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.

Genetic Profiling of Malaria and Lipid Mediator Quantification of Mouse Striated Muscles Infected with Malaria Parasites

Malaria remains one of the most important infectious diseases in the world. The pathogenesis of malaria parasites causes illness, abnormal function and damage in the hosts. Among the several complications, the effects of malaria seem to target the skeletal muscle system, leading to symptoms, such as muscle aches, muscle contractures, muscle fatigue, muscle pain, and muscle weakness. Long‐term impacts of malaria include death, disability, and significant socioeconomic burden on societies where the disease is prevalent. A better understanding of the biological processes underlying the progression of infection to disease is urgently needed to reduce the morbidity and mortality of malaria. Considering the vital importance of the skeletal muscles as the largest organ‐system in the body, comprising nearly 50% of its mass and largely responsible for the regulation and modulation of overall metabolism, we investigated the potential molecular mechanisms of plasmodia infection affecting skeletal muscles of mice infected with Plasmodium berghei and P. chabaudi, using the Mouse Signal Transduction Pathway Finder PCR Array, which was implemented to monitor gene expression changes of 10 essential signaling pathways, and also applied our new targeted lipidomic approach using liquid chromatography with tandem mass spectrometry (LC‐MS/MS) to profile total 158 LMs, mostly eicosanoids and docosanoids, and further quantify 16 key LMs directly associated with inflammation and tissue healing in skeletal muscles. Our results showed that seven common genes (Acsl4, Bmp2, Cebpd, Cdkn1a, Sqstm1, Stat1 and Txnrd1) had their expression significantly altered, being some of them related to the inflammatory and immune response of the host. We hypothesized that the higher number of altered genes in skeletal muscles of P. chabaudi infected mice when compared to P. berghei infected mice be related to the plasmodia strains used, that were selected according to their characteristics and effect on the experimental mice models. In addition to those genes expression results, the lipids found on higher concentrations, compared to the controls, are directly related to inflammatory responses, while the lipids found on lower concentrations help to resolve inflammatory processes. The AA/EPA ratio, an indicator of inflammation and one of the top biomarkers for the morbidity and mortality, was severely higher (by 5–6‐fold) in muscles from malaria infected mice. Our results shed new insights into the mechanisms underlying the molecular mechanisms of malaria‐induced muscle damage and might be useful in future works monitoring of disease progression and development of specific interventions for the mitigation of long‐term chronic effects on skeletal muscle function. We postulate that these results are relevant to other vector borne diseases.Support or Funding InformationThis work was funding by Sabbatical grant from the University of São Paulo, Brazil (MM), and The George W. and Hazel M. Jay and Evanston Research Endowments (MB)

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.

Dissemination of non-typhoidal Salmonella during Plasmodium chabaudi infection affects anti-malarial immunity.

Malaria-associated bacteremia accounts for up to one-third of deaths from severe malaria, and non-typhoidal Salmonella (NTS) has been reported as a major complication of severe malarial infection. Patients who develop NTS bacteremia during Plasmodium infection show higher mortality rates than individuals with malaria alone. Systemic bacteremia can be caused by a wound or translocation from epithelial or endothelialsites. NTS is an intestinal pathogen, however the contribution of bacterial translocation from the intestinal tract during Plasmodium infection is not well studied. Here, we investigated the integrity of the intestinal barrier function of P. chabaudi-infected mice using large molecules and Salmonella infection. Intestinal histology and the adaptive immune response to malaria were also studied using light microscopy and flow cytometry. P. chabaudi infection compromised intestinal barrier function, which led to increased intestinal cellular infiltration. In addition, we observed increased serum lipopolysaccharide binding protein and leakage of soluble molecules from the intestine into the blood in infected mice. Plasmodium infection also increased intestinal translocation and dissemination of NTS to the liver. The adaptive immune response to P. chabaudi infection was also significantly impacted by NTS translocation. Reduced B and T cell activation were observed in co-infected animals, suggesting interference in the malaria-specific immune responses by bacteremia. These studies demonstrate that P. chabaudi infection induces failure of the barrier function of the intestinal wall and enhanced intestinal bacterial translocation, affecting anti-malarial immunity.

Daily Rhythms of TNFα Expression and Food Intake Regulate Synchrony of Plasmodium Stages with the Host Circadian Cycle

The Plasmodium cell cycle, wherein millions of parasites differentiate and proliferate, occurs in synchrony with the vertebrate host's circadian cycle. The underlying mechanisms are unknown. Here we addressed this question in a mouse model of Plasmodium chabaudi infection. Inflammatory gene expression and carbohydrate metabolism are both enhanced in interferon-γ (IFNγ)-primed leukocytes and liver cells from P. chabaudi-infected mice. Tumor necrosis factor α (TNFα) expression oscillates across the host circadian cycle, and increased TNFαcorrelates with hypoglycemia and a higher frequency of non-replicative ring forms of trophozoites. Conversely, parasites proliferate and acquire biomass during food intake by the host. Importantly, cyclic hypoglycemia is attenuated and synchronization of P. chabaudi stages is disrupted in IFNγ-/-, TNF receptor-/-, or diabetic mice. Hence, the daily rhythm of systemic TNFα production and host food intake set the pace for Plasmodium synchronization with the host's circadian cycle. This mechanism indicates that Plasmodium parasites take advantage of the host's feeding habits.

Novel M-CSF-producing γδ T cells protect against recurrent malaria

Abstract In 2016, there were 216 million malaria cases – 445,000 of which resulted in deaths. Despite overwhelming evidence that γδ T cells strongly respond during malaria infection and vaccination, their functional and phenotypic characteristics remain the least understood facets of the adaptive immune response. Therefore, we studied the role of these cells in human and mouse malaria. In both Plasmodium falciparum-infected subjects and in P. chabaudi-infected mice, we found γδ T cells expanding rapidly after resolution of acute parasitemia, in contrast to αβ T cells that expanded at the acute stage and then declined. Silencing the murine γδ T cells led to recurrent rounds of Plasmodium parasitemia. Single-cell T cell receptor sequencing of the expanded mouse cells revealed oligoclonal γδ T cells restricted to the TRAV15N-1 (Vδ6.3) V-region and converging complementarity-determining region 3 (CDR3) motifs. Also, RNA-seq of the expanded γδ T cells showed an unexpected transcriptional profile characterized by myeloid-modulating factors, previously unseen in γδ T cells. The expanded TRAV15N-1 γδ T cells abundantly produced M-CSF, which was necessary for preventing parasitemic recurrence. Interestingly, αβ T cells were the major source of M-CSF during acute infection, while γδ T cells filled that role during the post-acute stage. We have uncovered a novel γδ T cell subset that fills a protective role in the late stage of malaria. These cells could provide the mechanism for other observed correlations between γδ T cell and myeloid activity in cancer and infectious disease.

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.

Antiplasmodial activity of tick defensins in a mouse model of malaria

Malaria is a mosquito-borne disease affecting millions of people mainly in Sub-Saharan Africa, Asia and some South American countries. Drug resistance to first-line antimalarial drugs (e.g. chloroquine, sulfadoxine–pyrimethamine and artemisinin) is a major constrain in malaria control. Antimicrobial peptides (AMPs) have shown promising results in controlling Plasmodium spp. parasitemia in in vitro and in vivo models of infection. Defensins are AMPs that act primarily by disrupting the integrity of cell membranes of invasive microbes. We previously showed that defensins from the tick Ixodes ricinus inhibited significantly the growth of P. falciparum in vitro, a property that was conserved during evolution. Here, we tested the activity of three I. ricinus defensins against P. chabaudi in mice. A single dose of defensin (120 μl of 1 mg/ml solution) was administered intravenously to P. chabaudi-infected mice, and the parasitemia was followed for 24 h post-treatment. Defensin treatment inhibited significantly the replication (measured as increases in parasitemia) of P. chabaudi after 1 h and 12 h of treatment. Furthermore, defensin injection was not associated with toxicity. These results agreed with the previous report of antiplasmodial activity of tick defensins against P. falciparum in vitro and justify further studies for the use of tick defensins to control malaria.

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

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

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

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

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.

Immunomodulation induced by the aqueous extract obtained from Ampelozizyphus amazonicus

Ampelozizyphus amazonicus Ducke (Rhamnaceae) (popularly known as “Saracura-mirá”) is a medicinal plant from Amazonia (Brazil). An aqueous drink is prepared from its barks and roots which displays a very bitter taste and forms abundant foam when shaken, due to the high content of dammarane-type saponins in the species. The plant is used in the region to treat and prevent malaria, as well as a stimulant and energetic. Previous studies did not show a direct action of this plant on Plasmodium blood forms, however, it is effective on controlling infection induced by sporozoite forms. In this study, we investigated whether the aqueous extract (SM) obtained from the stem barks of A. amazonicus collected in “quilombola” communities of Oriximiná (Para), Brazil, could control malaria infection through an overall augmentation of the immunological response. Daily oral treatment and dose of SM was based on its traditional use (Peçanha et al. 2013). Oral treatment with SM induces an increase in both IgM and IgG antibody titers in mice immunized with the T-independent type 2 (TI-2) antigen TNP-ficoll. We investigated the effect of SM treatment on the course of B cell response in P. chabaudi-infected mice and observed that SM treatment induced an increase in the levels of circulating total IgM and IgG in Plasmodium-infected mice when compared to infected untreated animals. There was, however, a decrease in the number of antibody-producing plasma cells (CD138+ cells) in the spleen of P. chabaudi-infected SM-treated mice. The data obtained in our study indicates that SM could amplify the response of murine B cells to a TI-2 antigen and could also increase immunoglobulin production during malaria infection and regulate the emergence of antibody secreting cells. Future studies on the role of saponins isolated from SM on both B cell response and resistance to malaria infection will be performed.

Immunobiologic and Antiinflammatory Properties of a Bark Extract fromAmpelozizyphus amazonicusDucke

Ampelozizyphus amazonicus is used in the treatment and prevention of malaria. The effect of an aqueous extract from this plant (SART) on the immune response was investigated by measuring immunoglobulin production induced by immunization with the antigen TNP-Ficoll in Plasmodium chabaudi-infected mice. SART treatment increased antigen-specific IgM and IgG levels in TNP-Ficoll-immunized mice. The B cell response during malarial infection was also modified by SART. There was an increase in total serum IgM and IgG and a decrease in the percentage of splenic plasma cells (CD138+ cells) in P. chabaudi-infected, SART-treated animals. SART (1, 3 or 10 mg/kg, p.o.) and the reference drug dexamethasone (5 mg/kg) were also tested in carrageenan-induced leukocyte migration to the subcutaneous air pouch (SAP). All SART doses significantly reduced leukocyte migration into the SAP. The protein concentration resulting from extravasation into the peritoneum was also significantly reduced. Our data indicate that SART possesses immunomodulatory properties, inducing an in vivo modification of the B lymphocyte response and anti-inflammatory properties, which are partly due to a reduction in cell migration and are most likely due to an inhibition of the production of inflammatory mediators. Preliminary HPLC-ESI-MS/MS analysis of SART shows a complex saponin profile with deprotonated molecule [M-H]− ions in the range of m/z 800–1000.

Transient Deficiency of Dendritic Cells Results in Lack of a Merozoite Surface Protein 1-Specific CD4 T Cell Response during Peak Plasmodium chabaudi Blood-Stage Infection

Splenic dendritic cells are crucial for controlling the immune response to malaria by initiating a CD4 gamma interferon (IFN-γ) response early in a blood-stage infection, which contributes to parasite clearance as well as to acute-stage immunopathology. CD8(-) CD11c(high) dendritic cells have been described previously to be important antigen-presenting cells for induction of these CD4 T cell responses in the spleens of Plasmodium chabaudi-infected mice. However, when isolated during the period of maximum parasitemia and shortly thereafter, the dendritic cells transiently lose their ability to stimulate T cells, recovering only as the parasitemia is controlled. This loss of a CD4 T cell response is also observed in vivo during this part of the infection. CD4 T cells from a T cell receptor-transgenic mouse recognizing a peptide of merozoite surface protein 1 (MSP1) injected into BALB/c mice during peak parasitemia proliferate poorly, and very few cells produce IFN-γ and interleukin-2 (IL-2), compared with transgenic T cells injected earlier in the blood-stage infection. CD8(-) dendritic cells at day 10 can process and present peptides on major histocompatibility complex (MHC) class II with an efficiency similar to that of dendritic cells from earlier in infection. The failure of the day 10 dendritic cells to activate MSP1-specific CD4 T cells fully in vitro is associated with reduced expression of CD86 and lower production of IL-12 rather than with induction of inhibitory DC receptors or production of IL-10.

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.