Plasmodium Berghei NK65 Research Articles

Anti-malarial activity of Holarrhena antidysenterica and Viola canescens, plants traditionally used against malaria in the Garhwal region of north-west Himalaya

BackgroundThe increasing number of multidrug-resistant Plasmodium strains warrants exploration of new anti-malarials. Medicinal plant research has become more important, particularly after the development of Chinese anti-malarial drug artemisnin from Artemisia annua. The present study shows evaluation of anti-malarial effects of two plants commonly used against malaria in the Garhwal region of north-west Himalaya, in order to discover the herbal-based medicine.MethodsIn vitro anti-plasmodial sensitivity of plant extracts was assessed using schizont maturation and parasite lactate dehydrogenase (pLDH) assay. Cytotoxic activities of the examined extracts were determined on L-6 cells of rat skeletal muscle myoblast. The 4-day test for anti-malarial activity against a chloroquine sensitive Plasmodium berghei NK65 strain in Swiss albino mice was used for monitoring in vivo activity of plant extracts.ResultsChloroform extract of H. antidysenterica (HA-2) and petroleum ether extract of V. canescens (VC-1) plants significantly reduced parasitaemia in P. berghei infected mice. The extract HA-2 showed in vitro anti-plasmodial activity with its IC50 value 5.5 μg/ml using pLDH assay and ED50 value 18.29 mg/kg in P. berghei infected Swiss albino mice. Similarly petroleum ether extract of V. canescens (VC-1) showed in vitro anti-plasmodial activity with its IC50 value 2.76 μg/ml using pLDH assay and ED50 15.8 mg/kg in P. berghei infected mice. The extracts coded as HA-2 at 30 mg/kg and VC-1 at 20 mg/kg exhibited parasite inhibition in mice: 73.2% and 63.0% respectively. Of these two plant extracts, petroleum ether extract of V. canescens was found slightly cytotoxic.ConclusionThe present investigation reflects the use of these traditional medicinal plants against malaria and these plants may work as potential source in the development of variety of herbal formulations for the treatment of malaria.

Suppressive, Curative and Prophylactic Potentials of Morinda lucida (Benth) against Erythrocytic Stage of Mice Infective Chloroquine Sensitive Plasmodium berghei NK-65

Suppressive, Curative and Prophylactic Potentials of Morinda lucida (Benth) against Erythrocytic Stage of Mice Infective Chloroquine Sensitive Plasmodium berghei NK-65

Immunopathology and dexamethasone therapy in a new model for malaria-associated acute respiratory distress syndrome

Malaria infection is often complicated by malaria-associated acute respiratory distress syndrome (MA-ARDS), characterized by pulmonary edema and hemorrhages. No efficient treatments are available for MA-ARDS and its pathogenesis remains poorly understood. To develop a new animal model for MA-ARDS, mice were infected with Plasmodium berghei NK65, and the development of MA-ARDS was characterized by increased lung weight, edema, leukocyte infiltration and hemorrhages (Figure ​(Figure1).1). The pulmonary expression of several cytokines and chemokines was increased to a higher level than in mice infected with P. chabaudi AS, which does not cause MA-ARDS. By depletion experiments, CD8+ T lymphocytes were shown to be pathogenic. High doses of dexamethasone blocked MA-ARDS, even when administered after appearance of the complication, and reduced pulmonary leukocyte accumulation. Figure 1 Histopathology of P. berghei NK65-induced MA-ARDS. Frozen sections of lungs of mice infected for 10 days with P. berghei NK65 or control mice were stained with H&E.The black bar corresponds with 100 μm. We developed a novel model of MA-ARDS with many similarities to human MA-ARDS and without cerebral complications. This contrasts with the more classical model with P. berghei ANKA, characterized by fulminant cerebral malaria. Hence, infection with P. berghei NK65 generates a broader time window to study the pathogenesis and to evaluate candidate treatments. The finding that high doses of dexamethasone cured MA-ARDS suggests that it might be more effective against MA-ARDS than it was in the clinical trials for cerebral malaria.

Essential Role for IL-27 Receptor Signaling in Prevention of Th1-Mediated Immunopathology during Malaria Infection

Successful resolution of malaria infection requires induction of proinflammatory immune responses that facilitate parasite clearance; however, failure to regulate this inflammation leads to immune-mediated pathology. The pathways that maintain this immunological balance during malaria infection remain poorly defined. In this study, we demonstrate that IL-27R-deficient (WSX-1(-/-)) mice are highly susceptible to Plasmodium berghei NK65 infection, developing exacerbated Th1-mediated immune responses, which, despite highly efficient parasite clearance, lead directly to severe liver pathology. Depletion of CD4(+) T cells---but not CD8(+) T cells---prevented liver pathology in infected WSX-1(-/-) mice. Although WSX-1 signaling was required for optimal IL-10 production by CD4(+) T cells, administration of rIL-10 failed to ameliorate liver damage in WSX-1(-/-) mice, indicating that additional, IL-10-independent, protective pathways are modulated by IL-27R signaling during malaria infection. These data are the first to demonstrate the essential role of IL-27R signaling in regulating effector T cell function during malaria infection and reveal a novel pathway that might be amenable to manipulation by drugs or vaccines.

Alpha-tocopherol transfer protein disruption confers resistance to malarial infection in mice

BackgroundVarious factors impact the severity of malaria, including the nutritional status of the host. Vitamin E, an intra and extracellular anti-oxidant, is one such nutrient whose absence was shown previously to negatively affect Plasmodium development. However, mechanisms of this Plasmodium inhibition, in addition to means by which to exploit this finding as a therapeutic strategy, remain unclear.Methodsα-TTP knockout mice were infected with Plasmodium berghei NK65 or Plasmodium yoelii XL-17, parasitaemia, survival rate were monitored. In one part of the experiments mice were fed with a supplemented diet of vitamin E and then infected. In addition, parasite DNA damage was monitored by means of comet assay and 8-OHdG test. Moreover, infected mice were treated with chloroquine and parasitaemia and survival rate were monitored.ResultsInhibition of α-tocopherol transfer protein (α-TTP), a determinant of vitamin E concentration in circulation, confers resistance to malarial infection as a result of oxidative damage to the parasites. Furthermore, in combination with the anti-malarial drug chloroquine results were even more dramatic.ConclusionConsidering that these knockout mice lack observable negative impacts typical of vitamin E deficiency, these results suggest that inhibition of α-TTP activity in the liver may be a useful strategy in the prevention and treatment of malaria infection. Moreover, a combined strategy of α-TTP inhibition and chloroquine treatment might be effective against drug resistant parasites.

Immunopathology and Dexamethasone Therapy in a New Model for Malaria-associated Acute Respiratory Distress Syndrome

Malaria infection is often complicated by malaria-associated acute respiratory distress syndrome (MA-ARDS), characterized by pulmonary edema and hemorrhages. No efficient treatments are available for MA-ARDS and its pathogenesis remains poorly understood. Development of a new animal model for MA-ARDS to explore the pathogenesis and possible treatments. C57BL/6 mice were infected with Plasmodium berghei NK65, and the development of MA-ARDS was evaluated by the analysis of lung weight, histopathology, and bronchoalveolar lavages. Cytokine and chemokine expression in the lungs was analyzed by reverse transcription-polymerase chain reaction, and the accumulation of leukocyte subclasses was determined by flow cytometric analysis. In this model, the pulmonary expression of several cytokines and chemokines was increased to a higher level than in mice infected with Plasmodium chabaudi AS, which does not cause MA-ARDS. By depletion experiments, CD8(+) T lymphocytes were shown to be pathogenic. High doses of dexamethasone blocked MA-ARDS, even when administered after appearance of the complication, and reduced pulmonary leukocyte accumulation and the expression of a monocyte/macrophage-attracting chemokine. We developed a novel model of MA-ARDS with many similarities to human MA-ARDS and without cerebral complications. This contrasts with the more classical model with P. berghei ANKA, characterized by fulminant cerebral malaria. Hence, infection with P. berghei NK65 generates a broader time window to study the pathogenesis and to evaluate candidate treatments. The finding that high doses of dexamethasone cured MA-ARDS suggests that it might be more effective against MA-ARDS than it was in the clinical trials for cerebral malaria.

Bioassay-guided fractionation and in vivo antiplasmodial effect of fractions of chloroform extract of Artemisia maciverae Linn

In the search for new plant-derived anti-malarial compounds, chromatographic fractions of chloroform extract of whole plants of Artemisia maciverae were tested in vivo using chloroquine resistant and chloroquine sensitive Plasmodium berghei NK 65 infected Swiss albino mice. One fraction and a sub-fraction of this were most active at 10/mg and cleared parasitemia in mice within 3 days. The different fractions and sub-fractions were tested with different reagents to determine the broad classes of compounds present. The active fraction tested positive for triterpenes and alkaloids, and the sub-fraction for only triterpenes. These tests suggest that the anti-malarial activities observed with these fractions may be due to these classes of compounds in the chloroform extract of the A. maciverae. Further chemical work is however required to characterize the active constituents.

Plasmodium berghei: Efficacy of 5-fluoroorotate in combination with commonly used antimalarial drugs in a mouse model

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.

In vivo antiplasmodial effect of chloroform extracts of Artemisia maciverae Linn and Artemisia maritima Linn

Chloroform extracts of Artemisia maciverae and Artemisia maritima (whole plants) were tested in vivo for anti-malarial activity in Swiss albino mice experimentally infected with chloroquine resistant Plasmodium berghei NK 65 at a dose of 100 mg/kg. The 2 plant extracts showed high anti-malarial activity. The parasitemia in the infected mice treated with the extracts were significantly reduced (P < 0.05) when compared with the untreated negative control and the pre-treatment period (Day 0). The plant extracts were also screened for phytochemicals and secondary metabolites. Some phytochemicals like flavonoids, steroids, terpenoids, tannins, phlobatannins, alkaloids and anthraqinones were detected in the two plant extracts. The anti-malarial activity of these extracts might be attributed to these phytochemicals/secondary metabolites. This study suggests that antiplasmodial activity of Artemisia may be widely distributed within the genus.

Plasmodium berghei: Lack of antimalarial activity of an analogue of folate precursor, 2,4-diamino-6-hydroxymethylpteridine in a mouse model

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 20 mg/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.

Antimalarial activity of 1-aryl-3,3-dialkyltriazenes

The antimalarial activity of 1-aryl-3,3-dialkyltriazenes to Plasmodium berghei NK-65 in infected mice was evaluated at an intraperitoneal dose of 100 mg/kgbw. Some of these compounds were found to possess potent antimalarial activity.

In Vivo antimalarial activity of aqueous extracts from Kenyan medicinal plants and their Chloroquine (CQ) potentiation effects against a blood‐induced CQ‐resistant rodent parasite in mice

Hot water extracts from eight medicinal plants representing five families, used for malaria treatment in Kenya were screened for their in vivo antimalarial activity in mice against a chloroquine (CQ) resistant Plasmodium berghei NK65, either alone or in combination with CQ. Extracts of three plants, Toddalia asiatica (root bark), Rhamnus prinoides (leaves and root bark) and Vernonia lasiopus (root bark) showed high chemosuppression in the range 51%-75%. Maytenus acuminata, M. heterophylla, M. senegalensis and Rhamnus staddo had moderate activities of 33%-49% parasitaemia suppression in the root bark and/or leaf extracts, while Withania somnifera (root bark) had a non-significant suppression (21%). In combination with CQ, extracts of V. lasiopus (all parts), leaf extracts of M. senegalensis, R. prinoides and T. asiatica as well as root barks of M. heterophylla, R. staddo and T. asiatica had improved parasitaemia suppression in the range 38%-66%, indicating synergistic interactions. Remarkable parasitaemia suppression by the extracts, either alone or in combination with CQ resulted into longer survival of mice relative to the controls, in some cases by more than 2 weeks. Plants, which showed significant antimalarial activity including V. lasiopus, T. asiatica and R. prinoides, should further be evaluated in the search for novel agents against drug-resistant malaria.

Antimalarial activity of methanolic extracts from plants used in Kenyan ethnomedicine and their interactions with chloroquine (CQ) against a CQ-tolerant rodent parasite, in mice

Methanolic extracts from 15 medicinal plants representing 11 families, used traditionally for malaria treatment in Kenya were screened for their in vivo antimalarial activity in mice against a chloroquine (CQ)-tolerant Plasmodium berghei NK65, either alone or in combination with CQ. The plant parts used ranged from leaves (L), stem bark (SB), root bark (RB), seeds (S) and whole plant (W). When used alone, extracts from seven plants, Clerodendrum myricoides (RB), Ficus sur (L/SB/RB), Maytenus acuminata (L/RB), Rhamnus prinoides (L/RB), Rhamnus staddo (RB), Toddalia asiatica (RB) and Vernonia lasiopus (RB) had statistically significant parasitaemia suppressions of 31.7–59.3%. In combination with CQ, methanolic extracts of Albizia gummifera (SB), Ficus sur (RB), Rhamnus prinoides and Rhamnus staddo (L/RB), Caesalpinia volkensii (L), Maytenus senegalensis (L/RB), Withania somnifera (RB), Ekebergia capensis (L/SB), Toddalia asiatica (L/RB) and Vernonia lasiopus (L/SB/RB) gave statistically significant and improved suppressions which ranged from 45.5 to 85.1%. The fact that these activities were up to five-fold higher than that of extract alone may suggest synergistic interactions. Remarkable parasitaemia suppression by the extracts, either alone or in combination with CQ mostly resulted into longer mouse survival relative to the controls, in some cases by a further 2 weeks. Plants, which showed significant antimalarial activity including Vernonia lasiopus, Toddalia asiatica, Ficus sur, Rhamnus prinoides and Rhamnus staddo warrant further evaluation in the search for novel antimalarial agents against drug-resistant malaria.

Antimalarial potential of xestoquinone, a protein kinase inhibitor isolated from a Vanuatu marine sponge Xestospongia sp.

As part of our search for new antimalarial drugs, we have screened for inhibitors of Pfnek-1, a protein kinase of Plasmodium falciparum, in south Pacific marine sponges. On the basis of a preliminary screening, the ethanolic crude extract of a new species of Xestospongia collected in Vanuatu was selected for its promising activity. A bioassay-guided fractionation led us to isolate xestoquinone which inhibits Pfnek-1 with an IC 50 around 1 μM. Among a small panel of plasmodial protein kinases, xestoquinone showed modest protein kinase inhibitory activity toward PfPK5 and no activity toward PfPK7 and PfGSK-3. Xestoquinone showed in vitro antiplasmodial activity against a FCB1 P. falciparum strain with an IC 50 of 3 μM and a weak selectivity index (SI 7). Xestoquinone exhibited a weak in vivo activity at 5 mg/kg in Plasmodium berghei NK65 infected mice and was toxic at higher doses.

The chemotherapy of rodent malaria. LXIII. Drug combinations to impede the selection of drug resistance, part 6: the potential value of chlorproguanil and dapsone in combination, and with the addition of artesunate

Resistance is readily produced in rodent malaria using the single-dose, '2%-relapse technique' (2%RT) against the individual compounds chlorproguanil (CPG), chlorcycloguanil (CCG), cycloguanil, dapsone (DDS) and artesunate (ASN). Using the '4-day test', a low level of synergism or a simple additional action between CPG and DDS was observed with multiple dosing of these two compounds in a combination. Resistance to a 1 : 3 combination of CPG-DDS was selected in each of three parasite lines: Plasmodium berghei NK65, P. yoelii ssp. NS and P. chabaudi AS. Of these lines, P. chabaudi AS was found to be the most sensitive to the 1 : 3 combination in the 2%RT (and was also previously found to be the most sensitive when the compounds were used individually). Plasmodium chabaudi AS was also the line found most sensitive to a 7 : 21 : 300 combination of CPG-DDS-ASN (CDA). In mice infected with P. chabaudi AS, compared with the use of the individual components, the CPG-DDS combination only a gave a modest level of protection (as indicated by the increase in the time required to select resistance in the 2%RT) but the triple CDA combination was totally effective over the duration of the experiment. New pharmacokinetic data to be reported elsewhere indicate, however, that the antimalarial action of CPG in mice is exerted by a mechanism that is not associated with the drug's conversion to the antifolate triazine, CCG. The question thus arises as to how, in the present model, the protective action of CDA was effected. The present results nevertheless reinforce the hypothesis that a CDA combination, appropriately proportioned for human use, should be of practical value, in protecting the individual components, when used for the treatment of multidrug-resistant P. falciparum, and possibly other Plasmodium species, in endemic areas. Clinical trials, both with a CPG-DDS combination (Lapdap) and CDA, are currently under way in tropical Africa. Further studies are now required to determine whether DDS, CPG or an as-yet unidentified metabolite of CPG interact with ASN, and whether a simple double combination of ASN with one or other of these would be as protective, against the selection of resistance, as CDA.

Plasmodium berghei NK65: Studies on the effect of treatment duration and inoculum size on recrudescence

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.

Mulinane-type diterpenoids from Azorella compacta display antiplasmodial activity

Two mulinane-type diterpenoids were isolated from Azorella compacta; namely 20-hydroxymulin-11,13-dienyl acetate and 13,14-dihydroxymulin-11-en-20-oic acid. The structures were elucidated by analysis of their spectroscopic data. These compounds, as well as three previously isolated diterpenes, were evaluated as potential in vivo growth inhibitors of Plasmodium berghei NK 65 on infected mice at an intraperitoneal dose of 10 mg/kg/day. Sixty percent and forty-two percent growth inhibition were obtained with 17-acetoxymulin-11,13-dien-20-oic acid and 13, 14-dihydroxymulin-11-en-20-oic acid, respectively.

Influence of CD4 +CD25 + T cells on Plasmodium berghei NK65 infection in BALB/c mice

CD4 + T cells co-expressing CD25 (CD4 +CD25 + T cells) have been identified as immunoregulatory suppressors modulating autoimmune response. Beside that, autoimmune response was supposed to be associated with malaria infection. Based on these data, we hypothesised that CD4 +CD25 + T cells may influence protective immunity to malaria parasites, while suppressing autoimmune response arising throughout the course of malarial infection. To test this possibility, we evaluated the kinetics of CD4 +CD25 + T cells during malaria infection and investigated the influence of CD25 depletion by anti-mouse CD25 monoclonal antibody (PC61) on the infection, using a mouse model of premunition to Plasmodium berghei NK65 malaria. The results showed that, during exacerbation of P. berghei NK65 infection, the proportion of CD4 +CD25 + T cells among CD4 + T cells decreased, although that of CD4 + T cells increased. CD25 depletion clearly delayed the growth of parasitaemia during parasite challenge, particularly in immunised mice. These findings demonstrated that CD4 +CD25 + T cells are able to influence protective immunity underlying premunition to P. berghei NK65 parasites.

Influence of antimalarial treatment on acquisition of immunity in Plasmodium berghei NK65 malaria.

Antimalarial treatments during primary Plasmodium berghei NK65 infection in BALB/c mice influenced the acquisition of protective immunity against reinfection. Among subcurative treatments, lower doses better enable mice to acquire protective immunity than do higher doses. Eradication of parasites from the start of infection did not promote protective immunity.

Superoxide-dependent and -independent pathways are involved in the transmission blocking of malaria.

Superoxide plays a crucial role in innate immunity to various pathogens. We examined the role of superoxides in the transmission of malaria using gp91phox knockout (X-CGD) mice that lack the ability to produce superoxide. Mosquitoes that fed on X-CGD mice infected intraperitoneally with Plasmodium berghei NK65 ANKA formed more oocysts than did those that fed on control mice at any day after infection. The number of oocysts peaked on day 5 post-infection in X-CGD and control mice and then decreased significantly after day 5 post-infection. However, on day 7 post-infection, the infectivity of gametocytes in X-CGD mice was significantly higher than that in control mice. These results show that two pathways, superoxide-dependent and -independent, are involved in the host systems regulating the transmission of malaria and inhibiting gametocyte development.