Plasmodium Chabaudi-infected Mice Research Articles

Antimalarial effect of synthetic endoperoxide on synchronized Plasmodium chabaudi infected mice.

The discovery of new antimalarial drugs can be developed using asynchronized Plasmodium berghei malaria parasites in vivo in mice. Studies on a particular stage are also required to assess the effectiveness and mode of action of drugs. In this report, we used endoperoxide 6-(1,2,6,7-tetraoxaspiro [7.11] nonadec-4-yl) hexan-1-ol (N-251) as a model antimalarial compound on P. chabaudi parasites. We examined the antimalarial effect of N-251 against ring-stage- and trophozoite-stage-rich P. chabaudi parasites and asynchronized P. berghei parasites using the 4-day suppressive test. The ED50 values were 27, 22, and 22 mg/kg, respectively, and the antimalarial activity of N-251 was verified in both rodent malaria parasites. To assess the stage-specific effect of N-251 in vivo, we evaluated the change of parasitemia and distribution of parasite stages using ring-stage- and trophozoite-stage-rich P. chabaudi parasites with one-day drug administration for one life cycle. We discovered that the parasitemias decreased after 13 and 9 hours post-treatment in the ring-stage- and trophozoite-stage-rich groups, respectively. Additionally, in the ring-stage-rich N-251 treated group, the ring-stage parasites hindered trophozoite parasite development. For the trophozoite-stage-rich N-251 treated group, the distribution of the trophozoite stage was maintained without a change in parasitemia until 9 hours. Because of these findings, it can be concluded that N-251 suppressed the trophozoite stage but not the ring stage. We report for the first time that N-251 specifically suppresses the trophozoite stage using P. chabaudi in mice. The results show that P. chabaudi is a reliable model for the characterization of stage-specific antimalarial effects.

The effect of Eucalyptus camaldulensis leaf extracts from different environmental harvesting locations on Plasmodium chabaudi-induced malaria outcome

In every country in the globe, malaria is still regarded as a health risk. In terms of therapeutic medicines against parasites, medicinal plants are a promising source, although the types and concentrations of their active compounds can vary depending on the environment. In this study, the effect of Eucalyptus camaldulensis leaf extracts (ECE) collected from two different habitats (sandy and muddy) on the antioxidant and antimalarial activity was examined. Phytochemical analysis of ECE from muddy (ECEM) and sandy (ECES) habitats were compared. ECEM contained 13 expected compounds while ECES contained only 10 compounds. This was evidenced through infrared spectroscopy. Also, ECEM contained more phenolics and flavonoids than ECES as well as processes higher antioxidant activity that reached 55%. Plasmodium chabaudi-infected mice were treated with ECEM and ECES and the parasitemia was compared. ECEM could significantly suppress the parasitemia by approximately 84% and also was able to decrease the spleen index better than EXCS. Moreover, ECEM was better than ECES in ameliorating the induced decrease in erythrocyte number and hemoglobin content in mice infected with P. chabaudi. It is possible to use E. camaldulensis, which grows in muddy environments, as an antimalarial drug with the largest efficacy gradients.

Role of human group IIA secreted phospholipase A2 in malaria pathophysiology: Insights from a transgenic mouse model

We previously showed that injection of recombinant human group IIA secreted phospholipase A2 (hGIIA sPLA2) to Plasmodium chabaudi-infected mice lowers parasitaemia by 20%. Here, we show that transgenic (TG) mice overexpressing hGIIA sPLA2 have a peak of parasitaemia about 30% lower than WT littermates. During infection, levels of circulating sPLA2, enzymatic activity and plasma lipid peroxidation were maximal at day-14, the peak of parasitaemia. Levels of hGIIA mRNA increased in liver but not in spleen and blood cells, suggesting that liver may contribute as a source of circulating hGIIA sPLA2. Before infection, baseline levels of leukocytes and pro-inflammatory cytokines were higher in TG mice than WT littermates. Upon infection, the number of neutrophils, lymphocytes and monocytes increased and were maximal at the peak of parasitaemia in both WT and TG mice, but were higher in TG mice. Similarly, levels of the Th1 cytokines IFN-γ and IL-2 increased in WT and TG mice, but were 7.7- and 1.7-fold higher in TG mice. The characteristic shift towards Th2 cytokines was observed during infection in both WT and TG mice, with increased levels of IL-10 and IL-4 at day-14. The current data are in accordance with our previous in vitro findings showing that hGIIA kills parasites by releasing toxic lipids from oxidized lipoproteins. They further show that hGIIA sPLA2 is induced during mouse experimental malaria and has a protective in vivo role, lowering parasitaemia by likely releasing toxic lipids from oxidized lipoproteins but also indirectly by promoting a more sustained innate immune response.

Caspase-8 mediates inflammation and disease in rodent malaria

Earlier studies indicate that either the canonical or non-canonical pathways of inflammasome activation have a limited role on malaria pathogenesis. Here, we report that caspase-8 is a central mediator of systemic inflammation, septic shock in the Plasmodium chabaudi-infected mice and the P. berghei-induced experimental cerebral malaria (ECM). Importantly, our results indicate that the combined deficiencies of caspases-8/1/11 or caspase-8/gasdermin-D (GSDM-D) renders mice impaired to produce both TNFα and IL-1β and highly resistant to lethality in these models, disclosing a complementary, but independent role of caspase-8 and caspases-1/11/GSDM-D in the pathogenesis of malaria. Further, we find that monocytes from malaria patients express active caspases-1, -4 and -8 suggesting that these inflammatory caspases may also play a role in the pathogenesis of human disease.

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

BackgroundMalaria 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.MethodsUsing 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.ResultsMoringa-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.ConclusionsTaken 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.

Chemical Genomic Profiling Unveils the in Vitro and in Vivo Antiplasmodial Mechanism of Açaí (Euterpe oleracea Mart.) Polyphenols.

Malaria remains a major detrimental parasitic disease in the developing world, with more than 200 million cases annually. Widespread drug-resistant parasite strains push for the development of novel antimalarial drugs. Plant-derived natural products are key sources of antimalarial molecules. Euterpe oleracea Martius (“açaí”) originates from Brazil and has anti-inflammatory and antineoplasic properties. Here, we evaluated the antimalarial efficacy of three phenolic fractions of açaí; total phenolics (1), nonanthocyanin phenolics (2), and total anthocyanins (3). In vitro, fraction 2 moderately inhibited parasite growth in chloroquine-sensitive (HB3) and multiresistant (Dd2) Plasmodium falciparum strains, while none of the fractions was toxic to noncancer cells. Despite the limited activity in vitro, the oral treatment with 20 mg/kg of fraction 1 reduced parasitemia by 89.4% in Plasmodium chabaudi-infected mice and prolonged survival. Contrasting in vitro and in vivo activities of 1 suggest key antiplasmodial roles for polyphenol metabolites rather than the fraction itself. Finally, we performed haploinsufficiency chemical genomic profiling (HIP) utilizing heterozygous Saccharomyces cerevisiae deletion mutants to identify molecular mechanisms of açaí fractions. HIP results indicate proteostasis as the main cellular pathway affected by fraction 2. These results open avenues to develop açaí polyphenols as potential new antimalarial candidates.

Potential Role for Regulatory B Cells as a Major Source of Interleukin-10 in Spleen from Plasmodium chabaudi-Infected Mice.

Interleukin-10 (IL-10)-producing regulatory B (Breg) cells were found to be induced in a variety of infectious diseases. However, its importance in the regulation of immune response to malaria is still unclear. Here, we investigated the dynamics, phenotype, and function of Breg cells using Plasmodium chabaudi chabaudi AS-infected C57BL/6 and BALB/c mice. BALB/c mice were more susceptible to infection and had a stronger IL-10 response in spleen than C57BL/6 mice. Analysis of the surface markers of IL-10-producing cells with flow cytometry showed that CD19+ B cells were one of the primary IL-10-producing populations in P. c. chabaudi AS-infected C57BL/6 and BALB/c mice, especially in the latter one. The Breg cells had a heterogeneous phenotype which shifted during infection. The well-established Breg subset, CD19+ CD5+ CD1dhi cells, accounted for less than 20% of IL-10-producing B cells in both strains during the course of infection. Most Breg cells were IgG+ and CD138- from day 0 to day 8 postinfection. Adoptive transfer of Breg cells to C57BL/6 mice infected with P. c. chabaudi AS led to a transient increase of parasitemia without an impact on survival rate. Our finding reveals that B cells play an active and important regulatory role in addition to mediating humoral immunity in immune response against malaria, which should be paid more attention in developing therapeutic or vaccine strategies against malaria involving stimulation of B cells.

Pharmacokinetics of artemisinin delivered by oral consumption of Artemisia annua dried leaves in healthy vs. Plasmodium chabaudi-infected mice

Ethnopharmacological relevanceThe Chinese have used Artemisia annua as a tea infusion to treat fever for >2000 years. The active component is artemisinin. Previously we showed that when compared to mice fed an equal amount of pure artemisinin, a single oral dose of dried leaves of Artemisia annua (pACT) delivered to Plasmodium chabaudi-infected mice reduced parasitemia at least fivefold. Dried leaves also delivered >40 times more artemisinin in the blood with no toxicity. The pharmacokinetics (PK) of artemisinin delivered from dried plant material has not been adequately studied. Materials and methodsHealthy and Plasmodium chabaudi-infected mice were oral gavaged with pACT to deliver a 100mgkg−1 body weight dose of artemisinin. Concentrations of serum artemisinin and one of its liver metabolites, deoxyartemisinin, were measured over two hours by GCMS. ResultsThe first order elimination rate constant for artemisinin in pACT-treated healthy mice was estimated to be 0.80h−1 with an elimination half-life (T½) of 51.6min. The first order absorption rate constant was estimated at 1.39h−1. Cmax and Tmax were 4.33mgL−1 and 60min, respectively. The area under the curve (AUC) was 299.5mgminL−1. In contrast, the AUC for pACT-treated infected mice was significantly greater at 435.6mgminL−1. Metabolism of artemisinin to deoxyartemisinin was suppressed in infected mice over the period of observation. Serum levels of artemisinin in the infected mice continued to rise over the 120min of the study period, and as a result, the T½ was not determined; the Cmax and Tmax were estimated at ≥6.64mgL−1 and ≥120min, respectively. Groups of healthy mice were also fed either artemisinin or artemisinin mixed in mouse chow. When compared at 60min, artemisinin was undetectable in the serum of mice fed 100mgANkg−1 body weight. When plant material was present either as mouse chow or Artemisia annua pACT, artemisinin levels in the serum rose to 2.44 and 4.32mgL−1, respectively, indicating that the presence of the plant matrix, even that of mouse chow, had a positive impact on the appearance of artemisinin in the blood. ConclusionsThese results showed that artemisinin and one of its drug metabolites were processed differently in healthy and infected mice. The results have implications for possible therapeutic use of pACT in treating malaria and other artemisinin-susceptible diseases.

Dried-leaf Artemisia annua: A practical malaria therapeutic for developing countries?

Artemisinin from the plant Artemisia annua (A. annua) L, and used as artemisinin combination therapy (ACT), is the current best therapeutic for treating malaria, a disease that hits children and adults especially in developing countries. Traditionally, A. annua was used by the Chinese as a tea to treat "fever". More recently, investigators have shown that tea infusions and oral consumption of the dried leaves of the plant have prophylactic and therapeutic efficacy. The presence of a complex matrix of chemicals within the leaves seems to enhance both the bioavailability and efficacy of artemisinin. Although about 1000-fold less potent than artemisinin in their antiplasmodial activity, these plant chemicals are mainly small molecules that include other artemisinic compounds, terpenes (mainly mono and sesqui), flavonoids, and polyphenolic acids. In addition, polysaccharide constituents of A. annua may enhance bioavailability of artemisinin. Rodent pharmacokinetics showed longer T1/2 and Tmax and greater Cmax and AUC in Plasmodium chabaudi-infected mice treated with A. annua dried leaves than in healthy mice. Pharmacokinetics of deoxyartemisinin, a liver metabolite of artemisinin, was more inhibited in infected than in healthy mice. In healthy mice, artemisinin serum levels were > 40-fold greater in dried leaf fed mice than those fed with pure artemisinin. Human trial data showed that when delivered as dried leaves, 40-fold less artemisinin was required to obtain a therapeutic response compared to pure artemisinin. ACTs are still unaffordable for many malaria patients, and cost estimates for A. annua dried leaf tablet production are orders of magnitude less than for ACT, despite improvements in the production capacity. Considering that for > 2000 years this plant was used in traditional Chinese medicine for treatment of fever with no apparent appearance of artemisinin drug resistance, the evidence argues for inclusion of affordable A. annua dried leaf tablets into the arsenal of drugs to combat malaria and other artemisinin-susceptible diseases.

In vitro and in vivo assessment of the anti-malarial activity of Caesalpinia pluviosa

BackgroundTo overcome the problem of increasing drug resistance, traditional medicines are an important source for potential new anti-malarials. Caesalpinia pluviosa, commonly named "sibipiruna", originates from Brazil and possess multiple therapeutic properties, including anti-malarial activity.MethodsCrude extract (CE) was obtained from stem bark by purification using different solvents, resulting in seven fractions. An MTT assay was performed to evaluate cytotoxicity in MCF-7 cells. The CE and its fractions were tested in vitro against chloroquine-sensitive (3D7) and -resistant (S20) strains of Plasmodium falciparum and in vivo in Plasmodium chabaudi-infected mice. In vitro interaction with artesunate and the active C. pluviosa fractions was assessed, and mass spectrometry analyses were conducted.ResultsAt non-toxic concentrations, the 100% ethanolic (F4) and 50% methanolic (F5) fractions possessed significant anti-malarial activity against both 3D7 and S20 strains. Drug interaction assays with artesunate showed a synergistic interaction with the F4. Four days of treatment with this fraction significantly inhibited parasitaemia in mice in a dose-dependent manner. Mass spectrometry analyses revealed the presence of an ion corresponding to m/z 303.0450, suggesting the presence of quercetin. However, a second set of analyses, with a quercetin standard, showed distinct ions of m/z 137 and 153.ConclusionsThe findings show that the F4 fraction of C. pluviosa exhibits anti-malarial activity in vitro at non-toxic concentrations, which was potentiated in the presence of artesunate. Moreover, this anti-malarial activity was also sustained in vivo after treatment of infected mice. Finally, mass spectrometry analyses suggest that a new compound, most likely an isomer of quercetin, is responsible for the anti-malarial activity of the F4.

Synthesis, Stability, and Antimalarial Activity of New Hydrolytically Stable and Water-Soluble (+)-Deoxoartelinic Acid

(+)-Deoxoartelinic acid (13), a new hydrolytically stable, water-soluble, and potent non-acetal-type antimalarial drug candidate, was successfully prepared from artemisinic acid by using sulfur ylide and photooxygenative cyclization in seven steps. This compound showed superior in vitro antimalarial activity against the chloroquine-resistant K1 strain of Plasmodium falciparum and higher suppression (98.7%) than arteether in vivo against Plasmodium chabaudi infected mice. (+)-Deoxoartelinic acid also showed remarkable stability with a half-life of 258.66 h, 23 times more stable than clinically useful arteether in simulated stomach acid, and improved solubility, 4 times more soluble than artemisinin in water.

Testosterone-induced changes in phosphatidylcholine molecular species composition of Plasmodium chabaudi-infected erythrocytes.

This study is concerned with the influence of testosterone on the phospholipid class and the phosphatidylcholine molecular species composition of various fractions obtained from the blood of Plasmodium chabaudi-infected mice. Blood plasma, infected erythrocytes, isolated parasites and erythrocyte membranes isolated from both non-infected and infected erythrocytes in the form of ghosts were analysed. In general, the phospholipid classes remained unaffected, while the phosphatidylcholine (PC) molecular species composition showed differences after testosterone treatment. In infected erythrocytes, there was a decrease in 16:0/20:4-PC and 18:0/20:4-PC and an increase in 16:0/18:2(16:0/20:3)-PC. The decrease of 16:0/20:4-PC was exclusively confined to parasites. The rise in 16:0/18:2(16:0/20:3)-PC and the diminution of 18:0/20:4-PC occurred in the erythrocyte membrane of both infected ghosts and non-infected ghosts as well as in the blood plasma. It is suggested that these changes occur primarily in the plasma thereby influencing the erythrocyte membranes. The decrease in 16:0/20:4-PC supports the view of the independence of the parasite from the biosynthetic lipid pathways of its host cell.

Requirement for Lipopolysaccharide for EnhancedIn VitroSuperoxide Producing Competence in Macrophages from Normal and Malaria (Plasmodium Chabaudi) Infected Mouse Spleen

Nucleated cells from mouse spleens were suspended in RPMI-1640 medium and plated in polystyrene tissue culture dishes with or without lipoplysaccharide (LPS, endotoxin) and incubated at 37 degrees C for 3 h in a humid incubator under 5% CO2. Adherent spleen cells obtained from uninfected control or Plasmodium chabaudi-infected mice showed negligible superoxide (O2) production in vitro, in response to triggering with phorbol myristate acetate (PMA). However, O2 release increased in a concentration-dependent fashion as LPS was introduced above a threshold of about 10 ng/ml. This LPS dependence is at least 100 times more than that required by mouse peritoneal macrophages and 10(6) times greater than the requirement reported for human monocytes. These findings emphasize the differences among the three mononuclear cells, and also suggest that LPS could prove useful in the isolation and culture of other metabolically competent cells.

Lipoproteins and malaria. II. Immunoregulatory role of immunoglobulin-lipoprotein complexes in mice infected with Plasmodium chabaudi on antibody producing cells

Injection of lipoproteins from Plasmodium chabaudi infected mice into mice previously immunized with either human serum transferrin, bovine serum albumin, polyvinyl-pyrrolidone or tetanus toxoid, was followed by a decrease in the levels of antibodies directed against these antigens, suggesting a blockade of antibody secreting cells (Goumard et al., 1982). However, lipoproteins in P. chabaudi infected mice are complexed with immunoglobulins during the second week of infection (Demonchy et al., 1982). In this study, the effects of lipoproteins and Ig-lipoprotein complexes (Ig-Lp) on antibody secreting cells was investigated in vitro. Spleen cells from mice immunized with tetanus toxoid were cultured in microplates and the antitetanus antibodies (anti-TT Ab) were measured in the culture supernatants using a radioimmunoassay (Goumard et al., 1984). Ig-Lp purified from day-11 or day-13 P. chabaudi infected mice inhibited the secretion of anti-TT Ab when introduced into microcultures. On the contrary, lipoproteins purified from either day-5, day-7, day-21 or day-28 infected mice as well as lipoproteins from uninfected mice did not inhibit anti-TT Ab secreting cells. Ig-Lp formed in vitro with lipoproteins purified from day-7 infected mice (Lp J-7) and day-20 infected mice sera, inhibited anti-TT Ab secreting cells. IgG purified from day-20 sera and incubated with Lp J-7, inhibited anti-TT Ab secreting cells but no inhibitory effect was observed with the F(ab')2 fragments of these Ig. Pre-incubation of anti-TT Ab secreting cells with Fc fragments of mouse IgG blocked the inhibitory effect of Ig-Lp purified from infected mouse sera or formed in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal deposits of lipoprotein-immunoglobulin complexes in Plasmodium chabaudi-infected mice.

Lipoprotein metabolism is altered and immunoglobulin-lipoprotein complexes (Ig-Lp) are formed during malaria infection (1-5). Ig-Lp were detected in the sera of Plasmodium chabaudi-infected mice 9 days post-infection (1 or 2 days after parasitemia had peaked at about 50%) and reached a maximum on day 13 (when the parasitemia had decreased to less than 1%). Renal glomerular deposits of IgM were first detected at day 3 and were heavy from day 9 to day 29; deposits of IgG and low density lipoprotein (LDL) were present from days 9 to 62, and were more dense from days 22 to 29; deposits of C3 were observed from day 13 to day 29. Apoprotein B component was found in heparin eluates of kidneys on day 10, 14, and 29. Fractionated Ig-Lp, as well as whole sera from day-13 infected mice, were injected into uninfected mice that developed LDL glomerular deposits only when pre-treated with histamine. LDL glomerular deposits were also observed after i.v. injection of day-29 sera (containing free anti-lipoprotein antibody) into day-7 infected mice, but not when a mixture of day-29 and day-7 sera was injected into normal recipient mice. LDL glomerular deposits, however, were observed when recipient mice were treated with the Plasmodium-derived Insoluble Material (PDIM) 3 days before the injection of the day-29-day-7 sera mixture or day-13 serum. Two hours after the i.v. injection of 125I-Ig-Lp, the radioactivity of the kidneys was higher in histamine-treated, PDIM-treated, and P. chabaudi-infected mice than in controls. The clearance of 125I-Ig-Lp was higher in infected and in PDIM-treated mice than in controls. We suggest that the glomerular deposit of Ig-Lp that occurs during P. chabaudi infection requires an enhancing factor such as PDIM that is released during infection.

Suppressive activity of splenic adherent cells from Plasmodium chabaudi-infected mice.

The immune response of splenic cells to SRBC and LPS or Con A mitogens is suppressed in P. chabaudi-infected mice. When mixed with cells from normal donors, spleen cells from infected mice also suppressed anti-SRBC and mitogenic responses. Suppression was not detected in lymph node cells, bone marrow, or thymocytes from infected mice. Removal of infected red cells had no effect on suppression. The cell population responsible for the suppressive activity was adherent to plastic, resistant to irradiation and anti-Thy-1 plus complement treatment.