Shotgun Characterization of the Circulating IgM Antigenome of an Infectious Pathogen by Immunocapture-LC-MS/MS from Dried Serum Spots.

One of the main challenges in compiling the complete collection of protein antigens from pathogens for the selection of vaccine candidates or intervention targets is to acquire a broad enough representation of them to be recognized by the highly diversified immunoglobulin repertoire in human populations. Dried serum spot sampling (DSS) retains a large repertoire of circulating immunoglobulins from each individual that can be representative of a population, according to the sample size. In this work, shotgun proteomics of an infectious pathogen based on DSS sampling coupled with IgM immunoprecipitation, liquid chromatography-mass spectrometry (LC-MS/MS), and bioinformatic analyses was combined to characterize the circulating IgM antigenome. Serum samples from a malaria endemic region at different clinical statuses were studied to optimize IgM binding efficiency and antibody leaching by varying serum/immunomagnetic bead ratios and elution conditions. The method was validated using Plasmodium falciparum extracts identifying 110 of its IgM-reactive antigens while minimizing the presence of human proteins and antibodies. Furthermore, the IgM antigen recognition profile differentiated between malaria-infected and noninfected individuals at the time of sampling. We conclude that a shotgun proteomics approach offers advantages in providing a high-throughput, reliable, and clean way to identify IgM-recognized antigens from trace amounts of serum. The mass spectrometry raw data and metadata have been deposited with ProteomeXchange via MassIVE with the PXD identifier PXD043800.

Isolation and Characterization of NpCI, a New Metallocarboxypeptidase Inhibitor from the Marine Snail Nerita peloronta with Anti-Plasmodium falciparum Activity.

Metallocarboxypeptidases are zinc-dependent peptide-hydrolysing enzymes involved in several important physiological and pathological processes. They have been a target of growing interest in the search for natural or synthetic compound binders with biomedical and drug discovery purposes, i.e., with potential as antimicrobials or antiparasitics. Given that marine resources are an extraordinary source of bioactive molecules, we screened marine invertebrates for new inhibitory compounds with such capabilities. In this work, we report the isolation and molecular and functional characterization of NpCI, a novel strong metallocarboxypeptidase inhibitor from the marine snail Nerita peloronta. NpCI was purified until homogeneity using a combination of affinity chromatography and RP-HPLC. It appeared as a 5921.557 Da protein with 53 residues and six disulphide-linked cysteines, displaying a high sequence similarity with NvCI, a carboxypeptidase inhibitor isolated from Nerita versicolor, a mollusc of the same genus. The purified inhibitor was determined to be a slow- and tight-binding inhibitor of bovine CPA (Ki = 1.1·× 10-8 mol/L) and porcine CPB (Ki = 8.15·× 10-8 mol/L) and was not able to inhibit proteases from other mechanistic classes. Importantly, this inhibitor showed antiplasmodial activity against Plasmodium falciparum in an in vitro culture (IC50 = 5.5 µmol/L), reducing parasitaemia mainly by inhibiting the later stages of the parasite's intraerythrocytic cycle whilst having no cytotoxic effects on human fibroblasts. Interestingly, initial attempts with other related proteinaceous carboxypeptidase inhibitors also displayed similar antiplasmodial effects. Coincidentally, in recent years, a metallocarboxypeptidase named PfNna1, which is expressed in the schizont phase during the late intraerythrocytic stage of the parasite's life cycle, has been described. Given that NpCI showed a specific parasiticidal effect on P. falciparum, eliciting pyknotic/dead parasites, our results suggest that this and related inhibitors could be promising starting agents or lead compounds for antimalarial drug discovery strategies.

Protein Susceptibility to Peroxidation by 4-Hydroxynonenal in Hereditary Hemochromatosis.

Iron overload caused by hereditary hemochromatosis (HH) increases free reactive oxygen species that, in turn, induce lipid peroxidation. Its 4-hydroxynonenal (HNE) by-product is a well-established marker of lipid peroxidation since it reacts with accessible proteins with deleterious consequences. Indeed, elevated levels of HNE are often detected in a wide variety of human diseases related to oxidative stress. Here, we evaluated HNE-modified proteins in the membrane of erythrocytes from HH patients and in organs of Hfe-/- male and female mice, a mouse model of HH. For this purpose, we used one- and two-dimensional gel electrophoresis, immunoblotting and MALDI-TOF/TOF analysis. We identified cytoskeletal membrane proteins and membrane receptors of erythrocytes bound to HNE exclusively in HH patients. Furthermore, kidney and brain of Hfe-/- mice contained more HNE-adducted protein than healthy controls. Our results identified main HNE-modified proteins suggesting that HH favours preferred protein targets for oxidation by HNE.

Plasmodium species differentiation by non-expert on-line volunteers for remote malaria field diagnosis.

BACKGROUND: Routine field diagnosis of malaria is a considerable challenge in rural and low resources endemic areas mainly due to lack of personnel, training and sample processing capacity. In addition, differential diagnosis of Plasmodium species has a high level of misdiagnosis. Real time remote microscopical diagnosis through on-line crowdsourcing platforms could be converted into an agile network to support diagnosis-based treatment and malaria control in low resources areas. This study explores whether accurate Plasmodium species identification-a critical step during the diagnosis protocol in order to choose the appropriate medication-is possible through the information provided by non-trained on-line volunteers. METHODS: 88 volunteers have performed a series of questionnaires over 110 images to differentiate species (Plasmodium falciparum, Plasmodium ovale, Plasmodium vivax, Plasmodium malariae, Plasmodium knowlesi) and parasite staging from thin blood smear images digitalized with a smartphone camera adapted to the ocular of a conventional light microscope. Visual cues evaluated in the surveys include texture and colour, parasite shape and red blood size. RESULTS: On-line volunteers are able to discriminate Plasmodium species (P. falciparum, P. malariae, P. vivax, P. ovale, P. knowlesi) and stages in thin-blood smears according to visual cues observed on digitalized images of parasitized red blood cells. Friendly textual descriptions of the visual cues and specialized malaria terminology is key for volunteers learning and efficiency. CONCLUSIONS: On-line volunteers with short-training are able to differentiate malaria parasite species and parasite stages from digitalized thin smears based on simple visual cues (shape, size, texture and colour). While the accuracy of a single on-line expert is far from perfect, a single parasite classification obtained by combining the opinions of multiple on-line volunteers over the same smear, could improve accuracy and reliability of Plasmodium species identification in remote malaria diagnosis.

Gene expression and immunolocalization of low-affinity neurotrophin receptor (p75) in rabbit male reproductive tract during sexual maturation.

This study reports the gene expression and immunolocalization of the low-affinity neurotrophin receptor, p75, in accessory glands (prostate and bulbourethral glands) and epididymis (caput and cauda) of male rabbits during sexual maturation. We showed that p75 was expressed in all tissues studied with similar mRNA levels during this period. However, it was differentially immunolocalized in bulbourethral glands and stereocilia of epididymis. These findings may be related with some remodeling processes in the accessory glands during sexual maturation, which could be associated with sperm maturation. The interaction of p75 with neurotrophins in rabbit male reproductive tract suggests the possible implication of this system in sexual maturation in rabbits.

Iron supplementation in mouse expands cellular innate defences in spleen and defers lethal malaria infection.

The co-endemicity of malnutrition, erythrocytopathies, transmissible diseases and iron-deficiency contribute to the prevalence of chronic anaemia in many populations of the developing world. Although iron dietary supplementation is applied or recommended in at risk populations, its use is controversial due to undesirable outcomes, particularly regarding the response to infections, including highly prevalent malaria. We hypothesized that a boosted oxidative stress due to iron supplementation have a similar impact on malaria to that of hereditary anaemias, enhancing innate response and conditioning tissues to prevent damage during infection. Thus, we have analysed antioxidant and innate responses against lethal Plasmodium yoelii during the first five days of infection in an iron-supplemented mouse. This murine model showed high iron concentration in plasma with upregulated expression of hemoxygenase-1. The sustained homeostasis after this extrinsic iron conditioning, delayed parasitemia growth that, once installed, developed without anaemia. This protection was not conferred by the intrinsic iron overload of hereditary hemochromatosis. Upon iron-supplementation, a large increase of the macrophages/dendritic cells ratio and the antigen presenting cells was observed in the mouse spleen, independently of malaria infection. Complementary, malaria promoted the splenic B and T CD4 cells activation. Our results show that the iron supplementation in mice prepares host tissues for oxidative-stress and induces unspecific cellular immune responses, which could be seen as an advantage to promote early defences against malaria infection.

Analogs of natural aminoacyl-tRNA synthetase inhibitors clear malaria in vivo.

Malaria remains a major global health problem. Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Here we explore the potential of the aminoacyl-tRNA synthetase (ARS) family as a source of antimalarial drug targets. First, a battery of known and novel ARS inhibitors was tested against Plasmodium falciparum cultures, and their activities were compared. Borrelidin, a natural inhibitor of threonyl-tRNA synthetase (ThrRS), stands out for its potent antimalarial effect. However, it also inhibits human ThrRS and is highly toxic to human cells. To circumvent this problem, we tested a library of bioengineered and semisynthetic borrelidin analogs for their antimalarial activity and toxicity. We found that some analogs effectively lose their toxicity against human cells while retaining a potent antiparasitic activity both in vitro and in vivo and cleared malaria from Plasmodium yoelii-infected mice, resulting in 100% mice survival rates. Our work identifies borrelidin analogs as potent, selective, and unexplored scaffolds that efficiently clear malaria both in vitro and in vivo.

Epigenetic therapy reprograms hereditary disease.

In this issue of Blood, Makarona et al demonstrate that histone deacetylase (HDAC) inhibitors (HDACis) in glucose-6-phosphate dehydrogenase (G6PD)-deficient cells reinstates enzyme activity by boosting gene transcription. This therapeutic approach opens new avenues for preclinical and clinical studies to treat not only chronic nonspherocytic hemolytic anemia caused by severe G6PD variants, but also other genetic diseases.

Glutathione peroxidase contributes with heme oxygenase-1 to redox balance in mouse brain during the course of cerebral malaria.

Oxidative stress has been attributed both a key pathogenic and rescuing role in cerebral malaria (CM). In a Plasmodium berghei ANKA murine model of CM, host redox signaling and functioning were examined during the course of neurological damage. Host antioxidant defenses were early altered at the transcriptional level indicated by the gradually diminished expression of superoxide dismutase-1 (sod-1), sod-2, sod-3 and catalase genes. During severe disease, this led to the dysfunctional activity of superoxide dismutase and catalase enzymes in damaged brain regions. Vitagene associated markers (heat shock protein 70 and thioredoxin-1) also showed a decaying expression pattern that paralleled reduced expression of the transcription factors Parkinson disease 7, Forkhead box O 3 and X-box binding protein 1 with a role in preserving brain redox status. However, the oxidative stress markers reactive oxygen/nitrogen species were not accumulated in the brains of CM mice and redox proteomics and immunohistochemistry failed to detect quantitative or qualitative differences in protein carbonylation. Thus, the loss of antioxidant capacity was compensated for in all cerebral regions by progressive upregulation of heme oxygenase-1, and in specific regions by early glutathione peroxidase-1 induction. This study shows for the first time a scenario of cooperative glutathione peroxidase and heme oxygenase-1 upregulation to suppress superoxide dismutase, catalase, heat shock protein-70 and thioredoxin-1 downregulation effects in experimental CM, counteracting oxidative damage and maintaining redox equilibrium. Our findings reconcile the apparent inconsistency between the lack of oxidative metabolite build up and reported protective effect of antioxidant therapy against CM.

Functional segregation and emerging role of cilia-related cytosolic carboxypeptidases (CCPs).

Recent experimental data indicating axonal regeneration, axogenesis, dendritogenesis, and ciliary axoneme assembly and wellness have linked the role of cytosolic metallocarboxypeptidase 1 (CCP1/AGTPBP1/Nna1) to the microtubule network. In addition, 5 of the 6 mammalian ccp genes have been shown to participate in post-translational modifications of tubulin, which occur in the microtubules of neurons, mitotic spindles, cilia, and basal bodies. Here, we compile evidence for the idea that the occurrence of CCPs strongly correlates with the presence of cilia, suggesting that CCP functions might be primarily related to cilia and basal bodies (CBBs). In agreement with this hypothesis, CCPs were localized in centrioles, basal bodies, and mitotic spindles in HeLa cells by confocal microscopy. By reconstructing the evolutionary history of CCPs, we show their presence in the last eukaryotic common ancestor and relate each group of CCP orthologs to specific roles in CBBs. The clues presented in this study suggest that during the evolution of eukaryotes, mechanisms mediated by CCPs through tubulin post-translational modifications controlling assembly, trafficking, and signaling in the microtubules, were transferred from cilia to cell and axon microtubules.

Immune checkpoints and cancer immunotherapies: insights into newly potential receptors and ligands.

Checkpoint markers and immune checkpoint inhibitors have been increasingly identified and developed as potential immunotherapeutic targets in various human cancers. Despite valuable efforts to discover novel immune checkpoints and their ligands, the precise roles of their therapeutic functions, as well as the broad identification of their counterpart receptors, remain to be addressed. In this context, it has been suggested that various putative checkpoint receptors can be induced upon activation. In the tumor microenvironment, T cells, as crucial immune response against malignant diseases as well as other immune central effector cells, such as natural killer cells, are regulated via co-stimulatory or co-inhibitory signals from immune or tumor cells. Studies have shown that exposure of T cells to tumor antigens upregulates the expression of inhibitory checkpoint receptors, leading to T-cell dysfunction or exhaustion. Although targeting immune checkpoint regulators has shown relative clinical efficacy in some tumor types, most trials in the field of cancer immunotherapies have revealed unsatisfactory results due to de novo or adaptive resistance in cancer patients. To overcome these obstacles, combinational therapies with newly discovered inhibitory molecules or combined blockage of several checkpoints provide a rationale for further research. Moreover, precise identification of their receptors counterparts at crucial checkpoints is likely to promise effective therapies. In this review, we examine the prospects for the application of newly emerging checkpoints, such as T-cell immunoglobulin and mucin domain 3, lymphocyte activation gene-3, T-cell immunoreceptor with Ig and ITIM domains (TIGIT), V-domain Ig suppressor of T-cell activation (VISTA), new B7 family proteins, and B- and T-cell lymphocyte attenuator, in association with immunotherapy of malignancies. In addition, their clinical and biological significance is discussed, including their expression in various human cancers, along with their roles in T-cell-mediated immune responses.

Co-Occurrence of G6PD Deficiency and SCT among Pregnant Women Exposed to Infectious Diseases.

During pregnancy, women have an increased relative risk of exposure to infectious diseases. This study was designed to assess the prevalence of the co-occurrence of glucose-6-phosphate dehydrogenase deficiency (G6PDd) and sickle cell trait (SCT) and the impact on anemia outcomes among pregnant women exposed to frequent infectious diseases. Over a six-year period (March 2013 to October 2019), 8473 pregnant women attending antenatal clinics (ANCs) at major referral hospitals in Northern Ghana were recruited and diagnosed for common infectious diseases (malaria, syphilis, hepatitis B, and HIV), G6PDd, and SCT. The prevalence of all the infections and anemia did not differ between women with and without G6PDd (χ2 < 3.6, p > 0.05 for all comparisons). Regression analysis revealed a significantly higher proportion of SCT in pregnant women with G6PDd than those without G6PDd (AOR = 1.58; p < 0.011). The interaction between malaria and SCT was observed to be associated with anemia outcomes among the G6PDd women (F-statistic = 10.9, p < 0.001). Our findings show that anemia is a common condition among G6PDd women attending ANCs in northern Ghana, and its outcome is impacted by malaria and SCT. This warrants further studies to understand the impact of antimalarial treatment and the blood transfusion outcomes in G6PDd/SCT pregnant women.

Evolutionary history of the genus Trisopterus.

The group of small poor cods and pouts from the genus Trisopterus, belonging to the Gadidae family, comprises four described benthopelagic species that occur across the North-eastern Atlantic, from the Baltic Sea to the coast of Morocco, and the Mediterranean. Here, we combined molecular data from mitochondrial (cytochrome b) and nuclear (rhodopsin) genes to confirm the taxonomic status of the described species and to disentangle the evolutionary history of the genus. Our analyses supported the monophyly of the genus Trisopterus and confirmed the recently described species Trisopterus capelanus. A relaxed molecular clock analysis estimated an Oligocene origin for the group (~30 million years ago; mya) indicating this genus as one of the most ancestral within the Gadidae family. The closure and re-opening of the Strait of Gibraltar after the Messinian Salinity Crisis (MSC) probably triggered the speciation process that resulted in the recently described T. capelanus.

In vitro antiplasmodial activity of selected plants from the Colombian North Coast with low cytotoxicity.

Background: Plants are an important option in the treatment of malaria, especially in endemic regions, and are a less expensive and more accessible alternative with a lower risk of toxicity. Colombia has a great diversity of plants, and evaluation of natural extracts could result in the discovery of new compounds for the development of antimalarial drugs. The purpose of this work was to evaluate the in vitro antiplasmodial activity and the cytotoxicity of plant extracts from the Colombian North Coast against Plasmodium falciparum. Materials and Methods: The antiplasmodial activity of 12 plant species from the Colombian North Coast that are used in traditional medicine was evaluated through in vitro cultures of P. falciparum, and the cytotoxicity of extracts of these species to human cells was determined. Plant extracts with high antiplasmodial activity were subjected to preliminary phytochemical screening. Results: Extracts from five plants had promising antiplasmodial activity. Specifically, Bursera simaruba (Burseraceae) (bark), Guazuma ulmifolia Lam. (Malvaceae) (whole plant), Murraya exotica L. (Rutaceae) (leaves), Hippomane mancinella L. (Euphorbiaceae) (seeds), and Capparis odoratissima Jacq. (Capparaceae) (leaves). Extracts presented 50% inhibitory concentration values between 1 and 9 µg/ml. Compared to no extract, these active plant extracts did not show cytotoxic effects on mononuclear cells or hemolytic activity in healthy human erythrocytes. Conclusions: The results obtained from this in vitro study of antiplasmodial activity suggest that active plant extracts from the Colombian North Coast are promising for future bioassay-guided fractionation to allow the isolation of active compounds and to elucidate their mechanism of action against Plasmodium spp.

Microscopic and submicroscopic infection by Plasmodium falciparum: Immunoglobulin M and A profiles as markers of intensity and exposure.

Assessment of serological Plasmodium falciparum-specific antibodies in highly endemic areas provides valuable information about malaria status and parasite exposure in the population. Although serological evidence of Plasmodium exposure is commonly determined by Plasmodium-specific immunoglobulin G (IgG) levels; IgM and IgA are likely markers of malaria status that remain relatively unexplored. Previous studies on IgM and IgA responses have been based on their affinity for single antigens with shortage of immune responses analysis against the whole Plasmodium proteome. Here, we provide evidence of how P. falciparum infection triggers the production of specific IgM and IgA in plasma and its relationship with parasite density and changes in hematological parameters. A total of 201 individuals attending a hospital in Breman Asikuma, Ghana, were recruited into this study. Total and P. falciparum-specific IgM, IgA, and IgG were assessed by ELISA and examined in relation to age (0-5, 14-49, and ≥50 age ranges); infection (submicroscopic vs. microscopic malaria); pregnancy and hematological parameters. Well-known IgG response was used as baseline control. P. falciparum-specific IgM and IgA levels increased in the population with the age, similarly to IgG. These data confirm that acquired humoral immunity develops by repeated infections through the years endorsing IgM and IgA as exposure markers in endemic malaria regions. High levels of specific IgA and IgM in children were associated with microscopic malaria and worse prognosis, because most of them showed severe anemia. This new finding shows that IgM and IgA may be used as diagnostic markers in this age group. We also found an extremely high prevalence of submicroscopic malaria (46.27% on average) accompanied by IgM and IgA levels indistinguishable from those of uninfected individuals. These data, together with the observed lack of sensitivity of rapid diagnostic tests (RDTs) compared to PCR, invoke the urgent need to implement diagnostic markers for submicroscopic malaria. Overall, this study opens the potential use of P. falciparum-specific IgM and IgA as new serological markers to predict malaria status in children and parasite exposure in endemic populations. The difficulties in finding markers of submicroscopic malaria are highlighted, emphasizing the need to explore this field in depth.

Hypothyroidism confers tolerance to cerebral malaria.

The modulation of the host's metabolism to protect tissue from damage induces tolerance to infections increasing survival. Here, we examined the role of the thyroid hormones, key metabolic regulators, in the outcome of malaria. Hypothyroidism confers protection to experimental cerebral malaria by a disease tolerance mechanism. Hypothyroid mice display increased survival after infection with Plasmodium berghei ANKA, diminishing intracranial pressure and brain damage, without altering pathogen burden, blood-brain barrier disruption, or immune cell infiltration. This protection is reversed by treatment with a Sirtuin 1 inhibitor, while treatment of euthyroid mice with a Sirtuin 1 activator induces tolerance and reduces intracranial pressure and lethality. This indicates that thyroid hormones and Sirtuin 1 are previously unknown targets for cerebral malaria treatment, a major killer of children in endemic malaria areas.

Proteomic approaches to identifying carbonylated proteins in brain tissue.

Oxidative stress plays a critical role in the pathogenesis of a number of diseases. The carbonyl end products of protein oxidation are among the most commonly measured markers of oxidation in biological samples. Protein carbonyl functional groups may be derivatized with 2,4-dinitrophenylhydrazine (DNPH) to render a stable 2,4-dinitrophenylhydrazone-protein (DNP-protein) and the carbonyl contents of individual proteins then determined by two-dimensional electrophoresis followed by immunoblotting using specific anti-DNP antibodies. Unfortunately, derivatization of proteins with DNPH could affect their mass spectrometry (MS) identification. This problem can be overcome using nontreated samples for protein identification. Nevertheless, derivatization could also affect their mobility, which might be solved by performing the derivatization step after the initial electrophoresis. Here, we compare two-dimensional redox proteome maps of mouse cerebellum acquired by performing the DNPH derivatization step before or after electrophoresis and detect differences in protein patterns. When the same approach is used for protein detection and identification, both methods were found to be useful to identify carbonylated proteins. However, whereas pre-DNPH derivatized proteins were successfully analyzed, high background staining complicated the analysis when the DNPH reaction was performed after transblotting. Comparative data on protein identification using both methods are provided.

Malaria hidden in a patient with diffuse large-B-cell lymphoma and sickle-cell trait.

We report a case of an African patient with sickle cell trait who was diagnosed in Spain with B-cell lymphoma. Blood smears were negative for malaria, and no plasmodium antigens were detected in the blood. To treat his lymphoma, the patient underwent chemotherapy and autologous stem cell transplantation. Following a splenectomy due to a worsening condition, he developed clinical malaria with detectable parasitemia. This case suggests that the humoral response and parasite removal by the spleen may afford protection from overt disease and may even help maintain subclinical human reservoirs of the disease.

Parasitostatic effect of maslinic acid. II. Survival increase and immune protection in lethal Plasmodium yoelii-infected mice.

BACKGROUND: The anti-malarial activity of maslinic acid (MA), a natural triterpene which has been previously shown to exert a parasitostatic action on Plasmodium falciparum cultures, was analysed in vivo by using the Plasmodium yoelii 17XL murine model. METHODS: ICR mice were infected with P. yoelii and treated with a single dose of MA by a intraperitoneal injection of MA (40 mg kg(-1) day(-1)) followed by identical dose administration for the following three days. Parasitaemia and accumulation of intraerythrocytic stages was monitored microscopically. To assess protective immunity, cured mice were challenged with the same dose of parasites 40 days after recovery from the primary infection and parasitaemia was further monitored for 30 days. Humoral response was tested by ELISA and visualization of specific anti-P. yoelii antibodies was performed by Western-blotting. RESULTS: ICR mice treated with MA increased the survival rate from 20% to 80%, showing an arrest of parasite maturation from day 3 to 7 after infection and leading to synchronization of the intraerythrocytic cycle and accumulation of schizonts by day 6, proving that MA also behaves as a parasitostatic agent in vivo. Mice which survived the primary infection displayed lower rates of parasitic growth, showing a decline of parasitaemia after day 15, and complete clearance at day 20. These mice remained immunoprotected, showing not malaria symptoms or detectable parasitaemia after rechallenge with the same lethal strain. The analysis of specific antibodies against P. yoelii, present in mice which survived the infection, showed a significant increase in the number and intensity of immunoreactive proteins, suggesting that the protected mice may trigger a strong humoral response. CONCLUSION: The survival increase observed in MA-treated mice can be explained considering that the parasitostatic effect exerted by this compound during the first days of infection increases the chances to develop effective innate and/or acquired immune responses. MA may represent a new class of anti-malarial compounds which, as a consequence of its parasitostatic action, favours the development of more effective sterilizing immune responses.

Parasitostatic effect of maslinic acid. I. Growth arrest of Plasmodium falciparum intraerythrocytic stages.

BACKGROUND: Natural products have played an important role as leads for the development of new drugs against malaria. Recent studies have shown that maslinic acid (MA), a natural triterpene obtained from olive pomace, which displays multiple biological and antimicrobial activities, also exerts inhibitory effects on the development of some Apicomplexan, including Eimeria, Toxoplasma and Neospora. To ascertain if MA displays anti-malarial activity, the main objective of this study was to asses the effect of MA on Plasmodium falciparum-infected erythrocytes in vitro. METHODS: Synchronized P. falciparum-infected erythrocyte cultures were incubated under different conditions with MA, and compared to chloroquine and atovaquone treated cultures. The effects on parasite growth were determined by monitoring the parasitaemia and the accumulation of the different infective stages visualized in thin blood smears. RESULTS: MA inhibits the growth of P. falciparum Dd2 and 3D7 strains in infected erythrocytes in, dose-dependent manner, leading to the accumulation of immature forms at IC50 concentrations, while higher doses produced non-viable parasite cells. MA-treated infected-erythrocyte cultures were compared to those treated with chloroquine or atovaquone, showing significant differences in the pattern of accumulation of parasitic stages. Transient MA treatment at different parasite stages showed that the compound targeted intra-erythrocytic processes from early-ring to schizont stage. These results indicate that MA has a parasitostatic effect, which does not inactivate permanently P. falciparum, as the removal of the compound allowed the infection to continue CONCLUSIONS: MA displays anti-malarial activity at multiple intraerythrocytic stages of the parasite and, depending on the dose and incubation time, behaves as a plasmodial parasitostatic compound. This novel parasitostatic effect appears to be unrelated to previous mechanisms proposed for current anti-malarial drugs, and may be relevant to uncover new prospective plasmodial targets and opens novel possibilities of therapies associated to host immune response.