Edmond Kahn

Peer reviewed article authored by (Kahn E, Tissot N, Frere P, Dauphin A, Boumhras M, Bachelet CM, Frouin F, Lizard G). Read article or submit your manuscript for publishing.

In the context of multiple sclerosis and X‐linked adrenoleukodystrophy, 7‐ketocholesterol (7KC) and very long chain fatty acids (C24:0, C26:0) are supposed to induce side effects respectively on oligodendrocytes which are myelin (which is a lipoproteic complex) synthesizing cells. The effects of 7KC (25, 50 μM), C24:0 and C26:0 (10, 20 μM) on cell viability and lipid membrane organization were investigated on 158N murine oligodendrocytes. Concerning 7KC and fatty acids (at 20 μM only): 1) cell growth was strongly inhibited; 2) marked induction of cell death was revealed with propidium iodide (PI); 3) no apoptotic cells were found with C24:0 and C26:0 (absence of cells with condensed and/or fragmented nuclei, of FLICA positive cells and of PI negative/SYTO16 negative cells); 4) some apoptotic cells were detected with 7KC. Fatty acids (at 20 μM only) and 7KC also induced a disorganization of lipid membranes revealed with Merocyanine 540. So, to point out the effects of 7KC (25 μM), C24:0 and C26:0 (20 μM) on the lateral organization of lipid membranes, we used LAURDAN, which gives simultaneous information about morphology and phase state of lipid domains: its emission is blue in the ordered lipid phase, green in the disordered lipid phase. To overcome the qualitative filtering settings of blue and green emission colors, data obtained by mono‐ and bi‐photon confocal microscopy were analyzed by spectral analysis. Sequences of emission images were obtained on both mono‐ and bi‐photon confocal microscopes and processed by means of Factor Analysis of Medical Image Sequences (FAMIS), which is a relevant tool to unmix emission spectra and provide pure color images. Only 7KC was capable to induce a green emission with LAURDAN. Thus, at concentrations inducing oligodendrocyte cell death, 7KC (25 μM) is more efficient than C24:0 and C26:0 (20 μM), to trigger lateral lipid membrane disorganization. © 2011 International Society for Advancement of Cytometry

Peer reviewed article authored by (Edmond Kahn, Mauhamad Baarine, Sophie Pelloux, et al). Read article or submit your manuscript for publishing.

OBJECTIVE: To evaluate the capture of nanoparticles (quantum dots [QDs], fluorospheres) by nonbeating mouse cardiac cells (HL1-NB) cultured without or with 7-ketocholesterol (7KC) found at an increased level in the plasma of atherosclerotic patients and to simultaneously analyze their cytotoxic, proinflammatory and oxidative properties. STUDY DESIGN: Flow cytometry (FCM), confocal laser scanning microscopy and subsequent factor analysis image processing were used to characterize the uptake of nanoparticles and to define their cytotoxicity, evaluated by enhanced permeability to SYTOX Green, release of lactate dehydrogenase (LDH) and morphologic nuclear changes determined with Hoechst 33342. Proinflammatory effects were estimated by enzyme linked immunoassay to quantify IL-8 and MCP-1 secretion. The overproduction of reactive oxygen species (ROS) was determined by FCM with hydroethidine. RESULTS: Whereas the nano particles had no cytotoxic or inflammatory effects, they could stimulate ROS production. QDs were not incorporated. When 7KC was used, LDH release was enhanced and QDs potentialized IL-8 secretion. The incorporation and exit dynamics of nano particles were visualized to differentiate the emission spectra of SYTOX Green and nano particles and to precisely determine the cellular localization of nano particles. CONCLUSION: The selected nanoparticles, which accumulate at the inner or outer cytoplasmic membrane level, can induce biologic activities and are able to interfere with those of chemically defined molecules such as 7KC. (Anal Quant Cytol Histol 2009;31:380-394)

Background: The 7‐ketocholesterol (7KC)‐induced cell death has some characteristics of apoptosis and is associated with polar lipid accumulation. So, we investigated the effects of the broad‐spectrum caspase inhibitor z‐VAD‐fmk and of the caspase‐2 inhibitor z‐VDVAD‐fmk on lipid profile evaluated by staining with Nile Red (NR).Methods: The 7KC‐treated human monocytic U937 cells were cultured in the absence or in the presence of the caspase inhibitors z‐VAD‐fmk or z‐VDVAD‐fmk. When staining with NR is performed, neutral and polar lipids have yellow and orange/red emission, respectively, and fluorescence was then analyzed by flow cytometry (FCM) and by confocal laser scanning microscopy (CLSM) combined with subsequent image processing. The 3D‐image sequences were obtained by means of CLSM using spectral analysis, and were analyzed by the factor analysis of medical image sequences algorithm to differentiate spectra inside mixed fluorescence emission and get corresponding specific images.Results: By FCM, comparatively to untreated cells, higher percentages of red fluorescent cells were identified in 7KC‐treated cells. Factor curves and images reveal orange and red fluorescence emissions in 7KC‐treated cells and show yellow, orange, and red fluorescence emissions in 7KC‐treated cells cultured in the presence of z‐VAD‐fmk or z‐VDVAD‐fmk.Conclusions: Our data support that investigation by FCM and by spectral analysis in CLSM associated with subsequent image processing provides useful tools to determine the effect of caspase inhibitors on lipid content evaluated with NR. They also favor the hypothesis of relationships between caspase activity and polar lipid accumulation. © 2007 International Society for Analytical Cytology

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.

OBJECTIVE: To characterize the behavior of magneto-fluorescent 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.

This article illustrates a methodology which can be used on cellular and tissular specimens prepared with fluorochromes and analyzed by laser scanning confocal microscopy. Fluorescent beads are used to simulate the fluorochromes and they determine their detectability inside the preparations. They can also be used to verify that the microscopes are set properly so that the resulting images can be analyzed and are reliable to evaluate possible co- localizations. Methods of factor analysis are applied to optical section series obtained on the microscope to characterize the fluorochromes that are used to stain different locations of the specimen. Photophysical properties (emission spectra, decay rates) of the fluorochromes are used to obtain the differentiation. Section series are obtained either by spectral selection via a sequence of filters or by successive scans of the same specimen. Concerning the three- dimensional analysis, differentiation of series that are obtained by z displacement leads to the restoration of focal planes and improves the legibility of specimens. These planes which are not at the same depth when the excitation sources are not aligned can the be superimposed to achieve the co-localization.

To demonstrate that cellular preparations requiring color analysis of different domains stained by molecular cytogenetic methods (fluorescence in situ hybridization) can be processed by spectral analysis of fluorescent emissions by either factor analysis of medical image sequences (FAMIS) or a META confocal configuration to isolate fluorescent probes.

OBJECTIVE: To analyze multilamellar cytoplasmic structures by confocal laser scanning microscopy (CLSM) combined with factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: After treatment of U937 cells with 7-ketocholesterol (7-keto), cytoplasmic alterations were assessed with monodansylcadaverine (MDC). By ultraviolet excitation of a confocal laser scanning microscope (UV-CLSM), spectral sequences were performed to characterize 7-keto and MDC distribution inside cells. FAMIS was used to transform the image sequences in factor curves and images. RESULTS: By UV-CLSM 7-keto fluorescence was detected together with MDC, which revealed morphologic cytoplasmic changes in cells. The factor images obtained from confocal image sequences emphasized the view of these results. These data are in agreement with biochemical characterizations of MDC-positive structures. CONCLUSION: The combined use of confocal microscopy and FAMIS allowed us to detect MDC-positive cytoplasmic structures in 7-keto-treated cells and to colocalize MDC and 7-keto distribution. This new method confirms the usefulness of MDC as a marker of oxysterol-induced cell death.