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Overhauser-enhanced MRI of elastase activity from in vitro human neutrophil degranulation.

Parzy E, Bouchaud V, Massot P, Voisin P, Koonjoo N, Moncelet D, Franconi JM, Thiaudière E, Mellet P - PLoS ONE (2013)

Bottom Line: However several diseases would benefit from an imaging technique able to specifically detect biochemical alterations.The sensitivity is sufficient to generate contrasted images of the degranulation of neutrophils induced by a calcium ionophore from 2×10(4) cells per milliliter, well under the physiological neutrophils concentrations.It also provides a long-expected method to monitor anti-protease treatments efficiency and help pharmaceutical research.

View Article: PubMed Central - PubMed

Affiliation: CRMSB, UMR 5536, University Bordeaux Segalen, CNRS, Bordeaux, France.

ABSTRACT

Background: Magnetic resonance imaging can reveal exquisite anatomical details. However several diseases would benefit from an imaging technique able to specifically detect biochemical alterations. In this context protease activity imaging is one of the most promising areas of research.

Methodology/principal findings: We designed an elastase substrate by grafting stable nitroxide free radicals on soluble elastin. This substrate generates a high Overhauser magnetic resonance imaging (OMRI) contrast upon digestion by the target proteases through the modulation of its rotational correlation time. The sensitivity is sufficient to generate contrasted images of the degranulation of neutrophils induced by a calcium ionophore from 2×10(4) cells per milliliter, well under the physiological neutrophils concentrations.

Conclusions/significance: These ex-vivo experiments give evidence that OMRI is suitable for imaging elastase activity from neutrophil degranulation. Provided that a fast protease-substrate is used these results open the door to better diagnoses of a number of important pathologies (cystic fibrosis, inflammation, pancreatitis) by OMRI or Electron Paramagnetic Resonance Imaging in vivo. It also provides a long-expected method to monitor anti-protease treatments efficiency and help pharmaceutical research.

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Related in: MedlinePlus

Comparative kinetics of hydrolysis of the elastin substrate by human neutrophil elastase (black squares), porcine pancreatic elastase (red circles), chymotrypsin (blue triangles) and trypsin (pink triangles) probed by EPR.0.36 mM equivalent nitroxide substrate was incubated at 37°C with 50 nM of each enzyme.
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pone-0057946-g002: Comparative kinetics of hydrolysis of the elastin substrate by human neutrophil elastase (black squares), porcine pancreatic elastase (red circles), chymotrypsin (blue triangles) and trypsin (pink triangles) probed by EPR.0.36 mM equivalent nitroxide substrate was incubated at 37°C with 50 nM of each enzyme.

Mentions: The ability of nitroxide-labeled elastin to generate an EPR signal upon digestion by human neutrophil elastase (HNE) was probed with increasing concentrations of protease. The peak to peak height of the central line of the nitroxide was measured at several incubation times for each concentration. As shown in figure 1, one hour incubation easily discriminates HNE concentrations between 5 and 50 nM. As seen in the inset increasing EPR signal is still detectable in the 0.5 to 5 nM range. Thus EPR detection shows that nitroxide-labeled elastin is a sensitive elastase substrate. The most remarkable property of elastases is their unique capability to release soluble peptides from insoluble elastin[8]. This is due in part to their P1 specificity in the Schechter and Berger nomenclature[16] for valine and alanine which, respectively, constitute 13% and 26% of the amino acid composition of elastin. But the most selective feature is their ability to bind to the fibrous structure of elastin. Since our labeled elastin was made from a solubilized form it was interesting to search whether it has kept some of its elastase substrate specificity. Figure 2 shows the kinetics of hydrolysis by 50 nM human neutrophil elastase, porcine pancreatic elastase, bovine trypsin and chymotrypsin. Nitroxide-labeled elastin retains a good selectivity for both elastases while trypsin is nearly inactive. The slight activity of chymotrypsin is probably due to the chemical denaturation of soluble elastin which unveiled newly accessible cleavage sites.


Overhauser-enhanced MRI of elastase activity from in vitro human neutrophil degranulation.

Parzy E, Bouchaud V, Massot P, Voisin P, Koonjoo N, Moncelet D, Franconi JM, Thiaudière E, Mellet P - PLoS ONE (2013)

Comparative kinetics of hydrolysis of the elastin substrate by human neutrophil elastase (black squares), porcine pancreatic elastase (red circles), chymotrypsin (blue triangles) and trypsin (pink triangles) probed by EPR.0.36 mM equivalent nitroxide substrate was incubated at 37°C with 50 nM of each enzyme.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585236&req=5

pone-0057946-g002: Comparative kinetics of hydrolysis of the elastin substrate by human neutrophil elastase (black squares), porcine pancreatic elastase (red circles), chymotrypsin (blue triangles) and trypsin (pink triangles) probed by EPR.0.36 mM equivalent nitroxide substrate was incubated at 37°C with 50 nM of each enzyme.
Mentions: The ability of nitroxide-labeled elastin to generate an EPR signal upon digestion by human neutrophil elastase (HNE) was probed with increasing concentrations of protease. The peak to peak height of the central line of the nitroxide was measured at several incubation times for each concentration. As shown in figure 1, one hour incubation easily discriminates HNE concentrations between 5 and 50 nM. As seen in the inset increasing EPR signal is still detectable in the 0.5 to 5 nM range. Thus EPR detection shows that nitroxide-labeled elastin is a sensitive elastase substrate. The most remarkable property of elastases is their unique capability to release soluble peptides from insoluble elastin[8]. This is due in part to their P1 specificity in the Schechter and Berger nomenclature[16] for valine and alanine which, respectively, constitute 13% and 26% of the amino acid composition of elastin. But the most selective feature is their ability to bind to the fibrous structure of elastin. Since our labeled elastin was made from a solubilized form it was interesting to search whether it has kept some of its elastase substrate specificity. Figure 2 shows the kinetics of hydrolysis by 50 nM human neutrophil elastase, porcine pancreatic elastase, bovine trypsin and chymotrypsin. Nitroxide-labeled elastin retains a good selectivity for both elastases while trypsin is nearly inactive. The slight activity of chymotrypsin is probably due to the chemical denaturation of soluble elastin which unveiled newly accessible cleavage sites.

Bottom Line: However several diseases would benefit from an imaging technique able to specifically detect biochemical alterations.The sensitivity is sufficient to generate contrasted images of the degranulation of neutrophils induced by a calcium ionophore from 2×10(4) cells per milliliter, well under the physiological neutrophils concentrations.It also provides a long-expected method to monitor anti-protease treatments efficiency and help pharmaceutical research.

View Article: PubMed Central - PubMed

Affiliation: CRMSB, UMR 5536, University Bordeaux Segalen, CNRS, Bordeaux, France.

ABSTRACT

Background: Magnetic resonance imaging can reveal exquisite anatomical details. However several diseases would benefit from an imaging technique able to specifically detect biochemical alterations. In this context protease activity imaging is one of the most promising areas of research.

Methodology/principal findings: We designed an elastase substrate by grafting stable nitroxide free radicals on soluble elastin. This substrate generates a high Overhauser magnetic resonance imaging (OMRI) contrast upon digestion by the target proteases through the modulation of its rotational correlation time. The sensitivity is sufficient to generate contrasted images of the degranulation of neutrophils induced by a calcium ionophore from 2×10(4) cells per milliliter, well under the physiological neutrophils concentrations.

Conclusions/significance: These ex-vivo experiments give evidence that OMRI is suitable for imaging elastase activity from neutrophil degranulation. Provided that a fast protease-substrate is used these results open the door to better diagnoses of a number of important pathologies (cystic fibrosis, inflammation, pancreatitis) by OMRI or Electron Paramagnetic Resonance Imaging in vivo. It also provides a long-expected method to monitor anti-protease treatments efficiency and help pharmaceutical research.

Show MeSH
Related in: MedlinePlus