oxygen species as toxic byproducts which are detoxified by a series of antioxidant mechanisms. Quinoline compounds have demonstrated activity against hemoglobin degradation with 5,8-dimethylthieno[2,3-b]quinoline-2-carboxylic acid (TQCA) representing a recent compound inhibiting this process. Thus, this study was undertaken to determine the ability of TQCA to modify the oxidative status in Plasmodium berghei-infected erythrocytes. After hemolysis, activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and dehydrogenase enzymes as well as lipid peroxidation were investigated by spectrophotometry. Saturated and unsaturated fatty acids were determined by gas-liquid chromatography and the in vivo effects of TQCA were confirmed by a malaria murine model (Rane test). The activity of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) in infected cells was diminished by this compound compared to control infection in 75.1 ± 3.5% and 26.5 ± 0.3%, respectively, while that of GPx and GR was also lowered (p <0.05). As an adaptive response we appreciated a 2.3-fold increase of SOD activity compared to control infection. Lipid peroxidation and the saturated/unsaturated fatty acids ratio were also decreased by this quinoline derivate in 49.2 ± 1.32% and 37 ± 0.06%, respectively, protecting the cells from hemolysis caused by the infection. The in vitro results were in concordance with the potential in vivo activity of this compound in an established malaria murine model in which TQCA showed significant decrease in the parasitemia levels and increased the mean survival days of infected mice. In conclusion, the antioxidant defense represents a biochemical target for TQCA actions as a potent antimalarial whose effects were also confirmed in vivo., (Copyright © 2015 Elsevier Inc. All rights reserved.)

We investigated the mechanisms of resistance of two antimalarial drugs piperaquine (PQ) and lumefantrine (LM) using the rodent parasite Plasmodium berghei as a surrogate of the human parasite, Plasmodium falciparum. We analyzed the whole coding sequence of Plasmodium berghei chloroquine resistance transporter (Pbcrt) and Plasmodium berghei multidrug resistance gene 1(Pbmdr-1) for polymorphisms. These genes are associated with quinoline resistance in Plasmodium falciparum. No polymorphic changes were detected in the coding sequences of Pbcrt and Pbmdr1 or in the mRNA transcript levels of Pbmdr1. However, our data demonstrated that PQ and LM resistance is achieved by multiple mechanisms that include elevated mRNA transcript levels of V-type H(+) pumping pyrophosphatase (vp2), Ca(2+)/H(+) antiporter (vcx1), gamma glutamylcysteine synthetase (ggcs) and glutathione-S-transferase (gst) genes, mechanisms also known to contribute to chloroquine resistance in P. falciparum and rodent malaria parasites. The increase in ggcs and gst transcript levels was accompanied by high glutathione (GSH) levels and elevated activity of glutathione-S-transferase (GST) enzyme. Taken together, these results demonstrate that Pbcrt and Pbmdr1 are not associated with PQ and LM resistance in P. berghei ANKA, while vp2, vcx1, ggcs and gst may mediate resistance directly or modulate functional mutations in other unknown genes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Curcuminoids are poorly water-soluble compounds with promising antimalarial activity. To overcome some of the drawbacks of curcuminoids, we explored the potential of liposomes for the intravenous delivery of curcuminoids in a model of mouse malaria. The curcuminoids-loaded liposomes were formulated from phosphatidylcholine (soy PC) by the thin-film hydration method. Antimalarial activity of curcuminoids-loaded liposomes alone and in combination with α/β arteether when administered intravenously, was evaluated in Plasmodium berghei infected mice. Animals treated with curcuminoids-loaded liposomes showed lower parasitemia and higher survival when compared to control group (no treatment). Importantly, the combination therapy of curcuminoids-loaded liposomes (40 mg/kg body wt) along with α/β arteether (30 mg/kg body wt) was able to not only cure infected mice but also prevented recrudescence. These data suggest that curcuminoids-loaded liposomes may show promise as a formulation for anti-malarial therapy., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Piperazine and pyrrolidine derivatives were synthesised and evaluated for their capacity to inhibit the growth of Plasmodium falciparum chloroquine-resistant (FCR-3) strain in culture. The combined presence of a hydroxyl group, a propane chain and a fluor were shown to be crucial for the antiplasmodial activity. Five compounds of the aryl-alcohol series inhibited 50% of parasite growth at doses ≤10 μM. The most active compound 1-(4-fluoronaphthyl)-3-[4-(4-nitro-2-trifluoromethylphenyl)piperazin-1-yl] propan-1-ol was almost 20-40 times more active on P. falciparum (IC(50): 0.5 μM) than on tumorogenic and non-tumorogenic cells. In vivo it has a very weak effect; inhibiting 35% of parasite growth only, at 10 mg/kg/day against Plasmodium berghei infected mice without any impact on survival time. In silico molecular docking study and molecular electrostatic potential calculation revealed that this compound bound to the active site of Plasmodium plasmepsin II enzyme., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The infection by the malaria parasite of its mammalian host is initiated by the asexual reproduction of the parasite within the host hepatocyte. Before the reproduction, the elongated sporozoites undergo a depolarizing morphogenesis to the spherical exo-erythrocytic form (EEF). This change can be induced in vitro by shifting the environmental conditions, in the absence of host hepatocytes. Using rodent malaria parasites expressing a FRET-based calcium sensor, YC3.60, we observed that the intracellular calcium increased at the center of the bulbous structure during sporozoite transformation. Modulators of intracellular calcium signaling (A23187 and W-7) accelerated the sporozoite-rounding process. These data suggest that calcium signaling regulates the morphological development of the malaria parasite sporozoite to the EEF, and support a fundamental role for calcium as a universal transducer of external stimuli in the parasitic life cycle., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Naphthoquine (NQ), as a component of ARCO® which composed of NQ and artemisinin, is a new 4-aminoquinoline antimalarial synthesized by our institute. Here, a naphthoquine-resistant line of rodent malaria parasite was selected through exposing Plasmodium berghei Keyberg 173 strain to progressively increased drug pressure. The selected strain showed a more than 200-fold decreased susceptibility to NQ with a stable resistance phenotype after 10 serial passages without drug pressure or when cryopreserved over a period of 12 months. In a cross-resistance assay, the susceptibility of NQ-resistant parasites to chloroquine was decreased by 14.5-fold. These findings imply NQ-resistant parasites might be selected by long-term usage of NQ in epidemic areas and the efficacy of NQ or ARCO® in chloroquine-resistant Plasmodium falciparum epidemic areas should be monitored closely., (Copyright © 2010 Elsevier Inc. All rights reserved.)

There is a great need of new drugs against malaria because of the increasing spread of parasite resistance against the most commonly used drugs in the field. We found that monensin, a common veterinary antibiotic, has a strong inhibitory effect in Plasmodium berghei and Plasmodium yoelii sporozoites hepatocyte infection in vitro. Infection of host cells by another apicomplexan parasite with a similar mechanism of host cell invasion, Toxoplasma tachyzoites, was also inhibited. Treatment of mice with monensin abrogates liver infection with P. berghei sporozoites in vivo. We also found that at low concentrations monensin inhibits the infection of Plasmodium sporozoites by rendering host cells resistant to infection, rather than having a direct effect on sporozoites. Monensin effect is targeted to the initial stages of parasite invasion of the host cell with little or no effect on development, suggesting that this antibiotic affects an essential host cell component that is required for Plasmodium sporozoite invasion., (Copyright 2010 Elsevier Inc. All rights reserved.)

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.

We have selected piperaquine (PQ) and lumefantrine (LM) resistant Plasmodium berghei ANKA parasite lines in mice by drug pressure. Effective doses that reduce parasitaemia by 90% (ED(90)) of PQ and LM against the parent line were 3.52 and 3.93 mg/kg, respectively. After drug pressure (more than 27 passages), the selected parasite lines had PQ and LM resistance indexes (I(90)) [ED(90) of resistant line/ED(90) of parent line] of 68.86 and 63.55, respectively. After growing them in the absence of drug for 10 passages and cryo-preserving them at -80 degrees C for at least 2 months, the resistance phenotypes remained stable. Cross-resistance studies showed that the PQ-resistant line was highly resistant to LM, while the LM-resistant line remained sensitive to PQ. Thus, if the mechanism of resistance is similar in P. berghei and Plasmodium falciparum, the use of LM (as part of Coartem) should not select for PQ resistance.

Resistance to antimalarial antifolates necessitates a search for new antimetabolites targeting other enzymes of the folate metabolic pathway. In this study, 5-fluoroorotate (FOA), reported to be an inhibitor of thymidylate synthase, was assayed against Plasmodium berghei NK 65 in mice, with(out) an oral uridine supplement. FOA (2.5 and 5.0 mg/kg bw.) was tested alone, or in a double and triple combination with a fixed oral dose of 1.25 and 2.5 mg/kg of pyrimethamine (PYR); 1.0 and 2.0 mg/kg of dapsone (DAP); 1.0 and 2.0 mg/kg of artesunate (ART). FOA achieved high suppression which ranged from 95.7% to aparasitaemic, activity that was dose-dependent. At the highest dosages used, FOA-PYR and FOA-DAP-ART combinations were synergistic with 100% cure rate, while FOA-PYR-ART was antagonistic. Drugs in a synergistic combination may exert less resistance selection pressure, thus FOA-PYR and FOA-DAP-ART warrant further evaluation with an ultimate object of possible clinical use against drug-resistant malaria.

It was earlier hypothesized that the malarial parasite may convert precursors of folate analogues to synthesize de novo inhibitors toxic to itself, but not to the mammalian cell. It was suggested that one such analogue, 2,4-diamino-6-hydroxymethylpteridine (DAP) may be converted to aminopterin (AMP), a known dihydrofolate reductase inhibitor. In the present study, we evaluated the ability of DAP to inhibit proliferation of Plasmodium berghei NK65 in mice, with(out) folinic acid rescue. Cumulative dosages of DAP ranging from 0.1 to 20mg/kg bw. administered either orally or intraperitoneally showed no suppression of parasite growth, or gave mild activities that were not statistically significant (P>0.05). Our findings do not seem to support the hypothesis of selective de novo metabolism of DAP to AMP by the malarial parasite.

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.

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.

While seeking strategies for interfering with Plasmodium development in vertebrate/invertebrate hosts, we tested the activity of gomesin, an antimicrobial peptide isolated from the hemocytes of the spider Acanthoscurria gomesiana. Gomesin was tested against asexual, sexual and pre-sporogonic forms of Plasmodium falciparum and Plasmodium berghei parasites. The peptide inhibited the in vitro growth of intraerythrocytic forms of P. falciparum. When gomesin was added to in vitro culture of P. berghei mature gametocytes, it significantly inhibited the exflagellation of male gametes and the formation of ookinetes. In vivo, the peptide reduced the number of oocysts of both Plasmodium species in Anopheles stephensi mosquitoes, and did not appear to affect the mosquitoes. These properties make gomesin an excellent candidate as a transmission blocking agent for the genetic engineering of mosquitoes.

RBx11160 (OZ277) is a promising antimalarial drug candidate that Ranbaxy Laboratories Limited and Medicines for Malaria Venture (MMV) are currently developing as a fixed combination with piperaquine. Here, we describe the in vitro (Plasmodium falciparum) and in vivo (Plasmodium berghei) activities of piperaquine in combination with RBx11160 and artemether. In vitro, both combinations demonstrated a slight tendency towards antagonism with mean sums of fractional inhibitory concentrations (mean Sigma FICs) of 1.5. In vivo, piperaquine and artemether were borderline antagonistic (mean Sigma FIC of 1.4). However, an additive in vivo interaction of piperaquine and RBx11160 (mean Sigma FIC of 1.1) was identified, suggesting that a RBx11160-piperaquine combination therapy in humans should allow each molecule to exert its full antimalarial effect.

Bisquinoline compounds have exhibited remarkable activity in vitro and in vivo against Plasmodium parasites by inhibition of heme detoxification. We have tested the ability of dequalinium 1,1'-(1,10-decanediyl)bis(4-amino-2-methylquinoline), a known antimicrobial agent, to inhibit beta-hematin synthesis using a non-emzymatic colorimetric assay and globin proteolysis by electrophoretic analysis (SDS-PAGE-15%). Dequalinium was able to inhibit both processes in vitro with close correlation to a murine malaria model, reducing parasitemia levels, prolonging the survival time post-infection and curing 40% of infected mice using a combination therapy with a loading dose of chloroquine. These results confirm that dequalinium is a promising lead for antimalarial drug development.

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.

Plasmodium berghei: The effect of five protease inhibitors, TPCK, TLCK, PMSF, leupeptin, and 1,10-phenanthroline on in vitro gametogenesis and early zygote development of P. berghei was investigated. PMSF and leupeptin showed no effect. Cysteine/serine protease inhibitors TPCK/TLCK at concentrations of 75 and 100 microM were effective on inhibiting exflagellation center formation, and this effect was reversible with the addition of l-cysteine. Exflagellation center formation was most effectively blocked by 1,10-phenanthroline (1mM), and exflagellation center numbers were restored by the addition of Zn(2+). A reduction of ookinete production was observed when TPCK/TLCK (100 microM) was added at 2h after gametogenesis, but no effect was observed with 1,10-phenanthroline (1mM). Our results suggest that proteolysis is important in both gametocyte activation and sexual development of P. berghei.

Recrudescence of Plasmodium berghei NK65 infection was studied to examine factors affecting recrudescence. Treatment with a high dose of chloroquine did not prevent recrudescence, but an extended duration of treatment suppressed the frequency of recrudescence. Infection with a larger number of parasites also resulted in more frequent recrudescences. Recrudescent parasites were as sensitive to chloroquine as those before treatment. Splenectomized mice were administered carbon particles, infected, and treated with chloroquine. Recrudescence was significantly more frequent in these mice than in mice given a sham operation and PBS. The results do not suggest that merozoite stages escape the effect of chloroquine by 'hiding' in phagocytes, but that latent parasites such as dormant ring stages may cause recrudescence.

We studied the transmission-blocking effect of isonicotinic acid hydrazide (INH), a widely used anti-tuberculosis drug, against Plasmodium gallinaceum and Plasmodium berghei. INH-treatment of infected animals did not inhibit parasite development in the blood of the vertebrate host, but did inhibit exflagellation, ookinete formation, and oocyst development in the mosquito. Oocyst development was inhibited in a dose-dependent manner. The ED(50) in the P. gallinaceum/chicken/Aedes aegypti model and P. berghei/mouse/Anopheles stephensi model was 72 and 109 mg/kg, respectively. In marked contrast, in vitro exflagellation and ookinete development were not directly affected by physiological concentrations of INH. We suggest that INH exerts its inhibitory effects on the mosquito stages of the malaria parasite by an indirect, and at present undefined mechanism. Further elucidation of the mechanism how INH inhibits parasite development specifically on mosquito stages may allow us to identify new targets for malaria control strategy.

The rapid emergence of multidrug-resistant Plasmodium falciparum is a worldwide concern. Despite the magnitude of the problem, the mechanisms involved in this phenomenon are not well understood. One current proposal suggests that toxic heme molecules are degraded by glutathione (GSH), and that anti-malarial drugs, such as chloroquine (CQ), inhibit this degradation, thus implicating GSH in drug resistance. Furthermore, in some strains of Plasmodium berghei and P. falciparum, chloroquine resistance is accompanied by an increase in glutathione levels and increased activity in GSH-related enzymes. We are investigating the relationship between the gamma-glutamylcysteine synthetase (ggcs) gene, the rate-limiting enzyme in de novo synthesis of GSH, and drug resistance in P. berghei at the molecular level. In this report, we have demonstrated an increase in pbggcs mRNA levels associated with CQ and mefloquine (MFQ) resistance. In addition, the pbggcs gene locus structure was shown to be similar and localized to chromosome 8 in four parasite lines of P. berghei with different drug resistance profiles. This work suggests a link between increased GSH levels and drug resistance in Plasmodium.

A rodent model of malaria, Plasmodium berghei was used to assess the antimalarial potential of dinitroaniline herbicides. Trifluralin, pendimethalin, oryzalin, and benfluralin were all active against P. berghei in vitro at, or close to, submicromolar concentrations, with a rank order of potency similar to that against other protozoa. The dinitroanilines did not elicit a cytotoxic effect against a mammalian cell line at concentrations 100-fold higher than those for activity against P. berghei. Neither trifluralin nor oryzalin exhibited any antimalarial activity in vivo after oral administration at the maximum dose tolerated by the host. In a pharmacokinetic study, it was found that the lack of in vivo antimalarial activity was due to poor absorption. Other DNs which have better absorption characteristics than either trifluralin or oryzalin may offer more scope for antimalarial activity in vivo.

The antimalarial activity of the hexane and methanol extracts derived from the Brazilian plants Pothomorphe peltata and Pothomorphe umbellata-whose leaves are popularly employed in medicinal folk remedies for the treatment of malaria-was assessed through in vivo tests with the Peters method. The extracts were delivered to Plasmodium berghei-infected mice via the oral or the subcutaneous route. A suppressive effect on the parasitemia seemed to be evident when data regarding the intraperitoneal injection of Pothomorphe umbellata extracts were analyzed. However, a definitive conclusion on an effective antimalarial activity is not possible, as two distinct-"standard" and "slow"-patterns of parasitemia occurring at similar frequencies in both treated and untreated intraperitoneally infected mice were observed. Nevertheless, the existence of two distinct profiles of parasitemia was not clear among the animals that were infected via the intravenous route. These data indicate the need for further studies on the biological features of the host/parasite interaction in the intraperitoneally P. berghei-infected Swiss mice system to standardize the model and to improve its usefulness in the screening of antimalarial compounds.

Amplification, mutations, or overexpression of the pfmdr1 gene have been associated with multiple drug resistance in some strains of Plasmodium falciparum. In order to better understand this potential mechanism of drug resistance, we are currently investigating putative mdr homologues in vivo in the rodent malaria Plasmodium berghei. We have identified and partially sequenced a gene that is amplified in a MFQ-resistant (MFQr) line. Using degenerate primers, a 579-bp fragment was amplified by PCR using P. berghei genomic DNA as template. The predicted amino acid sequence shares 66% identity with the previously reported pfmdr1 gene product (Pgh1) of P. falciparum. Southern blots and slot blots of genomic DNA suggest that this gene is amplified two- to threefold in a MFQr line (N/1100), as has been previously reported in some MFQr strains of P. falciparum. The P. berghei gene was mapped to chromosome 12 in all of the lines analyzed. Furthermore, the cloned PCR product also hybridizes to chromosome 5 of the MFQr strain.

The malaria circumsporozoite (CS) protein binds to glycosaminoglycans from heparan sulfate proteoglycans on the cell surface of hepatocytes and is specifically cleared from the bloodstream by the liver. We show here that the two conserved regions, I and II-plus, of the CS protein, in a concerted action, preferentially bind to highly sulfated heparin-like oligosaccharides in heparan sulfate. In a concentration-dependent manner, peptides representing region I and region II-plus inhibited the binding of recombinant CS protein to HepG2 cells by 62 and 84%, respectively. Furthermore, the action of endoproteinase Arg-C, which cleaves the recombinant CS constructs CS27IVC and CSFZ(Cys) predominantly at the conserved region I, was inhibited by heparin in a concentration-dependent fashion. CSFZ(Cys), which has a higher affinity to HSPGs than CS27IVC, was stabilized by heparin at a w/w ratio (CS protein:glycosaminoglycan) of 20/1, whereas full protection of CS27IVC required more heparin (5/1). Heparan sulfate provided full protection of CSFZ(Cys) only at a ratio of 1/10. Native fucoidan as well as normally sulfated fuco-oligosaccharides (0.76 mol sulfate/mol fucose) inhibited Plasmodium berghei development in HepG2 cells by 84 and 66%, respectively, in a concentration-dependent manner and sporozoite invasion into CHO cells by 80%. Desulfated fucoidan oligosaccharides were inactive. These results may explain the selective interaction between the CS protein and the unique heparan sulfate from liver, which is noted for its unusually high degree of sulfation, and may provide a plausible explanation for the selective targeting of the malaria CS protein to the liver.

The infectivity of P. berghei-infected TO mice to mosquitoes declines rapidly 2 to 5 days after blood inoculation, in spite of rising numbers of gametocytes in the blood. This pattern is typical of many malaria infections and various factors, particularly specific and nonspecific immune responses, have previously been implicated in the decline. Here we report that (1) simple physiological changes in the mouse blood, namely, falling pH and bicarbonate levels induced by high parasitaemias, are responsible for the sustained inhibition of infectivity; (2) the inhibition is reversible in vivo by the addition of sodium bicarbonate alone; (3) the inhibition occurs at the point of exflagellation; (4) contrary to previous observations (Kawamoto et al. 1992), exflagellation in P. berghei, like that in P. gallinaceum (Bishop and McConnachie 1956; Nijhout and Carter 1978; Nijhout 1979) and P. falciparum (Ogwan'g et al. 1993), is dependent on extracellular bicarbonate; and (5) induction of exflagellation by a mosquito factor is bicarbonate dependent. These new observations are critical to the design and interpretation of experiments on other transmission blocking phenomena.

Glutathione (GSH) plays a critical role in the detoxication and the protection of cells against oxidative stress. In the present study we examined the relationship between the intracellular GSH levels as well as glutathione S-transferase (GST), glutathione reductase (GR), and glutathione peroxidase (GPx) activities and how they relate to Plasmodium berghei resistance to chloroquine. Resistant strains (CQR30 and CQR60) were selected in vivo from a sensitive strain (NK65). Marked increases in GSH levels and GST activity within resistant parasites were observed, compared to sensitive parasites. On the other hand, GR and GPx activities were similar in sensitive and resistant parasites. Treatment with chloroquine did not influence the intracellular level of GSH, but it was found to significantly decrease GR activity. Intracellular depletion of GSH, by a nontoxic concentration of buthionine sulfoximine (BSO), significantly sensitized the resistant parasites to chloroquine. These results suggest that the P. berghei resistance results from altered GSH and GST levels and activity, respectively, which enable the detoxification of chloroquine in resistant parasites.

Mefloquine is an antimalarial drug with schizonticidal activity on blood-stage parasites. Studies of the role of mefloquine on the development of Plasmodium berghei ANKA in anopheles stephensi have been carried out that showed a dose-dependent effect on the sporogonic cycle of these parasites, with changes in the numbers of oocysts and the extent of sporozoite invasion of salivary glands. In this study, we show that mefloquine-resistant P. berghei ANKA blood stage parasites could be selected through drug pressure during continuous cyclical transmission of Anopheles gambiae s.l.

The effect of a synthetic cecropin-like peptide, Shiva-3, on in vitro ookinete development and on the early sporogonic stages of Plasmodium berghei in the midgut of Anopheles albimanus was investigated. Peptide concentrations of 75 and 100 microM were effective (P < 0.05) in reducing ookinete production and the number of infected mosquitoes in almost all experiments. These peptide concentrations in the midgut were not toxic for the survival of the mosquitoes. Complete inhibition was obtained if 100 microM Shiva-3 was applied in the first 8 hr of parasite development. The deleterious effect of the peptide on the parasite was effective after exposure for as short as 50 sec and the permanence of free peptide in the mosquito midgut was estimated to be of a minimum of 5 min. These observations indicate the possibility of using Shiva-like peptide genes to engineering malaria-resistant vectors as an alternative in malaria control strategies.

The in vitro growth of Plasmodium falciparum was reduced by 35 and 43% through high concentrations (5 mmole/liter) of magnesium in RPMI medium and magnesium-free medium, respectively, after 48 hr, whereas no significant inhibition could be observed under these conditions after 24 hr cultivation in the respective medium. Levels of magnesium between 0.5 and 3 mmole/liter showed no inhibitory effect on the in vitro growth of P. falciparum even after long-term exposure for 7 days. The 50 and 90% chloroquine inhibitory concentrations of the chloroquine-resistant strain K1 after 24 hr were reduced to some extent in the presence of magnesium at 5 mmole/liter, but less than in the presence of verapamil at 10 mumole/liter, which showed intrinsic activities at this concentration and which completely reversed resistance. However, high physiologic magnesium plasma levels were associated with a significantly longer survival time of NMRI mice infected with P. berghei strain ANKA, compared to normal physiological plasma magnesium levels. It is concluded that in the case of clinically symptomatic magnesium deficiency, supplementation of magnesium will not aggravate concomitant plasmodial infections and therefore should not be withheld.

A pyrimethamine-resistant line of Plasmodium berghei was derived by treating infected mice with high doses of pyrimethamine and selecting for recrudescence. This resistant line was compared with the parental pyrimethamine-sensitive line in order to ascertain whether drug resistance is associated with a biological advantage. Overall, the pyrimethamine-resistant line is quite similar to the sensitive line, except that it proceeds through sporogonic development more slowly than the pyrimethamine-sensitive parental line. However, under pyrimethamine pressure the sensitive line is unable to undergo the sporogonic cycle, whereas the resistant line is unaffected. These results indicate that the transmission of pyrimethamine resistance in this model is favored only under conditions of drug pressure.

Human complement was activated by rodent malaria, Plasmodium berghei, sporozoites through the alternative pathway, as revealed by C3 deposition on sporozoites using the fluorescent antibody technique. Sporozoites exposed to fresh human serum decreased in infectivity to HepG2 cells, but those exposed to heated or C3-deficient human serum showed normal infectivity to HepG2 cells. In contrast, C3 deposition was not observed on the sporozoites treated with mouse or rat serum even in the presence of specific polyclonal anti-sporozoite antibody. However, following treatment with trypsin (250 micrograms/ml), 81% of salivary gland sporozoites and 49% of oocyst sporozoites became reactive with mouse serum, and reactive sporozoites deposited mouse C3 on their surface in the presence of 30 mM EGTA and 1 mM Mg2+ without antibody. Concomitantly some sporozoites lost reactivity to anti-circumsporozoite protein monoclonal antibody. These results suggest that P. berghei sporozoites possibly express surface molecules that regulate the complement activation pathway of susceptible hosts but not of nonhosts, and that the putative structures consist of protease-sensitive molecule(s) which are closely associated with the circumsporozoite protein.

A stage-specific ribosomal RNA probe has been used to quantitate exoerythrocytic development of Plasmodium berghei in primary cultures of mouse hepatocytes. Parasite rRNA could be detected as soon as 6 hr after sporozoite invasion and was increased during schizogony to a maximum at 48 hr, when mature schizonts were identified by microscopy. As few as 10 exoerythrocytic schizonts could be detected by filter blot hybridization, followed by autoradiography and liquid scintillation counting. By hybridizing the culture rRNA samples with either parasite-specific or universal rRNA probes, the in vitro tissue schizonticidal activity and hepatotoxicity of primaquine, two of its analogues, and pyrimethamine, could be assessed. After a 48-hr exposure of the culture to serial dilutions of each drug, a quantitative relationship was demonstrated between the decrease of the parasite rRNA and the increase of the drug concentrations. No significant parasite-specific rRNA could be detected at the concentration achieving complete inhibition of schizont formation but causing no cytotoxic effects on host hepatocytes. In contrast to microscopic-based assays, this molecular approach provides an objective and quantitative in vitro method for rapid screening and evaluation of tissue schizonticidal antimalarials.

The role of ionic regulation in the induction of gametogenesis of Plasmodium berghei at 20 degrees C was investigated. A potent inhibitor of Na+/H+ exchange, amiloride, strongly inhibited exflagellation and subsequent ookinete formation induced by RPMI 1640 with 10% fetal calf serum at pH 8.0, whereas Na+ or K+ channel inhibitors, H(+)-ATPase inhibitors, and a protonophore had no significant effect. Amiloride-treated 'activated' microgametocytes synthesized DNA to levels consistent with the expected 8C, but failed to develop further. These results may suggest that an increase in intracellular pH induced by Na+/H+ exchange plays an important role in the induction of gametogenesis by cultivating at pH 8.0 and 20 degrees C. Cultivation at pH 8.0 and 37 degrees C did not induce the development, and microgametocytes remained as nonactivated forms, having the DNA content of 1.5C. By culturing at pH 7.3 and 20 degrees C, however, most of microgametocytes finished synthesis of DNA up to the 8C level, but ceased development at various stages. Additionally, exflagellation occurred in a simple medium composed of buffered saline with 10 mM glucose. Glucose was indispensable for exflagellation, presumably acting as an energy source. Exflagellation induced by this solution was also inhibited by amiloride. It is therefore suggested that the induction of microgametogenesis may be composed of two distinct mechanisms, one is a temperature-dependent DNA synthesis and the other is a pH-dependent control of developmental events leading to microgamete assembly and exflagellation.

Sodium artelinate, a new water-soluble and relatively stable derivative of artemisinin, and its parent compound were tested for their antimalarial action. Experiments were done in vitro with synchronous cultures of Plasmodium berghei. The inhibition of growth by different concentrations of sodium artelinate and artemisinin was determined using flow cytometry. In vivo testing was done by subcutaneous injection of each drug in mice infected with P. berghei. Sodium artelinate, being stable in aqueous solution, was also administered orally to infected mice by its addition to their drinking water. Comparison of the parent compound and the derivative showed that sodium artelinate was slightly less active than artemisinin both in culture and in vivo. However, after oral administration of sodium artelinate, parasites were cleared from the blood with one-half to one-tenth of the dose used in the experiments with subcutaneous injection. The number of mice which were cured by oral administration of sodium artelinate was greater than after subcutaneous injection, even with a total oral dose lower than the injected dose.

Five ornithine decarboxylase inhibitors: alpha-difluoromethylornithine (DFMO) (eflornithine); alpha-monofluoromethyl-3,4-dehydroornithine; alpha-monofluoromethyl-3,4-dehydroornithine methyl ester; alpha-monofluoromethyl-3,4-dehydroornithine ethyl ester; and (2R,5R)-delta-methyl-alpha-acetylenic putrescine were shown to inhibit erythrocytic schizogony of Plasmodium falciparum in vitro and reduced spermidine levels in infected erthrocytes. Only DFMO was effective at limiting erythrocytic schizogony of P. berghei in vivo. Administration of DFMO as a 2% solution in the drinking water for 4 days reduced parasitemia in mice by 50% in a 4-day suppression test but did not increase survival time of infected mice. This is the first demonstration of an effect of DFMO on plasmodial erythrocytic schizogony in vivo and suggests that interference with polyamine biosynthesis may, in fact, be a viable chemotherapeutic target in erythrocytic malaria.