Flow cytometry, a useful tool for detecting the lethal effect of pentamidine on leishmania viannia complex promastigote forms

Delgado, Gabriela; Puentes, Fabìola; Moreno, Alberto; Patarroyo, Manuel E.

doi:10.1006/phrs.2001.0825

Cited by 10 publications

(7 citation statements)

References 17 publications

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“…AmpB and Quillaja saponaria saponin (Saponin) were added as positive controls. Parasite viability was measured at 48 h by flow cytometry as previously described (Delgado et al, 2001). …”

Section: Materials and Methodologymentioning

confidence: 99%

Host-mediated Leishmania donovani treatment using AR-12 encapsulated in acetalated dextran microparticles

Collier

Peine

Gautam

et al. 2016

International Journal of Pharmaceutics

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Leishmaniasis is a disease caused by parasites of Leishmania sp., which effects nearly 12 million people worldwide and is associated with treatment complications due to widespread parasite resistance toward pathogen-directed therapeutics. The current treatments for visceral leishmaniasis (VL), the systemic form of the disease, involve pathogen-mediated drugs and have long treatment regimens, increasing the risk of forming resistant strains. One way to limit emergence of resistant pathogens is through the use of host-mediated therapeutics. The host-mediated therapeutic AR-12, which is FDA IND-approved for cancer treatment, has shown activity against a broad spectrum of intracellular pathogens; however, due to hydrophobicity and toxicity, it is difficult to reach therapeutic doses. We have formulated AR-12 into microparticles (AR-12/MPs) using the novel biodegradable polymer acetalated dextran (Ace-DEX) and used this formulation for the systemic treatment of VL. Treatment with AR-12/MPs significantly reduced liver, spleen, and bone marrow parasite loads in infected mice, while combinatorial therapies with amphotericin B had an even more significant effect. Overall, AR-12/MPs offer a unique, host-mediated therapy that could significantly reduce the emergence of drug resistance in the treatment of VL.

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“…AmpB and Quillaja saponaria saponin (Saponin) were added as positive controls. Parasite viability was measured at 48 h by flow cytometry as previously described (Delgado et al, 2001). …”

Section: Materials and Methodologymentioning

confidence: 99%

Host-mediated Leishmania donovani treatment using AR-12 encapsulated in acetalated dextran microparticles

Collier

Peine

Gautam

et al. 2016

International Journal of Pharmaceutics

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“…One million log phase promastigotes were seeded in 1 mL of complete RPMI-1640 medium, and parasite numbers, and their mobility and morphology were measured from 24 and 48 h. This was performed by flow cytometry measurements using experimental and control groups of parasites stained with propidium iodide and with Quillaja saponaria saponin as positive control, as reported in the literature (Delgado et al, 2001). The test compounds isolated from P. andrieuxii were compared with the antimonial drug, Pentostam.…”

Section: Methodsmentioning

confidence: 99%

Sterols with antileishmanial activity isolated from the roots of Pentalinon andrieuxii

Lezama-Dávila

Isaac-Márquez

et al. 2012

Phytochemistry

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A new cholesterol derivative, pentalinonsterol (cholest-4,20,24-trien-3-one, 1), and a new polyoxygenated pregnane sterol glycoside, pentalinonside (2), together with 18 known compounds, including 14 sterols (3–16), three coumarins (17–19), and a triterpene (20), were isolated from a n-hexane partition of a methanol extract of the roots of the Mexican medicinal plant Pentalinon andrieuxii. Structure elucidation of compounds 1 and 2 was accomplished by spectroscopic data interpretation. All isolates were evaluated in vitro for their antileishmanial activity. Among these compounds, 6,7-dihydroneridienone (15) was found to be the most potent principle against promastigotes of Leishmania mexicana (L. mexicana). The cholesterol analogue, pentalinonsterol (1), together with two known sterols, 24-methylcholest-4,24(28)-dien-3-one (3) and neridienone (16), also exhibited significant leishmanicidal activity in this same bioassay. Compounds 1, 3, 15, 16, cholest-4-en-3-one (4), and cholest-5,20,24-trien-3β-ol (7), showed strong antileishmanial activity against amastigotes of L. mexicana, and 4 was found to be the most potent agent with an IC50 value of 0.03 μM. All the isolates were also evaluated for their cytotoxicity in non-infected bone marrow-derived macrophages, but none of these compounds was found active towards this cell line. The intracellular parasites treated with compounds 1, 3, 4, 15, and 16 were further studied by electron microscopy; morphological abnormalities and destruction of the amastigotes were observed, as a result of treatment with these compounds.

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“…Assessment of novel drugs or treatment regimens for leishmaniasis requires reliable methods for high-throughput screening (HTS) against the clinically relevant stage of the disease, the intracellular amastigotes (4,5). For decades, anti-leishmanial drug discovery has rested upon the use of insect stage, free living promastigotes, for which simple and efficient in vitro assays have been made available (6)(7)(8)(9)(10)(11)(12). Nevertheless, failure to identify all active compounds and selection of numerous false-positive hits have recently been associated with the use of promastigotes in primary screenings, suggesting that host cell-mediated effects and stage-specific chemosensitivities should be addressed at early stages of drug discovery programs (13,14).…”

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confidence: 99%

Simple Colorimetric Trypanothione Reductase-Based Assay for High-Throughput Screening of Drugs against Leishmania Intracellular Amastigotes

Bogaart

Schoone

England³

et al. 2014

Antimicrob Agents Chemother

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f Critical to the search for new anti-leishmanial drugs is the availability of high-throughput screening (HTS) methods to test chemical compounds against the relevant stage for pathogenesis, the intracellular amastigotes. Recent progress in automated microscopy and genetic recombination has produced powerful tools for drug discovery. Nevertheless, a simple and efficient test for measuring drug activity against Leishmania clinical isolates is lacking. Here we describe a quantitative colorimetric assay in which the activity of a Leishmania native enzyme is used to assess parasite viability. Enzymatic reduction of disulfide trypanothione, monitored by a microtiter plate reader, was used to quantify the growth of Leishmania parasites. An excellent correlation was found between the optical density at 412 nm and the number of parasites inoculated. Pharmacological validation of the assay was performed against the conventional alamarBlue method for promastigotes and standard microscopy for intracellular amastigotes. The activity of a selected-compound panel, including several anti-leishmanial reference drugs, demonstrated high consistency between the newly developed assay and the reference method and corroborated previously published data. Quality assessment with standard measures confirmed the robustness and reproducibility of the assay, which performed in compliance with HTS requirements. This simple and rapid assay provides a reliable, accurate method for screening anti-leishmanial agents, with high throughput. The basic equipment and manipulation required to perform the assay make it easy to implement, simplifying the method for scoring inhibitor assays.

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Flow cytometry, a useful tool for detecting the lethal effect of pentamidine on leishmania viannia complex promastigote forms

Cited by 10 publications

References 17 publications

Host-mediated Leishmania donovani treatment using AR-12 encapsulated in acetalated dextran microparticles

Host-mediated Leishmania donovani treatment using AR-12 encapsulated in acetalated dextran microparticles

Sterols with antileishmanial activity isolated from the roots of Pentalinon andrieuxii

Simple Colorimetric Trypanothione Reductase-Based Assay for High-Throughput Screening of Drugs against Leishmania Intracellular Amastigotes

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