Albrecht Reith

Comparison of semiautomatic digitizer-tablet and simple point counting performance in morphometry

SummaryA comparison is made between the efficiency of an opto-manual digitizer-tablet (MOP-AM 03) and simple point counting in the analysis of drawings of basal cells in the normal and pathologically altered chonchal epithelium. The analysis included estimation of the areas of cellular, nuclear and nucleolar sectional profiles as well as the cell basal width, i.e. the length of cell profile attachment to the basement membrane.The morphometric results obtained with the two methods are virtually identical. The efficiency of point counting is somewhat higher than that of the digitizer, but the prices of the two sets of equipment differ by one or two orders of magnitude.In general, the unquestionable measuring precision of the digitizer is unlikely to be of much impact in most biological studies due to the inevitable biological variation between individuals under study and the large variation which is always added at the level of single features by random sectioning.

Discrimination of various epithelia by simple morphometric evaluation of the basal cell layer

SummaryThis study presents a simple morphometric method for objective classification of pseudostratified, various types of metaplastic, and dysplastic epithelium by evaluation of cellular features in the basal layer only. Fifty-four biopsy specimens were taken for diagnostic reasons from the nasal mucosa of nickel workers, and semithin toluidine-blue-stained sections were analysed.The most sensitive parameters in distinguishing between the various types of epithelium were : (i) the transverse nuclear diameter, (ii) the size of the nucleoli and (iii) the basal cell width expressed as an index weighted towards the cell profiles with the broadest attachment face on the basal lamina. A combination of these three parameters allows a clear separation between dysplastic, metaplastic and pseudostratified epithelium.The sequential increase in these parameters from pseudostratified epithelium through two histologically distinguishable stages of metaplasia (stratified cuboidal and mixed stratified cuboidal/stratified squamous epithelium) to fully developed squamous epithelium supports the concept that metaplasia develops gradually. The continuous increase in these parameters from metaplasia to dysplasia further suggests that metaplasia is a necessary step in the development of nasal epithelial dysplasia.This morphometric model appears especially useful in monitoring small sequential epithelial changes, and might also be used for evaluating other types of epithelia.

Intracytoplasmic Lumina with and without Cilia in Both Normal and Pathologically Altered Nasal Mucosa

Two different types of intracytoplasmic lumina have been observed electron microscopically in pseudostratified and metaplastic human nasal mucosa: one type contains microvilli and the other contains both microvilli and cilia. The latter type was seen only in pseudostratified columnar epithelium, while the former was also observed in different stages of metaplasia. From these observations we conclude that the type of intracytoplasmic lumina is determined by the differential potential of the cells in which they occur. Serial sectioning demonstrated that these structures sometimes communicate with the extracellular space. The distinctly different ultrastructure of microvilli lining the intracytoplasmic lumina and the foldlike extensions of the cell membrane favors an intracellular genesis rather than a formation by invagination of the cell membrane. It is suggested that intracytoplasmic lumina occur as a consequence of dysregulation in the process of epithelial maturation.

Stereological analysis of nasal mucosa. III. Stepwise alterations in cellular and subcellular components of pseudostratified, metaplastic and dysplastic epithelium in nickel workers.

SummaryNasal mucosal biopsies taken primarily for diagnostic purposes were graded as pseudostratified, metaplastic (consisting of stratified cuboidal, non-keratinizing and keratinizing stratified squamous epithelium) or dysplasic, and were analysed by simple stereological procedures at the electron microscopic level. For biological and methodological reasons the evaluation was focused on the basal cells of the epithelium. The transition of pseudostratified to metaplasic and dysplasic epithelium was accompanied by significant alterations in the karyoplasm and cytoplasm. The main changes in the karyoplasm were a gradual reduction of heterochromatin and an enlargement of the nucleoli. In the cytoplasm the main changes involved: (i) the mitochondria, with a threefold increase in size and a corresponding reduction in number. In addition the surface density of their cristae was reduced by 32%. (ii) The surface density of rough endoplasmic reticulum, which increased by 100%, (iii) the volume fraction of tonofilaments which decreased by 60%, and (iv) the number of desmosomes which increased by 60%.The morphologic changes, especially those involving the mitochondria and endoplasmic membranes, suggest that biochemical differences also exist between the original, and metaplasic and dysplasic epithelia. The stepwise alterations of several independent cellular and subcellular components support the hypothesis of a gradual transformation of pseudostratified epithelium to stratified squamous epithelium and dysplasia.

The surface structure of 7,12-dimethylbenz(a)anthracene transformed C3H/10T 1 2 cells. A quantitative scanning electron microscopical study

Abstract The mouse embryo fibroblast cells, termed C3H/10T12, developed three types of morphologically altered foci after exposure to 7,12 -dimethylbenz(a) anthracene. The types III and II cells showed oncogenic potential, whereas the type I cells remained non-oncogenic. By scanning electron microscopy the concentration of short microvilli was found to increase with increasing cell culture passage for all three types. Only on types II and III cells were long microvilli observed. There was a significant correlation between the oncogenic potential and the presence of long microvilli. This observation may be of value in screening for oncogenic transformation of cells in culture.

Numerical Aberrations of Chromosomes 1 and 17 Correlate with Tumor Site in Human Gastric Carcinoma of the Diffuse and Intestinal Types. Fluorescence In Situ Hybridization Analysis on Gastric Biopsies

Recent studies predict that tumor aneuploidy plays a direct role in tumor instability. The relationship between interphase cytogenetics, histology, grade, and tumor site was analyzed in 20 primary gastric carcinomas. Using fluorescence in-situ hybridization, the numerical changes of centromeric sequences of chromosomes 1, 3, 10, and 17 were directly analyzed in gastric biopsies. Polysomic copy numbers of chromosomes 1 and 17 were discovered in 63percnt; (10 of 16) and 59percnt; (10 of 17), respectively, of informative cancer cases. Chromosome 3 and 10 signal number changes were found in only 6percnt; (1 of 16) and 13percnt; (1 of 8), respectively, of informative cancer cases. There was a positive correlation between the appearance of polysomic nuclear target sites of chromosomes 1 and 17 (correlation coefficient r = 0.72; p < 0.005). Copy number changes were not significantly related to histologic subtypes of either the Laurén or WHO classifications. However, incidence of cancers having dual polysomic signal number abnormalities for both chromosomes 1 and 17 was significantly correlated to tumor location at the cardia. The data suggests that (i) human gastric cancer appears in two genomic groups that can be reliably diagnosed by fluorescence in-situ hybridization on routine biopsy sections, (ii) numerical aberrations of chromosomes 1, 3, 10, and 17 are largely independent of histologic subtypes, and (iii) polysomic copy number abnormalities of chromosomes 1 and 17 correlate to intragastric tumor site and are highest in cardia cancers, suggesting high tumor instability at this particular location.

Carcinogenesis testing of saccharin. No transformation or increased sister chromatid exchange observed in two mammalian cell systems

Abstract Saccharin was assayed in two mammalian cell systems for possible transforming or mutagenic effects. In the mouse embryo fibroblast C3H/10T 1 2 cells, saccharin was without transforming effects over a large concentration range. On transformed, but non-oncogenic C3H/10T 1 2 type I cells, a slight effect on transition to the growth pattern morphology of types II and III cells was found, but this was not accompanied by characteristic changes in plasma membrane structure when studied by scanning electron microscopy. In human lymphocytes, saccharin did not induce any sister chromatid exchange. Addition of a metabolic activation system did not change the sister chromatid exchange pattern as usually seen after positive control with 3,4-benzopyrene. Based on these studies, saccharin cannot be classified as a carcinogen, not even a weak one. Neither does it appear to be a mutagen.

Modification of surfactant metabolizing cells in rat lung by clofibrate, a hypolipidemic peroxisome proliferating agent. Evidence to suggest that clofibrate influences pulmonary surfactant metabolism.

SummaryThe influence of clofibrate (ethyl-α-pchlorophenoxy-isobutyrate), a hypolipidemic peroxisome proliferating agent, has been tested on the lungs of adult male rats. Drug administration for 7 days caused structural changes in two types of lung cells, both of which are involved in the metabolism of the pulmonary surfactant. By light microscopy the prominent features were the presence of enlarged type II alveolar epithelial cells and foamy intraalveolar macrophages. Compared with controls, type II cells in treated rats apparently contained more numerous surfactant-containing lamellar bodies, as visualized in semi-thin sections of Epon-embedded tissue. This difference was quantified morphometrically by light microscopy: the number of lamellar bodies was estimated as the profile number per individual type II alveolar cell, transsected at its nucleus. Clofibrate administration for 7 days resulted in a significant increase in the number of the lamellar inclusions. In contrast the number of type II alveolar cells per area of lung remained unchanged. There was no evidence of atelectasis or inflammatory infiltration in the drug-treated lungs, a finding confirmed in sections of perfusion-fixed, paraffin-embedded whole lung-lobes. By electron microscopy the lamellar inclusion bodies in the type II alveolar cells in treated rats, apart from being more numerous and sometimes smaller, were morphologically identical to those in controls. The vacuolated alveolar macrophages seen in treated rats also contained various lamellar phospholipid inclusions. Some of the alveoli in the treated animals showed a slightly increased acellular lining layer consisting of membranous bodies and lattices of tubular surfactant myelin. The data obtained support the view that clofibrate enhances the amount of intracellular pulmonary surfactant, although it is unclear if this results from an increase in the rate of surfactant synthesis or a decrease in the rate of surfactant secretion. Since clofibrate is known to increase the number of lipid metabolizing peroxisomes in rodent hepatocytes, it well be of interest in future studies to determine whether the changes observed in surfactant-metabolizing lung cells will be associated with the proliferation of pulmonary peroxisomes.

Stereoscopic back-scattered electron imaging of silver-stained proteins in nucleoli.

By using simultaneously the AgNOR silver staining method, back-scattered electron imaging mode and stereo-tilt in scanning electron microscopy (SEM), it is possible to observe the nucleus through the cell surface, the nucleolus, and the tri-dimensional distribution of the AgNOR-associated acidic proteins. In C3H10T1:2 cells and their 7-12-dimethylbenz-α-anthracene-treated transformants, the staining demonstrates several intranucleolar silver-staining granules (SSG), surrounded by a weakly staining region. The SSG may represent the fibrillar center (FC) and the weakly staining region, the fibrillar dense component (FD). This component can link several SSG together to form a “rope-like structure”. In cells with no visible nucleolus and inactive nucleolar organizer regions (NORs) the silver-staining granules are less numerous, close together and the presumed fibrillar dense components are not visible. The SSG are located more peripheraly, and the weakly staining region and the “rope-like structure” are less prominent in control cell nucleoli than in transformed cells with a comparatively high rate of RNA synthesis.