NoninvasiveIn VivoQuantification of Neutrophil Elastase Activity in Acute Experimental Mouse Lung Injury

Abstract

We developed a neutrophil elastase-specific near-infrared fluorescence imaging agent, which, combined with fluorescence molecular tomographic imaging, allowed us to detect and quantify neutrophil elastase activity in vivo, in real time, and noninvasively in an acute model of lung injury (ALI). Significantly higher fluorescent signal was quantified in mice with LPS/fMLP-induced ALI as compared to healthy controls, correlating with increases in the number of bronchoalveolar lavage cells, neutrophils, and elastase activity. The agent was significantly activated ex vivo in lung sections from ALI but not from control mice, and this activation was ablated by the specific inhibitor sivelestat. Treatment with the specific inhibitor sivelestat significantly reduced lung signal in mice with ALI. These results underscore the unique ability of fluorescence molecular imaging to quantify specific molecular processes in vivo, crucial for understanding the mechanisms underlying disease progression and for assessing and monitoring novel pharmacological interventions.

Highly Sensitive Real-Time In Vivo Imaging of an Influenza Reporter Virus Reveals Dynamics of Replication and Spread

“…For example, previous experiments monitoring inflammatory responses to influenza virus infection via PET imaging could be coupled with PASTN to determine the impact of the viral load and the site of replication on the severity of the response (55). Similarly, recruitment of discrete immune cell populations to sites of infection can be monitored by infecting mice with PASTN and detecting immune cells that have been labeled ex vivo with tracking dyes (56) or by detecting fluorogenic probes that are selectively activated by specific immune cell populations (57). Alternatively, innate immune responses and viral replication could be measured concurrently by using PASTN to infect reporter mice that drive firefly luciferase from an NF-B or IFN-␤ promoter(s), as NLuc and firefly luciferase exclusively use different substrates (29,58,59).…”

Enzyme-activatable imaging probe reveals enhanced neutrophil elastase activity in tumors following photodynamic therapy

“…Briefly, the NE selective probe consists of two NIR fluorophores (VivoTag-S680, PerkinElmer) that are linked to both the Cand N-termini of the peptide PMAVVQSVP, a highly NE-selective sequence. 22 The two fluorophores are placed in close enough proximity to each other for efficient quenching of fluorescence but become fluorescent upon cleavage of the substrate linker sequence. The linker sequence in the construct is designed to be preferentially cleaved by NE while remaining resistant to other proteases including mouse PR3.…”

“…The linker sequence in the construct is designed to be preferentially cleaved by NE while remaining resistant to other proteases including mouse PR3. 22 This is important as both PR3 and NE are abundant serine proteases with potentially overlapping substrates.…”

“…22,23 SCC VII and EMT6 tumors in C3H and BALB/c mice, respectively, were subjected to Photofrin-PDT and given 50 μl of 2 mg∕ml Sivelestat via direct intratumor injection immediately after irradiation and 15 min prior to NE680 probe administration.…”

Fluoromodule-based reporter/probes designed for in vivo fluorescence imaging

“…Signals from circulating probe can undesirably degrade contrast between the target and its surroundings. A powerful strategy for minimizing this source of background is to use activatable probes that produce signals only in response to specific cues such as low pH (8,9), endogenous enzyme activity (10,11), or interaction with reporter gene products. Luciferins, for example, generate light only after luciferase-mediated oxidation, providing exceptional contrast between reporter activity and circulating probe (12).…”