PubMed Health⌕ Search

Biomedical subjects

N Bonnet

Publications and source records attributed to N Bonnet.

At least 55 records · Page 3Linked to original sources
PubMed1998

Analysis of image sequences in fluorescence videomicroscopy of stationary objects.

Fluorescence videomicroscopy allows the temporal behavior of biological specimens to be studied at the cellular level. We describe two types of methods that can be used for extracting quantitative information from image sequences: the modeling approach, which is mainly local, and multivariate statistical analysis, which provides a global approach. The potentials for use of these two methods are illustrated through a simulation example and actual examples dealing with the study of chloride secretion by airway epithelial cells. We define some guidelines for making a choice between these two approaches, bearing in mind that a blend of these two methodologies is also possible.

Cells, Cultured↗
PubMed1998

Optimization of phosphorus localization by EFTEM of nucleic acid containing structures.

Energy Filtered Transmission Electron Microscopy (EFTEM) has been used to study nucleic acids localization in unstained thin sections of virus-infected cells. For this purpose, phosphorus maps (P-maps) have been obtained by applying the N-windows Egerton model for background subtraction from data acquired by a non-dedicated TEM Jeol 1200EXII equipped with a post-column PEELS Gatan 666-9000 and a Gatan Image Filter (GIF-100). To prevent possible errors in the evaluation of elemental maps and thus incorrect nucleic acid localization, we have studied different regions of swine testis (ST) cells with similar local density containing either high concentration of nucleic acids (condensed chromatin and ribosomes) or a very low concentration (mitochondria). Special care was taken to optimize the sample preparation conditions to avoid as much as possible the traditional artifacts derived from this source. Selection of the best set of pre-edge images for background fitting was also considered in order to produce "true P-maps". A new software for interactive processing of images series has been applied to estimate this set. Multivariate Statistical Analysis was used as a filtering tool to separate the "useful information" present in the inelastic image series (characteristic signal) from the "non-useful information" (noise and acquisition artifacts). The reconstitution of the original image series preserving mainly the useful information allowed the computation of P-maps with improved signal-to-noise ratio (SNR). This methodology has been applied to study the RNA content of maturation intermediate coronavirus particles found inside infected cells.

Animals↗
PubMed1998

Neutrophil elastase promotes rapid exocytosis in human airway gland cells by producing cytosolic Ca2+ oscillations.

The molecular and ionic mechanisms responsible for the regulation of mucus exocytosis in human airway gland cells remain poorly defined. To determine whether dynamic changes of intracellular free Ca2+ concentration [Ca2+]i can promote different exocytotic responses, we monitored dynamic changes in [Ca2+]i and secretory granule (SG) exocytosis in individual human tracheal submucosal serous gland (HTG) cells. These changes were in response to exposure of the cells to three different secretagogues associated with airway inflammation and disease: human neutrophil elastase (HNE), histamine, and ATP. Dynamic changes in [Ca2+]i from single cells were determined with Indo-1/AM using quantitative UV laser microspectrofluorometry. The rate of SG exocytosis was measured in single cells by fluorescence videomicroscopy of SG degranulation and by the ELISA method. Exposure of HTG cells to a low concentration of HNE (1.0 microM) caused a high rate of SG exocytosis (52% decrease in the initial quinacrine fluorescence) during the first 8-min stimulation period compared with that observed following exposure of the cells to 100 microM histamine (10% decrease) or 100 microM ATP (6% decrease). In contrast to a rapid and transient rise in [Ca2+]i induced by histamine (1.0-100 microM) and ATP (10-100 microM), HNE (0.01-1 microM) generated asynchronous oscillations in [Ca2+]i over the first 8-min period. Depletion of internal Ca2+ stores with thapsigargin (500 nM) induced a significant reduction (P < 0.01) in the observed increases in [Ca2+]i upon addition of each of the secretagogues, but did not greatly affect the SG exocytotic responses. Interestingly, the removal of extracellular Ca2+ (+5 mM EGTA) significantly reduced (P < 0.01) both the [Ca2+]i increases and the rate of SG exocytosis following exposure to the secretagogues. We also demonstrate that the influx of extracellular Ca2+ and [Ca2+]i oscillations rather than the absolute level of [Ca2+]i regulate the rapid onset and extent of exocytotic responses to HNE in airway gland cells. Taken together, these results provide strong evidence that [Ca2+]i is a critical intracellular messenger in the regulation of exocytosis process in human airway gland cells.

Adenosine Triphosphatases↗
PubMed1998

[A prospective comparative study of coronary angiography and endocoronary ultrasonography in the detection of coronary lesions after cardiac transplantation].

Coronary angiography is the reference method for the detection of coronary disease of the cardiac grafts which threatens the long-term prognosis of cardiac transplantation. The primary results of treatment for slowing, stabilising or even improving coronary transplant disease are encouraging and make necessary the development and evaluation of reliable diagnostic methods. The authors undertook a prospective study of 48 asymptomatic patients with normal graft wall motion between January 1995 and March 1997 to compare the results of coronary angiography and endocoronary ultrasonography. The patients had been transplanted in the 10 years preceding the study. The results of the two methods were concordant in 33 cases (69%) (NS), for the confirmation (9 cases) or the information of coronary transplant disease (24 cases). The results were contradictory in 15 cases (31%): in 12 cases, endocoronary ultrasonography showed signs of coronary disease whereas the coronary angiography was estimated to be normal: in the remaining 3 cases, coronary angiography was abnormal but no signs of coronary disease were found on endocoronary ultrasonography. The specificity of coronary angiographic detection was 89% and therefore very satisfactory, but its sensitivity (43%) was poor. In addition, endocoronary ultrasonography allows analysis of the extension of coronary lesions to unstenosed segments, the quantification of intimal thickening. Therefore, endocoronary ultrasonography should become the reference investigation for coronary disease of cardiac transplants.

Adult↗
PubMed1997

Induction of a cAMP-stimulated chloride secretion in regenerating poorly differentiated airway epithelial cells by adenovirus-mediated CFTR gene transfer.

In cystic fibrosis (CF), the airway epithelium is in the process of injury and regeneration. In the context of the CF gene therapy, we previously reported that regenerating poorly differentiated (PD) cells of human airway epithelium represent preferential cell targets for recombinant adenoviral gene vectors. To define whether PD non-CF and CF epithelial cells possess a functional cystic fibrosis transmembrane conductance regulator protein (CFTR) chloride channel, we analyzed the CFTR expression and the regulation of chloride secretion under cyclic (c)AMP stimulation in these regenerating PD epithelial cells of non-CF and CF airway tissue. Moreover, we studied the effects of CFTR gene transfer mediated by a replication-defective adenovirus containing the wild-type CFTR gene (AdCFTR) on CFTR expression and on cAMP-stimulated chloride secretion. Distribution of the CFTR protein was evaluated in regenerating PD airway cells by light fluorescence microscopy and scanning laser confocal microscopy. The cAMP-mediated regulation of cell membrane chloride secretion was investigated using the whole-cell patch clamp and SPQ (6-methoxy-N-[3-sulfopropyl]quinolinium) techniques. Compared with the absence of CFTR expression and cAMP-regulated chloride secretion in nontransduced regenerating PD cells of either non-CF or CF origin, transduction with AdCFTR induces a CFTR expression and a cAMP-regulated stimulation of the cell membrane chloride secretion in the regenerating PD cells. These results suggest that, out of the context of CF, remodeled and poorly differentiated airway epithelium may present abnormalities in ion transport. Moreover, our data suggest that, in the context of CF gene therapy, adenoviral vectors can be efficient in correcting, at least partially, the chloride secretion defect in the remodeled CF airway epithelium.

Adenoviridae↗
PubMed1997

Alteration of the neonatal pulmonary physiology after total cardiopulmonary bypass.

OBJECTIVES: The purpose of this study was to analyze the mechanisms associated with lung injury after cardiopulmonary bypass and to propose strategies of prevention. METHODS: Thirty-two neonatal piglets underwent 90 minutes of hypothermic cardiopulmonary bypass without aortic cross-clamping. Five experimental groups were defined: group I had standard cardiopulmonary bypass (control), group II received continuous low-flow lung perfusion during cardiopulmonary bypass, group III treatment was similar to that of group I with maintenance of ventilation, group IV received pneumoplegia, and group V received nitric oxide ventilation (30 ppm) after cardiopulmonary bypass. Data drawn from hemodynamic and gas exchange values and muscular and pulmonary tissular levels of adenosine triphosphate (in micromoles per gram) and myeloperoxidase (in international units per 100 mg) were used for comparisons before and 30 and 60 minutes after cardiopulmonary bypass. Pulmonary and systemic vascular endothelial functions were assessed in vitro after cardiopulmonary bypass on isolated rings of pulmonary and iliac arteries. RESULTS: Pulmonary vascular resistance index, cardiac index, and oxygen tension were better preserved in groups II, IV, and V. All groups disclosed a significant decrease in lung adenosine triphosphate levels and an increase in myeloperoxidase activity whereas these levels stayed within pre-cardiopulmonary bypass ranges in muscular beds. Endothelium-dependent relaxation was preserved in systemic arteries but was strongly affected in pulmonary arteries after cardiopulmonary bypass. None of the methods that aimed to protect the pulmonary vascular bed demonstrated any preservation of pulmonary endothelial function. CONCLUSION: Cardiopulmonary bypass results in ischemia-reperfusion injury of the pulmonary vascular bed. Lung protection by continuous perfusion, pneumoplegia, or nitric oxide ventilation can prevent hemodynamic alterations after cardiopulmonary bypass but failed to prevent any of the biochemical disturbances.

Adenosine Triphosphate↗
PubMed1997

Surgical management of atrio ventricular septal defects with normal caryotype.

OBJECTIVE: Atrio ventricular septal defects (AVSD) with normal caryotype represent in average 25% of AVSD. They constitute a more complex group of patients characterized by frequent left sided heart obstructive lesions, raising the problem of the appropriate indications between biventricular and univentricular procedures. METHODS: Sixty-nine consecutive patients, who had AVSD with normal caryotype underwent surgery. According to the anatomical complexity there were 22 intermediate AVSD, 36 complete AVSD and 11 complex AVSD. Associated lesions were present in 68% of the patients including left sided heart anomalies in 57%. According to the size of the left ventricle (LV) evaluated on the LV/RV end diastolic diameter ratio calculated at 2D echocardiogram: right ventricular (RV) dominance was found in 29%; with border line LV in 13 patients and truly hypoplastic LV in 7 patients. Biventricular repair was always favored in case of border line LV and precluded when the LV/RV ratio was less than 0.33. RESULTS: There were 57 biventricular repairs with 10 years actuarial survival of 70 +/- 8% and respectively, 100% in the complex AVSD, 86% in the intermediate AVSD and 51% in the complete AVSD (P = 0.006). The risk factors for biventricular repair were the association to a subaortic stenosis (P = 0.01) and the severity of the mitral valve lesions (P = 0.03) that led to 38% reoperation. There were 12 univentricular palliation with 10 years survival of 66 +/- 14%. The risk factor for univentricular palliation was the association to a severe pre-operative mitral regurgitation (P = 0.005). CONCLUSIONS: Biventricular repair should be precluded in patients presenting with subaortic stenosis. Severe mitral valve anomalies lead to elevated mortality and morbidity with frequent reoperations. Univentricular repair might have larger indications and cardiac transplantation might be considered in patients with truly hypoplastic LV presenting with severe pre-operative AV valve regurgitation.

Adolescent↗
PubMed1997

[A study of the expression of sarcoplasmic reticulum calcium ATP-ase gene in the left ventricle of the sheep's fetus submitted to chronic increased afterload in utero].

The messenger RNA of the cardiac sarcoplasmic reticulum ATP-ase (SERCA-2a) increases in the left ventricle (LV) during ontogenic development but decreases in hypertrophy induced by increased afterload. Because of the frequency of increased left ventricular overload in congenital heart disease, the authors investigated to see if these increases were likely to interfere with the normal ontogenic program of expression of the SERCA-2a gene in the LV of sheep's foetus. A preductal coarctation of the aorta was realised by banding the transverse aorta (AoT) at 93 days' gestation in 9 foetus (CoA) matched with 9 healthy twin foetus (T). All the foetus underwent haemodynamic, anatomical, histological and molecular biological investigations 4 weeks later. The concentration of SERCA-2a mRNA in the LV was measured by hybridation of a Northern blot with a rat DNAc probe normalised with RNAr 18S. A coarctation was observed in all the CoA group and in none of the T. The ratio of LV weight/body weight was increased in 65% of CoA (p < 0.0001). The concentration of SERCA-2a mRNA in the LV was much reduced in CoA (average -28.6%) of the values observed in T (p = 0.003). Left ventricular hypertrophy of the sheep's foetus induced by pathological increases of afterload surpassed or slowed down the physiological ontogenic maturation of expression of the SERCA-2a gene in abnormalities of cardiac pump function.

Animals↗
PubMed1996

Quantification and improvement of the signal-to-noise ratio in a magnetic resonance image acquisition procedure.

A procedure is developed to quantify and improve the signal-to-noise ratio (SNR) of magnetic resonance images. The image SNR is quantified using the correlation function of two independent acquisitions of an image. To test the performance of the quantification, SNR measurement data are fitted to theoretically expected curves. The proposed correlation technique is also used to improve the SNR by estimating the amplitude of the signal spectrum. The technique is applied to a set of MR images, and its performance in terms of gain in SNR, contrast-to-noise ratio (CNR), and resolution loss is compared to that of classical noise filters. The SNR as well as the CNR is improved significantly with minor loss of resolution. Finally, it is shown that the correlation technique can be implemented in a highly efficient way in almost any acquisition procedure of a magnetic resonance imaging system.

Image Processing, Computer-Assisted↗
PubMed1996

Quantitative imaging in electron and confocal microscopies for applications in biology.

Among the large number of topics related to the quantification of images in electron and confocal microscopies for applications in biology, we selected four subjects that we consider to be representative of some recent tendencies. The first is the quantification of three-dimensional data sets recorded routinely in scanning confocal microscopy. The second is the quantification of the textural and fractal appearance of images. The two other topics are related to image series, which are more and more often provided by imaging instruments. The first kind of series concerns electron energy-filtered images. We show that the parametric (modelling) approach can be complemented by non-parametric approaches (e.g., different variants of multivariate statistical techniques). The other kind of series consists of multiple mappings of a specimen. We describe several new tools for the study and quantification of the co-location, with potential application to multiple mappings in microanalysis or in fluorescence microscopy.

Image Processing, Computer-Assisted↗
PubMed1995

Regenerating cells in human airway surface epithelium represent preferential targets for recombinant adenovirus.

To investigate the efficiency of adenovirus-mediated gene delivery in regenerating human respiratory epithelium, we have performed infections with an E1- and E3-deleted type 5 recombinant adenovirus containing the Escherichia coli LacZ reporter gene on different culture models of regenerating human nasal polyp surface epithelium. These models included: (i) an ex vivo organ culture of nasal polyp tissue, (ii) an explant outgrowth cell culture, and (iii) an in vitro wound repair model, on dissociated cells. In ex vivo nasal polyp tissue, transduced cells were not detected in normal pseudostratified areas, but were found in areas of the surface epithelium with a morphology reminiscent of regenerating airway tissue. In the explant outgrowth cell culture, adenovirus-infected cells were preferentially detected at the periphery of the outgrowth. These transducible epithelial cells, representative of epithelial cells present in vivo during the process of surface airway epithelium regeneration, were shown to be migrating and poorly differentiated cells, which were proliferating or not. In the in vitro wound repair model, the efficiency of cell transduction was much higher in cells present in the wound area than in those far from the wound area. These results indicate that regenerating cells from human airway surface epithelium represent preferential targets for transgene expression, and suggest that efficiency of CFTR gene transfer by recombinant adenovirus vectors may be higher in regenerating CF airway mucosa than in normal tissue. However, since these cells do not show endogenous CFTR expression, the relevance of their preferential transduction for the functional correction of the ion transport defect in cystic fibrosis needs further investigations.

Adenoviridae↗
PubMed1995

Three-dimensional co-location of RNA polymerase I and DNA during interphase and mitosis by confocal microscopy.

The relative three-dimensional co-location of RNA polymerase I (RPI) and DNA was studied using confocal laser scanning microscopy during interphase and all the steps of mitosis in human cancerous cells. For each step of the cell cycle, immunolabeled RPI molecules and DNA specifically stained with chromomycin A3 were simultaneously imaged at high resolution through numerous optical sections. Then, all the data obtained were used to generate transverse sections, anaglyphs and volumic representations, which are all prerequisite approaches to a representative study of the three-dimensional organization of the nucleolus and the mitotic chromosomes. Our results indicated that in the interphasic nuclei, in which DNA is organized as a regular 3-D network, RPI was present within numerous irregular spheres arranged as several twisted necklaces. During metaphase, RPI labeling was segregated into pairs of spheres and typical crescent-shaped structures; both were centrally located within the set of chromosomes. During anaphase and telophase, a typical central and symmetric arrangement of labeled structures was systematically seen among the decondensing chromosomes, arranged as a regular cylinder and as a hollow half-sphere, respectively. This typical 3-D organization of structures containing RPI relative to DNA is another strong example of the non-random organization of the genome during interphase and mitosis.

Carcinoma, Squamous Cell↗
PubMed1994

Improvements in biological X-ray microanalysis: cryoembedding for specimen preparation and multivariate statistical analysis for data interpretation.

For biological X-ray microanalysis, cryoembedding (CE) combined with cryofixation (CF) and cryodehydration (CD) was already proposed as an alternative method to freeze-dried cryosections in 1984 by Wróblewski and Wroblewski. CD by freeze-drying (FD) is usually recommended because it provides better retention of diffusible elements. CD by freeze-substitution (FS) has the advantage of being simpler, giving more reproducible preservation of ultrastructure and causing fewer problems for resin infiltration. We have increased the retention of diffusible elements by using home-made devices for CS and CE in the new Lowicryl K11M and HM23 resins. These resins allow samples to be kept at a maximum temperature of 213K and 193K respectively. Application of multivariate statistical analysis (MSA) to X-ray data (spectra and maps) allows the study of correlations between the analyzed elements in different nuclear areas and in the cytoplasm. The "factorial" images, obtained with MSA, display the compartments of strong correlation between P and K (nucleic acids) and the compartments of strong correlation between S and K (proteins). We suggest that the future application of MSA methods will provide increased knowledge of the physio-pathological compartmentation of diffusible elements at the subcellular level.

Animals↗
PubMed1994

Multivariate statistical analysis applied to X-ray spectra and X-ray mapping of liver cell nuclei.

Principal Components Analysis (PCA) and Factorial Analysis of Correspondence (FAC), two Multivariate Statistical Analyses (MSA), were applied to the analysis of X-ray data. MSA are descriptive methods which graphically display the correlations and anticorrelations between a large number of elements. Series of X-ray spectral data and X-ray maps obtained from rat liver were analyzed with reference to the diffusible elements Na, Mg, Cl, K and Ca and also P and S. By using an in situ precipitation method, the pyroantimonate method, it was found that the free, precipitable cations Na+, Mg2+, and Ca2+ are, in the nuclei, mainly distributed throughout the nucleoplasm. Images obtained from FAC allow those areas rich in nucleic acids to be displayed as areas with a strong anticorrelation between P and Sb. In cryoprocessed tissues, the evaluated wet mass-fraction of diffusible elements corresponds to physiological values of total amounts (free and complexed). PCA makes it possible to graphically display the correlation between P and K in chromatin and nucleolus, the correlation between K, Cl and S in cytoplasm and nucleoplasm and the observation of two populations of nuclei according to different Na, Mg and K concentrations. Factorial images obtained from FAC allow those areas rich in nucleic acids to be displayed as areas with a strong correlation between P and K. Similarly those areas rich in proteins are displayed as areas with a strong correlation between S and K.

Animals↗
PubMed1993

Quantitative analysis and cartography in scanning electron microscopy: application to the study of bacterial adhesion to respiratory epithelium.

This paper describes the coupling between a scanning electron microscope (SEM) and an image analysis workstation. The system was designed in order to drive the SEM and to analyse any sample. It allows automatic (edge detection) or semiautomatic (pointing, marking, drawing) object detection. Two types of data can be obtained: (1) topographical information, such as the location of the object within a region of interest drawn at any magnification of the microscope, or (2) quantitative data, such as morphometric characteristics of objects. In addition, high resolution maps of the section, regions of interest, and objects can be obtained with a laser printer. This software was first applied to quantitate the adhesion of the bacteria Pseudomonas aeruginosa to human respiratory epithelial cells in culture. P. aeruginosa was shown associated with ciliated cells. The second application concerned the study of the distribution of specific carbohydrate residues at the surface of the respiratory cells. The gal residues were revealed using the lectin Ricinus communis agglutinin II, adsorbed to colloidal gold particles. A relationship between the presence of adherent bacteria and labelling was shown.

Bacterial Adhesion↗
PubMed1993

Immunodetection of low concentrations of ovalbumin in cryoprocessed quail oviduct cells by semi-automatic quantitative analysis.

In a previous paper (Quintana et al, Biol Cel 72, (1991) 167-180) we reported the anti-ovalbumin antibody immunolabeling in the cytoplasm of tubular gland cells from cryofixed, cryosubstituted and acrylic-resin embedded quail oviduct. To confirm these preliminary visual observations, we have carried out a semi-automatic quantitative study of immunogold labeling. The quantitative results confirm the previous data. In addition, we found a significant higher immunolabeling with low temperature Lowicryl K11M embedding procedure as compared with high temperature LR White embedding.

Animals↗