Neurons upregulate PD-L1 via IFN/STAT1/IRF1 to alleviate damage by CD8+ T cells in cerebral malaria.

BACKGROUND: Cerebral malaria (CM) is the most lethal complication of malaria, and survivors usually endure neurological sequelae. Notably, the cytotoxic effect of infiltrating Plasmodium-activated CD8+ T cells on cerebral microvasculature endothelial cells is a prominent feature of the experimental CM (ECM) model with blood-brain barrier disruption. However, the damage effect of CD8+ T cells infiltrating the brain parenchyma on neurons remains unclear. Based on the immunosuppressive effect of the PD-1/PD-L1 pathway on T cells, our previous study demonstrated that the systemic upregulation of PD-L1 to inhibit CD8+ T cell function could effectively alleviate the symptoms of ECM mice. However, it has not been reported whether neurons can suppress the pathogenic effect of CD8+ T cells through the PD-1/PD-L1 negative immunomodulatory pathway. As the important inflammatory factor of CM, interferons can induce the expression of PD-L1 via different molecular mechanisms according to the neuro-immune microenvironment. Therefore, this study aimed to investigate the direct interaction between CD8+ T cells and neurons, as well as the mechanism of neurons to alleviate the pathogenic effect of CD8+ T cells through up-regulating PD-L1 induced by IFNs. METHODS: Using the ECM model of C57BL/6J mice infected with Plasmodium berghei ANKA (PbA), morphological observations were conducted in vivo by electron microscope and IF staining. The interaction between the ECM CD8+ T cells (immune magnetic bead sorting from spleen of ECM mice) and primary cultured cortical neurons in vitro was observed by IF staining and time-lapse photography. RNA-seq was performed to analyze the signaling pathway of PD-L1 upregulation in neurons induced by IFNß or IFNγ, and verified through q-PCR, WB, IF staining, and flow cytometry both in vitro and in vivo using IFNAR or IFNGR gene knockout mice. The protective effect of adenovirus-mediated PD-L1 IgGFc fusion protein expression was verified in ECM mice with brain stereotaxic injection in vivo and in primary cultured neurons via viral infection in vitro. RESULTS: In vivo, ECM mice showed infiltration of activated CD8+ T cells and neuronal injury in the brain parenchyma. In vitro, ECM CD8+ T cells were in direct contact with neurons and induced axonal damage, as an active behavior. The PD-L1 protein level was elevated in neurons of ECM mice and in primary cultured neurons induced by IFNß, IFNγ, or ECM CD8+ T cells in vitro. Furthermore, the IFNß or IFNγ induced neuronal expression of PD-L1 was mediated by increasing STAT1/IRF1 pathway via IFN receptors. The increase of PD-L1 expression in neurons during PbA infection was weakened after deleting the IFNAR or IFNGR. Increased PD-L1 expression by adenovirus partially protected neurons from CD8+ T cell-mediated damage both in vitro and in vivo. CONCLUSION: Our study demonstrates that both type I and type II IFNs can induce neurons to upregulate PD-L1 via the STAT1/IRF1 pathway mediated by IFN receptors to protect against activated CD8+ T cell-mediated damage, providing a targeted pathway to alleviate neuroinflammation during ECM.

The influence of oviposition status on measures of transmission potential in malaria-infected mosquitoes depends on sugar availability.

BACKGROUND: Like other oviparous organisms, the gonotrophic cycle of mosquitoes is not complete until they have selected a suitable habitat to oviposit. In addition to the evolutionary constraints associated with selective oviposition behavior, the physiological demands relative to an organism's oviposition status also influence their nutrient requirement from the environment. Yet, studies that measure transmission potential (vectorial capacity or competence) of mosquito-borne parasites rarely consider whether the rates of parasite replication and development could be influenced by these constraints resulting from whether mosquitoes have completed their gonotrophic cycle. METHODS: Anopheles stephensi mosquitoes were infected with Plasmodium berghei, the rodent analog of human malaria, and maintained on 1% or 10% dextrose and either provided oviposition sites ('oviposited' herein) to complete their gonotrophic cycle or forced to retain eggs ('non-oviposited'). Transmission potential in the four groups was measured up to 27 days post-infection as the rates of (i) sporozoite appearance in the salivary glands ('extrinsic incubation period' or EIP), (ii) vector survival and (iii) sporozoite densities. RESULTS: In the two groups of oviposited mosquitoes, rates of sporozoite appearance and densities in the salivary glands were clearly dependent on sugar availability, with shorter EIP and higher sporozoite densities in mosquitoes fed 10% dextrose. In contrast, rates of appearance and densities in the salivary glands were independent of sugar concentrations in non-oviposited mosquitoes, although both measures were slightly lower than in oviposited mosquitoes fed 10% dextrose. Vector survival was higher in non-oviposited mosquitoes. CONCLUSIONS: Costs to parasite fitness and vector survival were buffered against changes in nutritional availability from the environment in non-oviposited but not oviposited mosquitoes. Taken together, these results suggest vectorial capacity for malaria parasites may be dependent on nutrient availability and oviposition/gonotrophic status and, as such, argue for more careful consideration of this interaction when estimating transmission potential. More broadly, the complex patterns resulting from physiological (nutrition) and evolutionary (egg-retention) trade-offs described here, combined with the ubiquity of selective oviposition behavior, implies the fitness of vector-borne pathogens could be shaped by selection for these traits, with implications for disease transmission and management. For instance, while reducing availability of oviposition sites and environmental sources of nutrition are key components of integrated vector management strategies, their abundance and distribution are under strong selection pressure from the patterns associated with climate change.

Effect of moringa extract on parasitemia, monocyte activation and organomegaly among Mus musculus infected by Plasmodium berghei ANKA.

In Indonesia, malaria remains a problem, with 94,610 active cases in 2021 and its current therapy includes chloroquine and artemisinin; however, resistance has been commonly reported. To overcome this problem, studies about potential medicinal plants that can be used as antimalaria, such as moringa (Moringa oleifera) started to receive more attention. The aim of this study was to investigate the effects of moringa in parasitemia, monocyte activation, and organomegaly on animal model malaria. This experimental study used male Mus musculus, infected by Plasmodium berghei ANKA, as an animal malaria model. The extract was made by maceration of dry moringa leaves, which were then divided into three concentrations: 25%, 50%, and 75%. Dihydroartemisinin-piperazine was used as a positive control treatment, and distilled water as a negative control treatment. The animals were observed for six days to assess the parasitemia count and the number of monocyte activation. On day 7, the animals were terminated, and the liver, spleen, and kidney were weighed. The results showed that the effective concentrations in reducing parasitemia and inducing monocyte activation were 50% and 25% of moringa leaf extract, respectively. The smallest liver and spleen enlargement was observed among animals within the group treated with a 50% concentration of M. oleifera extract. In contrast, the smallest kidney enlargement was observed in the group treated with 25% of M. oleifera extract. Further analysis is recommended to isolate compounds with antimalarial properties in moringa leaves.

In vivo antimalarial efficacy of Artemisia afra powder suspensions.

BACKGROUND: A global death toll of 608,000 in 2022 and emerging parasite resistance to artemisinin, the mainstay of antimalarial chemotherapy derived from the Chinese herb Artemisia annua, urge the development of novel antimalarials. A clinical trial has found high antimalarial potency for aqueous extracts of A. annua as well as its African counterpart Artemisia afra, which contains only trace amounts of artemisinin. The artemisinin-independent antimalarial activity of A. afra points to the existence of other antimalarials present in the plant. However, the publication was retracted due to ethical and methodological concerns in the trial, so the only evidence for antimalarial activity of A. afra is built on in vitro studies reporting efficacy only in the microgram per milliliter range. HYPOTHESIS: Our study aims to shed more light on the controversy around the antimalarial activity of A. afra by assessing its efficacy in mice. In particular, we are testing the hypothesis that A. afra contains a pro-drug that is inactive in vitro but active in vivo after metabolization by the mammalian host. METHODS: Plasmodium berghei-infected mice were treated once or thrice (on three consecutive days) with various doses of A. afra, A. annua, or pure artemisinin. RESULTS: Aqueous powder suspensions of A. annua but not A. afra showed antimalarial activity in mice. CONCLUSION: Our experiments conducted in mice do not support the pro-drug hypothesis.

Oral acute toxicity study and in vivo antimalarial activity of Strychnos lucida R. Br. tablet.

ETHNOPHARMACOLOGICAL RELEVANCE: Malaria eradication has been a major goal of the Indonesian government since 2020. Medicinal plants, such as Strychnos lucida R. Br., are empirically used to treat malaria through traditional preparation methods. However, the safety and efficacy of these plants have not yet been confirmed. Therefore, further investigations are necessary to confirm the safety and efficacy of S. lucida as an antimalarial agent. AIMS OF THE STUDY: To quantify the concentration of brucine in the S. lucida extract, determine the acute oral toxicity of the standardized extract, and evaluate the in vivo antimalarial potency of S. lucida tablet (SLT). MATERIALS AND METHODS: Acute oral toxicity of S.lucida extract was determined using the Organization for Economic Co-operation and Development 420 procedure, and the analytical method for brucine quantification was validated using high-performance liquid chromatography. In addition, antimalarial activity was determined using the Peter's four-day suppressive method. RESULTS: Acute toxicity analysis revealed S. lucida as a low-toxicity compound with a cut-off median lethal dose of 2000-5000 mg/kg body weight [BW], which was supported by the hematological and biochemical profiles of the kidneys, liver, and pancreas (p > 0.05). Extract standardization revealed that S. lucida contained 3.91 ± 0.074% w/w brucine, adhering to the limit specified in the Indonesian Herbal Pharmacopeia. Antimalarial test revealed that SLT inhibited the growth of Plasmodium berghei by 27.74-45.27%. Moreover, SLT improved the hemoglobin and hematocrit levels. White blood cell and lymphocyte counts were lower in the SLT-treated group than in the K (+) group (p < 0.05). CONCLUSION: Histopathological and biochemical evaluations revealed that S. lucida extract was safe at a dose of 2000 mg/kg BW with low toxicity. SLT inhibited Plasmodium growth and improved the hemoglobin, hematocrit, and red blood cell profiles. Additionally, SLT reduced the lymphocyte and WBC counts and increased the monocyte and thrombocyte counts as part of the immune system response against Plasmodium infection.

Mechanism of antimalarial action and mitigation of infection-mediated mitochondrial dysfunction by phyto-constituents of Andrographis paniculata ((Burm f.) Wall. ex Nees) in Plasmodium berghei-infected mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Andrographis paniculata (AP) ((Burm f.) Wall. ex Nees) is a medicinal plant, documented for its folkloric use in the treatment of malaria. AIM: This study was designed to determine the potency of extract and fractions of A. paniculata (AP) as a curative, both for susceptible and resistant malaria and to also determine the plant's mechanism of action. This study was also designed to determine whether AP extract and its most potent fraction will mitigate infection-mediated mitochondrial dysfunction, and to assess the phytochemical constituents of the most potent fraction. MATERIALS AND METHODS: n-Hexane, dichloromethane, ethylacetate and methanol were used to partition the methanol extract of A. paniculata. Graded doses of these extract and fractions were used to treat mice infected with chloroquine-sensitive strain of P. berghei in a curative model. The most potent fraction was used to treat mice infected with resistant (ANKA strain) P. berghei. Inhibition of hemozoin formation, reversal of mitochondrial dysfunction and antiinflammatory potentials were determined. A combination of ultraperformance liquid chromatography-quadrupole time of flight-mass spectrometry and nuclear magnetic resonance spectroscopy were used for chemical analysis. RESULTS: Microscopy revealed that the dichloromethane fraction decreased the parasite burden the most, and inhibition of the hemozoin formation is one of its mechanisms of action. The dichloromethane fraction reversed parasite-induced mitochondrial pore opening in the host, enzyme-dependent ATP hydrolysis and peroxidation of host mitochondrial membrane phospholipids as well as its antiinflammatory potentials. The UPLC-qTOF-MS report and NMR fingerprints of the dichloromethane fraction of A. paniculata yielded fourteen compounds of which sibiricinone C was identified from the plant for the first time. CONCLUSION: Fractions of A. paniculata possess antiplasmodial effects with the dichloromethane fraction having the highest potency. The potent effect of this fraction may be attributed to the phytochemicals present because it contains terpenes implicated with antimalarial and antiinflammatory activities.

Searching for new molecules involved in Anopheles mosquitoes' response to Plasmodium infection.

Plasmodium parasites within mosquitoes are exposed to various physiological processes, such as blood meal digestion activity, the gonotrophic cycle, and host responses preventing the entry of parasites into the midgut wall. However, when in vitro-cultured ookinetes are injected into the hemocoel of mosquitoes, Plasmodium parasites are not affected by the vertebrate host's blood contents and do not pass through the midgut epithelial cells. This infection method might aid in identifying mosquito-derived factors affecting Plasmodium development within mosquitoes. This study investigated novel mosquito-derived molecules related to parasite development in Anopheles mosquitoes. We injected in vitro-cultured Plasmodium berghei (ANKA strain) ookinetes into female and male Anopheles stephensi (STE2 strain) mosquitoes and found that the oocyst number was significantly higher in males than in females, suggesting that male mosquitoes better support the development of parasites. Next, RNA-seq analysis was performed on the injected female and male mosquitoes to identify genes exhibiting changes in expression. Five genes with different expression patterns between sexes and greatest expression changes were identified as being potentially associated with Plasmodium infection. Two of the five genes also showed expression changes with infection by blood-feeding, indicating that these genes could affect the development of Plasmodium parasites in mosquitoes.

Extracellular vesicles derived from plasmodium-infected red blood cells alleviate cerebral malaria in plasmodium berghei ANKA-infected C57BL/6J mice.

RTS,S is the first malaria vaccine recommended for implementation among young children at risk. However, vaccine efficacy is modest and short-lived. To mitigate the risk of cerebral malaria (CM) among children under the age of 5, it is imperative to develop new vaccines. EVs are potential vaccine candidates as they obtain the ability of brain-targeted delivery and transfer plasmodium antigens and immunomodulators during infections. This study extracted EVs from BALB/c mice infected with Plasmodium yoelii 17XNL (P.y17XNL). C57BL/6J mice were intravenously immunized with EVs (EV-I.V. + CM group) or subcutaneously vaccinated with the combination of EVs and CpG ODN-1826 (EV + CPG ODN-S.C. + CM group) on days 0 and 20, followed by infection with Plasmodium berghei ANKA (P.bANKA) on day 20 post-second immunization. We monitored Parasitemia and survival rate. The integrity of the Blood-brain barrier (BBB) was examined using Evans blue staining.The levels of cytokines and adhesion molecules were evaluated using Luminex, RT-qPCR, and WB. Brain pathology was evaluated by hematoxylin and eosin and immunohistochemical staining. The serum levels of IgG, IgG1, and IgG2a were analyzed by enzyme-linked immunosorbent assay. Compared with those in the P.bANKA-infected group, parasitemia increased slowly, death was delayed (day 10 post-infection), and the survival rate reached 75 %-83.3 % in the EV-I.V. + ECM and EV + CPG ODN-S.C. + ECM groups. Meanwhile, compared with the EV + CPG ODN-S.C. + ECM group, although parasitemia was almost the same, the survival rate increased in the EV-I.V. + ECM group.Additionally, EVs immunization markedly downregulated inflammatory responses in the spleen and brain and ameliorated brain pathological changes, including BBB disruption and infected red blood cell (iRBC) sequestration. Furthermore, the EVs immunization group exhibited enhanced antibody responses (upregulation of IgG1 and IgG2a production) compared to the normal control group. EV immunization exerted protective effects, improving the integrity of the BBB, downregulating inflammation response of brain tissue, result in reduces the incidence of CM. The protective effects were determined by immunological pathways and brain targets elicited by EVs. Intravenous immunization exhibited better performance than subcutaneous immunization, which perhaps correlated with EVs, which can naturally cross BBB to play a better role in brain protection.

Let it glow: genetically encoded fluorescent reporters in Plasmodium.

The use of fluorescent proteins (FPs) in Plasmodium parasites has been key to understand the biology of this obligate intracellular protozoon. FPs like the green fluorescent protein (GFP) enabled to explore protein localization, promoter activity as well as dynamic processes like protein export and endocytosis. Furthermore, FP biosensors have provided detailed information on physiological parameters at the subcellular level, and fluorescent reporter lines greatly extended the malariology toolbox. Still, in order to achieve optimal results, it is crucial to know exactly the properties of the FP of choice and the genetic scenario in which it will be used. This review highlights advantages and disadvantages of available landing sites and promoters that have been successfully applied for the ectopic expression of FPs in Plasmodium berghei and Plasmodium falciparum. Furthermore, the properties of newly developed FPs beyond DsRed and EGFP, in the visualization of cells and cellular structures as well as in the sensing of small molecules are discussed.

Disclosure of cinnamic acid/4,9-diaminoacridine conjugates as multi-stage antiplasmodial hits.

4,9-diaminoacridines with reported antiplasmodial activity were coupled to different trans-cinnamic acids, delivering a new series of conjugates inspired by the covalent bitherapy concept. The new compounds were more potent than primaquine against hepatic stages of Plasmodium berghei, although this was accompanied by cytotoxic effects on Huh-7 hepatocytes. Relevantly, the conjugates displayed nanomolar activities against blood stage P. falciparum parasites, with no evidence of hemolytic effects below 100 µM. Moreover, the new compounds were at least 25-fold more potent than primaquine against P. falciparum gametocytes. Thus, the new antiplasmodial hits disclosed herein emerge as valuable templates for the development of multi-stage antiplasmodial drug candidates.

Differential effects of translation inhibitors on Plasmodium berghei liver stage parasites.

Increasing numbers of antimalarial compounds are being identified that converge mechanistically at inhibition of cytoplasmic translation, regardless of the molecular target or mechanism. A deeper understanding of how their effectiveness as liver stage translation inhibitors relates to their chemoprotective potential could prove useful. Here, we probed that relationship using the Plasmodium berghei-HepG2 liver stage infection model. After determining translation inhibition EC50s for five compounds, we tested them at equivalent effective concentrations to compare the parasite response to, and recovery from, a brief period of translation inhibition in early schizogony, followed by parasites to 120 h post-infection to assess antiplasmodial effects of the treatment. We show compound-specific heterogeneity in single parasite and population responses to translation inhibitor treatment, with no single metric strongly correlated to the release of hepatic merozoites for all compounds. We also demonstrate that DDD107498 is capable of exerting antiplasmodial effects on translationally arrested liver stage parasites and uncover unexpected growth dynamics during the liver stage. Our results demonstrate that translation inhibition efficacy does not determine antiplasmodial efficacy for these compounds.

Evaluation of the antimalarial activity of SAM13-2HCl with morpholine amide (SKM13 derivative) against antimalarial drug-resistant Plasmodium falciparum and Plasmodium berghei infected ICR mice.

Antimalarial drugs are an urgently need and crucial tool in the campaign against malaria, which can threaten public health. In this study, we examined the cytotoxicity of the 9 antimalarial compounds chemically synthesized using SKM13-2HCl. Except for SKM13-2HCl, the 5 newly synthesized compounds had a 50% cytotoxic concentration (CC50) > 100 µM, indicating that they would be less cytotoxic than SKM13-2HCl. Among the 5 compounds, only SAM13-2HCl outperformed SKM13-2HCl for antimalarial activity, showing a 3- and 1.3-fold greater selective index (SI) (CC50/IC50) than SKM13-2HCl in vitro against both chloroquine-sensitive (3D7) and chloroquine -resistant (K1) Plasmodium falciparum strains, respectively. Thus, the presence of morpholine amide may help to effectively suppress human-infectious P. falciparum parasites. However, the antimalarial activity of SAM13-2HCl was inferior to that of the SKM13-2HCl template compound in the P. berghei NK65-infected mouse model, possibly because SAM13-2HCl had a lower polarity and less efficient pharmacokinetics than SKM13-2HCl. SAM13-2HCl was more toxic in the rodent model. Consequently, SAM13-2HCl containing morpholine was selected from screening a combination of pharmacologically significant structures as being the most effective in vitro against human-infectious P. falciparum but was less efficient in vivo in a P. berghei-infected animal model when compared with SKM13-2HCl. Therefore, SAM13-2HCl containing morpholine could be considered a promising compound to treat chloroquine-resistant P. falciparum infections, although further optimization is crucial to maintain antimalarial activity while reducing toxicity in animals.

Characterisation of the merozoite thrombospondin related anonymous protein (MTRAP) of Plasmodium berghei as a transmission-blocking antigen.

BACKGROUND: Malaria is an infectious disease caused by protozoan parasites belonging to the genus Plasmodium. Human-to-human transmission depends on a mosquito vector; thus, the interruption of parasite transmission from humans to mosquitoes is an important approach in the fight against malaria. The parasite stages infectious to mosquitoes are the gametocytes, sexual stages that are ingested by the vector during a blood meal and transform into male and female gametes in the midgut. Immunity against sexual stage antigens expressed by gametocytes, gametes, and the zygote formed after fertilisation can interrupt the parasite sexual cycle in the mosquito. This transmission blocking immunity is mediated by specific antibodies ingested during the mosquito blood feed, inhibiting the parasite development in the midgut. Merozoite thrombospondin related anonymous protein (MTRAP) is a merozoite and gametocyte surface protein essential for gamete egress from erythrocytes and for parasite transmission to mosquitoes. OBJECTIVES: Here, we evaluated the potential of the P. berghei MTRAP to elicit antibodies with the ability to inhibit gamete fertilisation in vitro. METHODS: We expressed a soluble recombinant PbMTRAP and used it to immunise BALB/c mice. The transmission blocking activity of the anti-rPbMTRAP antibodies was tested through in vivo challenge experiments followed by in vitro conversion assays. FINDINGS: Immunisations with the rPbMTRAP induced a strong antibody response and the antibodies recognised the native protein by Western Blot and IFA. Anti-rPbMTRAP present in the blood stream of immunised mice partially inhibited gamete conversion into ookinetes. CONCLUSION: Our results indicate that antibodies to PbMTRAP may reduce but are not sufficient to completely block transmission.

Lycopene possess an antimalarial effect on chloroquine-resistant malaria and its hematological aberrations in murine model.

Malaria remains a major public health issue worldwide, with high rates of morbidity and mortality. The resistance of Plasmodium parasites to commonly used antimalarial drugs has necessitated the development of novel drugs and targets for malaria treatment. Lycopene is a natural compound present in tomatoes and other red fruits and vegetables. This study aimed to evaluate the antimalarial activity of lycopene and its co-administration with chloroquine against chloroquine-resistant malaria, as well as to assess its impact on hematological abnormalities associated with malaria infection. The experimental animals for this study were infected with 10 7 NK65 Plasmodium berghei-infected red blood cells via intraperitoneal injection. The animals were then treated with artemether-lumefantrine, chloroquine, and varying doses of lycopene. The study evaluated percentage parasitemia, mean survival time, and various hematological parameters, including red blood cell count, hematocrit, hemoglobin concentration, mean corpuscular volume, mean corpuscular hemoglobin, red blood cell distribution width - coefficient of variation, red blood cell distribution width - standard deviation, white blood cell count, granulocyte count, lymphocyte count, monocyte count, and procalcitonin level. The study revealed that lycopene demonstrated significant (p < 0.05) antimalarial activity and the ability to ameliorate hematological abnormalities associated with acute malaria infection. The findings of this study highlight the potential of lycopene as a novel antimalarial agent. The results of this study may contribute to the development of new drugs for malaria treatment, particularly in low- and middle-income countries.

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.

Correlative light-electron microscopy methods to characterize the ultrastructural features of the replicative and dormant liver stages of Plasmodium parasites.

BACKGROUND: The infection of the liver by Plasmodium parasites is an obligatory step leading to malaria disease. Following hepatocyte invasion, parasites differentiate into replicative liver stage schizonts and, in the case of Plasmodium species causing relapsing malaria, into hypnozoites that can lie dormant for extended periods of time before activating. The liver stages of Plasmodium remain elusive because of technical challenges, including low infection rate. This has been hindering experimentations with well-established technologies, such as electron microscopy. A deeper understanding of hypnozoite biology could prove essential in the development of radical cure therapeutics against malaria. RESULTS: The liver stages of the rodent parasite Plasmodium berghei, causing non-relapsing malaria, and the simian parasite Plasmodium cynomolgi, causing relapsing malaria, were characterized in human Huh7 cells or primary non-human primate hepatocytes using Correlative Light-Electron Microscopy (CLEM). Specifically, CLEM approaches that rely on GFP-expressing parasites (GFP-CLEM) or on an immunofluorescence assay (IFA-CLEM) were used for imaging liver stages. The results from P. berghei showed that host and parasite organelles can be identified and imaged at high resolution using both CLEM approaches. While IFA-CLEM was associated with more pronounced extraction of cellular content, samples' features were generally well preserved. Using IFA-CLEM, a collection of micrographs was acquired for P. cynomolgi liver stage schizonts and hypnozoites, demonstrating the potential of this approach for characterizing the liver stages of Plasmodium species causing relapsing malaria. CONCLUSIONS: A CLEM approach that does not rely on parasites expressing genetically encoded tags was developed, therefore suitable for imaging the liver stages of Plasmodium species that lack established protocols to perform genetic engineering. This study also provides a dataset that characterizes the ultrastructural features of liver stage schizonts and hypnozoites from the simian parasite species P. cynomolgi.

Evaluation of the antimalarial and CD4+ T-cell modulatory effects of leaf methanol extract of Phyllanthus muellerianus (Kuntze) Exell (Phyllanthaceae) in Plasmodium berghei-infected mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Phyllanthus muellerianus (Kunze) Exell, a member of the Phyllanthaceae family, is a medicinal plant widely distributed in Africa. Decoctions from the leaves are used in Nigeria to treat fevers, convulsions, some neurological disorders and malaria. AIM OF THE STUDY: This study is to evaluate the anti-malarial properties of methanol extract of Phyllanthus muellerianus (MEPM) leaves and its ethyl acetate fraction using a murine malaria model infected with Plasmodium berghei. Additionally, we seek to investigate the potential modulatory effects of this extract and fraction on CD4+ T-cell populations in the context of malaria infection. MATERIALS AND METHODS: The anti-malarial effects of the leaf methanol extract of Phyllanthus muellerianus (MEPM) were screened using three established in vivo models of anti-plasmodial screening namely the curative, suppressive and prophylactic models. The methanol extract (MEPM) was afterwards fractionated into hexane (HFPM), ethyl acetate (EAFPM), and methanol (MFPM) fractions. In the pilot anti-malarial screening of the fractions, EAFPM exhibited the best antiparasitic activity. Subsequently, EAFPM was screened for anti-malarial activity using the three models above. The effects of the MEPM and EAFPM on haematological indices (Hb and PCV) of the inoculated animals were further screened and the mean survival time (MST) of the animals was monitored. CD4+ T cells of various groups were counted before and after treatment using a flow cytometer. The EAFPM was further subjected to HPLC analysis for identification of its major compounds. RESULTS: The EAFPM (100 and 200 mg/kg) elicited 88% and 93% cure respectively in the curative model, while artesunate (5 mg/kg,- the positive control) gave 87% protection. The MEPM and EAFPM also gave significant suppression of parasitemia in the suppressive model. The treated groups survived beyond 28 days as against 11 days by the control group (infected but not treated). The treated groups also prevented anaemia seen in the negative control. The EAFPM group significantly modulated the CD4+ T cell. Compounds identified were Gallocatechin, Quercetin -3-O-gallate, Ellagic acid, and Methylellagic acid rhamnoside). CONCLUSION: The study established that the leaf of Phyllanthus muellerianus possesses antimalarial activity, thus lending support to its use in the folkloric treatment of malaria.

The Anopheles leucine-rich repeat protein APL1C is a pathogen binding factor recognizing Plasmodium ookinetes and sporozoites.

Leucine-rich repeat (LRR) proteins are commonly involved in innate immunity of animals and plants, including for pattern recognition of pathogen-derived elicitors. The Anopheles secreted LRR proteins APL1C and LRIM1 are required for malaria ookinete killing in conjunction with the complement-like TEP1 protein. However, the mechanism of parasite immune recognition by the mosquito remains unclear, although it is known that TEP1 lacks inherent binding specificity. Here, we find that APL1C and LRIM1 bind specifically to Plasmodium berghei ookinetes, even after depletion of TEP1 transcript and protein, consistent with a role for the LRR proteins in pathogen recognition. Moreover, APL1C does not bind to ookinetes of the human malaria parasite Plasmodium falciparum, and is not required for killing of this parasite, which correlates LRR binding specificity and immune protection. Most of the live P. berghei ookinetes that migrated into the extracellular space exposed to mosquito hemolymph, and almost all dead ookinetes, are bound by APL1C, thus associating LRR protein binding with parasite killing. We also find that APL1C binds to the surface of P. berghei sporozoites released from oocysts into the mosquito hemocoel and forms a potent barrier limiting salivary gland invasion and mosquito infectivity. Pathogen binding by APL1C provides the first functional explanation for the long-known requirement of APL1C for P. berghei ookinete killing in the mosquito midgut. We propose that secreted mosquito LRR proteins are required for pathogen discrimination and orientation of immune effector activity, potentially as functional counterparts of the immunoglobulin-based receptors used by vertebrates for antigen recognition.

Stearoyl-CoA desaturase regulates organelle biogenesis and hepatic merozoite formation in Plasmodium berghei.

Plasmodium is an obligate intracellular parasite that requires intense lipid synthesis for membrane biogenesis and survival. One of the principal membrane components is oleic acid, which is needed to maintain the membrane's biophysical properties and fluidity. The malaria parasite can modify fatty acids, and stearoyl-CoA Δ9-desaturase (Scd) is an enzyme that catalyzes the synthesis of oleic acid by desaturation of stearic acid. Scd is dispensable in P. falciparum blood stages; however, its role in mosquito and liver stages remains unknown. We show that P. berghei Scd localizes to the ER in the blood and liver stages. Disruption of Scd in the rodent malaria parasite P. berghei did not affect parasite blood stage propagation, mosquito stage development, or early liver-stage development. However, when Scd KO sporozoites were inoculated intravenously or by mosquito bite into mice, they failed to initiate blood-stage infection. Immunofluorescence analysis revealed that organelle biogenesis was impaired and merozoite formation was abolished, which initiates blood-stage infections. Genetic complementation of the KO parasites restored merozoite formation to a level similar to that of WT parasites. Mice immunized with Scd KO sporozoites confer long-lasting sterile protection against infectious sporozoite challenge. Thus, the Scd KO parasite is an appealing candidate for inducing protective pre-erythrocytic immunity and hence its utility as a GAP.

Synthetic angiotensin II peptide derivatives confer protection against cerebral and severe non-cerebral malaria in murine models.

Malaria can have severe long-term effects. Even after treatment with antimalarial drugs eliminates the parasite, survivors of cerebral malaria may suffer from irreversible brain damage, leading to cognitive deficits. Angiotensin II, a natural human peptide hormone that regulates blood pressure, has been shown to be active against Plasmodium spp., the etiologic agent of malaria. Here, we tested two Ang II derivatives that do not elicit vasoconstriction in mice: VIPF, a linear tetrapeptide, which constitutes part of the hydrophobic portion of Ang II; and Ang II-SS, a disulfide-bridged derivative. The antiplasmodial potential of both peptides was evaluated with two mouse models: an experimental cerebral malaria model and a mouse model of non-cerebral malaria. The latter consisted of BALB/c mice infected with Plasmodium berghei ANKA. The peptides had no effect on mean blood pressure and significantly reduced parasitemia in both mouse models. Both peptides reduced the SHIRPA score, an assay used to assess murine health and behavior. However, only the constrained derivative (Ang II-SS), which was also resistant to proteolytic degradation, significantly increased mouse survival. Here, we show that synthetic peptides derived from Ang II are capable of conferring protection against severe manifestations of malaria in mouse models while overcoming the vasoconstrictive side effects of the parent peptide.