Dataset Information

Cathepsin G Activity as a New Marker for Detecting Airway Inflammation by Microscopy and Flow Cytometry.

ABSTRACT: Muco-obstructive lung diseases feature extensive bronchiectasis due to the uncontrolled release of neutrophil serine proteases into the airways. To assess if cathepsin G (CG) is a novel key player in chronic lung inflammation, we developed membrane-bound (mSAM) and soluble (sSAM) FRET reporters. The probes quantitatively revealed elevated CG activity in samples from 46 patients. For future basic science and personalized clinical applications, we developed a rapid, highly informative, and easily translatable small-molecule FRET flow cytometry assay for monitoring protease activity including cathepsin G. We demonstrated that mSAM distinguished healthy from patient cells by FRET-based flow cytometry with excellent correlation to confocal microscopy data.

SUBMITTER: Guerra M

PROVIDER: S-EPMC6439450 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Cathepsin G Activity as a New Marker for Detecting Airway Inflammation by Microscopy and Flow Cytometry.

Guerra Matteo M Frey Dario D Hagner Matthias M Dittrich Susanne S Paulsen Michelle M Mall Marcus A MA Schultz Carsten C

ACS central science 20190219 3

Muco-obstructive lung diseases feature extensive bronchiectasis due to the uncontrolled release of neutrophil serine proteases into the airways. To assess if cathepsin G (CG) is a novel key player in chronic lung inflammation, we developed membrane-bound (mSAM) and soluble (sSAM) FRET reporters. The probes quantitatively revealed elevated CG activity in samples from 46 patients. For future basic science and personalized clinical applications, we developed a rapid, highly informative, and easily ...[more]

PMID: 30937381

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Project description:This study compared the relative cellular uptake of 80 nm silver nanoparticles (AgNP) with four different coatings including: branched polyethyleneimine (bPEI), citrate (CIT), polyvinylpyrrolidone (PVP), and polyethylene glycol (PEG). A gold nanoparticle PVP was also compared to the silver nanoparticles. Biophysical parameters of cellular uptake and effects included flow cytometry side scatter (SSC) intensity, nuclear light scatter, cell cycle distributions, surface plasmonic resonance (SPR), fluorescence microscopy of mitochondrial gross structure, and darkfield hyperspectral imaging. The AgNP-bPEI were positively charged and entered cells at a higher rate than the negatively or neutrally charged particles. The AgNP-bPEI were toxic to the cells at lower doses than the other coatings which resulted in mitochondria being transformed from a normal string-like appearance to small round beaded structures. Hyperspectral imaging showed that AgNP-bPEI and AgNP-CIT agglomerated in the cells and on the slides, which was evident by longer spectral wavelengths of scattered light compared to AgNP-PEG and AgNP-PVP particles. In unfixed cells, AgNP-CIT and AgNP-bPEI had higher SPR than either AgNP-PEG or AgNP-PVP particles, presumably due to greater intracellular agglomeration. After 24 hr. incubation with AgNP-bPEI, there was a dose-dependent decrease in the G1 phase and an increase in the G2/M and S phases of the cell cycle suggestive of cell cycle inhibition. The nuclei of all the AgNP treated cells showed a dose-dependent increase in nanoparticles following non-ionic detergent treatment in which the nuclei retained extra-nuclear AgNP, suggesting that nanoparticles were attached to the nuclei or cytoplasm and not removed by detergent lysis. In summary, positively charged AgNP-bPEI increased particle cellular uptake. Particles agglomerated in the peri-nuclear region, increased mitochondrial toxicity, disturbed the cell cycle, and caused abnormal adherence of extranuclear material to the nucleus after detergent lysis of cells. These results illustrate the importance of nanoparticle surface coatings and charge in determining potentially toxic cellular interactions.

Dataset Information

Cathepsin G Activity as a New Marker for Detecting Airway Inflammation by Microscopy and Flow Cytometry.

Publications

Cathepsin G Activity as a New Marker for Detecting Airway Inflammation by Microscopy and Flow Cytometry.

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