O2 Level Controls Hematopoietic Circulating Progenitor Cells Differentiation into Endothelial or Smooth Muscle Cells

BACKGROUND:Recent studies showed that progenitor cells could differentiate into mature vascular cells. The main physiological factors implicated in cell differentiation are specific growth factors. We hypothesized that simply by varying the oxygen content, progenitor cells can be differentiated either in mature endothelial cells (ECs) or contractile smooth muscle cells (SMCs) while keeping exactly the same culture medium. METHODOLOGY/PRINCIPAL FINDINGS:Mononuclear cells were isolated by density gradient were cultivated under hypoxic (5% O2) or normoxic (21% O2) environment. Differentiated cells characterization was performed by confocal microscopy examination and flow cytometry analyses. The phenotype stability over a longer time period was also performed. The morphological examination of the confluent obtained cells after several weeks (between 2 and 4 weeks) showed two distinct morphologies: cobblestone shape in normoxia and a spindle like shape in hypoxia. The cell characterization showed that cobblestone cells were positive to ECs markers while spindle like shape cells were positive to contractile SMCs markers. Moreover, after several further amplification (until 3(rd) passage) in hypoxic or normoxic conditions of the previously differentiated SMC, immunofluorescence studies showed that more than 80% cells continued to express SMCs markers whatever the cell environmental culture conditions with a higher contractile markers expression compared to control (aorta SMCs) signature of phenotype stability. CONCLUSION/SIGNIFICANCE:We demonstrate in this paper that in vitro culture of peripheral blood mononuclear cells with specific angiogenic growth factors under hypoxic conditions leads to SMCs differentiation into a contractile phenotype, signature of their physiological state. Moreover after amplification, the differentiated SMC did not reverse and keep their contractile phenotype after the 3rd passage performed under hypoxic and normoxic conditions. These aspects are of the highest importance for tissue engineering strategies. These results highlight also the determinant role of the tissue environment in the differentiation process of vascular progenitor cells

Evaluation of Virological Microparticle Enzyme Immunoassay According to the ISO 15189: Real-Life Experience in a University Hospital

International audienceThe International Standard ISO 15189 based on the ISO 9001:2008 emphasizes specific requirements for quality and ability of medical laboratories. The accreditation of medical laboratories according to ISO 15189 includes the validation of biological methods, which depends on collection of bibliographic data and experimental proofs. Moreover, these results must be compared to provider data sheets and independent scientific data. In the immunodiagnostic field, independent published data are deeply lacking. The aim of our work was to share experience of method validation for virological immune markers on the widely used Architect i2000sr

Interaction de l’hémoglobine octamérique recombinante avec les cellules endothéliales

International audienceHemoglobin-based oxygen carriers (HBOCs) may generate oxidative stress, vasoconstriction and inflammation. To reduce these undesirable vasoactive properties, we increased hemoglobin (Hb) molecular size by genetic engineering with octameric Hb, recombinant (r) HbβG83C. We investigate the potential side effects of rHbβG83C on endothelial cells. The rHbβG83C has no impact on cell viability, and induces a huge repression of endothelial nitric oxide synthase gene transcription, a marker of vasomotion. No induction of Intermolecular-Adhesion Molecule 1 and E-selectin (inflammatory markers) transcription was seen. In the presence of rHbβG83C, the transcription of heme oxygenase-1 (oxidative stress marker) is weakly increased compared to the two other HBOCs (references) or Voluven (control). This genetically engineered octameric Hb, based on a human Hb βG83C mutant, leads to little impact at the level of endothelial cell inflammatory response and thus appears as an interesting molecule for HBOC development.Les transporteurs d’oxygène à base d’hémoglobine (HBOCs) peuvent induire stress oxydant, vasoconstriction et inflammation. Afin de réduire ces propriétés vasoactives indésirables, nous avons augmenté, par génie génétique, la taille moléculaire de l’hémoglobine (Hb) produisant une Hb octamérique recombinante (r), la rHbβG83C. La rHbβG83C n’a pas d’impact sur la viabilité des cellules endothéliales et induit une répression très importante de la transcription du gène de la NO synthase (marqueur de vasoactivité). Aucune induction de la transcription de gènes des molécules d’adhésion ICAM-1 et E-sélectine (marqueurs d’inflammation) n’a été mise en évidence. En présence de rHbβG83C, la transcription du gène de l’hème oxygénase-I (marqueur de stress oxydant) est faiblement augmentée en comparaison du cas des deux autres HBOCs références et au Voluven (témoin). La rHbβG83C, basée sur un mutant de l’Hb humaine, présente moins d’impact au niveau de la réponse inflammatoire des cellules endothéliales et semble donc être une molécule intéressante pour le développement d’un HBOC

Severe acute respiratory coronavirus virus 2 (SARS-CoV-2) seroprevalence among laboratory staff: Safe handling of coronavirus disease 2019 (COVID-19) samples

International audienceNo abstract availabl

The importance of the effect of shear stress on endothelial cells in determining the performance of hemoglobin based oxygen carriers

The lack of blood donations and the threat of infections from blood and blood products have led to extensive research into the development of blood substitutes. The latest generation of hemoglobin based oxygen carriers (HBOC) has been shown to induce side effects like hypertension, vasoconstriction, inflammation and oxidative stress. HBOC are able to restore volemia and transport oxygen after a hemorrhagic shock, the reperfusion leading to the restoration of the blood flow in vessels. We propose an innovative approach, more closely emulating clinical situations, to assess the impact of HBOC perfusion on endothelial cells (EC) in vitro. Through this approach we quantified levels of oxidative stress, vasoactive factors and inflammation. EC were cultivated under a laminar flow to reproduce the return of shear stress (SS) during the reperfusion. We showed that heme oxygenase I transcription correlated with changes in oxidatively modified heme and methemoglobin; all were lower under SS. SS induced increased nitric oxide production, which may have implications for the mechanism of in vivo vasoconstriction and hypertension. E-selectin changes under SS were greater than those of ICAM-1. Our results demonstrate how it is essential to include SS in assays attempting to understand the potential vascular side effects of HBOC perfusion. © 2008 Elsevier Ltd. All rights reserved

Extracellular matrix synthesis.

<p>The confocal microscopy observations of Extracellular matrix protein: laminin (A, B) and type IV collagen (C, D) of cells seeded on both coated surfaces (type I collagen and Polyelectrolyte Multilayer films (PEMs)). Objective×40, NA = 0.8, scale bars 75 µm.</p

Anti-HBs Cellular Immune Response in Kidney Recipients before and 4 Months after Transplantation

Patients with renal failure represent a population at risk for hepatitis B, since only 50 to 60% of them develop protective humoral responses after vaccination. As this could be due to an altered regulation of cellular immune responses, the objectives of the present study were to evaluate the proliferative abilities of lymphocytes from patients with chronic renal failure after stimulation in vitro with a mitogen (pokeweed mitogen [PWM]) or HBsAg. In order to differentiate between the immunodeficiency associated with renal failure and that due to immunosuppression posttransplantation, the same subjects were tested before and 4 months after kidney transplantation. The lymphoproliferation assay used was performed by flow cytometry, which is based on sequential analysis of the cell cycle and which allows analysis of cytokine production. Serologically, the group of 36 patients tested comprised 22% nonresponders, 30% poor responders, and 48% responders. Lymphocyte growth was observed for all patients after stimulation with PWM, indicating that these cells had the capacity to proliferate in vitro. The level of lymphoproliferation in response to PWM was significantly reduced after transplantation, yet both before and after transplantation, all serologic nonresponders developed cellular responses to at least two vaccines. No correlation between humoral and cellular responses was shown. Proliferating cells were lymphocytes, which mostly secreted interleukin 4 (IL-4) and IL-10 for the three serologic groups. This study suggests that even when repeated vaccination fails to induce significant antibody levels in patients with renal failure, specific HBs cellular responses develop, and these may prove to be efficient in protecting these patients against hepatitis B