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J P Rigaut

Publications and source records attributed to J P Rigaut.

At least 19 recordsLinked to original sources
PubMed2001

Mitochondria transfection by oligonucleotides containing a signal peptide and vectorized by cationic liposomes.

The progress of research in gene therapy allows hope for treatment of mitochondrial genetic disorders provided that efficient methods for gene transfer into mitochondria can be found. In this work, we have used an oligonucleotide coupled covalently to a mitochondria-targeted peptide at one end and a cationic liposome prepared from trimethyl aminoethane carbamoyl cholesterol iodide (TMAEC-Chol) to carry it in living cells. With a fluorescent probe to label the oligonucleotide at the other end and by means of confocal microscopy, we show that such modified oligonucleotides complexed to liposomes enter into the cytoplasm of human fibroblasts in primary culture, and then, after dissociation from the complexes, they penetrate into the mitochondria. The fluorescence was still observed after 8 days, suggesting the continued presence of oligonucleotides. At the concentrations used for this study, the cationic liposomes have practically no effect on cell growth, as revealed by the MTT assay.

Amino Acid Sequence↗
PubMed2000

Differences in the microtubule organization between normal and cml granulocytes after stimulation with chemotactic peptide.

Chemotaxis of polymorphonuclear leukocytes (PMNL) from chronic myeloid leukemia (CML) patients followed in a gradient of a chemotactic peptide n-formyl-methionyl-leucyl-phenylalanine (fMLP) is consistently defective in all the phases of the disease. Chemoattractant-induced polymerization of cytoskeletal proteins (actin and tubulin) plays a major role in regulation of cell shape and cellular motility. To study the role of microtubules in defective chemotaxis, we have compared fMLP-induced alterations in organization of microtubules in PMNL from CML patients with those from normal subjects by laser confocal microscopy. Our analysis shows differences in microtubule organization between normal and CML PMNL and suggests that both nucleation of new microtubule and elongation of pre-existing microtubules are essential for PMNL chemotaxis.

Humans↗
PubMed2000

Delivery and pathway in MCF7 cells of DNA vectorized by cationic liposomes derived from cholesterol.

We have investigated the delivery and the pathway in tumoral MCF7 cells of DNA carried by liposomes prepared from (trimethyl aminoethane carbamoyl cholesterol iodide (TMAE-Chol), a cholesterol-based cationic lipid with a quaternary ammonium on the polar head. The structure of DNA-liposome complexes depends on the length of DNA and on the lipid-DNA charge ratio X. Spherical beads constitute fine structures of the observed complexes even when they appear as aggregates. For oligonucleotide transfer, dissociation from liposomes after transfection, penetration of the oligonucleotides into nuclei, and a long resident time were observed. For plasmid transfer, a correlation between the variation in the transfection level and the ultrastructure of complexes was demonstrated. The results showed a cellular route of lipid/plasmid complexes from the beginning by endocytosis, entrapped into endosomes, released by the latter until entry in the perinuclear area, and then penetration of plasmids inside the nuclei resulting in the observed expression of the beta-galactosidase gene.

Biological Transport↗
PubMed2000

Breast carcinoma, intratumour heterogeneity and histological grading, using geostatistics.

Tumour progression is currently believed to result from genetic instability. Chromosomal patterns specific of a type of cancer are frequent even though phenotypic spatial heterogeneity is omnipresent. The latter is the usual cause of histological grading imprecision, a well documented problem, without any fully satisfactory solution up to now. The present article addresses this problem in breast carcinoma. The assessment of a genetic marker for human tumours requires quantifiable measures of intratumoral heterogeneity. If any invariance paradigm representing a stochastic or geostatistic function could be discovered, this might help in solving the grading problem. A novel methodological approach using geostatistics to measure heterogeneity is used. Twenty tumours from the three usual (Scarff-Bloom and Richardson) grades were obtained and paraffin sections stained by MIB-1 (Ki-67) and peroxidase staining. Whole two-dimensional sections were sampled. Morphometric grids of variable sizes allowed a simple and fast recording of positions of epithelial nuclei, marked or not by MIB-1. The geostatistical method is based here upon the asymptotic behaviour of dispersion variance. Measure of asymptotic exponent of dispersion variance shows an increase from grade 1 to grade 3. Preliminary results are encouraging: grades 1 and 3 on one hand and 2 and 3 on the other hand are totally separated. The final proof of an improved grading using this measure will of course require a confrontation with the results of survival studies.

Antigens, Nuclear↗
PubMed1999

Quantitative analysis of cytokeratin network topology in the MCF7 cell line.

BACKGROUND: In the MCF7 human breast cancer cell line, several patterns of cytokeratin networks are observed, depending on the intracellular localization. Our hypothesis is that architectural variations of cytokeratin networks depend on local tensions or forces appearing spontaneously in the cytoplasm. The aim of this work was to discriminate between the different patterns and to quantitate these variations. MATERIALS AND METHODS: Image analysis procedures were developed to extract cytokeratin filament networks visualized by immunofluorescence and confocal microscopy. Two methods were used to segment sets of curvilinear objects. The first, the "mesh-approach," based on classical methods of mathematical morphology, takes into account global network topology. The second, the "filament-approach" (novel), is meant to account for individual element morphology. These methods and their combination allow the computation of several features at two levels of geometry: global (network topology) and local (filament morphology). RESULTS: Variations in cytokeratin networks are characterized by their connectivity, density, mesh structure, and filament shape. The connectivity and the density of a network describe its location in a local "stress-force" zone or in a "relaxed" zone. The mesh structure characterizes the intracellular localization of the network. Moreover, the filament shape reflects the intracellular localization and the occurrence of a "stress-force" zone. CONCLUSIONS: These features permitted the quantitation of differences within the network patterns and within the specific filament shapes according to the intracellular localization. Further experiments on cells submitted to external forces will test the hypothesis that the architectural variations of intermediate filaments reflect intracytoplasmic tensions.

Algorithms↗
PubMed1999

Computerized image analysis of lung expansion patterns in surfactant treated immature newborn rabbits.

This study evaluates the alveolar stereological profile of immature rabbit lungs after treatment with various surfactant preparations, deliberately modified to represent different rates of film adsorption, minimum surface tension and compressibility. Surfactant was isolated from porcine or bovine lungs, and some of these preparations were enriched with dipalmitoylphosphatidylcholine and other synthetic lipids. Alveolar stereological parameters were evaluated in histological lung sections by computerized interactive image analysis. Surfactant treatment enhanced lung expansion, with a significant correlation coefficient between dynamic compliance and alveolar volume density in all groups of animals. Surfactant treatment also significantly increased alveolar image area. Enrichment of surfactant with synthetic lipids did not improve stereological parameters, but caused a significant increase in the coefficient of variation for alveolar perimeter and image area, suggesting more heterogeneous expansion pattern. Experimental evaluation of exogenous surfactants in immature newborn animals should include assessment of alveolar stereological parameters to detect deviations from the uniform expansion pattern seen after treatment with an optimal surfactant substitute.

Animals↗
PubMed1998

Asymptotic fractals in the context of grey-scale images.

The estimation of the fractal dimension in the case of concave log-log Richardson-Mandelbrot plots can be obtained by using asymptotic fractal equations. We demonstrate here, under asymptotic fractal conditions, that additional derivations making use of the Minkowski dilation in grey-scales lead to two asymptotes, one having a slope of 1 and the other a slope of DT-D + 1 (where DT is the topological dimension and D the fractal dimension). The resulting equation offers important advantages. It allows: (i) evaluation of scaling properties of a grey-scale image; (ii) estimation of D without any iteration and (iii) generation of texture and heterogeneity models. We concentrate here on the first two possibilities. Images from cultured cells in studies of cytoskeleton intermediate filaments and kinetic deformability of endothelial cells were used as examples.

Endothelium, Vascular↗
PubMed1998

Application of confocal scanning laser microscopy to 3-D DNA image cytometry of prostatic lesions.

OBJECTIVE: To explore the potential of three-dimensional (3-D) image cytometry for the measurement of DNA content in prostatic specimens using confocal scanning laser microscopy (CSLM) and 3-D image analysis. STUDY DESIGN: Thick tissue slices (100 microns), stained for DNA with chromomycin A3, from four patients with benign hyperplasia, prostatic intraepithelial neoplasia (PIN), well- and poorly differentiated adenocarcinoma of the prostate, were studied. Two different blocks from the same slice were studied for each case. Cell nuclei were segmented automatically. DNA content and nuclear volume were measured. RESULTS: DNA histograms showed a single peak in the diploid range for the hyperplasia and PIN cases. For the case of well-differentiated carcinoma, two peaks were observed, one in the diploid range and one in the tetraploid range. The two peaks were observed on two different blocks of the same tissue slice. Poorly differentiated carcinoma was characterized by an aneuploid distribution. For the cases of PIN and carcinoma, we observed a considerable variation in nuclear volume. CONCLUSION: The results indicate the potential of 3-D image cytometry for the measurement of DNA content in prostatic specimens while preserving tissue architecture.

Cell Nucleus↗
PubMed1997

Spatial distribution of cytoskeleton intermediate filaments during fetal rat hepatocyte differentiation.

The construction of the liver parenchyma throughout fetal development depends on the elaboration of intercellular contacts between epithelial cells and between epithelial and mesenchymal cells. During this time, the spatial distribution of cytokeratins in hepatocytes shows a striking evolution as demonstrated by confocal microscopy and image analysis. In the early stages of fetal rat development, the liver is mainly a hematopoietic organ and hepatocytes represent fewer than 40% of all liver cells. At this time, cytokeratin filaments are scarce and are randomly distributed inside the cytoplasm. A coexpression of desmin and cytokeratin is found in some cells. Intercellular contacts between epithelial and mesenchymal cells are more numerous than between epithelial cells. Later in development, hepatocytes are arranged in a "muralium duplex" architecture (two-cell-thick sheets). Contacts between hepatocytes become more numerous and bile canaliculi become well developed. The density of cytokeratin filaments increases and appears to be very high near the bile canaliculi. In adult liver, hepatocytes are arranged in a "muralium simplex" architecture. Cytokeratin filaments show a symmetrical distribution in relation to the nuclear region. The highest density of filaments is found near the cytoplasmic membrane. Variations of the spatial distribution of intermediate filaments throughout hepatocyte differentiation were investigated in a pilot study using computerized image analysis. We found significant differences between the filament networks in fetal and adult hepatocytes.

Animals↗
PubMed1997

Strong decrease in biotin content may correlate with metabolic alterations in colorectal adenocarcinoma.

Short-chain fatty acids are an important source of energy for colonocytes. One of these is propionate, which is metabolized through carboxylation by propionyl-CoA carboxylase (PCC), an enzyme encoded by 2 genes, PCCA and PCCB. The co-factor of this reaction is biotin, a product of intestinal bacterial metabolism, as is propionate. Despite detailed knowledge about the metabolic effects and physiology of biotin, the relative amounts of this vitamin in normal colonic mucosae and in tumour tissue remains quite unknown. The biotin content in normal and cancerous cells from the distal digestive tract was examined on 10 pairs of tissue specimens of colorectal cancer and adjacent normal mucosae using reflectance in situ hybridization (RISH). Having observed a high biotin content in colon mucosae and a low content in colorectal-cancer cells, we then studied the transcription levels of PCCA and PCCB genes in 9 colorectal cancers and the corresponding mucosae. In all cases, the levels of mRNA were lower in colorectal cancers than in normal mucosae, the decrease being always more marked for PCCB than for PCCA. In normal mucosae and in adenocarcinoma cancer cells, PCCA and PCCB transcription levels were strongly related to the amount of biotin detected, but not to the number of chromosomes 13 (which carries PCCA) or 3 (which carries PCCB).

Adenocarcinoma↗
PubMed1997

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

DNA ploidy provides important information for the evaluation of the prognosis of prostate cancer. For the purpose of DNA cytometry and nuclear measurements, we developed an image processing system for the acquisition and processing of three-dimensional (3D) images based on confocal scanning laser microscopy (CSLM). The advantage of the CSLM is the preservation of the tissue architecture and the possibility of multilabeling. It is possible to determine both individual nuclear features and cellular features and the degree of the spatial heterogeneity of several markers. Special attention was paid to the development of the automatic method for the 3D segmentation of cell nuclei. Thick tissue slides (100 microm), stained for DNA with chromomycin A3, from 4 patients (with benign hyperplasia, prostatic intraepithelial neoplasia (PIN), and well-and poorly-differentiated adenocarcinoma of the prostate), were studied in order to test the practicability of the developed methodology. DNA histograms showed a single peak in the diploid range for the hyperplasia and PIN cases. For the case of well-differentiated carcinoma, 2 peaks were observed, 1 in the diploid range and I in the tetraploid range. The case of poorly-differentiated carcinoma was characterized by an aneuploid distribution. For the cases of PIN and carcinomas, we observed a considerable variation of the volume of nuclei.

Adenocarcinoma↗
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↗
PubMed1997

Dynamic changes in quantitative features of human gastric lesions.

Quantitative DNA, morphometric cellular and nuclear variables were evaluated in surgical biopsies from patients with various gastric lesions: chronic gastritis, chronic ulcers, adenomatous polyps (gastric adenomas), primary carcinomas and their corresponding lymph-node metastases. Paraffin-embedded tissue sections were studied by static cytophotometry (plug method), karyometry (measurements with a graduated eyepiece micrometer of the major and minor axes of the elliptic nuclear profiles, and calculation of profile areas), and measurements of cellular profiles (largest and smallest caliper diameters). Tissue lymphocytes from the same slide were used as diploid controls for the DNA evaluations. An increase of both cellular and nuclear dimensions and DNA content was noted in all pathological tissues, as compared to normal mucosa; the highest values are found in primary gastric carcinomas. A progressive pattern increase of cellular and nuclear dimensions and of DNA content was observed through normal to cancerous tissues, chronic gastritis, chronic ulcers and adenomatous polyps (adenomas). Lymph-node metastases, in our study, had smaller nuclear (cellular) dimensions than primary cancers.

Adenocarcinoma↗
PubMed1997

A three-dimensional dynamic simulation model of epithelial tissue renewal.

OBJECTIVE: Based on the study of existing simulation and mathematical models, a dynamic simulation and three-dimensional (3-D) visualization model of the normal and pathologic architecture of the nasal epithelium is proposed. STUDY DESIGN: Positions, sizes, shapes and orientations of the nuclei, basal lamina and lumen were used for 3-D representation of the epithelium. Static modeling was applied to simulate the normal, metaplastic and dysplastic stages of the nasal epithelium. Then, dynamic modeling, starting from a static representation of a normal or a pathologic stage, used the starting values of cell proliferation parameters to simulate a tissue growth process. The basic hypothesis is that consecutive transformations through stages are mainly due to increased cell growth. RESULTS: Normal tissue renewal and progressive transitions between normality and hyperplasia after exposure to formaldehyde were obtained. CONCLUSION: A model that allows study of the evolution of the 3-D tissue architecture during the preneoplastic process in external epithelium is now available. Improvement of the model, consisting of adding information concerning cell communication, extracellular matrix and cell cycle modeling, should help to formulate and sharpen hypotheses concerning structural and kinetic dynamics of this tissue during the preneoplastic process.

Epithelial Cells↗
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↗