scholarly journals A Male and Female Gametocyte Functional Viability Assay To Identify Biologically Relevant Malaria Transmission-Blocking Drugs

2014 ◽  
Vol 58 (12) ◽  
pp. 7292-7302 ◽  
Author(s):  
A. Ruecker ◽  
D. K. Mathias ◽  
U. Straschil ◽  
T. S. Churcher ◽  
R. R. Dinglasan ◽  
...  
Keyword(s):  
Mature Stage ◽  
Male And Female ◽  
Content Type ◽  
Biologically Relevant ◽  
Viability Assay ◽  
High Throughput Drug Screening ◽  
Membrane Feeding ◽  
Transmission Stage ◽  
Target Male

ABSTRACTMalaria elimination will require interventions that prevent parasite transmission from the human host to the mosquito. Experimentally, this is usually determined by the expensive and laboriousPlasmodium falciparumstandard membrane feeding assay (PfSMFA), which has limited utility for high-throughput drug screening. In response, we developed theP. falciparumdual gamete formation assay (PfDGFA), which faithfully simulates the initial stages of the PfSMFAin vitro. It utilizes a dual readout that individually and simultaneously reports on the functional viability of male and female mature stage V gametocytes. To validate, we screen the Medicines for Malaria Venture (MMV) Malaria Box library with the PfDGFA. Unique to this assay, we find compounds that target male gametocytes only and also compounds with reversible and irreversible activity. Most importantly, we show that compound activity in the PfDGFA accurately predicts activity in PfSMFAs, which validates and supports its adoption into the transmission-stage screening pipeline.

scholarly journals High Accumulation and In Vivo Recycling of the New Antimalarial Albitiazolium Lead to Rapid Parasite Death

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Sharon Wein ◽  
Nicolas Taudon ◽  
Marjorie Maynadier ◽  
Christophe Tran Van Ba ◽  
Delphine Margout ◽  
...  
Keyword(s):  
Pharmacological Activity ◽  
Plasma Concentrations ◽  
Pharmacokinetic Studies ◽  
High Accumulation ◽  
Content Type ◽  
Viability Assay ◽  
Parasite Viability ◽  
Two Parameters

ABSTRACT Albitiazolium is the lead compound of bisthiazolium choline analogues and exerts powerful in vitro and in vivo antimalarial activities. Here we provide new insight into the fate of albitiazolium in vivo in mice and how it exerts its pharmacological activity. We show that the drug exhibits rapid and potent activity and has very favorable pharmacokinetic and pharmacodynamic properties. Pharmacokinetic studies in Plasmodium vinckei-infected mice indicated that albitiazolium rapidly and specifically accumulates to a great extent (cellular accumulation ratio, >150) in infected erythrocytes. Unexpectedly, plasma concentrations and the area under concentration-time curves increased by 15% and 69% when mice were infected at 0.9% and 8.9% parasitemia, respectively. Albitiazolium that had accumulated in infected erythrocytes and in the spleen was released into the plasma, where it was then available for another round of pharmacological activity. This recycling of the accumulated drug, after the rupture of the infected erythrocytes, likely extends its pharmacological effect. We also established a new viability assay in the P. vinckei-infected mouse model to discriminate between fast- and slow-acting antimalarials. We found that albitiazolium impaired parasite viability in less than 6 and 3 h at the ring and late stages, respectively, while parasite morphology was affected more belatedly. This highlights that viability and morphology are two parameters that can be differentially affected by a drug treatment, an element that should be taken into account when screening new antimalarial drugs.

scholarly journals Assessment in vitro of the antimalarial and transmission blocking activities of Cipargamin and Ganaplacide in artemisinin resistant Plasmodium falciparum

2022 ◽  
Author(s):  
Achaporn Yipsirimetee ◽  
Pornpawee Chiewpoo ◽  
Rupam Tripura ◽  
Dysoley Lek ◽  
Nicholas P. J. Day ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Antimalarial Activity ◽  
Artemisinin Resistance ◽  
Blood Stage ◽  
Mature Stage ◽  
Asexual Blood Stage ◽  
Male And Female ◽  
Transmission Blocking ◽  
Combination Treatments

Artemisinin resistance in Plasmodium falciparum has emerged and spread widely in the Greater Mekong Subregion threatening current first line artemisinin combination treatments. New antimalarial drugs are needed urgently. Cipargamin (KAE609) and ganaplacide (KAF156) are promising novel antimalarial compounds in advanced stages of development. Both compounds have potent asexual blood stage activities, inhibit P. falciparum gametocytogenesis and reduce oocyst development in anopheline mosquitoes. In this study, we compared the asexual and sexual stage activities of cipargamin, ganaplacide and artesunate in artemisinin resistant P. falciparum isolates (N=7, K13 mutation; C580Y, G449A and R539T) from Thailand and Cambodia. Asexual blood stage antimalarial activity was evaluated in a SYBR-green I based 72h in vitro assay, and the effects on male and female mature stage V gametocytes were assessed in the P. falciparum dual gamete formation assay. Ganaplacide had higher activities when compared to cipargamin and artesunate, with a mean (SD) IC50 against asexual stages of 5.5 (1.1) nM, 7.8 (3.9) nM for male gametocytes and 57.9 (59.6) nM for female gametocytes. Cipargamin had a similar potency against male and female gametocytes, with a mean (SD) IC50 of 123.1 (80.2) nM for male gametocytes, 88.5 (52.7) nM for female gametocytes and 2.4 (0.6) nM for asexual stages. Both cipargamin and ganaplacide showed significant transmission-blocking activities against artemisinin resistant P. falciparum in vitro .

scholarly journals The Spiroindolone Drug Candidate NITD609 Potently Inhibits Gametocytogenesis and Blocks Plasmodium falciparum Transmission to Anopheles Mosquito Vector

2012 ◽  
Vol 56 (7) ◽  
pp. 3544-3548 ◽  
Author(s):  
J. C. van Pelt-Koops ◽  
H. E. Pett ◽  
W. Graumans ◽  
M. van der Vegte-Bolmer ◽  
G. J. van Gemert ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Drug Candidate ◽  
Mosquito Vector ◽  
Content Type ◽  
Membrane Feeding ◽  
Dose Dependent Fashion ◽  
Dose Dependent ◽  
Membrane Feeding Assay ◽  
Reducing Potential

ABSTRACTThe global malaria agenda has undergone a reorientation from control of clinical cases to entirely eradicating malaria. For that purpose, a key objective is blocking transmission of malaria parasites from humans to mosquito vectors. The new antimalarial drug candidate NITD609 was evaluated for its transmission-reducing potential and compared to a few established antimalarials (lumefantrine, artemether, primaquine), using a suite ofin vitroassays. By the use of a microscopic readout, NITD609 was found to inhibit the early and late development ofPlasmodium falciparumgametocytesin vitroin a dose-dependent fashion over a range of 5 to 500 nM. In addition, using the standard membrane feeding assay, NITD609 was also found to be a very effective drug in reducing transmission to theAnopheles stephensimosquito vector. Collectively, our data suggest a strong transmission-reducing effect of NITD609 acting against differentP. falciparumtransmission stages.

scholarly journals The Fabp4-Cre-Model is Insufficient to Study Hoxc9 Function in Adipose Tissue

Biomedicines ◽  
2020 ◽  
Vol 8 (7) ◽  
pp. 184
Author(s):  
Sebastian Dommel ◽  
Claudia Berger ◽  
Anne Kunath ◽  
Matthias Kern ◽  
Martin Gericke ◽  
...  
Keyword(s):  
Body Weight ◽  
Adipose Tissue ◽  
High Fat Diet ◽  
Fat Distribution ◽  
Chow Diet ◽  
Littermate Control ◽  
Male And Female ◽  
Biologically Relevant

Developmental genes are important regulators of fat distribution and adipose tissue (AT) function. In humans, the expression of homeobox c9 (HOXC9) is significantly higher in subcutaneous compared to omental AT and correlates with body fat mass. To gain more mechanistic insights into the role of Hoxc9 in AT, we generated Fabp4-Cre-mediated Hoxc9 knockout mice (ATHoxc9-/-). Male and female ATHoxc9-/- mice were studied together with littermate controls both under chow diet (CD) and high-fat diet (HFD) conditions. Under HFD, only male ATHoxc9-/- mice gained less body weight and exhibited improved glucose tolerance. In both male and female mice, body weight, as well as the parameters of glucose metabolism and AT function were not significantly different between ATHoxc9-/- and littermate control CD fed mice. We found that crossing Hoxc9 floxed mice with Fabp4-Cre mice did not produce a biologically relevant ablation of Hoxc9 in AT. However, we hypothesized that even subtle reductions of the generally low AT Hoxc9 expression may cause the leaner and metabolically healthier phenotype of male HFD-challenged ATHoxc9-/- mice. Different models of in vitro adipogenesis revealed that Hoxc9 expression precedes the expression of Fabp4, suggesting that ablation of Hoxc9 expression in AT needs to be achieved by targeting earlier stages of AT development.

scholarly journals Ultrashort Peptide Bioconjugates Are Exclusively Antifungal Agents and Synergize with Cyclodextrin and Amphotericin B

2011 ◽  
Vol 56 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Christopher J. Arnusch ◽  
Hannah Ulm ◽  
Michaele Josten ◽  
Yana Shadkchan ◽  
Nir Osherov ◽  
...  
Keyword(s):  
Vitamin E ◽  
Amphotericin B ◽  
Selective Toxicity ◽  
Content Type ◽  
Biologically Relevant ◽  
Highly Active ◽  
Transmission Electron ◽  
Permeability Studies

ABSTRACTMany natural broad-spectrum cationic antimicrobial peptides (AMPs) possess a general mode of action that is dependent on lipophilicity and charge. Modulating the lipophilicity of AMPs by the addition of a fatty acid has been an effective strategy to increase the lytic activity and can further broaden the spectrum of AMPs. However, lipophilic modifications that narrow the spectrum of activity and exclusively direct peptides to fungi are less common. Here, we show that short peptide sequences can be targeted to fungi with structured lipophilic biomolecules, such as vitamin E and cholesterol. The conjugates were active againstAspergillus fumigatus,Cryptococcus neoformans, andCandida albicansbut not against bacteria and were observed to cause membrane perturbation by transmission electron microscopy and in membrane permeability studies. However, forC. albicans, selected compounds were effective without the perturbation of the cell membrane, and synergism was seen with a vitamin E conjugate and amphotericin B. Moreover, in combination with β-cyclodextrin, antibacterial activity emerged in selected compounds. Biocompatibility for selected active compounds was testedin vitroandin vivousing toxicity assays on erythrocytes, macrophages, and mice.In vitrocytotoxicity experiments led to selective toxicity ratios (50% lethal concentration/MIC) of up to 64 for highly active antifungal compounds, and noin vivomurine toxicity was seen. Taken together, these results highlight the importance of the conjugated lipophilic structure and suggest that the modulation of other biologically relevant peptides with hydrophobic moieties, such as cholesterol and vitamin E, generate compounds with unique bioactivity.

scholarly journals Lactic Acid Supplementation Increases Quantity and Quality of Gametocytes in Plasmodium falciparum Culture

2020 ◽  
Vol 89 (1) ◽  
pp. e00635-20
Author(s):  
Rachel West ◽  
David J. Sullivan
Keyword(s):  
Plasmodium Falciparum ◽  
Lactic Acid ◽  
Blood Film ◽  
Content Type ◽  
Acid Supplementation ◽  
Membrane Feeding ◽  
Medium Exchange ◽  
Passage Number ◽  
Low Passage

ABSTRACTMalaria infection by Plasmodium falciparum continues to afflict millions of people worldwide, with transmission being dependent upon mosquito ingestion of the parasite gametocyte stage. These sexually committed stages develop from the asexual stages, yet the factors behind this transition are not completely understood. Here, we found that lactic acid increases gametocyte quantity and quality in P. falciparum culture. Low-passage-number NF54 parasites exposed to 8.2 mM lactic acid for various times were monitored using blood film gametocyte counts and RNA analysis throughout 2 weeks of gametocyte development in vitro for a total of 5 biological cohorts. We found that daily continuous medium exchange and 8.2 mM lactic acid supplementation increased gametocytemia approximately 2- to 6-fold relative to controls after 5 days. In membrane feeding mosquito infection experiments, we found that gametocytes continuously exposed to 8.2 mM lactic acid supplementations were more infectious to Anopheles stephensi mosquitoes, essentially doubling prevalence of infected midguts and oocyst density. Supplementation on days 9 to 16 did not increase the quantity of gametocytes but did increase quality, as measured by oocyst density, by 2.4-fold. Lactic acid did not impact asexual growth, as measured by blood film counts and luciferase quantification, as well as radioactive hypoxanthine incorporation assays. These data indicate a novel role for lactic acid in sexual development of the parasite.

scholarly journals Phenotypic Changes in Artemisinin-Resistant Plasmodium falciparum LinesIn Vitro: Evidence for Decreased Sensitivity to Dormancy and Growth Inhibition

2011 ◽  
Vol 56 (1) ◽  
pp. 428-431 ◽  
Author(s):  
Franka Teuscher ◽  
Nanhua Chen ◽  
Dennis E. Kyle ◽  
Michelle L. Gatton ◽  
Qin Cheng
Keyword(s):  
Plasmodium Falciparum ◽  
Growth Arrest ◽  
Artemisinin Resistance ◽  
Mature Stage ◽  
Ring Stage ◽  
Content Type ◽  
Resistance Phenotype ◽  
Temporary Growth

ABSTRACTThe appearance ofPlasmodium falciparumparasites with decreasedin vivosensitivity but no measurablein vitroresistance to artemisinin has raised the urgent need to characterize the artemisinin resistance phenotype. Changes in the temporary growth arrest (dormancy) profile of parasites may be one aspect of this phenotype. In this study, we investigated the link between dormancy and resistance, using artelinic acid (AL)-resistant parasites. Our results demonstrate that the AL resistance phenotype has (i) decreased sensitivity of mature-stage parasites, (ii) decreased sensitivity of the ring stage to the induction of dormancy, and (iii) a faster recovery from dormancy.

scholarly journals Enterococcus faecalis Inhibits Hyphal Morphogenesis and Virulence of Candida albicans

2012 ◽  
Vol 81 (1) ◽  
pp. 189-200 ◽  
Author(s):  
Melissa R. Cruz ◽  
Carrie E. Graham ◽  
Bryce C. Gagliano ◽  
Michael C. Lorenz ◽  
Danielle A. Garsin
Keyword(s):  
Candida Albicans ◽  
Enterococcus Faecalis ◽  
Treatment Strategies ◽  
Content Type ◽  
Biologically Relevant ◽  
System A ◽  
Hyphal Morphogenesis ◽  
Negative Effect ◽  
Hyphal Formation

The Gram-positive bacteriumEnterococcus faecalisand the fungusCandida albicansare both found as commensals in many of the same niches of the human body, such as the oral cavity and gastrointestinal (GI) tract. However, both are opportunistic pathogens and have frequently been found to be coconstituents of polymicrobial infections. Despite these features in common, there has been little investigation into whether these microbes affect one another in a biologically significant manner. Using aCaenorhabditis elegansmodel of polymicrobial infection, we discovered thatE. faecalisandC. albicansnegatively impact each other's virulence. Much of the negative effect ofE. faecalisonC. albicanswas due to the inhibition ofC. albicanshyphal morphogenesis, a developmental program crucial toC. albicanspathogenicity. We discovered that the inhibition was partially dependent on the Fsr quorum-sensing system, a major regulator of virulence inE. faecalis. Specifically, two proteases regulated by Fsr, GelE and SerE, were partially required. Further characterization of the inhibitory signal revealed that it is secreted into the supernatant, is heat resistant, and is between 3 and 10 kDa. The substance was also shown to inhibitC. albicansfilamentation in the context of anin vitrobiofilm. Finally, a screen of anE. faecalistransposon mutant library identified other genes required for suppression ofC. albicanshyphal formation. Overall, we demonstrate a biologically relevant interaction between two clinically important microbes that could affect treatment strategies as well as impact our understanding of interkingdom signaling and sensing in the human-associated microbiome.

scholarly journals Aminophthalocyanine-Mediated Photodynamic Inactivation of Leishmania tropica

2016 ◽  
Vol 60 (4) ◽  
pp. 2003-2011 ◽  
Author(s):  
Ahmed Al-Qahtani ◽  
Saad Alkahtani ◽  
Bala Kolli ◽  
Pankaj Tripathi ◽  
Sujoy Dutta ◽  
...  
Keyword(s):  
Structural Integrity ◽  
Fluorescent Protein ◽  
Red Light ◽  
Host Cells ◽  
Leishmania Tropica ◽  
Photodynamic Inactivation ◽  
Cell Viability Assay ◽  
Content Type ◽  
Viability Assay

ABSTRACTPhotodynamic inactivation ofLeishmaniaspp. requires the cellular uptake of photosensitizers, e.g., endocytosis of silicon(IV)-phthalocyanines (PC) axially substituted with bulky ligands. We report here that when substituted with amino-containing ligands, the PCs (PC1 and PC2) were endocytosed and displayed improved potency againstLeishmania tropicapromastigotes and axenic amastigotesin vitro. The uptake of these PCs by bothLeishmaniastages followed saturation kinetics, as expected. Sensitive assays were developed for assessing the photodynamic inactivation ofLeishmaniaspp. by rendering them fluorescent in two ways: transfecting promastigotes to express green fluorescent protein (GFP) and loading them with carboxyfluorescein succinimidyl ester (CFSE). PC-sensitizedLeishmania tropicastrains were seen microscopically to lose their motility, structural integrity, and GFP/CFSE fluorescence after exposure to red light (wavelength, ∼650 nm) at a fluence of 1 to 2 J cm−2. Quantitative fluorescence assays based on the loss of GFP/CFSE from liveLeishmania tropicashowed that PC1 and PC2 dose dependently sensitized both stages for photoinactivation, consistent with the results of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay.Leishmania tropicastrains are >100 times more sensitive than their host cells or macrophages to PC1- and PC2-mediated photoinactivation, judging from the estimated 50% effective concentrations (EC50s) of these cells. Axial substitution of the PC with amino groups instead of other ligands appears to increase its leishmanial photolytic activity by up to 40-fold. PC1 and PC2 are thus potentially useful for photodynamic therapy of leishmaniasis and for oxidative photoinactivation ofLeishmaniaspp. for use as vaccines or vaccine carriers.

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