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Upstream binding transcription factor (UBTF) is a co-regulator of RNA polymerase I by constituting an initiation complex on rRNA genes. UBTF plays a role in rDNA bending and its maintenance in "open" state. It exists as two splicing variants, UBTF1 and UBTF2, which cannot be discerned with antibodies raised against UBTF. We investigated the ultrastructural localization of each variant in cells synthesizing GFP-tagged UBTF1 or UBTF2 by using anti-GFP antibodies and pre-embedding nanogold strategy. Detailed 3D distribution of UBTF1 and 2 was also studied by electron tomography. In control cells, the two isoforms are very abundant within fibrillar centers, but their repartition strongly differs. Electron tomography shows that UBTF1 is disposed as fibrils that are folded in coils whereas UBTF2 is localized homogenously, preferentially at their cortical area. As UBTF is a useful marker to trace rDNA genes, we used these data to improve our previous model of 3D organization of active transcribing rDNA gene within fibrillar centers. Finally, when rRNA synthesis is inhibited during actinomycin D treatment or entry in mitosis, UBTF1 and UBTF2 show a similar distribution along extended 3D loop-like structures. Altogether these data suggest new roles for UBTF1 and UBTF2 isoforms in the organization of active and inactive rDNA genes.

Background and aim Technological advances in the field of biological imaging now allow multi-modal studies of human embryo anatomy. The aim of this study was to assess the high magnetic field μMRI feasibility in the study of small human embryos (less than 21 mm crown-rump) as a new tool for the study of human descriptive embryology and to determine better sequence characteristics to obtain higher spatial resolution and higher signal/noise ratio. Methods Morphological study of four human embryos belonging to the historical collection of the Department of Anatomy in the Faculty of Medicine of Reims was undertaken by μMRI. These embryos had, successively, crown-rump lengths of 3 mm (Carnegie Stage, CS 10), 12 mm (CS 16), 17 mm (CS 18) and 21 mm (CS 20). Acquisition of images was performed using a vertical nuclear magnetic resonance spectrometer, a Bruker Avance III, 500 MHz, 11.7 T equipped for imaging. Results All images were acquired using 2D (transverse, sagittal and coronal) and 3D sequences, either T 1 -weighted or T 2 -weighted. Spatial resolution between 24 and 70 μm/pixel allowed clear visualization of all anatomical structures of the embryos. Conclusion The study of human embryos μMRI has already been reported in the literature and a few atlases exist for educational purposes. However, to our knowledge, descriptive or morphological studies of human developmental anatomy based on data collected these few μMRI studies of human embryos are rare. This morphological noninvasive imaging method coupled with other techniques already reported seems to offer new perspectives to descriptive studies of human embryology.

The colonization pattern of Vitis vinifera L. by Burkholderia phytofirmans strain PsJN was determined using grapevine fruiting cuttings with emphasis on putative inflorescence colonization under nonsterile conditions. Two-week-old rooted plants harbouring flower bud initials, grown in nonsterile soil, were inoculated with PsJN:gfp2x. Plant colonization was subsequently monitored at various times after inoculation with plate counts and epifluorescence and/or confocal microscopy. Strain PsJN was chronologically detected on the root surfaces, in the endorhiza, inside grape inflorescence stalks, not inside preflower buds and flowers but rather as an endophyte inside young berries. Data demonstrated low endophytic populations of strain PsJN in inflorescence organs, i.e. grape stalks and immature berries with inconsistency among plants for bacterial colonization of inflorescences. Nevertheless, endophytic colonization of inflorescences by strain PsJN was substantial for some plants. Microscopic analysis revealed PsJN as a thriving endophyte in inflorescence organs after the colonization process. Strain PsJN was visualized colonizing the root surface, entering the endorhiza and spreading to grape inflorescence stalks, pedicels and then to immature berries through xylem vessels. In parallel to these observations, a natural microbial communities was also detected on and inside plants, demonstrating the colonization of grapevine by strain PsJN in the presence of other microorganisms.

Glutamate excitotoxicity has been involved in the pathophysiology of epilepsy. Normal functioning of glutamate transporters clears the synaptically released glutamate to prevent excitotoxic neuronal death. Using densitometric immunohistochemical analysis, we examined the temporal expression of the neuronal glutamate transporter (EAAC1) in the lithium-pilocarpine rat model of temporal lobe epilepsy. During the acute period of lithium-pilocarpine-induced status epilepticus, EAAC1 transporter expression increased in the pyramidal neurons of cornus ammonis (CA)1, CA2 and CA3 (fields of the hippocampus), in dentate gyrus (DG) granule cells and in olfactory tubercle (Tu). During the latent period, EAAC1 expression was strongly expressed in the DG granular and molecular layers, Tu, cerebral cortex and septum, and went back to control levels in CA1, CA2 and CA3 layers. The overexpression of EAAC1 occurred mainly in structures prone to develop Fluoro-Jade-B-positive degenerating neurons. It is, however, not clear to what extent the overexpression of EAAC1 contributes to epileptogenesis and in which area it may represent a preventive or compensatory or response to injury.

Neuroendocrine tumours (NETs) of the lung represent a wide spectrum of phenotypically distinct entities, with differences in tumour progression and aggressiveness. The redistribution and/or the loss of various cell adhesion molecules, such as the E-cadherin-catenin complex, play a predominant role in carcinogenesis and in tumour invasion. Moreover, mutations in exon 3 of the beta-catenin gene, the adenomatous polyposis coli (APC) gene or the E-cadherin genes were previously found to result in intracytoplasmic and/or nuclear beta-catenin protein accumulation, activating nuclear transcription of target genes involved in tumour progression. In the present study, the distribution of the components of this E-cadherin-catenin complex has been investigated by immunohistochemistry and an attempt has been made to correlate the abnormal expression pattern with the eventual detection of mutations in the corresponding genes. This study included 27 primary NETs of the lung, with nine typical carcinoids (TCs), three atypical carcinoids (ACs), and 15 large cell neuroendocrine carcinomas (LCNECs). The E-cadherin-catenin complex remained expressed in most of these lung tumours, but with a cytoplasmic and/or nuclear redistribution of beta-catenin, E-cadherin, and alpha-catenin; abnormal positive immunoreactivity was observed in 24 (88.9%), in 21 (80.8%), and in 20 (76.9%) NETs, respectively. In the great majority of cases, there was a good correlation between the expression of these three proteins, but no significant association with histological classification or TNM stage. Thus, E-cadherin-complex redistribution cannot be considered a prognostic marker in NET of the lung. Of particular interest was the frequent focal beta-catenin nuclear immunostaining (55.5% in total), which was also unrelated to histological type or TNM stage. However, this study failed to detect any mutation in exon 3 of the beta-catenin gene, in the APC gene or in the E-cadherin gene. These data suggest another mechanism of regulation of beta-catenin in these tumours.

Cell spreading and migration associated with the expression of the 92-kD gelatinase (matrix metalloproteinase 9 or MMP-9) are important mechanisms involved in the repair of the respiratory epithelium. We investigated the location of MMP-9 and its potential role in migrating human bronchial epithelial cells (HBEC). In vivo and in vitro, MMP-9 accumulated in migrating HBEC located at the leading edge of a wound and MMP-9 expression paralleled cell migration speed. MMP-9 accumulated through an actin-dependent pathway in the advancing lamellipodia of migrating cells and was subsequently found active in the extracellular matrix (ECM). Lamellipodia became anchored through primordial contacts established with type IV collagen. MMP-9 became amassed behind collagen IV where there were fewer cell–ECM contacts. Both collagen IV and MMP-9 were involved in cell migration because when cell–collagen IV interaction was blocked, cells spread slightly but did not migrate; and when MMP-9 activation was prevented, cells remained fixed on primordial contacts and did not advance at all. These observations suggest that MMP-9 controls the migration of repairing HBEC by remodeling the provisional ECM implicated in primordial contacts.

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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 (P0.01) 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 (P0.01) 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.

International audience; Metaphase nucleolar organizer regions (NORs), one of four types of chromosome bands, are located on human acrocentric chromosomes. They contain r-chromatin, i.e., ribosomal genes complexed with proteins such as upstream binding factor and RNA polymerase I, which are argyrophilic NOR proteins. Immunocytochemical and cytochemical labelings of these proteins were used to reveal r-chromatin in situ and to investigate its spatial organization within NORs by confocal microscopy and by electron tomography. For each labeling, confocal microscopy revealed small and large double-spotted NORs and crescent-shaped NORs. Their internal three-dimensional (3D) organization was studied by using electron tomography on specifically silver-stained NORs. The 3D reconstructions allow us to conclude that the argyrophilic NOR proteins are grouped as a fiber of 60-80 nm in diameter that constitutes either one part of a turn or two or three turns of a helix within small and large double-spotted NORs, respectively. Within crescent-shaped NORs, virtual slices reveal that the fiber constitutes several longitudinally twisted loops, grouped as two helical 250- to 300-nm coils, each centered on a nonargyrophilic axis of condensed chromatin. We propose a model of the 3D organization of r-chromatin within elongated NORs, in which loops are twisted and bent to constitute one basic chromatid coil.

The respiratory epithelium is frequently injured by inhaled toxic agents or by micro-organisms. The epithelial wound repair represents a crucial process by which surface respiratory cells maintain the epithelial barrier integrity. The repair process involves both cell migration and proliferation, but as yet, the kinetic of these two mechanisms has not been extensively studied. Using an in vitro model of human respiratory epithelium wound repair, proliferative cell immunofluorescent staining and a computer-assisted technique allowing the tracking of living cells, we studied the cell proliferation and migration during the wound repair process. Respiratory epithelial cells were dissociated from human nasal polyps and cultured on a collagen I matrix. At confluency, a chemical wound was made on the culture. We observed that the cell mitotic activity peaked at 48 h after wounding (23% of the cells) and mainly concerned the cells located 160 to 400 microns from the wound edge. The migration speed was highest (35 to 45 microns/h) for the spreading cells at the wound edge and progressively decreased for the cells more and more distant from the wound edge. The temporal analysis of the cell migration speed during the wound repair showed that it was almost constant during the first 3 days of the repair mechanism and thereafter dropped down until the wound closure was completed (after 4 days). We also observed that over a 1-hour period, the intra-individual and interindividual variation of the cell migration speed was 43% and 37%, respectively. These results demonstrate that cell proliferation and cell migration during respiratory epithelial wound repair are differently expressed with regard to the cell location within the repairing area.

The nucleus is a complex volume constituted of numerous subcompartments in which specific functions take place due to a specific spatial organization of their molecular components. To understand how these molecules are spatially organized within these machineries, it is necessary to investigate their three-dimensional organization at high resolution. To reach this goal, electron tomography appears to be a method of choice; it can generate tomograms with a resolution of a few nanometers by using multiple projections of a tilted section several hundred to several thousand nanometers in thickness imaged by transmission electron microscopy (TEM).Specific identification of molecules of interest contained within such thick sections requires their specific immunocytochemical labelling using electron-dense markers. We recently demonstrated that electron tomography of proteins immunostained with nanogold particles before embedding, and subsequently amplified with silver, was very fruitful due to the inherently high spatial resolution of the medium-voltage scanning and transmission electron microscope (STEM). Here we describe this approach, which is very efficient for tracing the 3D organization of proteins within complex machineries by using antibodies raised against one of the proteins, or against GFP to analyse GFP-tagged proteins.

The nucleus is a complex volume constituted of numerous subcompartments in which specific functions take place due to a specific spatial organization of their molecular components. To understand how these molecules are spatially organized within these machineries, it is necessary to investigate their three-dimensional organization at high resolution. To reach this goal, electron tomography appears to be a method of choice; it can generate tomograms with a resolution of a few nanometers by using multiple projections of a tilted section several hundred to several thousand nanometers in thickness imaged by transmission electron microscopy (TEM).Specific identification of molecules of interest contained within such thick sections requires their specific immunocytochemical labelling using electron-dense markers. We recently demonstrated that electron tomography of proteins immunostained with nanogold particles before embedding, and subsequently amplified with silver, was very fruitful due to the inherently high spatial resolution of the medium-voltage scanning and transmission electron microscope (STEM). Here we describe this approach, which is very efficient for tracing the 3D organization of proteins within complex machineries by using antibodies raised against one of the proteins, or against GFP to analyse GFP-tagged proteins.

The colonization pattern of Vitis vinifera L. by Burkholderia phytofirmans strain PsJN was determined using grapevine fruiting cuttings with emphasis on putative inflorescence colonization under nonsterile conditions. Two-week-old rooted plants harbouring flower bud initials, grown in nonsterile soil, were inoculated with PsJN:gfp2x. Plant colonization was subsequently monitored at various times after inoculation with plate counts and epifluorescence and/or confocal microscopy. Strain PsJN was chronologically detected on the root surfaces, in the endorhiza, inside grape inflorescence stalks, not inside preflower buds and flowers but rather as an endophyte inside young berries. Data demonstrated low endophytic populations of strain PsJN in inflorescence organs, i.e. grape stalks and immature berries with inconsistency among plants for bacterial colonization of inflorescences. Nevertheless, endophytic colonization of inflorescences by strain PsJN was substantial for some plants. Microscopic analysis revealed PsJN as a thriving endophyte in inflorescence organs after the colonization process. Strain PsJN was visualized colonizing the root surface, entering the endorhiza and spreading to grape inflorescence stalks, pedicels and then to immature berries through xylem vessels. In parallel to these observations, a natural microbial communities was also detected on and inside plants, demonstrating the colonization of grapevine by strain PsJN in the presence of other microorganisms.

Telomestatin is a potent G-quadruplex ligand that specifically interacts with the 3′ telomeric overhang, leading to its degradation and that induces a delayed senescence and apoptosis of cancer cells. Protection of Telomere 1 (POT1) was recently identified as a specific single-stranded telomere-binding protein involved in telomere capping and T-loop maintenance. We showed here that a telomestatin treatment inhibits POT1 binding to the telomeric overhang in vitro. The treatment of human EcR293 cells by telomestatin induces a dramatic and rapid delocalization of POT1 from its normal telomere sites but does not affect the telomere localization of the double-stranded telomere-binding protein TRF2. Thus, we propose that G-quadruplex stabilization at telomeric G-overhang inactivates POT1 telomeric function, generating a telomere dysfunction in which chromosome ends are no longer properly protected. (Cancer Res 2006; 66(14): 6908-12)

Multicellular spheroids are known to be the most adapted model to keep the in vitro resistance properties of cells. This in vivo-like tissue-culture representation was applied to investigate the immune reactivity of MCF-7 cells by monocytes.Human blood monocytes, obtained by elutriation, were co-cultured with multicellular tumor spheroids of drug-sensitive (MCF-7S) and doxorubicin-resistant (MCF-7DXR) MCF-7 breast cancer cells.Tumor cells, according to their phenotype, induced differential recruitment and behavior of the immune cells towards the two types of spheroids. The secretion of various cytokines and the expression of several adhesion molecules were analysed. The MCF-7DXR/monocytes co-culture supernatant showed higher levels of IL-6 and IL-8 than the MCF-7S/monocytes co-culture supernatant. Cells from the MCF-7DXR spheroids expressed some adhesion molecules, CD-44 and CD-54, leading to a strong cellular cohesion in comparison with the sensitive spheroids.The two spheroid phenotypes represented an excellent model system for determining the precise tumor microenvironment in which cells move, the crucial molecular requirements and the mechanisms by which immunotherapeutic strategies could be developed to eradicate chemo-resistant tumors.

International audience; A methodology for simple convenient preparation of bright, negatively or positively charged, water-soluble CdSe/ZnS core/shellnanocrystals (NCs) and their stabilization in aqueous solution is described. Single NCs can be detected using a standard epifluo-rescent microscope, ensuring a detection limit of one molecule coupled with an NC. NCs solubilized in water by DL-Cys werestabilized, to avoid aggregation, by poly(allylamine) and conjugated with polyclonal anti-mouse antibodies (Abs). NC-Abs con-jugates were tested in dot-blots and exhibited retention of binding capacity within several nanograms of antigen detected. We furtherdemonstrated the advantages of NC-Abs conjugates in the immunofluorescent detection and three-dimensional (3D) confocalanalysis ofp-glycoprotein (p-gp), one of the main mediators of the MDR phenotype, overexpressed in the membrane of MCF7rbreast adenocarcinoma cells. Immunolabeling of p-gp with NC-Abs conjugates was 4200-, 2600-, and 420-fold more resistant tophotobleaching than its labeling with fluorescein isothiocyanate-Abs, R-phycoerythrin-Abs, and AlexaFluor488-Abs, respectively.The labeling of p-gp with NC-Abs conjugates was highly specific, and the data were used for confocal reconstruction of 3D imagesof the p-gp distribution in the MCF7r cell membrane. Finally, we demonstrated the applicability of NC-Abs conjugates obtained bythe method described to specific detection of antigens in paraffin-embedded formaldehyde-fixed cancer tissue specimens, usingimmunostaining of cytokeratin in skin basal carcinoma as an example. We conclude that the NC-Abs conjugates may serve as easy-to-do, highly sensitive, photostable labels for immunofluorescent analysis, immunohistochemical detection, and 3D confocal studiesof membrane proteins and cells.

International audience; The monoclonal antibody (MAb) Ki-67 is routinely used in clinical studies to estimate the growth fraction of tumors. However, the role of pKi-67, the protein detected by the Ki-67 MAb, remains elusive, although some biochemical data strongly suggest that it might organize chromatin. To better understand the functional organization of pKi-67, we studied its three-dimensional distribution in interphase cells by confocal microscopy and electron tomography. FluoroNanogold, a single probe combining a dense marker with a fluorescent dye, was used to investigate pKi-67 organization at the optical and ultrastructural levels. Observation by confocal microscopy followed by 3D reconstruction showed that pKi-67 forms a shell around the nucleoli. Double labeling experiments revealed that pKi-67 co-localizes with perinucleolar heterochromatin. Electron microscopy studies confirmed this close association and demonstrated that pKi-67 is located neither in the fibrillar nor in the granular components of the nucleolus. Finally, spatial analyses by electron tomography showed that pKi-67 forms cords 250-300 nm in diameter, which are themselves composed of 30-50-nm-thick fibers. These detailed comparative in situ analyses strongly suggest the involvement of pKi-67 in the higher-order organization of perinucleolar chromatin.

International audience; In this work, we have localized transcribing rRNA genes at the ultrastructural level and described their three-dimensional organization within the nucleolus by electron tomography. Isolated nucleoli, which exhibit a reduced transcriptional rate, were used to determine the sites of initial BrUTP incorporation (i.e. rRNA synthesis by the transcriptional machinery). Using pulse-chase experiments with BrUTP and an elongation inhibitor, cordycepin, it was possible to precisely localize the initial sites of BrUTP incorporation. Our data show that BrUTP incorporation initially takes place in the fibrillar centers and that elongating rRNAs rapidly enter the surrounding dense fibrillar component. Furthermore, we investigated the spatial arrangement of RNA polymerase I molecules within the whole volume of the fibrillar centers. Electron tomography was performed on thick sections of cells that had been labeled with anti-RNA polymerase I antibodies prior to embedding. Detailed tomographic analyses revealed that RNA polymerase I molecules are mainly localized within discrete clusters. In each of them, RNA polymerase I molecules were grouped as several coils, 60 nm in diameter. Overall, these findings have allowed us to propose a model for the three-dimensional organization of transcribing rDNA genes within the nucleolus.

We have previously shown that, in normal human airway tissue, localization of the cystic fibrosis transmembrane conductance regulator (CFTR) can be affected by epithelial maturation, polarity, and differentiation and that CFTR trafficking and apical localization depend on the integrity of the airway epithelium. In this study, we addressed the question of whether the three-dimensional (3-D) organization of adult human airway epithelial cells in suspension culture under rotation, leading to spheroid-like structures, could mimic the in vivo phenomenon of differentiation and polarization. The kinetics of the differentiation, polarity, and formation of the CFTR-ZO-1-ezrin complex was analyzed by transmission, scanning, and immunofluorescence microscopy. Functional activity of the airway surface epithelium was assessed by monitoring the degree of cAMP-stimulated chloride efflux from cultured cells. Our results show that after the initial step of dedifferentiation, characterized by a loss of ciliated cells and disappearance of epithelial subapical CFTR-ezrin-ZO-1 complex, the isolated cells formed 3-D spheroid structures within 24 hours. After 15 days, progressive ciliogenesis was observed and secretory cells could be identified. After 35 days of 3-D culture, ZO-1, CFTR, ezrin, and CD59 were apically or subapically located, and well-differentiated secretory and ciliated cells were identified. CFTR functionality was assessed by analyzing the Cl(-) secretion after amiloride and forskolin perfusion. After 35 days of culture of spheroids in suspension, a significant increase in Cl(-) efflux was observed in well-differentiated ciliated cells.

International audience; Although rRNA synthesis, maturation, and assembly into preribosomal particles occur within the nucleolus, the route taken by pre-rRNAs from their synthetic sites toward the cytoplasm remains largely unexplored. Here, we employed a nondestructive method for the incorporation of BrUTP into the RNA of living cells. By using pulse-chase experiments, three-dimensional image reconstructions of confocal optical sections, and electron microscopy analysis of ultrathin sections, we were able to describe topological and spatial dynamics of rRNAs within the nucleolus. We identified the precise location and the volumic organization of four typical subdomains, in which rRNAs are successively moving towards the nucleolar periphery during their synthesis and processing steps. The incorporation of BrUTP takes place simultaneously within several tiny spheres, centered on the fibrillar centers. Then, the structures containing the newly synthesized RNAs enlarge and appear as compact ringlets disposed around the fibrillar centers. Later, they form hollow spheres surrounding the latter components and begin to fuse together. Finally, these structures widen and form large rings reaching the limits of the nucleoli. These results clearly show that the transport of pre-rRNAs within the nucleolus does not occur randomly, but appears as a radial flow starting from the fibrillar centers that form concentric rings, which finally fuse together as they progress toward the nucleolar periphery.

International audience; The volumic rearrangement of both chromosomes and immunolabeled upstream binding factor in entire well-preserved mitotic cells was studied by confocal microscopy. By using high-quality three-dimensional visualization and tomography, it was possible to investigate interactively the volumic organization of chromosome sets and to focus on their internal characteristics. More particularly, this study demonstrates the nonrandom positioning of metaphase chromosomes bearing nucleolar organizer regions as revealed by their positive upstream binding factor immunolabeling. During the complex morphogenesis of the progeny nuclei from anaphase to late telophase, the equal partitioning of the nucleolar organizer regions is demonstrated by quantification, and their typical nonrandom central positioning within the chromosome sets is revealed.

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.

The cell migration that occurs during wound repair is dependent on modifications of the cell-matrix interaction in which extracellular matrix proteins and their receptors, the integrins, are involved. To study the interactions between airway epithelial cells and the extracellular matrix during the process of wound repair, we developed an in vitro wound model of human epithelial cells. Surface epithelial cells were dissociated from human nasal polyps and cultured on a type I collagen matrix. At confluency, a wound was made by the addition of 2 microliters of NaOH (1 N) to the cell culture. After the cell culture was washed, the wound area was recorded every 12 h for 96 h by a videomicroscopic technique. We calculated the wound-repair index that represents the decrease in the wound area per hour. Using immunofluorescence techniques, we first examined the localization, during wound repair, of fibronectin and of the beta 1-, alpha v-, alpha 2-, alpha 3-, and alpha 5-integrin subunits. Secondly, we carried out a series of wound-repair blocking experiments with the use of anti-integrin or anti-fibronectin antibodies diluted in the culture medium. We observed that fibronectin and the alpha 5- integrin subunit were exclusively expressed by the migratory cells in the wounded area. No difference in the localization of the alpha v-, alpha 2-, and alpha 3-integrin subunits was observed between the nonrepairing and repairing cells. The blocking experiments showed a significant decrease in the wound-repair index in the presence of either the anti-beta 1, -alpha 3, alpha 5, or the anti-fibronectin antibodies. Furthermore, the addition of fibronectin to the culture medium induced a significant increase in the wound repair index. These results suggest that fibronectin and the corresponding alpha 5 beta 1-integrin play an important role in the process of airway epithelium wound repair.

Silver dots deposited specifically on proteins of the nucleolar organizer regions (Ag-NOR proteins) after a one-step silver staining technique were visualized in cells in culture, in cells in smears, and in tissue sections, with a scanning laser confocal microscope working in the reflectance mode. After specific labeling of DNA with the fluorescent dye chromomycin A3, DNA and silver dots could be observed either individually or simultaneously. Therefore, it was possible to study the three-dimensional organization of nucleolar silver-stained structures relative to DNA with a high X, Y, and Z resolution. Our results showed that the argyrophilic components are organized as a twisted necklace structure within interphase nucleoli of cells in culture. We also demonstrated a striking three-dimensional symmetric disposition of NORs within the two sets of chromosomes in telophase cells. Similar results were obtained for cells in smears, although their three-dimensional organization was somewhat disturbed due to air-drying. We also demonstrated that silver dots cannot be visualized in the reflectance mode within sections of paraffin-embedded tissues. However, their simultaneous demonstration in non-confocal transmitted light, together with that of DNA in confocal mode, appeared very useful to study their localization within nuclei and mitotic chromosomes.

Scanning transmission electron microscopy at 300kV enables the visualization of nucleolar silver-stained structures within thick sections (3-8 microns) of Epon-embedded cells at high tilt angles (-50 degrees; +50 degrees). Thick sections coated with gold particles were used to determine the best conditions for obtaining images with high contrast and good resolution. For a 6-microns-thick section the values of thinning and shrinkage under the beam are 35 to 10%, respectively. At the electron density used in these experiments (100e-/A2/s) it is estimated that these modifications of the section stabilized in less than 10 min. The broadening of the beam through the section was measured and calculations indicated that the subsequent resolution reached 100 nm for objects localized near the lower side of 4-microns-thick sections with a spot-size of 5.6 nm. Comparing the same biological samples, viewed alternately in CTEM and STEM, demonstrated that images obtained in STEM have a better resolution and contrast for sections thicker than 3 microns. Therefore, the visualization of densely stained structures, observed through very thick sections in the STEM mode, will be very useful in the near future for microtomographic reconstruction of cellular organelles.

We developed a biomedical image database (BDIM) which proposes a standardized representation of value arrays such as images and curves, and of their associated parameters, independently of their acquisition mode to make their transmission and processing easier. It includes three kinds of interactions, oriented to the users. The network concept was kept as a constraint to incorporate the BDIM in a distributed structure and we maintained compatibility with the ACR/NEMA communication protocol. The management of arrays and their associated parameters includes two distinct bases of objects, linked together via a gateway. The first one manages arrays according to their storage mode: long term storage on optionally on-line mass storage devices, and, for consultations, partial copies of long term stored arrays on hard disk. The second one manages the associated parameters and the gateway by means of the relational DBMS ORACLE. Parameters are grouped into relations. Some of them are in agreement with groups defined by the ACR/NEMA. The other relations describe objects resulting from processed initial objects. These new objects are not described by the ACR/NEMA but they can be inserted as shadow groups of ACR/NEMA description. The relations describing the storage and their pathname constitute the gateway. ORACLE distributed tools and the two-level storage technique will allow the integration of the BDIM into a distributed structure, Queries and array (alone or in sequences) retrieval module has access to the relations via a level in which a dictionary managed by ORACLE is included. This dictionary translates ACR/NEMA objects into objects that can be handled by the DBMS.(ABSTRACT TRUNCATED AT 250 WORDS)

The processing of medical images requires the handling of complex structured sets of elementary objects (images, curves,... and their associated parameters). Usually, an elementary object cannot be interpreted without information concerning the structure to which it belongs (e.g image sequences). Then it is necessary to consider the whole structure like an atomic semantic entity, object of an image data base. As specific tools are necessary to manage these objects, an object oriented handling system (OHS), part of our medical image data base project (BDIM), was developed to perform : i) the array storage management, ii) the interface between applications and the BDIM to have access to objects (create, update, delete...) and components (navigation inside object structures, access to arrays and parameters). The image handling system (IHS) decribed here is the user level part of the OHS. IHS allows the evolution of the data base environment by adding or updating acquisition and/or processing functionalities. To unify data access methods, the concept of logical file is introduced as a special class of BDIM objects. The logical file does not necessitate the use of a specific declaration for the different kinds of images because it is possible, for a desired processing , to have access to the only concerned data.