Plasmodium Berghei Infection Research Articles

Host 5-HT affects Plasmodium transmission in mosquitoes via modulating mosquito mitochondrial homeostasis.

Malaria parasites hijack the metabolism of their mammalian host during the blood-stage cycle. Anopheles mosquitoes depend on mammalian blood to lay eggs and to transmit malaria parasites. However, it remains understudied whether changes in host metabolism affect parasite transmission in mosquitoes. In this study, we discovered that Plasmodium infection significantly decreased the levels of the tryptophan metabolite, 5-hydroxytryptamine (5-HT), in both humans and mice. The reduction led to the decrease of 5-HT in mosquitoes. Oral supplementation of 5-HT to Anopheles stephensi enhanced its resistance to Plasmodium berghei infection by promoting the generation of mitochondrial reactive oxygen species. This effect was due to the accumulation of dysfunctional mitochondria caused by 5-HT-mediated inhibition of mitophagy. Elevating 5-HT levels in mouse serum significantly suppressed parasite infection in mosquitoes. In summary, our data highlight the critical role of metabolites in animal blood in determining the capacity of mosquitoes to control parasite infection.

Phenotypic screening reveals a highly selective phthalimide-based compound with antileishmanial activity.

Pharmacophores such as hydroxyethylamine (HEA) and phthalimide (PHT) have been identified as potential synthons for the development of compounds against various parasitic infections. In order to further advance our progress, we conducted an experiment utilising a collection of PHT and HEA derivatives through phenotypic screening against a diverse set of protist parasites. This approach led to the identification of a number of compounds that exhibited significant effects on the survival of Entamoeba histolytica, Trypanosoma brucei, and multiple life-cycle stages of Leishmania spp. The Leishmania hits were pursued due to the pressing necessity to expand our repertoire of reliable, cost-effective, and efficient medications for the treatment of leishmaniases. Antileishmanials must possess the essential capability to efficiently penetrate the host cells and their compartments in the disease context, to effectively eliminate the intracellular parasite. Hence, we performed a study to assess the effectiveness of eradicating L. infantum intracellular amastigotes in a model of macrophage infection. Among eleven L. infantum growth inhibitors with low-micromolar potency, PHT-39, which carries a trifluoromethyl substitution, demonstrated the highest efficacy in the intramacrophage assay, with an EC50 of 1.2 +/- 3.2 μM. Cytotoxicity testing of PHT-39 in HepG2 cells indicated a promising selectivity of over 90-fold. A chemogenomic profiling approach was conducted using an orthology-based method to elucidate the mode of action of PHT-39. This genome-wide RNA interference library of T. brucei identified sensitivity determinants for PHT-39, which included a P-type ATPase that is crucial for the uptake of miltefosine and amphotericin, strongly indicating a shared route for cellular entry. Notwithstanding the favourable properties and demonstrated efficacy in the Plasmodium berghei infection model, PHT-39 was unable to eradicate L. major infection in a murine infection model of cutaneous leishmaniasis. Currently, PHT-39 is undergoing derivatization to optimize its pharmacological characteristics.

Mosquito midgut stem cell cellular defense response limits Plasmodium parasite infection

A novel cellular response of midgut progenitors (stem cells and enteroblasts) to Plasmodium berghei infection was investigated in Anopheles stephensi. The presence of developing oocysts triggers proliferation of midgut progenitors that is modulated by the Jak/STAT pathway and is proportional to the number of oocysts on individual midguts. The percentage of parasites in direct contact with enteroblasts increases over time, as progenitors proliferate. Silencing components of key signaling pathways through RNA interference (RNAi) that enhance proliferation of progenitor cells significantly decreased oocyst numbers, while limiting proliferation of progenitors increased oocyst survival. Live imaging revealed that enteroblasts interact directly with oocysts and eliminate them. Midgut progenitors sense the presence of Plasmodium oocysts and mount a cellular defense response that involves extensive proliferation and tissue remodeling, followed by oocysts lysis and phagocytosis of parasite remnants by enteroblasts.

PRL2 regulates neutrophil extracellular trap formation which contributes to severe malaria and acute lung injury

Excessive host immune responses contribute to severe malaria with high mortality. Here, we show that PRL2 in innate immune cells is highly related to experimental malaria disease progression, especially the development of murine severe malaria. In the absence of PRL2 in myeloid cells, Plasmodium berghei infection results in augmented lung injury, leading to significantly increased mortality. Intravital imaging revealed greater neutrophilic inflammation and NET formation in the lungs of PRL2 myeloid conditional knockout mice. Depletion of neutrophils prior to the onset of severe disease protected mice from NETs associated lung injury, and eliminated the difference between WT and PRL2 CKO mice. PRL2 regulates neutrophil activation and NET accumulation via the Rac-ROS pathway, thus contributing to NETs associated ALI. Hydroxychloroquine, an inhibitor of PRL2 degradation alleviates NETs associated tissue damage in vivo. Our findings suggest that PRL2 serves as an indicator of progression to severe malaria and ALI. In addition, our study indicated the importance of PRL2 in NET formation and tissue injury. It might open a promising path for adjunctive treatment of NET-associated disease.

Evaluation of the antimalarial properties of Solanum incanum L. leaf extract fractions and its ability to downregulate delta aminolevulinate dehydratase to prevent the establishment of malaria infection

Ethnopharmacological relevanceSolanum incanum L. is commonly used in traditional herbal medicine (THM) in Kenya for treating various ailments. Recent developments in disease treatment have introduced the concept of host-directed therapy (HDT). This approach involves targeting factors within the host cell that can impede the growth or replication of a pathogen. One such host factor is delta aminolevulinate dehydratase (δ-ALAD), the second enzyme in the heme biosynthesis pathway utilized by Plasmodium for growth. Studies using mice models have shown an increase in δ-ALAD expression during Plasmodium berghei infection. Another plant in the Solanum genus, S. guaranticum, has been found to inhibit δ-ALAD in red blood cells in vitro and in the brain in vivo. Is it possible that the bioactive compounds in S. incanum extracts could also be effective in HDT for malaria treatment? Aim of studyTo better assess the effectiveness of S. incanum leaf extracts as a curative and prophylaxis in malaria parasite infection, and to test the plant’s ability to decrease δ-ALAD expression. Materials and methodsThe leaves of S. incanum were collected, dried, and pulverized before being subjected to a successive extraction protocol to obtain crude, hexane, ethyl acetate, and aqueous extract fractions. Phytochemical analysis was conducted on all extract fractions, followed by GC-MS analysis of the fraction with the most potent antimalarial activity. An acute toxicity study was also performed on the extracted fractions. The potency of the extract fractions as curative and prophylactic antimalarial was then evaluated in THM using Plasmodium berghei-infected mice at a dose of 100 mg/kg. The extract fraction with the highest activity was further evaluated at varying doses and its effect on δ-ALAD was measured using RT-qPCR. The percentage of parasitemia and chemosuppression, and mean survival time were used as indices of activity. ResultsPhytochemical analysis revealed that the ethyl acetate and aqueous extract fractions contained high terpenoids, flavonoids, and phenols levels. However, alkaloids were only present in moderate quantities in the aqueous extract, and quinones were found in high levels only in the crude extract. Additionally, all extract fractions contained saponins in high levels but lacked tannins. While the plant extracts were found to be non-toxic, they did not exhibit curative antimalarial activity. However, all extract fractions showed prophylactic antimalarial activity, with the ethyl acetate extract having the highest percentage of chemosuppression even at doses of 250 and 1000 mg/kg. In the negative control, the expression of δ-ALAD was 5.4-fold, but this was significantly reduced to 2.3-fold when mice were treated with 250 mg/kg of the ethyl acetate fraction. GC-MS analysis of the ethyl acetate fraction revealed high percentages of 2-methyloctacosane, tetracosane, and decane. ConclusionThe fractions extracted from S. incanum leaves have been found to possess only antimalarial prophylactic properties, with the ethyl acetate extract fraction showing the most effective results. The activity of this fraction may be attributed to its ability to decrease the expression of δ-ALAD, as it contains an alkane compound implicated with enzyme-inhibitory activity.

Antimalarial activity of borrelidin and fumagilin in Plasmodium berghei-infected mice.

Malaria remains a significant global health burden, with drug resistance posing a major challenge to its control. The emergence of resistance to antimalarial drugs represents a critical issue in malaria management, as it heightens the likelihood of morbidity and mortality associated with the disease. There is an urgent requirement for a novel candidate drug with a distinct mechanism of action. In light of the ongoing challenges in malaria management, particularly the emergence of drug resistance, this study aimed to investigate the efficacy of a novel combination therapy of borrelidin and fumagilin against Plasmodium berghei infection on Swiss Webster mice. The findings of this study could contribute to developing new and effective antimalarial treatments. This study employed a unique approach, using Swiss Webster mice aged 6-8 weeks and dividing them into five groups, each with five mice. The therapeutic efficacy of the combination treatment was evaluated through a comprehensive assessment of parasitemia levels, survival rates, and histological changes in the liver and spleen. This rigorous methodology ensures the reliability and validity of our findings. The combination of borrelidin and fumagilin led to the lowest parasitemia at 5%, contrasting with the control group reaching 15%. Moreover, the combination group exhibited the highest inhibition rate of 69.6% on day nine post-infection. Histopathological alterations were limited to sinusoid dilation, hepatocyte ballooning, and the presence of hemozoin. These findings suggest that the combination of borrelidin and fumagilin holds promise as a potential antimalarial therapy.

Combination of computational techniques and RNAi reveal targets in Anopheles gambiae for malaria vector control.

Increasing reports of insecticide resistance continue to hamper the gains of vector control strategies in curbing malaria transmission. This makes identifying new insecticide targets or alternative vector control strategies necessary. CLassifier of Essentiality AcRoss EukaRyote (CLEARER), a leave-one-organism-out cross-validation machine learning classifier for essential genes, was used to predict essential genes in Anopheles gambiae and selected predicted genes experimentally validated. The CLEARER algorithm was trained on six model organisms: Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens, Mus musculus, Saccharomyces cerevisiae and Schizosaccharomyces pombe, and employed to identify essential genes in An. gambiae. Of the 10,426 genes in An. gambiae, 1,946 genes (18.7%) were predicted to be Cellular Essential Genes (CEGs), 1716 (16.5%) to be Organism Essential Genes (OEGs), and 852 genes (8.2%) to be essential as both OEGs and CEGs. RNA interference (RNAi) was used to validate the top three highly expressed non-ribosomal predictions as probable vector control targets, by determining the effect of these genes on the survival of An. gambiae G3 mosquitoes. In addition, the effect of knockdown of arginase (AGAP008783) on Plasmodium berghei infection in mosquitoes was evaluated, an enzyme we computationally inferred earlier to be essential based on chokepoint analysis. Arginase and the top three genes, AGAP007406 (Elongation factor 1-alpha, Elf1), AGAP002076 (Heat shock 70kDa protein 1/8, HSP), AGAP009441 (Elongation factor 2, Elf2), had knockdown efficiencies of 91%, 75%, 63%, and 61%, respectively. While knockdown of HSP or Elf2 significantly reduced longevity of the mosquitoes (p<0.0001) compared to control groups, Elf1 or arginase knockdown had no effect on survival. However, arginase knockdown significantly reduced P. berghei oocytes counts in the midgut of mosquitoes when compared to LacZ-injected controls. The study reveals HSP and Elf2 as important contributors to mosquito survival and arginase as important for parasite development, hence placing them as possible targets for vector control.

Curcumin as a Stabilizer of Macrophage Polarization during Plasmodium Infection.

Malaria is a parasitic infection responsible for high morbidity and mortality rates worldwide. During the disease, phagocytosis of infected red blood cells by the macrophages induces the production of reactive oxygen (ROS) and nitrogen species (RNS), culminating in parasite death. Curcumin (CUR) is a bioactive compound that has been demonstrated to reduce the production of pro-inflammatory cytokines and chemokines produced by macrophages but to reduce parasitemia in infected mice. Hence, the main purpose of this study is to investigate whether curcumin may interfere with macrophage function and polarization after Plasmodium berghei infection in vitro. In our findings, non-polarized macrophage (M0), classically activated (M1), and alternatively activated (M2) phenotypes showed significantly increased phagocytosis of infected red blood cells (iRBCs) when compared to phagocytosis of uninfected red blood cells (RBCs) 3 h after infection. After 24 h, M1 macrophages exposed to RBCs + CUR showed greater elimination capacity when compared to macrophages exposed to iRBCs + CUR, suggesting the interference of curcumin with the microbicidal activity. Additionally, curcumin increased the phagocytic activity of macrophages when used in non-inflammatory conditions (M0) and reduced the inducible nitric oxide synthase (iNOS) and arginase activities in all macrophage phenotypes infected (M0, M1, and M2), suggesting interference in arginine availability by curcumin and balance promotion in macrophage polarization in neutral phenotype (M0). These results support the view of curcumin treatment in malaria as an adjuvant, promoting a balance between pro- and anti-inflammatory responses for a better clinical outcome.

Effects of Moringa oleifera Extract as an Immunomodulator of Lymphocyte Cells and Macrophages in BALB/c Mice Infected with Plasmodium berghei

Highlights: 1. This study assessed the immunomodulatory potential of Moringa oleifera, which may serve as a natural source forantimalarial treatment.2. Moringa oleifera extract can act as an immunomodulator due to its suppressive effect on Plasmodium berghei infection. Abstract Malaria is an infectious disease caused by protozoa of the genus Plasmodium. In Indonesia, this disease remains a health concern that must be resolved. Due to its high prevalence in eastern Indonesia, it is a challenge to eradicate this disease. Moringa oleifera contains various substances that are believed to have antimalarial activity. This study aimed to determine the effectiveness of Moringa oleifera leaf extract in increasing immune cells and eradicating parasites by using mice infected with Plasmodium berghei. The research was conducted in vivo on BALB/c strain mice (n=40) that were already infected with Plasmodium berghei. Moringa oleifera leaf extract at 25%, 50%, and 75% concentrations was administered orally every day to the mice, while a peripheral blood smear was performed to evaluate parasitemia levels and macrophage activation. A complete blood count was also performed after all tests on the mice were completed. Statistical analysis was performed using the one-way analysis of variance (ANOVA) test with a=0.05 and 95% confidence interval (CI). The results showed that the administration of Moringa oleifera leaf extract at 25%, 50%, and 75% concentrations caused varying degrees of parasitemia compared to the negative group (p&lt;0.05). The group that received the extract at 50% concentration differed significantly from the control groups in the number of activated macrophages. The results of the complete blood count indicated immunomodulatory effects through the presence of diverse immune cell types. In conclusion, Moringa oleifera leaf extract suppresses Plasmodium berghei infection and enhances immune cell stimulation.

Evaluation of the Efficacy of Artemether/Lumefantrine/Doxycycline Combination against Plasmodium berghei in Mice

Background: Malaria is common in regions like sub-Saharan Africa and South East Asia, where it results in millions of mortalities per year. Resistance to currently available antimalarials further compounds the problem. Drug combination has been demonstrated to be effective in overcoming resistance to antimalarials. This study aimed to assess the efficacy of the combination of Artemether/Lumefantrine (AL) and Doxycycline (DX) against Plasmodium berghei infection in mice. Methods: Thirty Adult Swiss albino mice were inoculated with the chloroquine (CQ)-sensitive P. berghei NK65 strain. The curative test involved oral administration of any of DX, AL, AL/DX, or Chloroquine (CQ) to mice with established infections, while the suppressive test was performed by treating mice shortly after parasite inoculation. Parasitemia levels were monitored using thin smears stained with Giemsa solution. Mean Survival Time (MST) was determined for each group. Results: In the curative test, DX, AL, and AL/DX exhibited parasitemia inhibitions of 81.4%, 94.5%, and 93.2%, respectively, while CQ achieved 93.5% inhibition. MST was significantly extended by DX, AL, and AL/DX treatments compared to negative control (NC). In the suppressive test, parasitemia inhibitions were 79.9%, 96.1%, 95.5%, and 94.9% for DX, AL, AL/DX, and CQ, respectively. The administration of DX, AL, and AL/DX also significantly prolonged MST compared to NC. Conclusion: The study demonstrates that the drug combination, particularly AL/DX, exhibits significant antimalarial effects in both curative and suppressive tests. The combination treatments led to substantial reductions in parasitemia levels and extended mean survival time, indicating their potential as effective antimalarial strategies. These findings highlight the promising role of drug combinations in addressing malaria infections and suggest avenues for further investigation and development of combination therapies.

Human nuclear hormone receptor activity contributes to malaria parasite liver stage development

Chemical genetic approaches have had a transformative impact on discovery of drug targets for malaria but have primarily been used for parasite targets. To identify human pathways required for intrahepatic development of parasite, we implemented multiplex cytological profiling of malaria infected hepatocytes treated with liver stage active compounds. Some compounds, including MMV1088447 and MMV1346624, exhibited profiles similar to cells treated with nuclear hormone receptor (NHR) agonist/antagonists. siRNAs targeting human NHRs, or their signaling partners identified eight genes that were critical for Plasmodium berghei infection. Knockdown of NR1D2, a host NHR, significantly impaired parasite growth by downregulation of host lipid metabolism. Importantly, treatment with MMV1088447 and MMV1346624 but not other antimalarials, phenocopied the lipid metabolism defect of NR1D2 knockdown. Our data underlines the use of high-content imaging for host-cellular pathway deconvolution, highlights host lipid metabolism as a drug-able human pathway and provides new chemical biology tools for studying host-parasite interactions.

Leukocyte Differentiation Mice (Mus Musculus) Infected with Plasmodium Berghei after Therapy With Sargassum Duplicatum Extract

Leukocytes are white blood cells produced by hematopoietic tissue that serves to help the body fight various infectious diseases as part of the immune system. Sargassum duplicatum contains steroid, alkaloid, phenol, flavonoid, saponin, and tannin compounds that can be immunomodulatory. The purpose of this research is to know the effect of methane extract of brown algae Sargassum duplicatum on the differentiation of leukocyte mice (Mus musculus) infected with Plasmodium berghei. Mice weighing 20-30 grams in Plasmodium berghei infection as much as 0.1 ml per head and left to the percentage of parasitemia reaches 1-5%. Then the mice (Mus musculus) were given brown algae methanol extract (Sargassum duplicatum) at doses 10, 100, and 200 mg / g BW for 4 consecutive days. From day 0 to 6 also made a blood smear to calculate the number of leukocytes. Data analysis will be done using ANOVA. The results showed that the administration of brown algae extracts of Sargassum duplicatum influenced in increasing the number of neutrophils, monocytes, and basophils and decreased the number of eosinophils and lymphocytes of mice infected with Plasmodium berghei to near normal range. Dose 200 mg/kg BB methanol extract of brown alga Sargassum duplicatum is a dose that can affect the number of neutrophils, eosinophils, lymphocytes, and monocytes of mice infected with Plasmodium berghei most closely to normal range.

Derivatives of Amaryllidaceae Alkaloid Ambelline as Selective Inhibitors of Hepatic Stage of Plasmodium berghei Infection In Vitro.

The incidence rate of malaria and the ensuing mortality prompts the development of novel antimalarial drugs. In this work, the activity of twenty-eight Amaryllidaceae alkaloids (1-28) belonging to seven different structural types was assessed, as well as twenty semisynthetic derivatives of the β-crinane alkaloid ambelline (28a-28t) and eleven derivatives of the α-crinane alkaloid haemanthamine (29a-29k) against the hepatic stage of Plasmodium infection. Six of these derivatives (28h, 28m, 28n and 28r-28t) were newly synthesized and structurally identified. The most active compounds, 11-O-(3,5-dimethoxybenzoyl)ambelline (28m) and 11-O-(3,4,5-trimethoxybenzoyl)ambelline (28n), displayed IC50 values in the nanomolar range of 48 and 47 nM, respectively. Strikingly, the derivatives of haemanthamine (29) with analogous substituents did not display any significant activity, even though their structures are quite similar. Interestingly, all active derivatives were strictly selective against the hepatic stage of infection, as they did not demonstrate any activity against the blood stage of Plasmodium infection. As the hepatic stage is a bottleneck of the plasmodial infection, liver-selective compounds can be considered crucial for further development of the malaria prophylactics.

Correlation of Parasitaemia and Anemia in Mice Infected with Plasmodium berghei ANKA

Malaria is an infectious disease caused by protozoa of the genus Plasmodium and transmitted through the bite of a female Anopheles mosquito. Plasmodium berghei ANKA is a species of rodent malaria parasite that is commonly used to study malaria pathology and the immune system against infections. Parasitaemia in malaria is the figure of malaria pathology due to some numbers of parasite-infected erythrocytes present in the peripheral blood. Hemoglobin (HGB) and hematocrit (HCT) levels are the parameters of anemia and some hematological changes caused by malaria infection. This study aimed to determine the correlation between parasitemia and anemia in BABL/c mice infected with Plasmodium berghei ANKA. Two uninfected and infected mice groups were compared for parasitemia, HGB, and HCT levels. Analysis statistics showed a significant difference in HGB and HCT between uninfected and infected groups. Pearson correlation analysis showed no significant correlation between parasitemia and HGB and HCT levels in infected mice. Anemia in mice infected with Plasmodium berghei ANKA can occur when parasitemia is even low; the higher parasitemia worsens the hamatological condition. Parasitemia plays a role independently in the severity of anemia. Plasmodium berghei infection in mice is useful for studying malaria anemia.

Bifidobacterium longum Administration Diminishes Parasitemia and Inflammation During Plasmodium berghei Infection in Mice.

During Plasmodium berghei (P. berghei) infection, infected erythrocytes are sequestered in gut tissues through microvascular circulation, leading to dysbiosis. This study aimed to investigate the effect of Lactobacillus casei (L. casei) and Bifidobacterium longum (B. longum) administration on the parasitemia level, gut microbiota composition, expression of cluster of differentiation 103 (CD103) in intestinal dendritic and T regulatory cells (T reg), plasma interferon gamma (IFN-γ) and tumor necrosis factor (TNF-α) levels in P. berghei infected mice. P. berghei was inoculated intraperitoneally. Infected mice were randomly divided into 5 groups and treated with either L. casei, B. longum, or the combination of both for 5 days before up to 6 days post-infection (p.i). The control group was treated with phosphate-buffered saline (PBS), while uninfected mice were used as negative control. Levels of CD103 and forkhead box P3 (FoxP3) expression were measured by direct immunofluorescense, while plasma IFN-γ and TNF-α level were determined using enzyme-linked immunosorbent assay (ELISA). All treated groups showed an increase in parasitemia from day 2 to day 6 p.i, which was significant at day 2 p.i (p = 0.001), with the group receiving B. longum displaying the lowest degree of parasitemia. Significant reduction in plasma IFN-γ and TNF-α levels was observed in the group receiving B. longum (p = 0.022 and p = 0.026, respectively). The CD103 and FoxP3 expression was highest in the group receiving B. longum (p = 0.01 and p = 0.02, respectively). B. longum showed the best protective effect against Plasmodium infection by reducing the degree of parasitemia and modulating the gut immunity. This provides a basis for further research involving probiotic supplementation in immunity modulation of infectious diseases.

Antimalarial activity of the 80%methanol extract and solvent fractions of Cucumis ficifolius A. rich roots against Plasmodium berghei in mice

Ethnopharmacological relevanceMalaria is still a known health threat, especially in parts of sub-Saharan Africa. It is one of the frequently mentioned issues with hospital admission and outpatient care in Ethiopia. Cucumis ficifolius A. Rich roots are historically used in Ethiopia to treat meningitis, inflammation, and malaria. However, the antimalarial activity of this plant has not been scientifically studied so far. Aim of the studyThis study aimed to determine the in vivo antimalarial activity of 80% methanol extract and solvent fractions of the roots of Cucumis ficifolius against Plasmodium berghei infection in mice. MethodsThe in vivo antimalarial activity of the 80% methanol extract and solvent fractions of Cucumis ficifolius A. Rich was evaluated by standard chemo suppressive, curative and repository tests using Plasmodium berghei (ANKA strain) in Swiss albino mice at doses of 100, 200 and 400 mg/kg/day. The level of parasitemia, survival time, variation in weight, rectal temperature, and packed cell volume of mice were determined to establish the activity of the extracts. ResultThe 80% methanol extract of Cucumis ficifolius A. Rich roots had a promising suppression of parasitemia at 400 mg/kg with a chemosuppression value of 65.21 ± 1.20%. Among the solvent fractions, the chloroform fraction showed the highest antimalarial activity in the four-day suppressive test with a chemosuppression value of 55.9 ± 0.28%, followed by the n-butanol (42.9 ± 0.24%), and aqueous (40.57 ± 0.52%) fractions at a dose of 400 mg/kg. The highest survival times were observed with crude extract (15.4 ± 0.24 days) at 400 mg/kg, and chloroform fraction (13.4 + 0.24 days), though all extracts increased survival time. ConclusionThe findings of the present study collectively indicate the root extract of Cucumis ficifolius has a promising antiplasmodial activity which substantiates the traditional claim of the plant.

Synergistic antimalarial treatment of Plasmodium berghei infection in mice with dihydroartemisinin and Gymnema inodorum leaf extract

BackgroundChemotherapy is crucial in the fight against malaria. The rise of resistance to most antimalarial medicines has been a serious hurdle to effective treatment. Artemisinin-based combination therapies (ACTs) are currently the most effective antimalarial medication. Malaria parasites are growing more resistant to ACTs, particularly in Southeast Asia. As a result, effective alternative antimalarials are in high demand. The leaf extract of Gymnema inodorum (GIE) has previously shown promise as an effective antimalarial. Therefore, this study evaluated the antimalarial potential of combination dihydroartemisinin (DHA) and GIE therapy against Plasmodium berghei in a mouse model.MethodsThe medications were evaluated using the standard 4-day test for determining the 50% effective dosage (ED50) of DHA and GIE on P. berghei ANKA (PbANKA). DHA and GIE were combined using a fixed-ratio approach, with DHA/GIE ED50s of 100/0, 80/20, 60/40, 40/60, 20/80, and 0/100, respectively.ResultsThe ED50 against PbANKA was determined to be 2 mg/kg of DHA and 100 mg/kg of GIE. The 60/40 (DHA/GIE) ratio demonstrated significantly higher antimalarial activity than the other ratios (p < 0.001) against PbANKA, with 88.95% inhibition, suggesting synergistic efficacy (combination index (CI) = 0.68695). Furthermore, this ratio protected PbANKA-infected mice against loss of body weight and packed cell volume decline, leading to a longer survival time over 30 days.ConclusionOur results suggest that GIE could be an effective adjuvant to DHA that can enhance the antimalarial effects in the treatment of PbANKA-infected mice.

Plasmodium berghei Purified Hemozoin Associated with DNA Strongly Inhibits P. berghei Liver-Stage Development in BALB/c Mice after Intravenous Inoculation.

In the acidic lysosome-like digestive vacuole, Plasmodium parasites crystallize heme from hemoglobin into hemozoin, or malaria pigment. Upon release of progeny merozoites, the residual hemozoin is phagocytized by macrophages principally in the liver and spleen where the heme crystals can persist for months to years, as heme oxygenase does not readily degrade the crystal. Previous studies demonstrated hemozoin modulation of monocytes and macrophages. Hemozoin modulates immune function activity of monocytes/macrophages. Here, we used purified/washed hemozoin (W-Hz) isolated from murine Plasmodium berghei infections and intravenously (i.v.) injected it back into naive mice. We characterized the modulating effect of W-Hz on liver-stage replication. Purified washed hemozoin decreases P. berghei liver levels both at 1 week and 1 month after i.v. injection in a dose and time dependent fashion. The injected hemozoin fully protected in nine out of 10 mice given a 50 sporozoite inoculum, and in 10 out of 10 mice against 2,000 sporozoites when they were infected an hour or a day after hemozoin inoculation. DNase treatment at the hemozoin reversed the observed liver load reduction. The liver load reduction was similar in mature B- and T-cell-deficient RAG-1 knockout (KO) mice suggesting an innate immune protection mechanism. This work indicates a role for residual hemozoin in down modulation of Plasmodium liver stages.

Malaria-derived exosomes exacerbate liver injury during blood stage of Plasmodium berghei infection

Liver injury is a common clinical feature of Plasmodium spp. infection and contributes to multi-organ failure of severe malaria. Malaria-derived exosomes (MD-Exos) have recently engaged as key mediators in parasite-host interactions, modulating the subsequent pathogenic process. However, the role of MD-Exos in malaria-related liver injury and the underlying mechanisms remain unclear. Herein, exosomes from C57BL/6 mice infected with or without P. berghei ANKA serum (namely inf-Exos or un-Exos) were isolated and characterized by transmission electron microscopy, western blotting, and nanoparticle tracking analysis. The miRNAs profiling between inf-Exos and un-Exos were generated using RNA-seq and qPCR. The functions of inf-Exos on liver injury were investigated after two types of exosomes injected into mice intravenously (i.v.), by examining histopathological and apoptotic changes, macrophage polarization, and pro-inflammatory response. The infected red blood cells-stimulated mouse Raw264.7 macrophage cells targeted by inf-Exos or un-Exos were cultured for further study and verification the potential mechanisms. We found that both inf-Exos and un-Exos displayed a typical cup-shaped structure with a diameter of 60–200 nm, and had a positive expression of exosomal markers (e.g., CD9, CD63, and CD81). Compared with infected control mice, the treatment of inf-Exos but not un-Exos dramatically enhanced peripheral blood parasitemia and ECM incidence, exacerbated liver histopathological damage, elevated numbers of liver apoptotic cells, CD68+and CD86+ macrophages. The CD68+-TREM-1+ macrophages in liver tissues and the mRNA levels of pro-inflammatory cytokines (e.g., iNOS, TNF-α, IL-1β, and IL-6) were increased by inf-Exos treatment in vivo. Meanwhile, the treatment of inf-Exos resulted in a substantial increase of the mRNA levels of CD86, iNOS, TNF-α, IL-1β, and IL-6, but led to a remarkable decrease of Bcl-6 and SOCS-1 in Raw264.7 cells stimulated with iRBC in vitro. Notably, compared to un-Exos, five types of miRNAs (including miR-10a-5p, miR-10b-5p, miR-155–5p, miR-205–5p, and miR-21a-5p), that were previously reported to target Bcl-6 or SOCS-1, present higher abundance on inf-Exos, as demonstrated by RNA-seq and qPCR. Collectively, our data suggest that inf-Exos exacerbate malaria-induced liver pathology via triggering excessive pro-inflammatory response and promoting macrophage M1 polarization. Our findings will provide new insights into the roles of inf-Exos in malaria parasite-host interaction and pathogenesis of liver injury.

Fetal Growth Retardation is Associated with High Apoptotic Cells and Low VEGF Expression in Placenta of Malarial Pregnant Mice.

During pregnancy, pregnant women are susceptible to malaria, contributing significantly to maternal and infant mortality. This research was conducted to study the effect of Plasmodium berghei infection in pregnant mice on fetal growth retardation through placental cell apoptosis and the change of local vascularization. Eighteen pregnant Balb/c strain mice resulting from simultanously mating were divided into two groups those were nine pregnant mice used as non infected group and nine pregnant mice infected with Plasmodium berghei on day 9th post mating used as infected group respectively. On day 15th of post mating, all of the pregnant mice were killed. Fetal weights were measured using analytic balance. Apoptosis of placental cells and VEGF expression in the placental tissue were measured using immunohistochemistry. Result showed that there was sequestration of parasite-infected red blood cells (PRBCs) in intervillous space. Statistical analysis showed that the fetal weights in infected pregnant mice group was significantly lower than non infected one (p = 0.01), and the placental cell apoptosis in placental tissue of infected pregnant mice was significantly higher than the non infected one (p=0.00).There was also a significant difference on VEGF expression between infected group and non infected group (p= 0,00). Plasmodium berghei infection in pregnant Balb/c mice can cause fetal growth retardation due to high of placental cell apoptosis and low VEGF expression.