SYTO-13, a Viability Marker as a New Tool to Monitor In Vitro Pharmacodynamic Parameters of Anti-Pneumocystis Drugs

Abstract

While Pneumocystis pneumonia (PcP) still impacts the AIDS patients, it has a growing importance in immunosuppressed HIV-negative patients. To determine the anti-Pneumocystis therapeutic efficacy of new compounds, animal and in vitro models have been developed. Indeed, well-designed mouse or rat experimental models of pneumocystosis can be used to describe the in vivo anti-Pneumocystis activity of new drugs. In vitro models, which enable the screening of a large panel of new molecules, have been developed using axenic cultures or co-culture with feeder cells; but no universally accepted standard method is currently available to evaluate anti-Pneumocystis molecules in vitro. Thus, we chose to explore the use of the SYTO-13 dye, as a new indicator of Pneumocystis viability. In the present work, we established the experimental conditions to define the in vitro pharmacodynamic parameters (EC50, Emax) of marketed compounds (trimethoprim/sulfamethoxazole, pentamidine, atovaquone) in order to specifically measure the intrinsic activity of these anti-P. carinii molecules using the SYTO-13 dye for the first time. Co-labelling the fungal organisms with anti-P. carinii specific antibodies enabled the measurement of viability of Pneumocystis organisms while excluding host debris from the analysis. Moreover, contrary to microscopic observation, large numbers of fungal cells can be analyzed by flow cytometry, thus increasing statistical significance and avoiding misreading during fastidious quantitation of stained organisms. In conclusion, the SYTO-13 dye allowed us to show a reproducible dose/effect relationship for the tested anti-Pneumocystis drugs.

Evaluation of Chemo‐ and Photo‐toxicity of a Live Fluorescent Dye for Cell Analysis

“…Since primary cells retain their endothelial characteristics for a limited number of passages in vitro, the natural choice for live cell studies is the use of chemical dyes that, once added to the cell culture, render the cell membrane, organelles or nucleus intensely fluorescent. A widely used compound for live cell staining is SYTO 13 (9,15,16). However, its viability in combination with endothelial cells has not yet been reported.…”

“…The method can be applied to calibrate the dye application in terms of optimal working concentration in protocols for staining and illumination. We present the case of SYTO 13, a commonly adopted chemical fluorophore (8,9), in combination with primary human endothelial cells (HUVECs). The motility behavior of these cells is relevant to study their response to flow and wall shear stress, subtending to their function in the maintenance of tissue homeostasis (10).…”

Using flow cytometry and light-induced fluorescence to characterize the variability and characteristics of bioaerosols in springtime in Metro Atlanta, Georgia

“…SYTO-13 is a highly permeable stain and effectively detects nucleic acids (DNA and RNA) of bacteria endospores and vegetative cells (Comas-Riu and Vives-Rego, 2002). Fungal spores have also been effectively stained by DNA/RNA probes (Bochdansky et al, 2016;Chen and Li et al, 2005), but some fungal spores might not be equally stained due to their harder cell wall and chromatin binding of DNA (Standaert-Vitse et al, 2015). Future work is needed to study this further.…”

“…Despite their low number concentration relative to abiotic particles, PBAP possess unique functional and compositional characteristics that differentiate them from abiotic aerosol. For example, certain PBAP constitute the most efficient of atmospheric ice nucleators, affecting the microphysics of mixed phase clouds and precipitation (Hoose and Möhler, 2012;Sullivan et al, 2018). The mass and nutrient content of PBAP may suffice to comprise an important supply of bioavailable phosphorous to oligotrophic marine ecosystems (Longo et al, 2014;Myriokefalitakis et al, 2016).…”