An Opsonic Phagocytosis Assay for Plasmodium falciparum Sporozoites

ABSTRACT Plasmodium falciparum malaria remains the deadliest parasitic disease worldwide. Vaccines targeting the preerythrocytic sporozoite and liver stages have the potential to entirely prevent blood-stage infection and disease, as well as onward transmission. Sporozoite surface and secreted proteins are leading candidates for inclusion in a preerythrocytic stage-specific, antibody-based vaccine. Preclinical functional assays to identify humoral correlates of protection in vitro and to validate novel sporozoite protein targets for inclusion in multisubunit vaccines currently do not consider the interaction of sporozoite-targeting antibodies with other components of the immune system. Here, we describe the development of a simple flow cytometric assay to quantitatively assess the ability of antibodies directed against P. falciparum sporozoites to facilitate their phagocytosis. We demonstrate that this sporozoite opsonic phagocytosis assay (SOPA) is compatible with both monoclonal antibodies and human immune serum and can be performed using cryopreserved P. falciparum sporozoites. This simple, accessible assay will aid with the assessment of antibody responses to vaccination with Plasmodium antigens and their interaction with phagocytic cells of the immune system.

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Stages of in vitro phagocytosis of Plasmodium falciparum-infected erythrocytes by human monocytes

Revista da Sociedade Brasileira de Medicina Tropical ◽

10.1590/s0037-86822009000200001 ◽

2009 ◽

Vol 42 (2) ◽

pp. 103-106 ◽

Cited By ~ 3

Author(s):

Maria Imaculada Muniz-Junqueira ◽

Carlos Eduardo Tosta

Keyword(s):

Plasmodium Falciparum ◽

Blood Circulation ◽

Defense Mechanisms ◽

Healthy Adult ◽

Critical Role ◽

Immune Serum ◽

Human Monocytes ◽

Malaria Parasites

Monocytes/macrophages play a critical role in the defense mechanisms against malaria parasites, and are the main cells responsible for the elimination of malaria parasites from the blood circulation. We carried out a microscope-aided evaluation of the stages of in vitro phagocytosis of Plasmodium falciparum-infected erythrocytes, by human monocytes. These cells were obtained from healthy adult individuals by means of centrifugation through a cushion of Percoll density medium and were incubated with erythrocytes infected with Plasmodium falciparum that had previously been incubated with a pool of anti-plasmodial immune serum. We described the stages of phagocytosis, starting from adherence of infected erythrocytes to the phagocyte membrane and ending with their destruction within the phagolisosomes of the monocytes. We observed that the different erythrocytic forms of the parasite were ingested by monocytes, and that the process of phagocytosis may be completed in around 30 minutes. Furthermore, we showed that phagocytosis may occur continuously, such that different phases of the process were observed in the same phagocyte.

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Effect of Fluorescent Dyes onIn Vitro-Differentiated, Late-Stage Plasmodium falciparum Gametocytes

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03772-14 ◽

2014 ◽

Vol 58 (12) ◽

pp. 7398-7404 ◽

Cited By ~ 14

Author(s):

Tamirat Gebru ◽

Benjamin Mordmüller ◽

Jana Held

Keyword(s):

Plasmodium Falciparum ◽

Late Stage ◽

Fluorescent Dyes ◽

Clinical Symptoms ◽

Laboratory Strain ◽

Clinical Isolates ◽

Synthetic Dyes ◽

Content Type ◽

Highly Active

ABSTRACTPlasmodium falciparumgametocytes are not associated with clinical symptoms, but they are responsible for transmitting the pathogen to mosquitoes. Therefore, gametocytocidal interventions are important for malaria control and resistance containment. Currently available drugs and vaccines are not well suited for that purpose. Several dyes have potent antimicrobial activity, but their use against gametocytes has not been investigated systematically. The gametocytocidal activity of nine synthetic dyes and four control compounds was tested against stage V gametocytes of the laboratory strain 3D7 and three clinical isolates ofP. falciparumwith a bioluminescence assay. Five of the fluorescent dyes had submicromolar 50% inhibitory concentration (IC50) values against mature gametocytes. Three mitochondrial dyes, MitoRed, dihexyloxacarbocyanine iodide (DiOC6), and rhodamine B, were highly active (IC50s < 200 nM). MitoRed showed the highest activity against gametocytes, with IC50s of 70 nM against 3D7 and 120 to 210 nM against clinical isolates. All compounds were more active against the laboratory strain 3D7 than against clinical isolates. In particular, the endoperoxides artesunate and dihydroartemisinin showed a 10-fold higher activity against 3D7 than against clinical isolates. In contrast to all clinically used antimalarials, several fluorescent dyes had surprisingly highin vitroactivity against late-stage gametocytes. Since they also act against asexual blood stages, they shall be considered starting points for the development of new antimalarial lead compounds.

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Plasmodium falciparum Line-Dependent Association ofIn VitroGrowth-Inhibitory Activity and Risk of Malaria

Infection and Immunity ◽

10.1128/iai.06190-11 ◽

2012 ◽

Vol 80 (5) ◽

pp. 1900-1908 ◽

Cited By ~ 11

Author(s):

Josea Rono ◽

Anna Färnert ◽

Daniel Olsson ◽

Faith Osier ◽

Ingegerd Rooth ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Inhibitory Activity ◽

Content Type ◽

Phenotypic Differences ◽

Erythrocyte Invasion ◽

Growth Inhibitory ◽

Growth Inhibitory Activity ◽

Control Study

ABSTRACTPlasmodium falciparum's ability to invade erythrocytes is essential for its survival within the human host. Immune mechanisms that impair this ability are therefore expected to contribute to immunity against the parasite. Plasma of humans who are naturally exposed to malaria has been shown to have growth-inhibitory activity (GIA)in vitro. However, the importance of GIA in relation to protection from malaria has been unclear. In a case-control study nested within a longitudinally followed population in Tanzania, plasma samples collected at baseline from 171 individuals (55 cases and 116 age-matched controls) were assayed for GIA using threeP. falciparumlines (3D7, K1, and W2mef) chosen based on their erythrocyte invasion phenotypes. Distribution of GIA differed between the lines, with most samples inhibiting the growth of 3D7 and K1 and enhancing the growth of W2mef. GIA to 3D7 was associated with a reduced risk of malaria within 40 weeks of follow-up (odds ratio, 0.45; 95% confidence interval [CI], 0.21 to 0.96;P= 0.04), whereas GIA to K1 and W2mef was not. These results show that GIA, as well as its association with protection from malaria, is dependent on theP. falciparumline and can be explained by differences in erythrocyte invasion phenotypes between parasite lines. Our study contributes knowledge on the biological importance of growth inhibition and the potential influence ofP. falciparumerythrocyte invasion phenotypic differences on its relationship to protective immunity against malaria.

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Mycobacterium tuberculosis Alters the Metalloprotease Activity of the COP9 Signalosome

mBio ◽

10.1128/mbio.01278-14 ◽

2014 ◽

Vol 5 (4) ◽

Cited By ~ 7

Author(s):

Lia Danelishvili ◽

Lmar Babrak ◽

Sasha J. Rose ◽

Jamie Everman ◽

Luiz E. Bermudez

Keyword(s):

Mycobacterium Tuberculosis ◽

Cop9 Signalosome ◽

Virulence Determinants ◽

Bacterial Survival ◽

Phagocytic Cells ◽

Content Type ◽

Metalloprotease Activity ◽

Macrophage Protein

ABSTRACT Inhibition of apoptotic death of macrophages by Mycobacterium tuberculosis represents an important mechanism of virulence that results in pathogen survival both in vitro and in vivo. To identify M. tuberculosis virulence determinants involved in the modulation of apoptosis, we previously screened a transposon bank of mutants in human macrophages, and an M. tuberculosis clone with a nonfunctional Rv3354 gene was identified as incompetent to suppress apoptosis. Here, we show that the Rv3354 gene encodes a protein kinase that is secreted within mononuclear phagocytic cells and is required for M. tuberculosis virulence. The Rv3354 effector targets the metalloprotease (JAMM) domain within subunit 5 of the COP9 signalosome (CSN5), resulting in suppression of apoptosis and in the destabilization of CSN function and regulatory cullin-RING ubiquitin E3 enzymatic activity. Our observation suggests that alteration of the metalloprotease activity of CSN by Rv3354 possibly prevents the ubiquitin-dependent proteolysis of M. tuberculosis-secreted proteins. IMPORTANCE Macrophage protein degradation is regulated by a protein complex called a signalosome. One of the signalosomes associated with activation of ubiquitin and protein labeling for degradation was found to interact with a secreted protein from M. tuberculosis, which binds to the complex and inactivates it. The interference with the ability to inactivate bacterial proteins secreted in the phagocyte cytosol may have crucial importance for bacterial survival within the phagocyte.

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Thrombospondin mediates the cytoadherence of Plasmodium falciparum- infected red cells to vascular endothelium in shear flow conditions

Blood ◽

10.1182/blood.v71.1.71.bloodjournal71171 ◽

1988 ◽

Vol 71 (1) ◽

pp. 71-75

Author(s):

EP Rock ◽

EF Jr Roth ◽

RR Rojas-Corona ◽

JA Sherwood ◽

RL Nagel ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Cerebral Malaria ◽

Ex Vivo ◽

Rabbit Antiserum ◽

Immune Serum ◽

Red Cells ◽

Specific Attachment ◽

Infected Cells ◽

Human Immune ◽

Venular Endothelium

Cerebral malaria is thought to involve specific attachment of Plasmodium falciparum-infected knobby red cells to venular endothelium. The nature of surface ligands on host endothelial cells that may mediate cytoadherence is poorly understood. We have investigated the effects of soluble thrombospondin, rabbit antiserum raised against thrombospondin, and human immune serum on cytoadherence of parasitized erythrocytes in ex vivo mesocecum vasculature. Preincubation of infected red cells with soluble thrombospondin or human immune serum inhibits binding of infected red cells to rat venular endothelium. Infusion of the microcirculatory preparation with rabbit antithrombospondin antibodies before perfusion of parasitized erythrocytes also resulted in decreased cytoadherence. In addition, incubation of infected cells with human immune sera obtained from malaria patients significantly inhibited the observed cytoadherence. Our results indicate that thrombospondin mediates binding of infected red cells to venular endothelium and may thus be involved in the pathogenesis of cerebral malaria.

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A Chemical Rescue Screen Identifies a Plasmodium falciparum Apicoplast Inhibitor Targeting MEP Isoprenoid Precursor Biosynthesis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03342-14 ◽

2014 ◽

Vol 59 (1) ◽

pp. 356-364 ◽

Cited By ~ 52

Author(s):

Wesley Wu ◽

Zachary Herrera ◽

Danny Ebert ◽

Katie Baska ◽

Seok H. Cho ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Antimalarial Drug ◽

Drug Targets ◽

Specific Activity ◽

Drug Candidate ◽

Content Type ◽

Chemical Rescue ◽

Growth Inhibitory ◽

Parasite Populations

ABSTRACTThe apicoplast is an essential plastid organelle found inPlasmodiumparasites which contains several clinically validated antimalarial-drug targets. A chemical rescue screen identified MMV-08138 from the “Malaria Box” library of growth-inhibitory antimalarial compounds as having specific activity against the apicoplast. MMV-08138 inhibition of blood-stagePlasmodium falciparumgrowth is stereospecific and potent, with the most active diastereomer demonstrating a 50% effective concentration (EC50) of 110 nM. Whole-genome sequencing of 3 drug-resistant parasite populations from two independent selections revealed E688Q and L244I mutations inP. falciparumIspD, an enzyme in the MEP (methyl-d-erythritol-4-phosphate) isoprenoid precursor biosynthesis pathway in the apicoplast. The active diastereomer of MMV-08138 directly inhibited PfIspD activityin vitrowith a 50% inhibitory concentration (IC50) of 7.0 nM. MMV-08138 is the first PfIspD inhibitor to be identified and, together with heterologously expressed PfIspD, provides the foundation for further development of this promising antimalarial drug candidate lead. Furthermore, this report validates the use of the apicoplast chemical rescue screen coupled with target elucidation as a discovery tool to identify specific apicoplast-targeting compounds with new mechanisms of action.

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Vitamin A Metabolism by Dendritic Cells Triggers an Antimicrobial Response againstMycobacterium tuberculosis

mSphere ◽

10.1128/msphere.00327-19 ◽

2019 ◽

Vol 4 (3) ◽

Cited By ~ 3

Author(s):

Elliot W. Kim ◽

Avelino De Leon ◽

Zhichun Jiang ◽

Roxana A. Radu ◽

Adrian R. Martineau ◽

...

Keyword(s):

Dendritic Cells ◽

Mycobacterium Tuberculosis ◽

Retinoic Acid ◽

Immune System ◽

Vitamin A ◽

Family Member ◽

Serum Vitamin ◽

Normal Lung ◽

Content Type

ABSTRACTEpidemiological evidence correlates low serum vitamin A (retinol) levels with increased susceptibility to active tuberculosis (TB); however, retinol is biologically inactive and must be converted into its bioactive form, all-transretinoic acid (ATRA). Given that ATRA triggers a Niemann-Pick type C2 (NPC2)-dependent antimicrobial response againstMycobacterium tuberculosis, we investigated the mechanism by which the immune system converts retinol into ATRA at the site of infection. We demonstrate that granulocyte-macrophage colony-stimulating factor (GM-CSF)-derived dendritic cells (DCs), but not macrophages, express enzymes in the vitamin A metabolic pathway, including aldehyde dehydrogenase 1 family, member a2 (ALDH1A2) and short-chain dehydrogenase/reductase family, member 9 (DHRS9), enzymes capable of the two-step conversion of retinol into ATRA, which is subsequently released from the cell. Additionally, mRNA and protein expression levels of ALDH1A2 and DC marker CD1B were lower in tuberculosis lung tissues than in normal lung. The conditioned medium from DCs cultured with retinol stimulated antimicrobial activity fromM. tuberculosis-infected macrophages, as well as the expression of NPC2 in monocytes, which was blocked by specific inhibitors, including retinoic acid receptor inhibitor (RARi) orN,N-diethylaminobenzaldehyde (DEAB), an ALDH1A2 inhibitor. These results indicate that metabolism of vitamin A by DCs transactivates macrophage antimicrobial responses.IMPORTANCETuberculosis (TB) is the leading cause of death by a single infectious agent worldwide. One factor that contributes to the success of the microbe is the deficiency in immunomodulatory nutrients, such as vitamin A (retinol), which are prevalent in areas where TB is endemic. Clinical trials show that restoration of systemic retinol levels in active TB patients is ineffective in mitigating the disease; however, laboratory studies demonstrate that activation of the vitamin A pathway inMycobacterium tuberculosis-infected macrophages triggers an antimicrobial response. Therefore, the goal of this study was to determine the link between host retinol levels and retinoic acid-mediated antimicrobial responses againstM. tuberculosis. By combining establishedin vitromodels within situstudies of lung tissue from TB patients, this study demonstrates that the innate immune system utilizes transcellular metabolism leading to activation between dendritic cells and macrophages as a means to combat the pathogen.

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Computational Chemogenomics Drug Repositioning Strategy Enables the Discovery of Epirubicin as a New Repurposed Hit for Plasmodium falciparum and P. vivax

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02041-19 ◽

2020 ◽

Vol 64 (9) ◽

Author(s):

Letícia Tiburcio Ferreira ◽

Juliana Rodrigues ◽

Gustavo Capatti Cassiano ◽

Tatyana Almeida Tavella ◽

Kaira Cristina Peralis Tomaz ◽

...

Keyword(s):

Plasmodium Falciparum ◽

In Silico ◽

Antimalarial Activity ◽

Drug Repositioning ◽

Dna Gyrase ◽

Plasmodium Yoelii ◽

Computer Assisted ◽

Content Type

ABSTRACT Widespread resistance against antimalarial drugs thwarts current efforts for controlling the disease and urges the discovery of new effective treatments. Drug repositioning is increasingly becoming an attractive strategy since it can reduce costs, risks, and time-to-market. Herein, we have used this strategy to identify novel antimalarial hits. We used a comparative in silico chemogenomics approach to select Plasmodium falciparum and Plasmodium vivax proteins as potential drug targets and analyzed them using a computer-assisted drug repositioning pipeline to identify approved drugs with potential antimalarial activity. Among the seven drugs identified as promising antimalarial candidates, the anthracycline epirubicin was selected for further experimental validation. Epirubicin was shown to be potent in vitro against sensitive and multidrug-resistant P. falciparum strains and P. vivax field isolates in the nanomolar range, as well as being effective against an in vivo murine model of Plasmodium yoelii. Transmission-blocking activity was observed for epirubicin in vitro and in vivo. Finally, using yeast-based haploinsufficiency chemical genomic profiling, we aimed to get insights into the mechanism of action of epirubicin. Beyond the target predicted in silico (a DNA gyrase in the apicoplast), functional assays suggested a GlcNac-1-P-transferase (GPT) enzyme as a potential target. Docking calculations predicted the binding mode of epirubicin with DNA gyrase and GPT proteins. Epirubicin is originally an antitumoral agent and presents associated toxicity. However, its antiplasmodial activity against not only P. falciparum but also P. vivax in different stages of the parasite life cycle supports the use of this drug as a scaffold for hit-to-lead optimization in malaria drug discovery.

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Mesenchymal Stem Cells in COVID-19: A Journey from Bench to Bedside

Laboratory Medicine ◽

10.1093/labmed/lmaa049 ◽

2020 ◽

Vol 52 (1) ◽

pp. 24-35

Author(s):

Kamal Kant Sahu ◽

Ahmad Daniyal Siddiqui ◽

Jan Cerny

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Immune System ◽

Therapeutic Option ◽

Antiviral Agent ◽

In Vitro Models ◽

Human Immune System ◽

Economic Sectors ◽

Human Immune

Abstract The COVID-19 pandemic has led to a major setback in both the health and economic sectors across the globe. The scale of the problem is enormous because we still do not have any specific anti-SARS-CoV-2 antiviral agent or vaccine. The human immune system has never been exposed to this novel virus, so the viral interactions with the human immune system are completely naive. New approaches are being studied at various levels, including animal in vitro models and human-based studies, to contain the COVID-19 pandemic as soon as possible. Many drugs are being tested for repurposing, but so far only remdesivir has shown some positive benefits based on preliminary reports, but these results also need further confirmation via ongoing trials. Otherwise, no other agents have shown an impactful response against COVID-19. Recently, research exploring the therapeutic application of mesenchymal stem cells (MSCs) in critically ill patients suffering from COVID-19 has gained momentum. The patients belonging to this subset are most likely beyond the point where they could benefit from an antiviral therapy because most of their illness at this stage of disease is driven by inflammatory (over)response of the immune system. In this review, we discuss the potential of MSCs as a therapeutic option for patients with COVID-19, based on the encouraging results from the preliminary data showing improved outcomes in the progression of COVID-19 disease.

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Ex VivoDrug Susceptibility of Ferroquine against Chloroquine-Resistant Isolates of Plasmodium falciparum and P. vivax

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01375-10 ◽

2011 ◽

Vol 55 (9) ◽

pp. 4461-4464 ◽

Cited By ~ 25

Author(s):

Jutta Marfurt ◽

Ferryanto Chalfein ◽

Pak Prayoga ◽

Frans Wabiser ◽

Enny Kenangalem ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Combination Therapy ◽

Drug Class ◽

Ex Vivo ◽

Multidrug Resistant ◽

Drug Action ◽

Drug Uptake ◽

Content Type ◽

Excellent Activity

ABSTRACTFerroquine (FQ; SSR97193), a ferrocene-containing 4-aminoquinoline derivate, has potentin vitroefficacy against chloroquine (CQ)-resistantPlasmodium falciparumand CQ-sensitiveP. vivax. In the current study,ex vivoFQ activity was tested in multidrug-resistantP. falciparumandP. vivaxfield isolates using a schizont maturation assay. Although FQ showed excellent activity against CQ-sensitive and -resistantP. falciparumandP. vivax(median 50% inhibitory concentrations [IC50s], 9.6 nM and 18.8 nM, respectively), there was significant cross-susceptibility with the quinoline-based drugs chloroquine, amodiaquine, and piperaquine (forP. falciparum,r= 0.546 to 0.700,P< 0.001; forP. vivax,r= 0.677 to 0.821,P< 0.001). The observedex vivocross-susceptibility is likely to reflect similar mechanisms of drug uptake/efflux and modes of drug action of this drug class. However, the potent activity of FQ against resistant isolates of bothP. falciparumandP. vivaxhighlights a promising role for FQ as a lead antimalarial against CQ-resistantPlasmodiumand a useful partner drug for artemisinin-based combination therapy.

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