[Epidermolysis bullosa acquisita of childhood]. / Epidermolyse bulleuse acquise de l'enfant.

BACKGROUND: Epidermolysis bullosa acquisita (EBA) is a subepidermal autoimmune blistering disease characterized immunologically by autoantibodies to type VII collagen. Its occurrence in childhood is rare. Thirty-five cases have been described to date in the literature. PATIENTS AND METHODS: We report the case of an 8-year-old girl presenting blistering lesions on the cheeks, extremities and limb extension areas. The diagnosis of EBA was confirmed by histology, direct immunofluorescence of a perilesional skin biopsy specimen, indirect immunofluorescence on salt-split skin substrate and direct electron microscopy. The patient was controlled clinically under treatment with dapsone alone. DISCUSSION: This 36th childhood case of EBA presented typical clinical features, a similar prognosis and comparable treatment response to other paediatric cases. Clinical presentation is inflammatory and affects the face. As in our case, in childhood, prognosis is often better than in adults without the need for immunosuppressive agents.

Electron tomography of metaphase nucleolar organizer regions: evidence for a twisted-loop organization.

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 three-dimensional study of chromosomes and upstream binding factor-immunolabeled nucleolar organizer regions demonstrates their nonrandom spatial arrangement during mitosis.

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.

Shrinkage of freeze-dried cryosections of cells: Investigations by EFTEM and cryo-CLEM.

Freeze-drying of cryosections of cells or tissues is considered to be the most efficient preparation for microanalysis purpose related to transmission electron microscopy. It allows the measurements of ions and water contents at the ultrastructural level. However an important drawback is associated to freeze-drying: the shrinkage of the cryosections. The aim of this paper is the investigation of this phenomenon by means of three different methods applied to both hydrated and dehydrated cryosections: direct distance measurements on fiducial points, thickness measurements by energy filtered transmission microscopy (EFTEM) and cryo-correlative light electron microscopy (cryo-CLEM). Measurements in our experimental conditions reveal a lateral shrinkage around 10% but the most important result concerns the lack of differential shrinkage between most of the cellular compartments.

Three-dimensional co-localization of nucleolar argyrophilic components and DNA in cell nuclei by confocal microscopy.

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.

Identification of phospholipids in secretory granules of human submucosal gland respiratory cells.

Although it has been shown that tracheal epithelial cells in culture synthesize and secrete phospholipids, no direct evidence for in situ phospholipid storage in human respiratory secretory epithelial cells has been demonstrated. We used a high-resolution cytochemical enzyme-gold technique to identify and precisely localize phospholipids in human submucosal gland secretory cells. In addition, lysozyme, a specific serous cell marker, was identified using the biotinstreptavidin gold technique with lysozyme antiserum. This double labeling of phospholipids and lysozyme was performed using gold particles of diameters 15 nm and 5 nm, respectively. Quantitation of phospholipid labeling was performed on an image analyzer. Phospholipids were identified in serous granules (8.87 +/- 2.21 gold particles/microns 2) in a significantly (p less than 0.05) higher density than in mucous granules (5.57 +/- 3.07 gold particles/microns 2). These results support the hypothesis that submucosal human airway serous and mucous secretory cells produce phospholipids which may be secreted in the airway lumen.

Viral integration sites in human papilloma virus-33-immortalized cervical keratinocyte cell lines.

The viral organization of HPV-33 was determined by Southern blotting in 2 HPV-33-immortalized cervical cell lines (CK11 and CK12) and compared to our previous results obtained on 10 other already characterized HPV-33-immortalized cell lines (CK1 to CK10). As observed in CK1 to CK10, the viral DNA was found integrated in the cellular genome of CK11 and CK12. However, in CK11 and CK12, the integrated viral genome was deleted and mostly limited to the URR and the E6-E7 ORFs, stressing the importance of those sequences in the immortalization process. Furthermore, CK11 and CK12 showed a unique and identical integration site, as observed in CK1 to CK10, which also harbored HPV-33 integrated at a unique and identical site (which was however different from the one evidenced in CK11 and CK12). Indeed, in situ hybridizations on chromosomes allowed the precise localization of the viral DNA on chromosome 13q33-34 in CK1 to CK10 whereas it was mapped to chromosome 9p13 in CK11 and CK12. We discuss the possibility that integration of HPV-33 at those two particular sites has conferred some growth advantages to the cells and could have thus played a crucial role in the immortalization.

Nuclear transport as an ultimate step of multidrug resistance.

Adriamycin (ADM) incorporation into nuclei of whole multidrug resistant (MDR) CEM cells is lower than into sensitive ones (S), that is mostly thought to be the consequence of a decrease of drug related to the activity of the multidrug resistance plasma membrane protein P 170. Isolated nuclei of the lymphoblastic tumor cell line CEM, which structures were controlled by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal microscopy, where incubated with 10(-6) mole/l of ADM. Incorporation into DNA was quantified by spectrofluorimetry. It was lower and slower into MDR nuclei than into S ones. Different modulators of active transport influence drug transfer into S nuclei and had no effect in MDR nuclei. The nuclear transfer into S nuclei appeared divided into two components: one was decreased by WGA, increased by cytosolic factors and an other part was purely passive in an identical intensity to MDR nuclei. Resistance of MDR nuclei seemed indebt to a defect, in these cells, of factors that mediate and/or activate nuclear transport of drug.

Proliferation assessment in breast cancer: a double-staining technique for AgNOR quantification in MIB-1 positive cells especially adapted for image cytometry.

There are two ways of measuring the cell proliferation. The first one consists of quantifying the number of cycling cells with the help of antibodies directed against cells either in G1, S, G2 or M phase. The second way is to assess the cell cycle duration by the quantification of AgNOR proteins. Measuring both the features on the same slide represents an attractive way to tackle the proliferating activity of a cell culture or a tumor. Here, we propose a MIB-1 and AgNOR double staining method especially adapted to image cytometry measurement using MIB-1 antibody coupled to FITC in order to avoid the thresholding problems encountered with such a multilabeling technique. We have applied this new method on a series of 39 breast cancer cases, with at least 4 years follow-up, in order to determine the prognosis significance of this measurement. MIB-1 alone is not linked to prognosis, while the global mean AgNOR area is significantly linked to prognosis in terms of development of visceral metastasis or death. However, the global mean AgNOR area is insufficient to determine the time limit of appearance of metastasis or relapse. Our results clearly demonstrate that a high mean AgNOR area within a cell population having a high MIB-1 index can discern tumors with a high metastatic potential. By multiplying AgNOR area by the percentage of MIB-1 positive cells we calculate the proliferative activity, P, which brings very important information concerning the time limit of relapse.

Ultrastructural comparative distribution of carbohydrates in human tracheal and frog palate mucosa using neuraminidase and lectin-colloidal gold complexes.

We have compared, at the ultrastructural level, the carbohydrate structure of glycoconjugates of the different types of secretory cells of the human tracheal mucosa (HTM) and the frog palate mucosa (FPM), proposed as a model for studying bacterial adherence to mucus-coated respiratory epithelium. In addition to reactivity with Concanavalin A and Lens cullinaris agglutinin, reactivity of Epon-embedded HTM and FPM secretory granules was studied by transmission electron microscopy using neuraminidase-gold complex and colloidal gold-adsorbed lectins with affinity for sugar residues of human mucins, namely the following: Helix pomatia, Lotus tetragonolobus, Ricinus communis II, Wheat germ and Limax flavus agglutinins. The affinity of HTM and FPM mucous and serous cells for the different colloidal-gold complexes was very similar, however Limax flavus agglutinin labelled only HTM and not FPM secretory granules. The FPM mucous and serous secretory granules were nevertheless intensely labelled by the neuraminidase-gold complex, demonstrating the presence of sialic acid residues. The close ultrastructural and histochemical similarities between HTM and FPM suggest that the FPM may be a valuable model for studying the specific interaction between microbial lectins and mucus glycoproteins in the bacterial adherence phenomenon.

[Silver staining of nucleolus organizer regions (NORs). Application to the study of nucleolar structure and value in pathology]. / Coloration des organisateurs nucléoraires (NORs) par l'argent. Application à l'étude de la structure du nucléole et intérêts en pathologie.

Nucleolus is the morphologic expression of synthesis and maturation of ribosomal RNA (rRNA) from amplified ribosomal DNA (rDNA). Nucleolar Organizer Regions (NORs) are functional subunits of the nucleolus in which actively transcribed rDNA is surrounded with numerous regulatory proteins. Some of them are argyrophilic non-histone proteins (Ag-NOR proteins). By using a cytochemical reaction based on this argyrophilia, active NORs may be stained by the precipitation, at their level, of metallic silver granules whose quantity is directly related to the nucleolar activity. In the present paper, we described various applications of a silver-staining method we developed in our laboratory. The Ag-NOR proteins were ultrastructurally localized within precise nucleolar components. Moreover by viewing thick-sections of silver-stained cells with high-voltage microscopes we were able to describe the three-dimensional structure of nucleoli. Finally, this silver-staining method may be applied at the optical level, to sections of routinely fixed and paraffin-embedded human tissues. With this simple staining method, it is now possible to study the relationships of the number of nucleolar argyrophilic structures with the diagnostic of neoplasms.

[Lymphadenopathy of dermatosis in the course of drug hypersensitivity. Cytologic, immunohisto- and immunocytologic, ultrastructural aspects. Report of a case]. / Lymphadénopathie des dermatoses au cours d'une hypersensibilité médicamenteuse. Aspect cytologique, immunohisto et immunocytologique, ultrastructural. A propos d'une observation.

In addition to the morphological details obtained from the imprints, a simple immunocytological study allowed us to diagnose one case of a dermopathic lymphadenopathy simulating a T cell lymphoma, following a drug-induced erythrodermia. We were able to identify the increase of CD1a+ and Prot. S100+ cells on acetone fixed imprints. The histological, immunohistological and ultrastructural investigations confirmed the value of the cytological study and that the dendritic cells were Langerhans cells (Birbeck granules+). Most of them were considered as migrating from the dermal lesions.

[Palmoplantar filiform parakeratotic hyperkeratosis and digestive adenocarcinoma]. / Hyperkératose palmo-plantaire filiforme parakératosique et adénocarcinome digestif.

INTRODUCTION: There are very few observations of filiform palmo-plantar hyperkeratosis reported. Nevertheless it's worth knowing this entity for his potential association with a visceral neoplasia. CASE REPORT: We report the first case of filiform palmo-plantar hyperkeratosis associated with a digestive adenocarcinoma and a polycystic kidney disease. DISCUSSION: After a review of palmar and plantar filiform hyperkeratosis in the literature, we will discuss the possible association with neoplasia or other pathologies. This pathology requires a strict clinical and paraclinical follow-up.

[Nucleolar organizers: NOR. New prognostic factors in prostatic cancer. Preliminary results in 20 cases]. / Les organisateurs nucléolaires: NOR. Nouveaux facteurs pronostiques dans le cancer de la prostate. Résultats préliminaires á propos de vingt observations.

The authors present a preliminary series if two groups of ten patients with advanced prostatic cancer with sufficient follow-up in whom the study of nucleolar organizers (NOR) was performed according to a new light microscopy argentaffin technique. This original technique was developed by D. Ploton at the CHU de Reims. The principles of the technique are presented. The interpretation of the results, very dispersed at the present time, leads the authors two us two new clinical and histological indicators estimating the degree of severity of the prostatic cancer: the index of clinical severity based on the quality of survival and its duration from time T0 and the multifactorial index of histological severity based on the WHO classification, Gleason grade and NOR.

[Nucleolar organizers in superficial bladder tumors. Prognostic analysis of 37 observations]. / Les organisateurs nucléolaires (NORs) dans les tumeurs superficielles de la vessie. Analyse pronostique à propos de 37 observations.

Nucleolar Organisers (NORs) are intranucleolar segments of DNA coding for ribosomal RNA. The agyrophilic proteins (AgNOR) associated with NORs allow them to be cytolabelled on paraffin sections. The number of NORs (NOR index) is correlated with cellular proliferation and has a diagnostic and prognostic value in neoplastic disease. The AgNOR method was analysed in a series of 37 superficial bladder tumours with different clinical courses. The NORs were counted in normal urothelium, on superficial tumours used to establish the diagnosis of the disease and on recurrent superficial tumours. The NOR index was 4.54 for normal urothelium, 5.89 for non-invasive superficial tumours, 7.33 for invasive superficial tumours, and 9.75 for invasive recurrences. These results demonstrate an increase in nucleolar argyrophilia with invasion and invasive potential of superficial bladder tumours which were initially homogeneous for stage and grade. The AfNOR method could constitute a new method of early histoprognostic evaluation for urothelial tumours.

Electron tomography of amplified nanogold immunolabelling: Improvement of quality based on alignment of projections with sinograms and use of post-reconstruction deconvolution.

Electron tomography of immunolabelled proteins identified with amplified nanogold particles imaged by Scanning and Transmission Electron Microscopy within thick sections is a powerful method to investigate the three-dimensional organization of complex cellular machineries. In order to increase the overall quality of the reconstructed cube, we have developed two methods that improve the tomographic reconstruction process. We first performed a very precise alignment of the projections before reconstruction with a technique using sinograms. After reconstruction, we propose to compute image restoration by calculating the Point Spread Function of the projection/back-projection system and to use it to deblur the reconstructed cubes. Improvement in the quality of the reconstructed cubes is demonstrated on images of nucleolar proteins tagged with EGFP and immunolabelled with nanogold particles.

Structure and molecular organization of the nucleolus.

The aim of this paper is to give a short review on the nucleolus. In particular, we focused on the relationship of nucleolar ultrastructure with rRNA synthesis and processing. We also gave rapid data on organization of rRNA genes. Finally, we also focused on AgNOR proteins and the organization of "AgNOR granules".

[Nucleolar structure during the prophase of Physarum polycephalum]. / Structures nucléolaires au cours de la prophase de Physarum polycephalum.

With Sugihara's fixation technique, electron microscopic study of early-prophase nucleolus in the Physarm polycephalum showed the existence of particular fibrillar structures. The characteristic feature of these spherical structures is an electron-lucid center surrounded by a dense fibrillar component. Their relationships between the "fibrillar centers" and between the rate of ribosomal RNA synthesis are studied.