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C Souchier

Publications and source records attributed to C Souchier.

At least 19 recordsLinked to original sources
PubMed1996

Evaluation of five green fluorescence-emitting streptavidin-conjugated fluorochromes for use in immunofluorescence microscopy.

Fluorescein isothiocyanate (FITC) is largely used in immunofluorescence methods. We propose to analyse the quality of some recent fluorochromes using image analysis. Fluorochromes tested include FITC and dichlorotriazinylaminofluorescein (DTAF), dipyrrometheneboron difluoride (BODIPY), Rhodol Green and cyanine 2. RAMOS cells were immunolabelled against the proliferating cell nuclear antigen (PCNA) revealed by the biotin-streptavidin technique. Slides were mounted in anhydrous glycerol or in buffered glycerol (pH 7.0 or pH 8.5). No antifading medium was added. Cell fluorescence emission intensity and bleaching characteristics were measured. Rhodol Green exhibited the highest fluorescence intensity and the best photobleaching resistance. Although BODIPY also resisted well during the photobleaching assay, its fluorescence intensity was weak. FITC, DTAF and cyanine 2 showed intermediate fluorescence intensity and a fast decay of fluorescence. Among the green emitting fluorochromes tested, Rhodol Green appeared to be the best.

Bacterial Proteins↗
PubMed1996

Mapping of corticotropic cells in the normal human pituitary.

We accomplished the first mapping of corticotropic cells in the whole human adult pituitary. Corticotropic cells were identified by immunocytochemistry (ICC) and quantified by image analysis on 12 pituitaries obtained from people who had died suddenly. An overall view of each pituitary was given by 15-21 sections (mean 18 sections) at 300-micron intervals on six slides. Each section was systematically treated by indirect immunoperoxidase using an anti-ACTH[17-39] polyclonal antiserum. All the measures were done with a x 6.3 objective lens, each field (0. 5 mm2) being considered as the unit area. The mean pituitary density (surface of labeled cells/total surface) of corticotropic cells (9.5 +/- 3.0% per 0. 5 mm2) is significantly higher in men (11.5 +/- 5.1%) than in women (7.0 +/- 1.3%). This difference is due to an inverse relationship between the corticotropic cell density and the weight of the pituitary, which is higher in women than in men. The mean diameter of corticotropic cells is 14.9 micron and their total number per pituitary is approximately 10(7) cells. We confirmed that the spatial distribution of corticotropic cells is nonuniform: they are mainly distributed in the anteromedian part of the anterior lobe. In addition, our results demonstrated that the inferior part of the pituitary contained three times more corticotropic cells than the superior part (mean density 18.0% vs 6.0%) and the anterior part twice as many as the posterior part (mean density 12.3% vs 6.8%). On the horizontal plane, the pituitary was divided into eight zones, in which the mean of area was 2.5-21.0%. The maximal cell density may reach 40-60%. The use of this map should help the pathologist to recognize if there is corticotropic hyperplasia in a small pituitary fragment surgically removed from a patient with Cushing's disease. On the basis of this study, we put forward some criteria for diagnosing corticotropic hyperplasia.

Adrenocorticotropic Hormone↗
PubMed1996

Quantitative evaluation of the distribution of cell density. Application to human pituitary corticotroph cells.

OBJECTIVE: An image analysis program was developed to analyze cell density in a whole organ. The method was applied to the evaluation of adrenocorticotrophic hormone (ACTH-storing cells in normal human anterior pituitary in order to help the pathologist to evaluate hyperplasia on small surgical biopsies. STUDY DESIGN: ACTH cells were labeled using the immunoperoxidase technique. Data were systematically acquired on a composite image that made possible two-dimensional cell pattern representation in sequential sections of the organ and measurements of cell density in selected histologic parts. RESULTS: ACTH-storing cells were located more in the anterior than posterior part and more in the lower than in the upper pituitary part. The probability of observing high-density value even in normal cases was precisely evaluated zone by zone. Moreover, a model was proposed to evaluate mean cell density from small surgical pieces. CONCLUSION: The same methodology could be applied to other endocrine cell types in the thyroid, pancreas or intestinal tract when hyperplasia is considered the cause of certain clinical syndromes.

Adrenocorticotropic Hormone↗
PubMed1995

Methods for cell proliferation analysis by fluorescent image cytometry.

Methods were developed for multimodal microscopic image analysis in order to identify and analyze one cell type under various microscopic conditions. Our purpose was to quantify both total DNA content using propidium iodide (PI) stain and S-phase fraction using the bromodeoxyuridine (BrdUrd) incorporation technique in cell population subsets. The model chosen was plasma cells in bone marrow triply labelled with fluorescein isothiocyanate (FITC) for intracytoplasmic immunoglobulins, with amino-methylcoumarin-acetate (AMCA) for BrdUrd, and with PI for DNA. Image analysis included three phases. First, plasma cells were recognized on FITC images, and the centroid positions were stored. Second, plasma cell nuclei were geodesically reconstructed from these stored positions using PI images in which DNA content was measured, and the nuclear mask outlines were stored. Third, BrdUrd incorporation level of plasma cells was measured on AMCA images inside PI nuclei masks and stored. Image DNA vs. BrdUrd scatterplots were obtained for cells selected according to the expression of intracytoplasmic immunoglobulin. Thus, both ploidy and proliferation could be independently evaluated on a subset of the cellular population.

Animals↗
PubMed1995

Cdk1 is a marker of proliferation in human lymphoid cells.

To better understand the relationship between the proliferation of human lymphoid cells and the expression of cdk1, a catalytic subunit of the histone H1 kinase (H1K), we examined its mRNA and protein content in 3 B-cell lines: Ramos, Reh-6 and IARC 963. Cells were elutriated according to their position in the cell cycle. Cell fractions were analyzed for cdk1 mRNA and protein cellular content by Northern blot and immunoblot, respectively, as well as for H1K activity. Both mRNA and protein amounts and H1K activity varied according to cell cycle phase, the lowest values being observed in G1-enriched fractions. For comparison, elutriated fractions were also tested for the expression of cdk2 and cdk4 proteins. Both showed some variations among fractions, but they were less clear than those of cdk1. We also tested 29 samples of lymphoid neoplastic and non-neoplastic tissues for proliferative activity (percentage of S and G2/M cells estimated by flow cytometry) and expression of cdk1, cdk2 and cdk4 proteins. We found a significant correlation between the percentage of cells in S or S + G2/M phases and cdk1 protein content but not cdk2 or cdk4 content. We conclude that cdk1 expression in human lymphoid cells varies during the cell cycle at both mRNA and protein levels.

Biomarkers↗
PubMed1995

Subregional mapping of the human gonadotropin-releasing hormone receptor (GnRH-R) gene to 4q between the markers D4S392 and D4S409.

We have isolated nine yeast artificial chromosomes (YACs) containing the gene that encodes the human gonadotropin-releasing hormone receptor (GnRH-R) gene by screening the YAC library of the Centre d'Etude du Polymorphisme Humain (Hôpital Saint-Louis, Paris, France) by the use of the polymerase chain reaction. We defined the location of the GnRH-R gene relative to 4q microsatellite markers D4S392 and D4S409. The genetic positions of these markers on chromosome 4 are 76 and 77 cM, respectively. This location was further established by chromosomal in situ hybridization.

Base Sequence↗
PubMed1995

Expression of cell cycle regulatory proteins in chronic lymphocytic leukemias. Comparison with non-Hodgkin's lymphomas and non-neoplastic lymphoid tissue.

The expression of certain cell cycle regulatory proteins: cdk1, cdk2, cdk4, cyclin A, cyclin B, cyclin E, Bcl2 and PCNA was examined in peripheral blood lymphocytes (PBL) from 25 cases of chronic lymphocytic leukemias (CLL) in order to analyze a possible cell cycle involvement of CLL lymphocytes. For comparison, we also studied the expression of these proteins in: 23 samples of non-Hodgkin's lymphoma (NHL) tissue of different histological types, 10 samples of non-neoplastic lymphoid tissue (NLT), non-stimulated PBL (NS-PBL) and PHA-stimulated PBL (PHA-PBL) from three healthy donors. Samples were lysed and proteins were resolved on polyacrylamide gel followed by Western blot. The expression of cdk4 and cyclin E, both known to act in early cell cycle stage, was approximately on the same level in all groups of lymphoid pathology examined. In particular, we found that that 19 out of 24 CLL cases were cyclin E positive and all but one were cdk4 positive, ie they expressed these markers over twice the level of non-stimulated healthy PBL. The cdk1 expression was above the level seen in NS-PBL in 14 (56%) cases, but the average expression was significantly lower than in the other tissues examined, including low-grade lymphomas. Cdk2 expression was comparable in CLL and in low malignancy grade NHL, but weaker than in other NHL and in NLT. Cyclins A and B, normally observed in advanced cell cycle phases, were not seen in any CLL case. The presence of cdk4 and cyclin E in the blood cells of the majority of CLL cases studied, as well as cdk1 and cdk2 in some cases, indicate that the CLL cells are not quiescent, but are blocked in an early stage of the G1 cell cycle phase, and/or that the expression of these proteins is pathologically deregulated.

Adult↗
PubMed1995

[Importance of image analysis in immunoelectron microscopy. Visualization of heterogeneous reactivity of sera from patients with linear IgA dermatitis bullosa with structures of the dermo-epidermal junction].

Interpretation of electron microscopy pictures illustrating immunoperoxidase labelling is often difficult for non-specialists. We used an image analysis system to facilitate the comprehension of the results obtained with indirect immunoperoxidase technique on normal human skin. Sera from patients with linear IgA bullous dermatosis (LABD) were selected on the basis of an exclusive IgA class reactivity with the dermo-epidermal junction. Various patterns of epidermal labelling could be demonstrated with 82% of the immunoreactive sera. Only 2 out of 11 sera reacted with sub-lamina densa antigens. These results confirm the heterogeneous nature of diseases diagnosed, according to the present criteria, as LABD.

Epidermis↗
PubMed1995

Quantitative immunochemistry of endothelial cells in cutaneous tissue.

In vivo endothelial cell exploration in human pathology has been limited by the lack of a suitable procedure for quantification of endothelial molecule expression. The objective of the present study was the development of a precise, simple and rapid image analysis method to quantify dermis endothelial cells and associated molecules. Cutaneous tissue sections were labelled by immunoperoxidase procedure using a monoclonal anti-von Willebrand factor (vWF) antibody, which identifies endothelial cells. The image analysis programme was tested to quantify final reaction deposit (DAB) with reference to different anti-vWF antibody concentrations. The entire biopsy section was acquired, field by field. Analysis was restricted to three dermis zones geodesically defined from epidermis. Optical density and area measurements were performed in whole area and in positive areas. This work demonstrated the feasibility of endothelial cell study assisted by image analysis. Surface labelled by anti-vWF antibody at saturating concentration may be considered as an endothelial reference surface, and thus may be useful for future quantification of other endothelial molecules.

Antibodies, Monoclonal↗
PubMed1995

Transverse-axial tubular system in guinea pig ventricular cardiomyocyte: 3D reconstruction, quantification and its possible role in K+ accumulation-depletion phenomenon in single cells.

K+ accumulation-depletion (AD) phenomena were found in single guinea pig ventricular myocytes using the patch-clamp method in whole cell configuration. We suggest that the cardiomyocyte transverse-axial tubular system (TATS) lumen is the restricted extracellular space where the K+ AD could take place. A three-dimensional (3D) reconstruction of the TATS in a cardiomyocyte segment from serial ultrafine sections was made by three-dimensional isosurface rendering and quantitative data were obtained from the image processing. This original approach of the TATS intricated network gave a new vision of this membrane system; moreover, quantitative data about the tubular membrane importance (52.6% of the total plasma membrane) and its surface area versus the tubular volume fraction (STATS/VTATS = 13.5 micron 2/micron 3) would fit in the electrophysiological results. The hypothesis whereby this 'extracellular' compartment could play, in single cells, a role as important as that of narrow clefts in the whole heart is discussed.

Animals↗
PubMed1994

Choice of fixation and denaturation for the triple labelling of intra-cytoplasmic antigen, bromodeoxyuridine and DNA. Application to bone marrow plasma cells.

A triple staining method of intra-cytoplasmic antigen, bromodeoxyuridine (BrdU), and DNA for fluorescence image analysis is described. Several kinds of fixation and DNA denaturation methods were tested to obtain a technique suitable for heterogeneous tissues. The model chosen was the analysis of plasma cells in bone marrow. The fluorochromes used were fluorescein isothiocyanate (FITC) for intra-cytoplasmic antigens (light chain immunoglobulins), aminomethylcoumarin acetic acid (AMCA) for BrdU, and propidium iodide (PI) for DNA. The quality of the staining was analysed according to: (1) cell morphology with a good preservation of the chromatin structure, (2) intensity of light chains and of BrdU labelling, and (3) the quality of DNA staining judged from a DNA histogram. For most of the analysed tissues, fixation with methanol followed by 0.5% paraformaldehyde and denaturation by an NaOH concentration adapted to the tissue gave good results. However, in our model fixation by methanol, followed by methanol/acetic acid and denaturation of DNA by 0.03 N NaOH was the solely satisfactory technique. A good correlation (P < 0.001) was found with the plasma cell BrdU labelling index obtained with our reference immuno-enzymatic technique. Quantification of DNA content showed a satisfactory G1 peak coefficient of variation (CV) in diploid cells and a 4C to 2C ratio equal to 2. With this technique, the nuclear and cytoplasmic structures of both myeloid cells and plasma cells were well preserved, while their sensitivity to DNA denaturation was quite different.

Antigens↗
PubMed1994

Laser scanning confocal microscopy and quantitative microscopy with a charge coupled device camera improve detection of human papillomavirus DNA revealed by fluorescence in situ hybridization.

Epithelial cervical CaSki, SiHa and HeLa cells containing respectively 600 copies of human papillomavirus (HPV) DNA type 16, 1-2 copies of HPV DNA type 16 and 10-50 copies of HPV DNA type 18 were used as model to detect different quantities of integrated HPV genome. The HPV DNA was identified on cell deposits with specific biotinylated DNA probes either by enzymatic in situ hybridization (EISH) or fluorescence in situ hybridization (FISH) involving successively a rabbit anti-biotin antibody, a biotinylated goat anti-rabbit antibody and streptavidin-alkaline phosphatase complex or streptavidin-fluorescein isothiocyanate complex. With brightfield microscopy and EISH, hybridization spots were observed in CaSki and HeLa cells but hardly any in SiHa cells. With fluorescence microscopy and FISH, hybridization spots were clearly seen only on CaSki cell nuclei. In an attempt to improve the detection of low quantities of HPV DNA signals revealed by FISH, laser scanning confocal microscopy (LSCM) and quantitative microscopy with an intensified charge coupled device (CCD) camera were used. With both LSCM and quantitative microscopy, as few as 1-2 copies of HPV DNA were detected and found to be confined to cell nuclei counterstained with propidium iodide. Under Nomarski phase contrast, a good preservation of the cell structure was observed. With quantitative microscopy, differences in the number, size, total area and integrated fluorescence intensity of hybridization spots per nucleus were revealed between CaSki, SiHa and HeLa cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Nucleus↗
PubMed1994

Fluorescence image analysis of plasma cells in monoclonal gammopathies.

The DNA content of plasma cells was estimated by fluorescence image analysis in monoclonal gammopathies. Image analysis provides the possibility of measuring the bone marrow plasma cell population separately from the other myeloid cells. Plasma cells were labeled, identified and then relocated for propidium iodide intensity measurements. Patients with DNA malignancy grade > 0.20 or with DNA index > 1.15 had a poorer survival rate than did patients with a low DNA malignancy grade or DNA index. DNA malignancy grade is based on the variance of DNA values around the normal diploid peak, as proposed by Böcking. Of all the biologic and clinical parameters investigated, the serum beta 2 microglobulin level was the most important prognostic factor for survival in the multivariate regression analysis, and the DNA entropy index provided the most significant additional information.

Aged↗
PubMed1993

Detection of human papillomavirus DNA in CaSki and HeLa cells by fluorescent in situ hybridization. Analysis by flow cytometry and confocal laser scanning microscopy.

CaSki and HeLa cell lines, isolated from human uterine carcinomas and containing integrated human papillomavirus (HPV) DNA type 16 and 18, respectively were used to evaluate the sensitivity of HPV-DNA detection on suspended cells by fluorescent in situ hybridization using flow cytometry and on corresponding cell deposits using confocal laser scanning microscopy (CLSM). HPV DNAs were detected in cell suspensions with biotinylated DNA probes and revealed with a three-step technique: a rabbit antibiotin antibody, a biotinylated goat anti-rabbit antibody and a streptavidin-fluorescein isothiocyanate complex. By flow cytometry, HPV DNA was detectable only in CaSki cells which contained about 600 copies of HPV DNA per cell. In HeLa cells, with only 20-50 copies of HPV DNA, flow cytometry could not detect HPV DNA, whereas CLSM permitted visualization of fluorescent labelling of HPV DNA hybrids. Furthermore, CLSM showed good preservation of cellular morphology and the nucleus was clearly recognizable after fluorescent in situ hybridization and counterstaining with propidium iodide. Moreover, this examination confirmed that the fluorescent foci were specifically confined to the cell nuclei.

DNA, Viral↗
PubMed1993

Comparison of anti-fading agents used in fluorescence microscopy: image analysis and laser confocal microscopy study.

To ascertain the ability of commercial and home-made anti-fading media to reduce the decrease of fluorescein isothiocyanate (FITC) fluorescence, we studied the bleaching characteristics of FITC-stained Reh 6 cells mounted in buffered glycerol and in anti-fading media. We measured the intensity of fluorescence over time with a confocal laser scanning microscope and a standard epifluorescence microscope coupled to an image analysis system. Most of the anti-fading media effectively retard fading but each has drawbacks. Better results were obtained with media containing p-phenylenediamine (solutions in buffered glycerol, Vectashield, Fluorstop). However, Mowiol, Slowfade, n-propyl gallate (20 g/liter) were also effective in retarding fading. Most of them, except Mowiol, reduced fluorescence intensity. We concluded that the choice of anti-fading medium would depend on the desired results: a slower decay of fluorescence despite an initial quenching of fluorescence or a lower retardant effect with no decrease in initial fluorescence intensity. Moreover, the combination of Mowiol with another anti-fading medium may be a useful compromise when a strong retardant effect is required without marked quenching of the initial fluorescence.

Animals↗
PubMed1993

Heterogeneity of islet cell cytoplasmic antibodies and autoantibodies to glutamate decarboxylase in relatives of patients with type 1 (insulin-dependent) diabetes.

The aims of our study were to correlate the specificities of islet cell cytoplasmic autoantibodies (ICA) observed in 14 first-degree relatives of diabetic patients with the development of clinical diabetes. To detect whether ICAs recognized in an homogeneous fashion the two major islet cell components, namely beta and alpha cells, the number of islet cells simultaneously recognized by ICAs and anti-glucagon antibodies was determined using a four layer indirect immunofluorescence technique and confocal laser microscopy. The different types of pattern, either whole islet cell staining (type I) or beta cell predominant staining (type II), was then correlated to the presence of autoantibodies to glutamate decarboxylase (GAD), as well as to metabolic outcome. Eight relatives (57.2%) had the type I pattern and 6 (42.8%) the type II pattern. Type 1 diabetes occurred in all ICA positive relatives with the type I pattern and in only 2 out of the 6 relatives with the type II pattern. Interestingly, this later pattern was significantly associated with the presence of autoantibodies to GAD although 2 relatives with antibodies with the type I pattern were also positive. Although more effective for 5 sera with the type II pattern, preincubation with rat brain homogenate significantly decreased the titres of 3/6 sera with the type I pattern. Altogether, our results suggest that autoimmunity to beta cells may be associated with antibodies directed to separate autoantigens with different cellular distributions. From these observations, additional autoantigens distinct from glutamate decarboxylase are also certainly involved.

Adolescent↗
PubMed1993

Image analysis of dendritic cells in the human fetal thymus.

The distribution and evolution of thymic dendritic cells (DC) were studied during human ontogeny. Immunochemical techniques were used to detect S100 protein-positive cells on fetal thymus sections, at different post-fecundation (PF) ages; an image analysis of these positive cells was then carried out. Variations in the percentage of these cells in the medulla were determined according to age: the higher percentages were seen at 12 and 16 weeks PF. Dendritic cells were present at an early stage in the thymic rudiment (7 weeks PF), a finding consistent with an origin of these cells in the fetal liver, and with the possible existence of local attraction and/or maturation factors. The expression of the CD54 adhesion molecule revealed the existence of particular interactions between DC and lymphocytes in the medullary area, and at the cortico-medullar junction. Diffuse CD11a (LFA-1) expression on lymphocytes was consistent throughout gestation. The monocyte/macrophage markers (CD11b, CD14) and the reaction of non-specific esterases showed that the distribution pattern of these cells differed from the DC pattern. These ontogenic data are related to the significant role played by DC throughout the different stages of thymopoiesis.

Antigens, CD↗