Biomedical subjects

J Vassy

Publications and source records attributed to J Vassy.

At least 37 records · Page 2Linked to original sources

PubMed1997

Epidermal localization and protective effects of topically applied superoxide dismutase.

Data from the literature, as well as our previous work, indicate a protective effect of superoxide dismutase (SOD) in topical application against UV-induced cutaneous damage. In the present article we show that pre-treatment of the skin with SOD protects against PUVA-induced inflammatory reactions not only in murine, but also in human skin. Using fluorescently labelled Cu,Zn SOD, epifluorescence microscopy and digital image processing, we demonstrate that the FITC fluorescence localizes in the stratum corneum and upper granulosa, as well as in the epidermal cell layer surrounding the lumina of the hair follicles. These findings were similar for murine and human skin. Since autofluorescence was eliminated by a special filter, it can be ascertained that the fluorescence observed in the tissues was due to FITC-labelled SOD.

Administration, Cutaneous↗

PubMed1996

Superoxide-mediated clastogenesis and anticlastogenic effects of exogenous superoxide dismutase.

Superoxide-mediated clastogenesis is characteristic for various chronic inflammatory diseases with autoimmune reactions and probably plays a role in radiation-induced clastogenesis and in the congenital breakage syndromes. It is consistently prevented by exogenous superoxide dismutase (SOD), but not by heat-inactivated SOD, indicating that the anticlastogenic effect is related to the catalytic function of the enzyme. Increased superoxide production by activated monocytes/macrophages is followed by release of more long-lived metabolites, so-called clastogenic factors, which contain lipid peroxidation products, unusual nucleotides of inosine, and cytokines such as tumor necrosis factor alpha. Since these components are not only clastogenic, but can stimulate further superoxide production by monocytes and neutrophils, the genotoxic effects are self-sustaining. It is shown here that anticlastogenic effects of exogenous SOD are preserved despite extensive washing of the cells and removal of all extracellular SOD. Using flow cytometry and confocal laser microscopy, rapid adherence of the fluorescently labeled enzyme to the cell surface could be observed with slow uptake into the cell during the following hours. The degree of labeling was concentration and time dependent. It was most important for monocytes, compared with lymphocytes, neutrophils, and fibroblasts. The cytochrome c assay showed significantly diminished O2- production by monocytes, pretreated with SOD and washed thereafter. The preferential and rapid binding of SOD to monocytes may be of importance not only for the superoxide-mediated genotoxic effects, described above, but also from a therapeutic standpoint. It can explain the observation that beneficial effects of injected SOD lasted for weeks and months despite rapid clearance of the enzyme from the blood stream according to pharmacodynamic studies.

Animals↗

PubMed1996

Temporary ex vivo inhibition of the expression of the human oncogene HER2 (NEU) by a triple helix-forming oligonucleotide.

A 28-base phosphodiester triple helix-forming oligonucleotide, mostly G and A containing, targeted to a polypurine tract interrupted by a purine-pyrimidine inversion, situated upstream from the TATA box of the promoter of the human HER2 gene, was conceived by computer modeling. The "energetically best choice" was oligo 28(C), which formed the triple helix in vitro, as proved by gel retardation and Fourier transform infrared spectroscopy. When administered as a complex with lipofectin, fluorescence confocal microscopy and electrophoresis confirmed the delivery and persistence of this unprotected oligonucleotide inside MCF7 (breast cancer) cells. At a concentration of 2 microM, the oligonucleotide reduced within 6 h the HER2 mRNA level to 42% (Northern blot) but did not interfere with the transcription of a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase. During the first day of administration at 0.22 microM, it lowered to 59% the HER2 protein in treated, as compared to nontreated, cells (ELISA). The effect was sequence specific when compared to that of five different negative controls, and it was target selective when compared to the expression of a related, nontargeted protein, the epidermal growth factor receptor. By day 2, the inhibitory effect was overcome by replenishment reactions.

Base Sequence↗

PubMed1996

Differential nuclear localization of p50, p52, and RelB proteins in human accessory cells of the immune response in situ.

The Rel/NF-kappa B proteins, p50, p52, p65, c-Rel, and RelB, constitute a family of transcription factors involved in the positive regulation of a variety of genes during the immune response. Recently, it has been shown that RelB knockout mice have no dendritic cells (DC). An overexpression of p50 has been described in follicular dendritic cells (FDC). A constitutive NF-kappa B activity has been reported in mature macrophages. This led to the hypothesis that some of the Rel/NF-kappa B proteins were key nuclear factors in functions of accessory cells of the immune response. Therefore, we investigated in situ the nuclear localization of Rel/NF-kappa B proteins in accessory cells of the immune system by immunohistochemistry and double labeling by immunofluorescence from five normal human tonsils and five lymph nodes with follicular hyperplasia. Nuclear p65 and c-Rel proteins were found in all cell types including lymphocytes. In germinal centers GC, p50, p52, and RelB were found in the nuclei of FDC only and were not detected in the nuclei of CD68+ cells. In T cell areas, p50, p52, and RelB were found in the nuclei of HLA-DR+ cells with an antigen-presenting cell (APC) morphology. p52 and RelB were detected in the nuclei in both CD1a+ and CD68+ cells from the T cell area, whereas p50 was found only in CD68- and CD1a- cells. Cells with nuclear p50 were negative for the CD38, CD20 and CD2 markers. These results show that, physiologically, high levels of nuclear of p50, p52 and RelB are restricted to accessory cells of the immune system, which include FDC in GC, and DC and macrophages in the T cell zone, that specialized scavenger macrophages from GC do not have detectable levels of p52 and RelB, whereas macrophages from the T cell area, known to present the antigen to T cells, do have both nuclear p52 and RelB, and that in the T cell zone, p52 and RelB are located in nuclei of both CD1a+, CD68+ or both, cells APC, whereas p50 is restricted to CD1a- and CD68- APC. The different patterns of p50, p52 and RelB protein nuclear localization may provide insight into their different roles during the immune response in vivo.

Adolescent↗

PubMed1996

Quantitative image analysis of cytokeratin filament distribution during fetal rat liver development.

Recent reports suggest that not only chemical but also mechanical influences from the cellular environment could have profound effects on gene activity and could act on cell differentiation and proliferation. These mechanisms involve a tissue matrix system, which includes extracellular matrix, nuclear matrix, and cytoskeleton. Supposing that the cytoskeleton mediates mechanical transduction from the cell environment to the nucleus, significant differences in the spatial distribution of the cytoskeleton network could reflect variations in environmental signals. Hepatocytes during fetal development provide a useful model to study such variations, because a progressive establishment of intercellular contact is reported. The aim of this work is to discriminate steps in hepatocyte differentiation from fetal to adult livers, using computerized quantitative image analysis of cytokeratin (C8) immunofluorescent localization, visualized by confocal scanning laser microscopy. The filament structure is represented by the gray-scale skeleton of the digital images obtained by specially designed segmentation methods. A set of line features was investigated, including number and length of lines, orientation of lines, and the fractal dimension of the filament network. The features studied showed highly significant differences throughout liver development, with an increase of the total amount of cytokeratin filaments. We could also demonstrate a modification in the structure of the network, being more and more dense, with an increase of connecting points. Moreover, it has been shown that filaments have some orientation in fetal hepatocytes, and that directionality. Thus, significant differences in the pattern of the cytokeratin filament network according to the stages of hepatocyte differentiation have been demonstrated with objective and quantitative methods.

Animals↗

PubMed1996

Application of confocal scanning laser microscopy for an automated nuclear grading of prostate lesions in three dimensions.

Confocal scanning laser microscopy provides the opportunity to obtain three-dimensional (3-D) images by piling up consecutive confocal planes. This technique was applied to capture 3-D images from 100-microns-thick tissue blocks from prostate lesions (hyperplasia, dysplasia, adenocarcinomas). Automated methods were implemented to perform a nuclear grading of 3-D cell nuclei from these specimens. Special attention was focused on the development of a new approach to 3-D chromatin texture analysis. This method uses mathematical morphology operations to tessellate the chromatin into homogeneous domains. The nuclear features (volume, shape, texture) were subjected to a discriminant analysis. Using a set of five features, the classification of cell nuclei yielded an accuracy of 96.3%. The results indicate the potential of 3-D imaging and analysis techniques for an automated nuclear grading of prostate lesions.

Cell Nucleus↗

PubMed1995

Combined analysis of in situ hybridization, cell cycle and structural markers using reflectance and immunofluorescence confocal microscopy.

A method for the simultaneous detection of mRNA by reflectance in situ hybridization (RISH), cell cycle and structural markers by immunofluorescence using confocal laser scanning microscopy is presented. The mRNA expression of two ras-related genes rhoB and rhoC was analysed in human breast cancer cell lines and human histological specimens (breast cancer tissues and skin biopsies). In breast cancer cell lines, the conditions were optimized to detect RNA-RNA hybrids and DNA synthesis after pulse-labelling with bromodeoxyuridine. Endonuclease-exonuclease digestion, which allows the accessibility to specific antibodies of halogenated pyrimidine molecules, was carried out following ISH. Finally, cytokeratin or vimentin staining was performed. The detection of signals, arising from 1-nm colloidal gold particles without silver enhancement, by reflectance confocal laser scanning microscopy is described. Bromodeoxybiridine DNA markers and cytokeratin/vimentin staining were detected concomitantly using different fluorochromes. To allow comparative expression of two related genes, the mRNA of rhoB and rhoC were detected using digoxigenin- or biotin-labelled riboprobes and, after 3-D imaging, a detailed analysis by optical horizontal (x, y) and vertical (x, z) sectioning was undertaken. The subsequent bromodeoxyuridine detection procedure permitted to us explore the specific transcription of these two genes during S and non-S phases. This method allows the identification and localization of several subcellular components in cells within a complex tissue structure and makes it possible to analyse further transcript localization in relation to the function of the encoded protein and to the cell cycle.

Biomarkers↗

PubMed1995

A dual approach to structural texture analysis in microscopic cell images.

The computer-based quantitative analysis of microscopic cell images provides important diagnostic information in clinical and experimental pathology. The arrangement of various cell structures can be described as texture. We developed a new approach to structural texture analysis. It assumes that texture consists of homogeneous regions (texture primitives). Texture can be regarded in a dual way--as a composition of regions or as a pattern composed of the regions' boundaries (lines). We implemented methods for the segmentation of regions and lines in grayscale images. The detection of regions is followed by a region-growing process to avoid an oversegmentation. The segmented regions and lines are stored in a uniform data structure which reflects their arrangement in the image. The presented methods were applied to study the chromatin distribution in cell nuclei and the development and differentiation of intermediate filaments in fetal liver cells.

Algorithms↗

PubMed1995

Chromatin texture analysis in three-dimensional images from confocal scanning laser microscopy.

OBJECTIVE: Three-dimensional (3D) images of tissue blocs can be obtained through optical sectioning by a confocal scanning laser microscope and by piling up of consecutive confocal planes. These images offer the opportunity to quantitate biologic structures (i.e., nuclear chromatin) in 3D. Chromatin texture analysis is regarded as a key element in nuclear grading of malignant lesions. The present study introduced a new method of structural texture analysis of 3D chromatin distribution in cell nuclei. The method was assessed for its validity in a pilot study. STUDY DESIGN: The study group consisted of 300 3D cell nuclei from prostatic lesions, 100 each of hyperplasia, dysplasia and well-differentiated carcinoma. A set of 15 texture features was studied for its classification capability in a discriminant analysis. RESULTS: A classifier that confined three texture features yielded a classification accuracy of 91.7% for the training set (60 nuclei). The classification accuracy for the test set (240 nuclei) was 93.3%. CONCLUSION: The new method demonstrated its potential for 3D chromatin analysis. Further studies on large numbers of cases are necessary to evaluate the use of 3D nuclear grading for diagnostic pathology.

Chromatin↗

PubMed1994

Reflectance in situ hybridization (RISH): detection, by confocal reflectance laser microscopy, of gold-labelled riboprobes in breast cancer cell lines and histological specimens.

A method for reflectance in situ hybridization (RISH) is presented. The importance of the method is demonstrated by results obtained on cytological and histological breast cancer specimens. Scattering reflectance signals from 1-nm colloidal-gold particles after RNA/RNA in situ hybridization, using digoxigenin-labelled riboprobes, were detected by confocal scanning laser microscopy. The mRNA expression of two ras-related genes, rho B and rho C, was analysed in human histological breast cancer specimens and in human breast cancer cell lines. Horizontal (x, y) and vertical (z) optical sections after three-dimensional imaging were used for visualization. A marked heterogeneity (between individual cells and between specimens) was noted for the expression of the rho B gene, both in cytological and in histological samples. On the other hand, rho C was always expressed and showed no heterogeneity. This method allows the identification of several cellular constituents in an heterogeneous tissue structure, as demonstrated by the simultaneous detection of rho B (or rho C) by reflectance and of DNA, cytokeratin and/or vimentin by fluorescence.

Antibodies, Monoclonal↗

PubMed1993

Confocal microscopy immunofluorescence localization of desmin and other intermediate filament proteins in fetal rat livers.

Immunolocalization of desmin in fetal rat livers shows that on day 12 of gestation a high number of liver cells express desmin. This number decreases from day 14 onward. On day 20 about the same density of desmin-containing cells is found in fetal rat livers as is found in adult rat livers. Desmin-containing cells show two types of labeling patterns, especially on days 12 and 13 of gestation: (a) a basketlike network of intermediate filaments throughout the whole cell, similar to the labeling pattern of cytokeratin in hepatocytes; and (b) more strongly labeled intermediate filaments developing long and slender processes between adjacent cells, close to the labeling pattern of Ito cells in adult rats. From day 14 of gestation, the first type becomes rare, and from day 18 only the second type remains. Double-labeling experiments show that coexpression of desmin and cytokeratin is found in cells of the first type on days 12, 13 and 14 of gestation. Cells containing desmin with the labeling pattern of the second type never express cytokeratin. Coexpression of vimentin and cytokeratin is never found in fetal hepatocytes, even on day 12 of gestation. Numerous nonhepatocyte cells coexpress desmin and vimentin, but some cells contain vimentin or desmin alone. In desmin-containing cells the labeling pattern is of the first type (basketlike network). These results suggest that in early stages of fetal liver development, desmin is found in two different types of liver cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Immunolocalization of a specific type of prosome close to the bile canaliculi in fetal and adult rat liver.

Prosomes are mRNA-associated RNP particles and cofactors of untranslated (ribosome-) free mRNP having a multicatalytic proteinase (MCP; proteasome) activity. The expression of prosomal proteins in fetal development of the rat liver was investigated by indirect immunofluorescence, using a panel of monoclonal antibodies to individual prosomal proteins (p-mAbs). In all fetal and adult stages tested, strong immunofluorescence staining was observed with the p31K-specific p-mAb exclusively, whilst Western blot analysis showed reactivity also with the p27K and p33K antigens. Double labeling with the 31K p-mAb and an anti-cytokeratin antibody showed that the prosome antigen superimposes partially onto this type of intermediate filaments (IF), confirming earlier observations made on cultured cell lines of various types. Most interestingly, the p31K antigen was found preferentially in the pericanalicular zone of hepatocytes in the developing liver, from day 17 onwards up to the adult state. This shows a preferential concentration of prosomes of a specific type, including the p31K antigen, in the morphologically and possibly functionally specialized apical domain of the hepatocyte, in a differentiation-related fashion.

Aging↗

PubMed1991

Three-dimensional DNA image cytometry by confocal scanning laser microscopy in thick tissue blocks.

A method for the quantification of nuclear DNA in thick tissue blocks by confocal scanning laser microscopy is presented. Tissues were stained en bloc for DNA by chromomycin A3. Three-dimensional images, 60 microns deep, were obtained by stacking up confocal fluorescent images obtained with an MRC-500 (Bio-Rad, Richmond, CA). The effects due to bleaching and attenuation by depth of fluorescence emission were corrected mathematically. The DNA contents were estimated by summing up the detected emission intensities (discretized into pixel gray levels) from each segmented nucleus. Applications to an adult rat liver and to a human in situ carcinoma of theesophagus are shown to demonstrate, respectively, the precision of the method and its potential usefulness in histopathology. Comparisons are made with DNA histograms obtained on the same materials by image cytometry on smears and by flow cytometry. Ploidy peaks obtained with the confocal method, although wider than with other methods, are well separated. Confocal image cytometry offers the invaluable advantage of preserving the tissue architecture and therefore allowing, for instance, the selection of histological regions and the evaluation of the degree of heterogeneity of a tumor.

Animals↗

PubMed1991

Modelling and analysis of 3-D arrangements of particles by point processes with examples of application to biological data obtained by confocal scanning light microscopy.

Within the concept of point processes, a review is presented of quantities which can be used in studies of three-dimensional (3-D) aggregates of particles. Suitable characteristics and estimators are given for both unmarked and marked point processes. To demonstrate the feasibility of such quantitative approaches, an application in histology, dealing with 3-D arrangements of cell nuclei in rat liver, is described. Using a confocal scanning light microscope, 3-D images are recorded and image analysis used to obtain the coordinates of the centroid, together with the volume and DNA content, of each cell nucleus. Examples of results are given, using both unmarked and marked point processes. In the latter case, cell type, nuclear volume and ploidy group are suitable marks.

Animals↗

PubMed1991

High-resolution three-dimensional images from confocal scanning laser microscopy. Quantitative study and mathematical correction of the effects from bleaching and fluorescence attenuation in depth.

Three-dimensional images can be assembled by piling up consecutive confocal fluorescent images obtained by confocal scanning laser microscopy. The present work was based on three-dimensional (50-microns-deep) images at high (x, y) resolution obtained with an MRC-500 after en bloc staining of thick slices of rat liver by chromomycin A3 for nuclear DNA. The results of studies on bleaching, fluorescence excitation and emission intensities at various depths of histologic preparations are described. These effects could be evaluated separately by acquiring piled-up ("brick-stepping") and non-piled-up ("side-stepping") (x, y) images at consecutive depths and also (x, z) images. Empirical equations allowed the fitting of experimental plots of bleaching versus time, at different laser intensities and at different depths, and of fluorescence emission intensity versus depth. The main conclusions were that under our experimental conditions: (1) there was no attenuation by depth of the fluorochrome penetration, (2) there was no attenuation of the exciting beam intensity up to at least 50 microns deep, (3) there was an attenuation of the fluorescence emission intensity by depth, (4) bleaching happened equally on all planes above and below any confocal plane being studied, and (5) the fluorescence bleaching half-life was independent of depth. A mathematical correction scheme designed to compensate for bleaching and for attenuation of fluorescence emission in depth is presented. This correction is required for obtaining three-dimensional images of better quality, for optimal three-dimensional image segmentation and for any quantitative analysis based upon voxel-discretized emission intensities (gray levels)--e.g., estimating, by confocal image cytometry, textural chromatin parameters and nuclear DNA amounts.

Animals↗

PubMed1990

Analysis by confocal scanning laser microscopy imaging of the spatial distribution of intermediate filaments in foetal and adult rat liver cells.

Confocal scanning laser microscopy has been used to make three-dimensional observations of the spatial distribution of cytoskeleton intermediate filaments in rat liver hepatocytes, at various stages during foetal development and in the adult. Single and double immuno-labelling with fluorescein and Texas Red fluorescence have been used to study the intracellular spatial distribution of C18 cytokeratin and vimentin. Simultaneous confocal imaging with double-fluorescence emission requires an image processing step for the correction of 'contamination' effects due to the overlap between fluorescein and Texas Red emission spectra. At the pre-natal period (day 20 of gestation) each type of intermediate filament labelling is only present in a certain cellular category, C18 cytokeratin in hepatocytes and vimentin in mesenchymal cells. However, at the earliest developmental stages (day 12 of gestation), vimentin and cytokeratin seem to be found in the same type of cells, probably mesenchymal cells. Some striking developmental changes, associated with the differentiation of the liver parenchyma, are observed for both C18 cytokeratin and vimentin. In earlier foetal stages, C18 filaments are scarce, hazily labelled and randomly distributed inside the hepatocytic cytoplasm. Late during foetal development (days 18-20 of gestation), hepatocytic cytokeratin filaments are abundant, well individualized and sharply labelled. The hepatocytes are arranged in a muralium duplex architecture (two-cell-thick sheets) and the labelling intensity measured in the hepatocytic cytoplasm at the basal pole is double that measured at the sinusoidal pole, while, in the adult, hepatocytes are arranged in a muralium simplex architecture (one-cell-thick sheets) and cytokeratin filaments have a symmetrical distribution in relation to the nuclear region.

Animals↗

PubMed1988

Development of the fetal rat liver: ultrastructural and stereological study of hepatocytes.

Qualitative and quantitative changes in the liver tissue composition have been studied during prenatal development of the Wistar rat by electron microscopy and stereologic methods. The absolute volume of the fetal liver is multiplied by 84 between days 13 and 20 of gestation. In the meantime, the average hepatocyte volume is multiplied by 1.5 between days 12 and 20. The volumetric fraction of hepatocytes increases from 35% of the volumetric fraction of the liver on day 12 to 66% on day 20 of gestation. The non-hepatocyte cells decrease from 49% on day 12 to 25% on day 20. By days 12 and 13, the rough endoplasmic reticulum and the Golgi apparatus are well differentiated, indicating that young fetal hepatocytes are able to synthesize and export plasma proteins. The volumetric fraction of free ribosomes decreases from 38% of the hepatocytic cytoplasm on days 12 and 13 to 6% on day 20. The mitochondrial compartment occupies about 10% of the hepatocyte cytoplasm. The mitochondria, small and round on days 12, 13 and 14, become oblong from day 18 of gestation. The shape of hepatocytes changes during the prenatal development, from potato-like on days 12, 13, 14 to cubic on day 20, with an intermediate, more spheric, stage on day 18.

Animals↗

PubMed1986

The effect of dexamethasone on transferrin secretion by cultured fetal rat hepatocytes.

Cultured fetal rat hepatocytes derived from 12, 15 and 19-day gestation rats are capable of secreting transferrin. When dexamethasone is added to the medium an increased secretion rate is observed. The changes in secretion rates in control as well as dexamethasone-treated cells during culture have been shown to correlate with the level of mRNA coding for transferrin. Immunocytochemical experiments show that initially all hepatocytes contain transferrin which is localized in the lumina of the perinuclear space, rough endoplasmic reticulum and in the saccules and vesicles of the Golgi apparatus. During culture, particularly in control cells, the intensity of labelling varies from cell to cell. In addition, adjacent cells are observed to label more intensely in different intracellular organelles.

Animals↗