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Evaluation of small molecule FRET reporter for the diagnosis and monitoring of proteolytic activity in a chronic obstructive lung disease model

: Reither, G.; Heath, N.; Holz, Olaf; Kahn, N.; Hohlfeld, Jens Michael; Schulz, C.; Mall, M.A.


Pneumologie 68 (2014), No.6, Art. A14
ISSN: 0934-8387
International Symposium "Lung Regeneration and Beyond - BREATH meets REBIRTH" <3, 2014, Hannover>
Fraunhofer ITEM ()

Proteases such as neutrophil elastase (NE) and matrix metalloprotease 12 (MMP-12) are key factors in inflammatory processes and contribute the gradual destruction of extracellular lung matrix in chronic inflammation. We now hypothesize that activity levels of inflammation-relevant proteases may be useful indicators for the onset and progression of obstructive lung diseases such as cystic fibrosis or COPD. With the recently published small molecule FRET protease reporters NEmo and LaRee, for detection of NE and MMP-12 activity, respectively, it is possible to monitor protease activity at the single cell level.
The goal of this study is to apply protease activity measurements to sputum specimens from patients with chronic obstructive lung diseases. We investigated the impact of sample generation and experimental conditions on the performance and outcome of diagnostic protease monitoring in sputum samples in general. We tested the new approach on a double blinded sample set from the Fraunhofer ITEM Hannover (BREATH). In this study healthy subjects were exposed to irritating conditions or control air in an incubation room. Sputum was produced and processed by standard operation procedure. Through comparison of by-hand analysis, we succeeded in receiving satisfactory data sets via automated cell analysis of hundreds of cells per sample using novel macros. This assay format is now expanded to employ additional small molecule protease probes for cathepsin B and S.
Our novel approach of the assessment of protease activity at the single cell level applied to multiple protease types may result in a new tool for diagnosis and monitoring. The detection of patient specific protease activity patterns may improve the differentiated diagnosis and therapeutic strategies.