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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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OMRI detection of neutrophil degranulation in the presence of nitroxide-labeled elastin (1 mM equivalent nitroxide concentration).(a) OMRI intensity from 11×104 resting (black circles) or activated (red squares) neutrophils in 0.5 ml versus time of incubation at 37 °C. (b) OMRI intensity at five hours incubation at 37°C versus the number of resting (black circles) or activated (red squares) neutrophils. In (c) the images corresponding to plot (b) are displayed. Gradient Echo (Fast Low Angle SHot) trans-axial images were acquired with the following parameters: TR: 300 ms, TE: 10 ms, RF nutation angle: 70 degrees, Field of View (FOV): 22 mm*22 mm, acquisition matrix: 64*64, in plane resolution: 0.34 mm*0.34 mm, slice thickness: 3 mm, 2 averages. OMRI HF irradiation was applied for 260 ms out of 300 ms TR.
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pone-0057946-g005: OMRI detection of neutrophil degranulation in the presence of nitroxide-labeled elastin (1 mM equivalent nitroxide concentration).(a) OMRI intensity from 11×104 resting (black circles) or activated (red squares) neutrophils in 0.5 ml versus time of incubation at 37 °C. (b) OMRI intensity at five hours incubation at 37°C versus the number of resting (black circles) or activated (red squares) neutrophils. In (c) the images corresponding to plot (b) are displayed. Gradient Echo (Fast Low Angle SHot) trans-axial images were acquired with the following parameters: TR: 300 ms, TE: 10 ms, RF nutation angle: 70 degrees, Field of View (FOV): 22 mm*22 mm, acquisition matrix: 64*64, in plane resolution: 0.34 mm*0.34 mm, slice thickness: 3 mm, 2 averages. OMRI HF irradiation was applied for 260 ms out of 300 ms TR.

Mentions: The potential capabilities of this substrate for imaging was probed using OMRI with the same neutrophil containing samples as in the previous EPR study. The kinetics shown in Figure 5a highlights a strong increase of the image intensity in the presence of the calcium ionophore, to be compared with the slow and moderate increase measured in the absence of ionophore. In figure 5b the image intensity versus the number of neutrophils undergoes a significantly steeper increase in the presence of calcium ionophore. In comparison to the EPR signal intensity curves OMRI intensity curves show less linearity at low enzymatic activity points which appear to be overestimated. This feature would be favorable to in vivo OMRI since low proteolytic activities would have a better chance to be detected.


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)

OMRI detection of neutrophil degranulation in the presence of nitroxide-labeled elastin (1 mM equivalent nitroxide concentration).(a) OMRI intensity from 11×104 resting (black circles) or activated (red squares) neutrophils in 0.5 ml versus time of incubation at 37 °C. (b) OMRI intensity at five hours incubation at 37°C versus the number of resting (black circles) or activated (red squares) neutrophils. In (c) the images corresponding to plot (b) are displayed. Gradient Echo (Fast Low Angle SHot) trans-axial images were acquired with the following parameters: TR: 300 ms, TE: 10 ms, RF nutation angle: 70 degrees, Field of View (FOV): 22 mm*22 mm, acquisition matrix: 64*64, in plane resolution: 0.34 mm*0.34 mm, slice thickness: 3 mm, 2 averages. OMRI HF irradiation was applied for 260 ms out of 300 ms TR.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585236&req=5

pone-0057946-g005: OMRI detection of neutrophil degranulation in the presence of nitroxide-labeled elastin (1 mM equivalent nitroxide concentration).(a) OMRI intensity from 11×104 resting (black circles) or activated (red squares) neutrophils in 0.5 ml versus time of incubation at 37 °C. (b) OMRI intensity at five hours incubation at 37°C versus the number of resting (black circles) or activated (red squares) neutrophils. In (c) the images corresponding to plot (b) are displayed. Gradient Echo (Fast Low Angle SHot) trans-axial images were acquired with the following parameters: TR: 300 ms, TE: 10 ms, RF nutation angle: 70 degrees, Field of View (FOV): 22 mm*22 mm, acquisition matrix: 64*64, in plane resolution: 0.34 mm*0.34 mm, slice thickness: 3 mm, 2 averages. OMRI HF irradiation was applied for 260 ms out of 300 ms TR.
Mentions: The potential capabilities of this substrate for imaging was probed using OMRI with the same neutrophil containing samples as in the previous EPR study. The kinetics shown in Figure 5a highlights a strong increase of the image intensity in the presence of the calcium ionophore, to be compared with the slow and moderate increase measured in the absence of ionophore. In figure 5b the image intensity versus the number of neutrophils undergoes a significantly steeper increase in the presence of calcium ionophore. In comparison to the EPR signal intensity curves OMRI intensity curves show less linearity at low enzymatic activity points which appear to be overestimated. This feature would be favorable to in vivo OMRI since low proteolytic activities would have a better chance to be detected.

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