Expression of Green Fluorescent Protein as a Marker for Effects of Antileishmanial Compounds In Vitro

Kamau, Sarah W.; Grimm, Felix; Hehl, Adrian B.

doi:10.1128/aac.45.12.3654-3656.2001

Cited by 42 publications

(30 citation statements)

References 29 publications

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“…We did not observe any colocalization or equal intensities of the two fluorescence signals after the treatment with SPLUNC1 because the GFP was not functionally expressed in the inhibited bacterial cells (a similar observation in GFP fluorescent reduction/elimination in inhibited cells was reported by Kamau et al [34]). …”

Section: Discussionmentioning

confidence: 57%

Multifunctional Role of Human SPLUNC1 in Pseudomonas aeruginosa Infection

et al. 2013

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The human short PLUNC1 (SPLUNC1) protein has been identified as a component of the pulmonary antimicrobial response based on its structural similarity to the bactericidal/permeability-increasing (BPI) protein. Using a genetically modified mouse model, we recently verified the antimicrobial activity of SPLUNC1 against Pseudomonas aeruginosa in vivo. To further define the mechanism of epithelial SPLUNC1-mediated antibacterial action, we carried out studies to determine how SPLUNC1 protects the host from acute respiratory infections. P. aeruginosa treated with recombinant human SPLUNC1 protein showed decreased growth in vitro. This antibacterial activity was due to growth inhibition, as a consequence of a SPLUNC1-induced increase in bacterial cell permeability. Removal of SPLUNC1 allowed the recovery of P. aeruginosa and suggested no permanent cell injury or direct killing of bacteria. Further investigation showed coating of bacterial cells by SPLUNC1. We suggest that this "bacterial cell coating" is necessary for the bacteriostatic function of SPLUNC1. Additionally, we demonstrated a novel role for SPLUNC1 as a chemoattractant that facilitated migration of macrophages and neutrophils. Taking the findings together, we propose synergistic roles for human SPLUNC1 as an antibacterial agent with bacteriostatic and chemotactic activities.

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Section: Discussionmentioning

confidence: 57%

Multifunctional Role of Human SPLUNC1 in Pseudomonas aeruginosa Infection

et al. 2013

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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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“…Recently, in an experimental model of malaria, imaging of Plasmodium berghei transfected with GFP demonstrated the dynamics of infection directly in live mice (1). GFP-transfected Leishmania parasites have been used to screen for antileishmanial activity in cell cultures by flow cytometry and microtiter plate assays (5,8,12,23). Herein we describe the use of episomally GFPtransfected Leishmania in a whole-body imaging system to follow the dynamics of infection.…”

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confidence: 99%

Real-Time In Vivo Green Fluorescent Protein Imaging of a Murine Leishmaniasis Model as a New Tool for Leishmania Vaccine and Drug Discovery

Mehta

Huang

Yang

et al. 2008

Clin Vaccine Immunol

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Leishmania species are obligate intracellular protozoan parasites that cause a broad spectrum of clinical diseases in mammalian hosts. The most frequently used approach to quantify parasites in murine model systems is based on thickness measurements of the footpad or ear after experimental infection. To overcome the limitations of this method, we used a Leishmania mutant episomally transfected with enhanced green fluorescent protein, enabling in vivo real-time whole-body fluorescence imaging, to follow the progression of Leishmania infection in parasitized tissues. Fluorescence correlated with the number of Leishmania parasites in the tissue and demonstrated the real-time efficacy of a therapeutic vaccine. This approach provides several substantial advantages over currently available animal model systems for the in vivo study of immunopathogenesis, prevention, and therapy of leishmaniasis. These include improvements in sensitivity and the ability to acquire real-time data on progression and spread of the infection.

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Expression of Green Fluorescent Protein as a Marker for Effects of Antileishmanial Compounds In Vitro

Cited by 42 publications

References 29 publications

Multifunctional Role of Human SPLUNC1 in Pseudomonas aeruginosa Infection

Multifunctional Role of Human SPLUNC1 in Pseudomonas aeruginosa Infection

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

Real-Time In Vivo Green Fluorescent Protein Imaging of a Murine Leishmaniasis Model as a New Tool for Leishmania Vaccine and Drug Discovery

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