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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

Representative optical microscope field from the May-Grünwald-Giemsa staining of the neutrophil-enriched preparation used for the degranulation experiments.
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pone-0057946-g007: Representative optical microscope field from the May-Grünwald-Giemsa staining of the neutrophil-enriched preparation used for the degranulation experiments.

Mentions: Neutrophils were isolated from a leukoreduction filter kindly provided by the French Blood Service (Bordeaux, France). The filter was back-flushed with 50 ml of DMEM (gibco) completed with BSA (40 g/l)(Sigma), Citrate-dextrose solution (10% vol/vol) (Sigma) and Pulmozyme, a dornase alpha commercial solution (10 µl/ml) at pH 7.4. The cells were diluted to 160 ml with the same solution and spun 20 mn at 110 g and 20°C in four tubes. The pellets were re-suspended in 90 ml of DMEM with dornase, layered on six tubes containing 15 ml of Granulosep (Eurobio) and spun 20 minutes at 1500 g and 20°C. The interface containing the white cells was harvested and washed in two times 50 ml of DMEM with dornase. Each pellet was resuspended in 20 ml DMEM with dornase, layered on 10 ml of Lymphocyte Separation Medium (Eurobio) and spun 40 mn at 400 g and 20°C. The pellet was highly enriched in granulocytes but still contained red cells and a few lymphocytes as seen in figure 7. It was harvested and washed with DMEM and was used as such in further experiments since neither red cells nor lymphocytes are able to release elastase. Neutrophils counting was carried out from a sample diluted in red cells lysing solution (Becton Dickinson) washed in phosphate buffer saline solution and analyzed on a Guava easyCyte flow cytometer/counter (Millipore). The neutrophil population was identified and counted according to its forward scatter versus side scatter properties.


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)

Representative optical microscope field from the May-Grünwald-Giemsa staining of the neutrophil-enriched preparation used for the degranulation experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057946-g007: Representative optical microscope field from the May-Grünwald-Giemsa staining of the neutrophil-enriched preparation used for the degranulation experiments.
Mentions: Neutrophils were isolated from a leukoreduction filter kindly provided by the French Blood Service (Bordeaux, France). The filter was back-flushed with 50 ml of DMEM (gibco) completed with BSA (40 g/l)(Sigma), Citrate-dextrose solution (10% vol/vol) (Sigma) and Pulmozyme, a dornase alpha commercial solution (10 µl/ml) at pH 7.4. The cells were diluted to 160 ml with the same solution and spun 20 mn at 110 g and 20°C in four tubes. The pellets were re-suspended in 90 ml of DMEM with dornase, layered on six tubes containing 15 ml of Granulosep (Eurobio) and spun 20 minutes at 1500 g and 20°C. The interface containing the white cells was harvested and washed in two times 50 ml of DMEM with dornase. Each pellet was resuspended in 20 ml DMEM with dornase, layered on 10 ml of Lymphocyte Separation Medium (Eurobio) and spun 40 mn at 400 g and 20°C. The pellet was highly enriched in granulocytes but still contained red cells and a few lymphocytes as seen in figure 7. It was harvested and washed with DMEM and was used as such in further experiments since neither red cells nor lymphocytes are able to release elastase. Neutrophils counting was carried out from a sample diluted in red cells lysing solution (Becton Dickinson) washed in phosphate buffer saline solution and analyzed on a Guava easyCyte flow cytometer/counter (Millipore). The neutrophil population was identified and counted according to its forward scatter versus side scatter properties.

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