The aim of the study is to evaluate the role of the endothelial barrier in the occurrence of stroke-like episode. And to study the protein C system on the surface of the endothelium.
The study consists in isolating endothelial progenitor cells in peripheral blood. After blood puncture, PBMC (peripheral blood mononuclear cell) are isolated by Ficoll gradient and then culturing in appropriated medium.
When cells are confluent, investigators can study the endothelial barrier by using permeability assay and transendothelial resistance test (iCELLigence technology).
Then, protein C system will be studied. Indeed, EPCR (Endothelial protein C receptor) and TM (thrombomodulin), are the two receptors responsible for the activation of protein C. The expression of these receptors will be assessed in flow cytometry. The activated protein C is able to interact with the third receptor PAR1 (Protease activated receptor 1) which is responsible for the protective effects of the on the endothelial barrier. The PAR1 expression will be studied in flow cytometry also.
In order to measure the capacity of the protein C system to generate activated protein C investigators will assay an activated protein C generation on the cell surface.
All these assays will be realized in basal condition and in heat stress (42°C), to mimic the stroke-like episode environment. Indeed, stroke-like episodes occur always during febrile illness.
- Peripheral blood puncture Biological
Intervention Desc: Endothelial progenitor cells will be isolated from peripheral blood by density gradient and cultured in 10%FBS EGM2 medium On the EPCs we will study: the endothelial permeability the protein C system ARM 1: Kind: Experimental Label: CDG with stroke-like history ARM 2: Kind: Experimental Label: CDG without stroke-like history
|Type||Measure||Time Frame||Safety Issue|
|Primary||Endothelial permeability assay in basal condition||at inclusion|
|Primary||Endothelial permeability assay after an heat shock||at inclusion|
|Secondary||Activated protein C generation assay on the endothelial cell surface||at inclusion|
|Secondary||Quantification of the endothelial receptors (EPCR, TM, PAR1)||at inclusion|
|Secondary||Endothelial permeability assay after an activated protein C incubation||at inclusion|