High-Content Assay for Measuring Intracellular Growth of<i>Leishmania</i>in Human Macrophages

Dagley, Michael J; Saunders, Eleanor; Simpson, Kaylene J.; McConville, Malcolm J.

doi:10.1089/adt.2015.652

Cited by 22 publications

(21 citation statements)

References 33 publications

(47 reference statements)

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“…Since microscopic enumeration is too tedious for routine evaluation of compounds and their combinations and because our ␤-lactamase parasites no longer reliably express this reporter gene, a high-content assay for intracellular L. donovani (see Fig. S1 in the supplemental material) was developed based on previously reported image-based assays (14,15). Employing overnight infection of macrophages as done previously to validate the assay, the Z= factor (16) for the infected versus uninfected controls was 0.74 Ϯ 0.03 (means Ϯ standard errors; n ϭ 6).…”

Section: Resultsmentioning

confidence: 99%

Antileishmanial Efficacy and Pharmacokinetics of DB766-Azole Combinations

Joice

Yang

Farahat

et al. 2018

Antimicrob Agents Chemother

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Given the limitations of current antileishmanial drugs and the utility of oral combination therapy for other infections, developing an oral combination against visceral leishmaniasis should be a high priority. combination studies with DB766 and antifungal azoles against intracellular showed that posaconazole and ketoconazole, but not fluconazole, enhanced DB766 potency. Pharmacokinetic analysis of DB766-azole combinations in uninfected Swiss Webster mice revealed that DB766 exposure was increased by higher posaconazole and ketoconazole doses, while DB766 decreased ketoconazole exposure. In -infected BALB/c mice, DB766-posaconazole combinations given orally for 5 days were more effective than DB766 or posaconazole alone. For example, 81% ± 1% (means ± standard errors) inhibition of liver parasite burden was observed for 37.5 mg/kg of body weight DB766 plus 15 mg/kg posaconazole, while 37.5 mg/kg DB766 and 15 mg/kg posaconazole administered as monotherapy gave 40% ± 5% and 21% ± 3% inhibition, respectively. Combination index (CI) analysis indicated that synergy or moderate synergy was observed in six of nine combined dose groups, while the other three were nearly additive. Liver concentrations of DB766 and posaconazole increased in almost all combination groups compared to monotherapy groups, although many increases were not statistically significant. For DB766-ketoconazole combinations evaluated in this model, two were antagonistic, one displayed synergy, and one was nearly additive. These data indicate that the efficacy of DB766-posaconazole and DB766-ketoconazole combinations is influenced in part by the pharmacokinetics of the combination, and that the former combination deserves further consideration in developing new treatment strategies against visceral leishmaniasis.

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Section: Resultsmentioning

confidence: 99%

Antileishmanial Efficacy and Pharmacokinetics of DB766-Azole Combinations

Joice

Yang

Farahat

et al. 2018

Antimicrob Agents Chemother

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“…L . infantum isolates were stained with CellTracker Violet BMQC dye (Thermo Fisher) as previously described [29]. The parasites were cultured axenically in Schneider's Drosophila medium (Thermo Fisher) plus 10% FBS prior to infection of cells.…”

Section: Methodsmentioning

confidence: 99%

sCD163 levels as a biomarker of disease severity in leprosy and visceral leishmaniasis

et al. 2017

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BackgroundCD163, receptor for the haptoglobin–hemoglobin complex, is expressed on monocytes/macrophages and neutrophils. A soluble form of CD163 (sCD163) has been associated with the M2 macrophage phenotype, and M2 macrophages have been shown to down-modulate inflammatory responses. In particular, previous studies have shown that M2 is closely associated with the most severe clinical presentation of leprosy (i.e. lepromatous leprosy (LL)), as well as tuberculosis. We hypothesized that sCD163 correlates with severity of diseases caused by intracellular pathogens.Methodology/Principal findingsTo assess this hypothesis, sCD163 levels were measured in the serum of leprosy and visceral leishmaniasis (VL) patients stratified by severity of the clinical presentation. sCD163 levels were significantly higher in patients with these diseases than those observed in healthy control individuals. Further analyses on infection and disease status of leprosy and VL patients revealed a clear association of sCD163 levels with clinical parameters of disease severity. In vitro culture assays revealed that Leishmania infection induced CD163 expression on the surface of both monocyte/macrophages and neutrophils, suggesting these cells as possible sources of sCD163. FACS analyses shows that the cells expressing CD163 produces both TNF-α and IL-4.Conclusions/SignificanceTaken together, our results reveal sCD163 as a potential biomarker of severity of diseases caused by intracellular pathogens M. leprae and Leishmania spp. and have a modulatory role, with a mix of an inflammatory property induced by TNF-α release, but that potentially induces an anti-inflammatory T cell response, related to IL-4 release.

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“…For in vitro screens and assays, this has ranged from the need to develop methods that (i) are adaptable to and enable high-throughput screens against the replicative intracellular-macrophage amastigote stage of Leishmania donovani, one of the causative species of VL (7); and (ii) include high-throughput technologies that enable the collection of more information compared to the traditionally used assays based on manual counting and reporter genes (8, 9). For example, highcontent screening (HCS) systems that permit the screening of large sets of compounds using imaging techniques that also capture information about compound toxicity against host cells and mode of action (10, 11) have been applied to antileishmanial drug discovery (12)(13)(14)(15)(16)(17).…”

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confidence: 99%

A Replicative In Vitro Assay for Drug Discovery against Leishmania donovani

Tegazzini

Díaz

Aguilar

et al. 2016

Antimicrob Agents Chemother

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The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis, a disease potentially fatal if not treated. Current available treatments have major limitations, and new and safer drugs are urgently needed. In recent years, advances in high-throughput screening technologies have enabled the screening of millions of compounds to identify new antileishmanial agents. However, most of the compounds identified in vitro did not translate their activities when tested in in vivo models, highlighting the need to develop more predictive in vitro assays. In the present work, we describe the development of a robust replicative, high-content, in vitro intracellular L. donovani assay. Horse serum was included in the assay media to replace standard fetal bovine serum, to completely eliminate the extracellular parasites derived from the infection process. A novel phenotypic in vitro infection model has been developed, complemented with the identification of the proliferation of intracellular amastigotes measured by EdU incorporation. In vitro and in vivo results for miltefosine, amphotericin B, and the selected compound 1 have been included to validate the assay.T he leishmaniases are a complex of diseases, with visceral and cutaneous manifestations caused by protozoan parasites of the genus Leishmania. Visceral leishmaniasis (VL) has been the main focus for drug research and development over the past 2 decades, due to the large disease burden in East Africa and South Asia (1) and potential patient death if not treated. For VL, there has been progress in treatment over the past decade, with clinical evidence for efficacy of and registration for use of oral miltefosine, paromomycin, and the liposomal formulation of amphotericin B (AmBisome, Gilead, USA) in South Asia (2), as well as combinations of these standard drugs (3). The need for new drugs to treat VL remains, as (i) miltefosine is the only approved oral treatment but requires 28 days of treatment and potential teratogenicity limits its use (4), (ii) paromomycin requires 21 days of treatment and intramuscular administration (http://www.dndi.org/diseases-projects/diseases /vl/current-treatment/current-treatment-vl.html), and (iii) liposomal amphotericin B formulations, which have successful cure rates with a single dose (5), require intravenous (i.v.) infusion, have a high cost if not donated, and have a requirement for cold storage, limiting use in countries where the disease is endemic (6). As part of the drive to find new treatments, there has been a refocus on the assays and models used to identify and develop new molecules as antileishmanial drugs. For in vitro screens and assays, this has ranged from the need to develop methods that (i) are adaptable to and enable high-throughput screens against the replicative intracellular-macrophage amastigote stage of Leishmania donovani, one of the causative species of VL (7); and (ii) include high-throughput technologies that enable the collection of more information compared to the traditionally use...

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High-Content Assay for Measuring Intracellular Growth ofLeishmaniain Human Macrophages

Cited by 22 publications

References 33 publications

Antileishmanial Efficacy and Pharmacokinetics of DB766-Azole Combinations

Antileishmanial Efficacy and Pharmacokinetics of DB766-Azole Combinations

sCD163 levels as a biomarker of disease severity in leprosy and visceral leishmaniasis

A Replicative In Vitro Assay for Drug Discovery against Leishmania donovani

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