Biomedical subjects

Jean-François Stoltz

Publications and source records attributed to Jean-François Stoltz.

At least 19 recordsLinked to original sources

PubMed2007

Efficient generation of CD34+ progenitor-derived dendritic cells from G-CSF-mobilized peripheral mononuclear cells does not require hematopoietic stem cell enrichment.

As a result of their potent antigen-presentation function, dendritic cells (DC) are important tools for cell therapy programs. In vitro-generated DC from enriched CD34+ hematopoietic stem cells (HSC; enriched CD34 DC) have already proven their efficiency in Phase I/II clinical trials. Here, we investigated whether enrichment of CD34+ HSC before the onset of culture was absolutely required for their differentiation into DC. With this aim, we developed a new two-step culture method. PBMC harvested from G-CSF-mobilized, healthy patients were expanded for 7 days during the first step, with early acting cytokines, such as stem cell factor, fetal liver tyrosine kinase 3 ligand (Flt-3L), and thrombopoietin. During the second step, expanded cells were then induced to differentiate into mature DC in the presence of GM-CSF, Flt-3L, and TNF-alpha for 8 days, followed by LPS exposure for 2 additional days. Our results showed that the rate of CD34+/CD38+/lineageneg cells increased 19.5+/-10-fold (mean+/-sd) during the first step, and the expression of CD14, CD1a, CD86, CD80, and CD83 molecules was up-regulated markedly following the second step. When compared with DC generated from enriched CD34+ cells, which were expanded for 7 days before differentiation, DC derived from nonenriched peripheral blood stem cells showed a similar phenotye but higher yields of production. Accordingly, the allogeneic stimulatory capacity of the two-step-cultured DC was as at least as efficient as that of enriched CD34 DC. In conclusion, we report herein a new two-step culture method that leads to high yields of mature DC without any need of CD34+ HSC enrichment.

Antigens, CD34↗

PubMed2006

A simple method for quantifying high density antigens in erythrocyte membrane by flow cytometry.

RBC flow cytometric analysis is usually used to quantify antigen content. Calibration systems enable antigen content determination by relating mean fluorescence intensity with the number of bound antibody molecules (equivalent to the number of antigen molecules). For that reason, antibodies must be used at saturating concentration, which may lead to agglutination when working with high density antigens. Then, forward scattering, side scattering and fluorescence will be increased, thus obtaining wrong results. In this work, the simple Langmuir adhesion model was applied. Flow cytometry was used to quantify GPA, a transmembrane protein present at high density on RBC. The fluorescence intensity of samples at different anti-GPA sub-saturating concentrations was measured. Sometimes, agglutinates were present and two peaks of fluorescence were observed, the principal one corresponding to isolated cells and the secondary one corresponding to agglutinated cells. In those cases, the principal peak was taken into account for the analysis. The GPA antigen content obtained for nine analyzed samples ranged from 3 to 13 x 10(5) sites per cell, which is similar to those values found in literature. Therefore, the Langmuir adsorption model enables us to determine the antigen content for the anti-GPA/GPA system on RBC membrane. This model could be used to quantify high density antigens in RBC and in other cells.

Antibodies, Monoclonal↗

PubMed2006

Effects of a new perfluorocarbon emulsion on human plasma and whole-blood viscosity in the presence of albumin, hydroxyethyl starch, or modified fluid gelatin: an in vitro rheologic approach.

BACKGROUND: Artificial oxygen carriers such as perfluorocarbon (PFC) emulsions have reached Phase III clinical trials as alternatives to homologous blood, but their rheologic effects have not been characterized. In this study, the rheologic effects of PFC emulsion in the presence of clinically used volume expanders were investigated. STUDY DESIGN AND METHODS: The effects of a new PFC emulsion (small droplet size with narrow size distribution) at two PFC concentrations (4 and 8 g/dL) on plasma and whole-blood viscosity in the presence of human albumin solution (HAS), hydroxyethyl starch (HES), or modified fluid gelatin (MFG) were investigated. Three hematocrit (Hct) levels were investigated: 30, 20, and 13 percent. Plasma, PFC emulsions, and whole-blood viscosity, with a Couette viscometer, and RBC elongation, with an ektacytometer, were measured for shear rates of 0.2 to 128 per second. RESULTS: The two PFC concentrations increased plasma and whole-blood viscosities. Viscosity values similar to physiologic ones (Hct level, 40%) were observed at: 1) Hct level of 13 percent, with 4 or 8 g per dL MFG-PFC; 2) Hct level of 20 percent, with 4 g per dL MFG-PFC; and 3) Hct level of 30 percent, with 4 g per dL HES-PFC and 4 and 8 g per dL HAS-PFC. RBC deformability was unchanged. CONCLUSION: It is concluded that this new PFC emulsion increases plasma and blood viscosity and that among the three studied volume expanders, the interaction with MFG can result in viscosity values above the physiologic one even at low Hct values. The possible consequences of the increased viscosity at low Hct values are discussed.

Albumins↗

PubMed2006

Short-term heart retention and distribution of intramyocardial delivered mesenchymal cells within necrotic or intact myocardium.

Cell therapy with bone marrow mesenchymal stem cells (BMSCs) is a new strategy for treating ischemic heart failure, but data concerning the distribution and retention of transplanted cells remain poor. We investigated the short-term myocardial retention of BMSCs when these cells are directly injected within necrotic or intact myocardium. 111Indium-oxine-labeled autologous BMSCs were injected within either 1-month-old infarction (n = 6) or normal myocardium (n = 6) from rats. Serial in vivo pinhole scintigraphy was scheduled during 1 week in order to track the implanted cells. The myocardial retention of BMSCs was definitely higher in myocardial infarction than in normal myocardial area (estimated percent retention at 2 h: 63 +/- 3% vs. 25 +/- 4%, p < 0.001) and the estimated cardiac retention values were unchanged in both groups along the 7 days of follow-up. On heart sections at day 7, labeled BMSCs were still around the injection site and appeared confined to the scarred tissue corresponding either to the infarct area or to the myocardium damaged by needle insertion. BMSCs have a higher retention when they are injected in necrotic than in normal myocardial areas and these cells appear to stay around the injection site for at least a 7-day period.

Animals↗

PubMed2006

Effect of cyclic stretching and foetal bovine serum (FBS) on proliferation and extra cellular matrix synthesis of fibroblast.

It is well known today that mechanical forces are one of the important factors that induce a variety of cellular responses including morphological changes, protein synthesis, and gene expression and which are involve in tissue remodelling. We studied the effect of uniaxial cyclic stretching on the proliferation, collagens, and tenascin C mRNA expression of fibroblasts under different concentrations of foetal bovine serum. Proliferation was studied by cell cycle analysis, mRNA expression of collagen and tenascin C was studied by RT-PCR. Human fibroblasts were grown in silicon sheet coated with 1% gelatin. Cyclic stretching (5% elongation) was applied at 0.5 Hz (30 cycle/min), for 24 h with two concentrations of the serum (0.5%, 10% FBS). We showed that stretching enhances the synthesis of collagen and tenascin C, but do not act on the proliferation. In contrast, higher concentration of serum enhances the proliferation. These findings suggest that both mechanical stretching and serum concentration can modulate proliferation and extra cellular matrix synthesis in human fibroblasts.

Animals↗

PubMed2006

The regulatory role of dendritic cells in the immune tolerance.

Immune homeostasis is important for the protection of a host from pathogen aggression, as well as for preventing autoimmunity. Dendritic cells (DCs), the most potent antigen presenting cells, are critical in innate, adaptive immunity and in central tolerance. Recently, their involvement in peripheral tolerance has been shown. Whether DCs induce immunity or tolerance depends on their state of maturation. Different subsets of tolerogenic DCs have been identified in vivo, either in physiological, or pathological conditions, such as tumors, or GVHD. Moreover, tolerogenic DCs can be generated in vitro, by using different culture conditions, such as IL-10 or TGF-beta. In our study, we obtained tolerogenic DCs, by culturing them in the presence of human mesenchymal stem cells (MSCs).

Animals↗

PubMed2006

Hydroxyethylstarch 200 and 240 differently affect aortic distensibility but not viscosity and blood pressure upon acute isovolumic hemodilution.

OBJECTIVES: It has been shown that a hydroxyethylstarch solution significantly increases the aortic distensibility coefficient (ADC) as compared to other non-hydroxyethylstarch colloid solutions. In order to investigate whether the effect of hydroxyethylstarch on ADC is class-specific, we investigated the effect of two hydroxyethylstarch solutions (HES 200: Elohes and HES 240: Hesteril) on the ADC and compared them with two other colloid solutions: 5% albumin and fluid gelatin (Gelofusin) in a rabbit model of acute isovolumic hemodilution. METHODS: Twenty-eight male New Zealand white rabbits were anesthetized and randomly allocated to receive (n=7, each): albumin, hydroxyethylstarch-200, hydroxyethylstarch-240 and gelatin for acute isovolumic hemodilution by exchanging 13 ml.kg(-1) body weight of blood with an identical volume of the test solution. Blood viscosity, mean arterial pressure, aortic blood flow and heart rate were measured and ADC was calculated. RESULTS: All groups were comparable with respect to arterial pressure, heart rate and aortic blood flow velocity before and after isovolumic hemodilution. After hemodilution, ADC coefficient remained unchanged as compared with pre-hemodilution values with albumin, hydroxyethylstarch-240 and gelatin, whereas a sustained 3 fold increase was observed with hydroxyethylstarch-200. CONCLUSION: These results demonstrate that minor physicochemical differences between two hydroxyethylstarch solutions result in measurable differences in ADC and suggest that the clinical effects of colloids should probably be analyzed for each type of colloid and not for classes of colloids (e.g. hydroxyethylstarch or fluid gelatins).

Animals↗

PubMed2006

Flow cytometry and spectral imaging multiphoton microscopy analysis of CD36 expression with quantum dots 605 of untreated and 7-ketocholesterol-treated human monocytic cells.

OBJECTIVE: To evaluate CD36 expression with quantum dots 605 (QDs 605) on untreated and 7-ketocholesterol (7KC)-treated monocytic U937 cells by flow cytometry (FCM) and confocal and multiphoton laser scanning microscopy (CLSM). STUDY DESIGN: Cells were analyzed by CLSM, following flow cytometric quantification of CD36 expression and 7KC uptake. Image sequences were obtained by spectral analysis in monophoton and multiphoton CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm to differentiate emission spectra. In CLSM analysis, cell deposits were screened in ultraviolet excitation modes to optimize the possibilities of QDs 605 and have the benefit of nuclei counterstaining by DAPI. RESULTS: FCM and CLSM reveal the expression of CD36 by means of QDs 605. FCM provides information on 7KC uptake. CLSM provides the localization of 7KC vs. DAPI. As factor curves and images show the red, narrow emission of QDs 605 vs. violet and blue emissions of 7KC and DAPI, respectively, a reliable identification of CD36 is obtained. CONCLUSION: QDs 605 are useful tools to perform antigenic expression in FCM and CLSM. Moreover, CLSM and subsequent spectral analysis provide a more specific characterization of QDs 605 fluorescent emission in the UV excitation mode and a simultaneous identification of 7KC.

Algorithms↗

PubMed2005

Polyelectrolyte multilayers and degradable polymer layers as multicompartment films.

Polyelectrolyte multilayers are now a well established concept with numerous potential applications in particular as biomaterial coatings. To timely control the biological activity of cells in contact with a substrate, multicompartment films made of different polyelectrolyte multilayers deposited sequentially on the solid substrate constitute a promising new approach. In a first paper (Langmuir 2004, 20, 7298) we showed that such multicompartment films can be designed by alternating exponentially growing polyelectrolyte multilayers acting as reservoirs and linearly growing ones acting as barriers. In the present study, we first demonstrate however that these barriers composed of synthetic polyelectrolytes are not degraded despite the presence of phagocytic cells. We propose an alternative approach where exponentially growing poly(L-lysine)/hyaluronic acid (PLL/HA) multilayers, used as reservoirs, are alternated with biodegradable polymer layers consisting in poly(lactic-co-glycolic acid) (PLGA) and acting as barriers for PLL chains that diffuse within the PLL/HA reservoirs. We first show that these PLGA layers can be deposited alternatively with PLL/HA multilayers leading to polyelectrolyte multilayer/hydrolyzable polymeric layer films and acting as a reservoirs/barriers system. Bone marrow cells seeded on these films ending by a PLL/HA reservoir rapidly degrade it and internalize the PLL chains confined in this reservoir. Then the cells degraded locally the PLGA barrier and internalize the PLL localized in a lower (PLL/HA) compartment after 5 days of seeding. By changing the thickness of the PLGA layer, we hope to be able to tune the time delay of degradation. Such mixed architectures made of polyelectrolyte multilayers and hydrolyzable polymeric layers could act as coatings allowing us to induce a time scheduled cascade of biological activities. We are currently working on the use of comparable films with compartments filled by proteins or peptides and in which the degradation of the barriers results from a hydrolysis over tunable time scales.

Journal Article↗

PubMed2005

7-Ketocholesterol favors lipid accumulation and colocalizes with Nile Red positive cytoplasmic structures formed during 7-ketocholesterol-induced apoptosis: analysis by flow cytometry, FRET biphoton spectral imaging microscopy, and subcellular fractionation.

BACKGROUND: Oxidized low-density lipoproteins play key roles in atherosclerosis. Their toxicity is at least in part due to 7-ketocholesterol (7KC), which is a potent inducer of apoptosis. In this study on human promonocytic U937 cells, we determined the effects and the interactions of 7KC with cellular lipids during 7KC-induced apoptosis. METHODS: Morphologic and functional changes were investigated by microscopic and flow cytometric methods after staining with propidium iodide, 3,3'-dihexyloxacarbocyanine iodide, and Hoechst 33342. Cellular lipid content was identified by using filipin to quantify free cholesterol and Nile Red (NR), which emit a yellow or orange-red fluorescence in the presence of neutral and polar lipids, respectively. After staining with NR, interactions of 7KC with cellular lipids were identified by fluorescence resonance energy transfer biphoton spectral imaging confocal microscopy and by subcellular fractionation, gas chromatography, and mass spectrometry. RESULTS: During 7KC-induced apoptosis the fluorescence from filipin and the ratio of measured (orange-red vs. yellow) fluorescence of NR were enhanced. Spectral analysis of images obtained in biphoton mode and resulting factor images demonstrated the occurrence of fluorescence resonance energy transfer between 7KC and NR and the subsequent colocalization of 7KC and NR. These data were in agreement with biochemical characterization and demonstrated that 7KC and neutral and polar lipids accumulate in NR-stained cytoplasmic structures. CONCLUSIONS: During 7KC-induced apoptosis, 7KC modifies the cellular content of neutral and polar lipids, favors free cholesterol accumulation, and colocalizes with neutral and polar lipids that are inside NR-stained cytoplasmic structures.

Apoptosis↗

PubMed2005

Experimental comparison between autofluorescence spectra of constrained fresh and cryopreserved arteries.

The study of mechanical properties of the arterial wall is an important step in the comprehension of the vascular physiopathological functioning. However, cryopreserving biological tissues using very low temperatures can induce biological and structural modifications which may involve complications (dilatation, bursting, stenosis) after reimplantation. Many procedures of mechanical tests (traction, dilatation) developed in research allow us to comprehend and analyse rheological behaviour of the arterial wall. The study presented in this article offers a new perspective to detect changes of mechanical properties of cryopreserved arterial samples. In fact, the original idea is to couple a mechanical test bed (uniaxial traction of arterial rings) with spectroscopic measurements (autofluorescence) for the purpose of correlating mechanical modifications and spectral variations. Ultimately, this new approach could lead to develop a device allowing atraumatic and contactless optical examinations of arterial graft to determine its mechanical state before reimplantation.

Animals↗

PubMed2005

Autologous cell based therapy for treating chronic infarct myocardium.

Recent experimental and clinical studies have shown that autologous cell based therapy using skeletal myoblasts or bone marrow-derived stem cells might have beneficial effects in chronic ischemic heart disease. The underlying concept is based on the repopulation of necrotic tissue by either readily contractile myoblasts or by bone marrow-derived stem cells. However, there is a need to resolve a number of issues for determining the better way to perform these treatments and, moreover, for assessing the real beneficial functional effect of each of these cell therapies. In this mini-review, we will discuss (i) the issues of the selection of chronic infarct animal to truly determine the impact of cell therapy on cardiac function recovery, and (ii) the evaluation of the bio-availability and the bio-distribution of transplanted cells. Some new investigational methodologies based on clinical end-points are also proposed.

Animals↗

PubMed2004

Multicompartment films made of alternate polyelectrolyte multilayers of exponential and linear growth.

The layer by layer deposition process of polyelectrolytes is used to construct films equipped with several compartments containing "free polyelectrolytes". Each compartment corresponds to a stratum of an exponentially growing polyelectrolyte multilayer film, and two consecutive compartments are separated by a stratum composed of a linearly growing multilayer that acts as a barrier preventing polyelectrolyte diffusion from one compartment to another. We use hyaluronic acid/poly(L-lysine) as the system to build the compartments and the poly(styrene sulfonate)/poly(allylamine) system for the barrier. Using confocal microscopy, it is shown that poly(L-lysine) diffuses only within the compartment in which it was initially introduced during the film construction and is thus unable to cross the barriers. Using fluorescein isothiocyanate as a pH indicator, it is also shown that although poly(styrene sulfonate)/poly(allylamine) multilayers act as a barrier for polyelectrolytes, they do not prevent proton diffusion through the film. Such films open the route for multiple functionalization of biomaterial coatings.

Journal Article↗

PubMed2004

Layer by layer buildup of polysaccharide films: physical chemistry and cellular adhesion aspects.

The formation ofpolysaccharide films based on the alternate deposition of chitosan (CHI) and hyaluronan (HA) was investigated by several techniques. The multilayer buildup takes place in two stages: during the first stage, the surface is covered by isolated islets that grow and coalesce as the construction goes on. After several deposition steps, a continuous film is formed and the second stage of the buildup process takes place. The whole process is characterized by an exponential increase of the mass and thickness of the film with the number of deposition steps. This exponential growth mechanism is related to the ability of the polycation to diffuse "in" and "out" of the whole film at each deposition step. Using confocal laser microscopy and fluorescently labeled CHI, we show that such a diffusion behavior, already observed with poly(L-lysine) as a polycation, is also found with CHI, a polycation presenting a large persistence length. We also analyze the effect of the molecular weight (MW) of the diffusing polyelectrolyte (CHI) on the buildup process and observe a faster growth for low MW chitosan. The influence of the salt concentration during buildup is also investigated. Whereas the CHI/HA films grow rapidly at high salt concentration (0.15 M NaCl) with the formation of a uniform film after only a few deposition steps, it is very difficult to build the film at 10(-4) M NaCl. In this latter case, the deposited mass increases linearly with the number of deposition steps and the first deposition stage, where the surface is covered by islets, lasts at least up to 50 bilayer deposition steps. However, even at these low salt concentrations and in the islet configuration, CHI chains seem to diffuse in and out of the CHI/HA complexes. The linear mass increase of the film with the number of deposition steps despite the CHI diffusion is explained by a partial redissolution of the CHI/HA complexes forming the film during different steps of the buildup process. Finally, the uniform films built at high salt concentrations were also found to be chondrocyte resistant and, more interestingly, bacterial resistant. Therefore, the (CHI/HA) films may be used as an antimicrobial coating.

Animals↗

PubMed2004

Analysis of fluorescent MRI contrast agent behavior in the liver and thoracic aorta of mice.

OBJECTIVE: To characterize the behavior of magnetofluorescent products injected in mice intravenously. STUDY DESIGN: The magnetic resonance imaging (MRI) products were labelled with fluorescent molecules to examine the biodistribution process in vivo and observe them at the cellular level by means of confocal microscopy. Three-dimensional (3D) sequences of images were obtained by spectral analysis of sample preparations in a multiphoton confocal microscope and analyzed by the factor analysis of medical image sequence algorithm, which provides factor curves. Factor images are the result of image-processing methods that utilize information from emission spectra. Preparations are also screened in the counting mode to provide fluorescent lifetime imaging microscopy (FLIM) characterizations. RESULTS: Factor images and FLIM images can help to analyze MRI targeting inside the liver and thoracic aorta of mice. They show positive detection of Fe-Texas red and BOPTA-Eu in the liver and positive detection of Fe-Texas red and negative detection of BOPTA-Eu inside the thoracic aorta. CONCLUSION: This investigation established the utility of fluorescent MRI contrast agents as in vivo staining tools for cellular sites.

Algorithms↗

PubMed2004

FRET multiphoton spectral imaging microscopy of 7-ketocholesterol and Nile Red in U937 monocytic cells loaded with 7-ketocholesterol.

OBJECTIVE: To show the effect of 7-ketocholesterol (7KC) on cellular lipid content by means of flow cytometry and the interaction of 7KC with Nile Red (NR) via ultraviolet fluorescence resonance energy transfer (FRET) excitation of NR on U937 monocytic cells by means of 2-photon excitation confocal laser scanning microscopy (CLSM). STUDY DESIGN: Untreated and 7KC-treated U937 cells were stained with NR and analyzed by flow cytometry and CLSM. 3D sequences of images were obtained by spectral analysis in a 2-photon excitation CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm, which provides factor curves and images. Factor images are the result of the FAMIS image processing method, which handles emission spectra. In FRET analysis, preparations are screened at selected UV wavelengths to avoid emission of NR in the absence of 7KC. RESULTS: During 7KC-induced cell death,flow cytometry and CLSM revealed a modification of the cellular lipid content. Factor images show FRET occurrence and subsequent colocalization of 7KC and NR. CONCLUSION: This investigation established the utility of 2-photon excitation CLSM to assess colocalization of 7KC with NR by FRET and to identify and distinguish polar and neutral lipids stained by NR that accumulate from the effect of 7KC.

Cell Death↗

PubMed2003

Interactions of mechanotransduction pathways.

Integrins may serve as mechanosensors in endothelial cells (ECs): shear stress causes integrin-Shc association, assembly of the signaling complex and then leads to JNK activation. Flow also mediates selective and cell-specific alterations in vascular cell G-protein expression that correlate with changes in cell-signalling, G-protein functionality and modulate Ca2+ concentration. In this study, we explored the cross-talks between EC membrane mechanosensors, such as integrins, ion channels, and G-proteins in shear stress-induced signal transduction by their specific inhibition. Confluent monolayer of bovine aortic endothelial cells (BAECs) were incubated with or without specific inhibitors prior to shearing experiments. Our results showed an attenuation of integrin-Shc association under shear stress with RGD, and with PTX, but not with BAPTA/AM. The inhibitions of shear-activated JNK are similar for RGD and PTX. However, unlike for integrin association, the chelation of calcium reduced JNK activation. These results provide several lines of evidence of the interactions between different mechanosensors in ECs. First, integrin-Shc association required cell attachment and G-protein activity, but not intracellular calcium. Second, shear-induced JNK activation is regulated by multiple mechano-sensing mechanisms such as integrin, G-protein and calcium concentration.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Spatial distribution of the beta2 integrin (CD11b/CD18) and L-selectin (CD62L) adherence receptors on human neutrophils by Conventional Optical Scanning Microscopy (COSM).

Receptors such as CD62L and CD11b/CD18, are transmembrane glycoproteins which regulate leukocyte adhesive phenotype. Flow cytometry (FCM) makes it possible to assess a characterization of the cell activation level by receptor quantifying, but that technique does not integrate other factors of adherence regulation, such as spatial distribution and molecular conformation. Our study consisted in exploring the main adherence receptors on Polymorphonuclear Neutrophils (PMN) that were simultaneously analyzed by FCM and Conventional Optical Scanning Microscopy (COSM). FCM analysis showed that TNFalpha induce a decrease in CD62L expression and an increase in beta2 integrins. COSM analysis distinguished three stages of cellular distribution of CD11b/CD18 within resting PMN: most of them (about 80%) had homogeneous distribution (heterogeneous spots distributed over the entire cell surface), for 10-15% of the cells, there was a crown distribution around the widest cell diameter and in less that 10% of them receptor distribution was polarized. CD62L was in the form of heterogeneous spots distributed in a circle on the surface on non-stimulated PMN. PMN stimulation by TNFalpha was associated to a randomized clustering involving both selectin and beta2 integrin. Three-dimensional analysis elicited data not shown by quantitative cytometry. For a single averaged value of the density determined by FMC, various spatial distributions of adherence receptors are found on the surface of non-stimulated PMN. The characterization of the leukocyte adhesive phenotype has to integrate adherence receptors density as well as their spatial distribution.

CD18 Antigens↗