Guidelines of AgNOR quantitation. Committee on AgNOR Quantitation within the European Society of Pathology.
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Freezing↗
Biomedical subjects
D Ploton
Publications and source records attributed to D Ploton.
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".
Animals↗
Ultrastructural distribution of DNA within the nucleolus of various animal cell lines or tissues revealed by terminal deoxynucleotidyl transferase.
We have used the highly sensitive in situ terminal deoxynucleotidyl transferase method, applied to ultrathin sections, to investigate the location of DNA within nucleoli of various animal cells. In all the nucleoli studied, intense labelling is revealed over the peri- and intranucleolar condensed chromatin. Gold particles are also consistently found over the fibrillar centres, especially at their periphery, namely in the border area between the fibrillar centres and the dense fibrillar component, whereas the dense fibrillar component itself seems to be free of label in nucleoli in which these two compartments can be distinguished. We conclude that, in transcriptionally active nucleoli of this type, DNA is a characteristic constituent of the fibrillar centres, distinguishing them functionally from the dense fibrillar component. Some nucleoli exhibit neither fibrillar centres nor a dense fibrillar component, but have a single, albeit heterogeneous accumulation of fibrillar material; gold particles are consistently seen over some parts of this fibrillar compartment. This suggests that certain parts of the fibrillar material are functionally similar to the fibrillar centres of those nucleoli that possess them.
Animals↗
Applications of medium-voltage STEM for the 3-D study of organelles within very thick sections.
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.
Animals↗
Adriamycin resistance is characterized by ultrastructural changes in human leukaemic K562 cells in vitro.
Ultrastructural changes associated with adriamycin (ADM) resistance have been investigated in the human K562 leukaemic cell line: sensitive K562 cells, a resistant subline cultured in the continuous presence of ADM and resistant cells without ADM Transmission electron microscopy (TEM) study revealed that K562-resistant cells displayed ultrastructural modifications of the cell surface, chromatin and nucleolus conformation. Alterations were not directly related to the presence of adriamycin as deprivated cells exhibited modificated characters through a slow progressive recovery phenomenon.
Acetylation↗
A new marker for early detection and indicator of progression of cancer of the bladder. Preliminary results with Ag-NOR index in 38 cases of superficial bladder cancer.
The multiform biology of superficial bladder tumors, both morphologically and evolutively, and the lack of reliable predictors of progression have led the authors to study the Ag-NOR proteins as a new marker of these tumors. It is well known that particularly the low-grade superficial tumors frequently relapse on the same histologic and proliferative module. Their potential of progression is probably present at the time of the first manifestation of the disease or it can show itself along the relapsing evolution with classic modifications translating the cellular dedifferentiation. The NOR index, set up by the authors, has several advantages: firstly, it corresponds to a functional value of normal and neoplastic cells; secondly, it can be used also with paraffin blocks. Another advantage is the semiautomatic lecture, reproducible also in the urinary cytology, mainly of low-grade tumors, reducing the number of false-negatives. The conclusion of the study of 38 cases of superficial bladder cancer has induced the authors to believe that an increased NOR index is a reliable 'marker' of their progression. Therefore, the authors suggest the use of the NOR activity for the surveillance of the urothelial disease and for a more logical therapeutic strategy.
Adult↗
Nucleolar organizer regions: preliminary results of the clinical use of a new marker for prostatic carcinoma (40 cases).
It is a widely diffused opinion that moving backwards in time the moment of the diagnosis of cancer of prostate, so that the tumor is detected earlier than normal, means that the treatment would be more effective than the one adopted in the usual times of diagnosis. For this reason the earlier diagnosis of prostate cancer has become more and more a compulsory target of the modern urologist, at a time of booming of the third age, of increased lifetime expectancy, of significant elevation of prostate cancer rate and of the persistent uncertainty of the efficacy of available treatments. Theoretically the mortality rate of prostate cancer can be reduced by the prevention programs and by the improvements of treatment methods, but the 'earlier' diagnosis is certainly an easier and less expensive strategy to achieve the same objective. The authors have evaluated the argyrophilic-nucleolar organizer region (Ag-NOR) proteins on 40 cases of adenocarcinoma of prostate collected through a multicentric program in France and in Italy. The Ag-NOR have been stained with silver technique set up by Ploton and Derenzini while the quantitative index has been evaluated by a semiautomatic system partially commercially available, partially modified by the authors. The conclusions: (a) the Ag-NOR index is a simple and reproducible method; (b) the Ag-NOR staging system corresponds to Gleason's grading; (c) the Ag-NOR elevation is a reliable marker of increased cell proliferation and is detectable much earlier than the morphologic changes of Gleason's classification.
Adenocarcinoma↗
Differential localization of the cystic fibrosis transmembrane conductance regulator in normal and cystic fibrosis airway epithelium.
Deletion of the amino acid residue Phe 508 of the cystic fibrosis transmembrane conductance regulator (CFTR) protein represents the most common mutation identified in cystic fibrosis (CF) patients. A monoclonal and a polyclonal antibody directed against different regions of CFTR were used to localize the CFTR protein in normal and CF airway epithelium derived from polyps of non-CF and CF subjects homozygous for the delta Phe 508 CFTR mutation. To identify the cellular and subcellular localization of CFTR, immunofluorescent light microscopy, confocal scanning microscopy, and immunogold transmission electron microscopy were performed on cryofixed tissue. A markedly different subcellular distribution was identified between normal and CF airway epithelial cells. In normal epithelium, labeling was restricted to the surface apical compartment of the ciliated cells. In contrast, in the epithelium from homozygous delta Phe 508 CF patients, CFTR markedly accumulated in the cytosol of all the epithelial cells. These findings are consistent with the concept that the CFTR delta Phe 508 mutation modifies the intracellular maturation and trafficking of the protein, leading to an altered subcellular distribution of the delta Phe 508 mutant CFTR.
Cystic Fibrosis↗
Semiautomatic quantification of silver-stained nucleolar organizer regions in tissue sections and cellular smears.
Silver staining of nucleoli reveals argyrophilic proteins associated with nucleolar organizer region (Ag-NOR) proteins. Argyrophilic components appear as dots about 1 micron in diameter dispersed throughout the nucleolus (Ag-NOR dots). The count of Ag-NOR dots is a useful index for improving the cancer diagnosis and determination of prognosis. Here we describe software developed on a medium-cost image analyzer in order to evaluate the mean area of NORs and their number relative to an internal reference, the number and areas of clusters of NORs and the area of the nucleus. Statistical analysis of the data was performed during counting. The first application concerned counting NOR dots during mitosis in cell imprints; those counts were 2.3, 15.3 and 55.56 for the metaphase, telophase and interphase, respectively (relative to unitary dots of metaphase cells). In the second application we demonstrated a significant difference in NOR numbers between two groups of prostatic cancers with good and poor prognoses (6.05 +/- 2.79 SD and 7.96 +/- 3.01, respectively; with Student's t test, = 1.999; P = .05).
Animals↗
Three-dimensional imaging of the mucus secretory process in the cryofixed frog respiratory epithelium.
Using the frog palate as a representative model of human mucociliary epithelium, we analyzed, after quick freezing fixation, the three-dimensional (3-D) respiratory mucus secretory release with high voltage (200-300 kV) transmission electron microscopy (TEM). The 3-D vision of the mucus release from the secretory cells was obtained as stereo-pairs and "bas-relief" images after analysis of stereo-pairs using an image analyzer. After standard glutaraldehyde fixation, the secretory cells showed a typical goblet shape with secretory granules heterogeneous in size and electron-density which often fuse together. On the other hand, quick-frozen secretory cells exhibited a columnar shape and their membrane-bound secretory granules contained a homogeneously dark matrix. The expanded gel mucus layer was preserved and its depth never exceeded 2 microns. When the epithelium was immersed in culture medium in presence of cholinergic agonist, a marked discharge of mucus was observed and the granules swelled at the apex of the secretory cell before being discharged in the lumen. In native cryofixed epithelium, the secretory granules exhibited a marked deformability during the process of their extrusion from the secretory cell. Clusters of secretory granules surrounded by cytoplasmic material were observed in the extracellular lumen, suggesting an apocrine-type secretion. These observations indicate that rapid cryofixation and 3-D stereoscopic imaging enable a unique opportunity to analyze, without artifact, the mucous secretory process. We speculate that, apart from the classical merocrine-type secretion mechanism, the respiratory mucus may be released, at least partly by an apocrine-type secretion.
Animals↗
Interphase nucleolar organizer regions in cancer cells.
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Cell Division↗
Digital three-dimensional visualization of cellular organelles studied by medium- and high-voltage electron microscopy.
Tilted thick sections (one-half to several micrometers) of biological specimens observed with medium- to high-voltage electron microscopes are extremely useful for the study of the three-dimensional (3-D) structure of organelles. If high resolution in 3-D visualization and 3-D reconstruction is needed, many images corresponding to various angles of rotation and tilt must be recorded. This necessitates very time-consuming work--including eventual photographic processing--before good positioning of the object is defined. We have developed software which permits very rapid and precise determination of the tilt-axis, the registration of tilted views, 3-D measurements and 3-D visualization. Images are digitized either from negative films or directly with a camera fitted to the microscope. The application of the software is performed in minutes and allows for a rapid check of the quality of the tilt-series and of the features of interest of the object. Application of the software to the study of the 3-D structure of active components of the nucleolus stained with silver is shown.
Cell Nucleolus↗
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.
Bronchoalveolar Lavage Fluid↗
[Nucleolar organizers in superficial bladder tumors. Prognostic analysis of 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.
Actuarial Analysis↗
[Nucleolar organizers: NOR. New prognostic factors in prostatic cancer. Preliminary results in 20 cases].
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.
Humans↗
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.
Animals↗
Growth and characterization of isolated bovine tracheal gland cells in culture. Influence of a reconstituted basement membrane matrix.
We describe a method for establishing the culture of bovine tracheal submucosal gland (BTG) cells, in which we have also examined the influence of a reconstituted basement membrane matrix derived from the Engelbreth-Holm-Swarm tumor (EHS) on the growth and morphological differentiation of these cells. BTG cells have been isolated by tissue enzymatic digestion using trypsin, deoxyribonuclease I, elastase, hyaluronidase and EGTA for 1 hr at 37 degrees C. Afterwards, cells and tissue were collected by centrifugation and were incubated for 15 min with 15% newborn calf serum to inactivate the proteolytic enzymes. Enzymatic digestion using only trypsin, centrifugation and inactivation steps were repeated three times. Using this protocol, we obtained 15 +/- 4 (X 10(6] cells per g of tracheal submucosa with 72 +/- 2% (n = 5) cell viability. On microscopic observation, isolated cells were mainly composed of serous type glandular cells. Cells were cultured in a 1:1 medium of Dulbecco's Modified Eagle's/Ham's F12 supplemented with 10% fetal calf serum and subcultured in either plastic flasks or flasks coated with EHS matrix. On the plastic, the BTG cells exhibited at confluency an epithelioid appearance. They stained positively with the immunofluorescent anticytokeratin antibody and contained PAS-staining granules. By electron microscopy, lactoferrin, a protein marker specific to the serous cells, was demonstrated immunocytochemically in small secretory vesicles. BTG cells cultured on EHS matrix revealed a significantly increased growth in comparison to those cultured on plastic. In post-confluent culture of BTG cells on EHS matrix, we observed numerous dome-like structures formed by differentiated cells which were joined together around luminal spaces.
Animals↗
[Silver staining of nucleolus organizer regions (NORs). Application to the study of nucleolar structure and value in pathology].
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.
Humans↗