Species cross-reactive membrane-associated urothelial differentiation antigen.

An antigen, termed "urothelium membrane antigen" (UMA), confined to urothelium and most abundantly associated with the asymmetric unit membrane of the terminally differentiated luminal cells was identified with a monospecific (absorbed or affinity purified) rabbit antiserum obtained by immunization with membranes from normal bovine urothelium. A cross-species immunofluorescence-positive reaction was observed in normal bladder: Luminal urothelial membrane reacted most intensely. Underlying normal urothelial cells also showed a weaker cytoplasmic reaction. Specific luminal membrane labeling was confirmed by transmission and scanning electron microscopy. Specificity of the anti-UMA differed from the general epithelial reactivity obtained with antibodies to bovine urothelium cytosol. Anti-UMA did not cross-react with keratin, although there was some evidence of a filamentous localization in the cytoplasm of permeabilized cells. UMA was variably expressed by urothelial carcinoma-derived cell lines to a degree apparently related to the degree of cell differentiation, showing the highest positivity on the well-differentiated RT4 and RT112 lines and only a very weak reaction with the anaplastic MGHU-1 (EJ) and T24 lines. On immunoblots, anti-UMA reacted with a peptide of approximately 54,000 daltons.

Characterization of the P-glycoprotein over-expressing drug resistance phenotype exhibited by Chinese hamster ovary cells following their in-vitro exposure to fractionated X-irradiation.

Exposure of the Chinese hamster ovarian AuxB1 cell line in vitro to fractionated X-irradiation generated sublines designated DXR-10, which proved resistant to multiple drugs and overexpressed P-glycoprotein (Pgp), as judged by Western blotting using the C219 monoclonal antibody. Further characterization of these irradiated DXR-10 sublines has provided evidence for: (i) the expression of cross-resistance to gramacidin D, taxol, puromycin and Navelbine, but not to daunomycin or mitoxantrone; (ii) overexpression of the class I Pgp, as judged by Western blotting using the C494 monoclonal antibody; (iii) decreased accumulation of 3H-vincristine, which could be enhanced by verapamil addition; (iv) unaltered accumulation and subcellular distribution of adriamycin; (v) significantly increased rhodamine 123 accumulation in the presence of verapamil; (vi) plasma-membrane ultrastructural modifications resulting in a significantly increased surface area; (vii) numerous clonal karyotypic alterations, with abnormalities involving the long arm of chromosome 1 being consistently identified; (viii) a lack of overexpression of sorcin; (ix) increased total glutathione levels and overexpression of glutathione S-transferase pi. The fact that only certain of these features are considered characteristic of the 'classic' multidrug-resistant CHRC5 cell line supports our earlier proposal that exposure to fractionated X-irradiation results in the expression of a unique drug-resistance phenotype.

Radiation effects in the small bowel of the diabetic mouse.

PURPOSE: Irradiation of the small intestine in the mouse induces damaging structural alterations to the architecture of the enteric mucosa. There is growing interest in the possible relevance of underlying additional pathology when appreciating the total response of tissues to irradiation. The possibility that small intestinal mucosal abnormalities in the streptozotocin-induced diabetic mouse may exacerbate radiation-induced injury was tested by examining the combined effects of the two treatments. MATERIALS AND METHODS: Streptozotocin-diabetic and -non-diabetic mice were exposed to 10 Gy abdominal X-radiation. Profiles of mucosal epithelial cell populations were quantified and comparisons with corresponding groups of unirradiated mice made on the third day post-irradiation. RESULTS: The histological appearances of the small intestinal mucosa were similar in both groups of irradiated mice, but the numbers of profiles of crypts and of columnar, goblet, Paneth and entero-endocrine cells were depressed in these groups when compared with values in corresponding groups of unirradiated mice. However, the expression of radiation damage in the diabetic mouse was less severe than in the non-diabetic mouse, particularly in the jejunum where the changes attendant on the onset of diabetes were most marked. CONCLUSION: These findings suggest that the response of mouse to radiation may be moderated by the presence of this type of pathophysiology. However, there is no evidence that the damage produced by streptozotocin-induced diabetes and radiation is additive.

A commentary on morphological and quantitative aspects of microparticle translocation across the gastrointestinal mucosa.

It is now generally accepted that particulates in the nano-range (< 1 micron) can and do cross the intestinal mucosa. However, the issue is less well resolved for particles in the micro-range (> 1 micron) and this is discussed in relation to the variety of experimental designs present in the literature. Emphasis is placed on the relative contributions of quantitative bulk tissue analysis with respect to qualitative and quantitative morphological analysis. The discussion is extended to observations on factors influencing the particle translocation process including variation in particle uptake in relation to intestinal region and time post-dose administration based on data for uptake of -2 microns latex particles by rat Peyer's patch tissue. Although a significant body of data now identifies the intestinal processus of particle translocation it is underlined that discrepancies may arise as a consequence of different analytical approaches and that this is an issue to be addressed for valid comparisons of data.

Morphogenesis and ultrastructure of the mouse embryonic salivary gland in tissue culture. Normal, and following exposure to trypsin.

The primordial submandibular glands of 12-day-old mouse embryos were studied in tissue culture before and after treatment with trypsin under the electron microscope. In vitro differentiation proceeded normally and reached a high level of differentiation. Following a soak in trypsin for 15 or 30 minutes, considerable changes were noted in the basal lamina and in the mesenchymatous cells. There often occurred bizarre bullous protrusions of the cytoplasm through the apparently weakened basal lamina and the mesenchymatous cells were converted into so-called "ropalocytes". Subsequently the cells regained their normal appearance and the basement lamina was covered by a thick layer of amorphous electron-opaque basement membrane like material. It is concluded that the basal lamina (the basement membrane under the light microscope) might be the keystone in the differentiation of an organ and its maintenance in the adult. The development of innervation has also been studied and it was shown that the developing submandibular galnd is endowed with large bundles of nerve axons surrounded by Schwann cells lying in the epithelial-mesenchymal region. Intra-epithelial nerves were conspicuous and occasional synaptic bars or rings could be seen contributing to thedifferentiation of the secretory cell.

Organ culture of the avian and mammalian otocyst.

A chemically defined medium supplemented with serum has proved suitable for the growth of the isolated embryonic otocyst of both avian and mammalian provenience. The results lend further support to the value of the technique and confirm the findings of previous authors. [A film was presented.]

Tympanosclerosis. An electron microscopic study of matrix vesicles.

The ultrastructure of tympanosclerotic tissue has been studied and compared with other processes of fibrogenesis and calcification. Evidence is presented of the presence of large numbers of extracellular membrane-bound matrix vesicles and their role as the site of primary calcification in tympanosclerosis. Calcified spherular structures are formed and fuse into larger structures lined by a lamina limitans indicating the lines of arrested calcification in the collagenous matrix.

A morphological and microanalytical investigation into the uptake of particulate iron across the gastrointestinal tract of rats.

The uptake and translocation of particulate iron across the gastrointestinal (GI) mucosa of young adult rats has been investigated using a range of morphological techniques and X-ray microanalysis (XRMA). In animals fed a suspension of iron powder constituted of metallic iron particles ranging in size from 6-9 microns down to 5-30 nm, light microscopic histochemistry has clearly revealed iron deposits within the tissues of the duodenum. Scanning electron microscopy of the duodenal tissue by back-scattered electron imaging has complemented the light microscopic observations and revealed a selective localization of iron in the villi with variation in levels of iron uptake by the mucosal cells. Ultrastructural and XRMA analysis of duodenum has established the presence of metallic iron nanoparticles within the brush border, lateral intercellular spaces of the mucosal cells, mitochondrial cristae and cytoplasm of both mucosal and stromal cells. The observations indicate that metallic iron particles, in the nano-size range, may be taken up by the GI mucosa and that the passage of such particles across the epithelial barrier may take place through both a paracellular as well as a transcytotic process.

On the molecular profiling of cell surfaces by SEM.

Cell surfaces interface with a variety of environments and, as a consequence, cell surface properties are of considerable functional importance to the biological organism. SEM immunocytochemistry (SEM-IC) is one of a range of techniques used to analyse cell surface properties. A major goal of SEM-IC centres on extended survey or high-magnification morphological analysis of cell and tissue surfaces combined with molecular profiles of these surfaces as established by gold-labelling. The properties of colloidal gold make it the marker of choice for SEM-IC and a representative gold-labelling protocol is outlined. The SEM-IC gold-labelling technique has been applied advantageously to the analysis both of cell surfaces and cytoskeletal and extracellular matrix elements: a tabulation of the main SEM-IC biomedical applications is given. Illustrated examples demonstrate how SEM-IC provides a highly effective approach for analysis both of cell and tissue differentiation-maturation sequences, and of pathological change involving not only the entire tissue or cell surface but also minute changes in microdomain characteristics of the individual cell surface. Steps in exploiting the technique of colloidal gold SEM-IC have been several-fold and include: use of backscattered electron imaging; accurate localization of gold particles by superimposition on topographical maps of the cell surface; and use of small (1-10 nm) gold probes followed by silver enhancement in order to minimize steric hindrance. Factors under assessment include: use of low voltage SEM; BE imaging of samples coated with ultrathin metal films; and use of gold-labelled SEM-IC for direct quantification of the numbers of target molecules exposed on cell surfaces by automated image analysis of the digitized BE image.

Chemically-defined medium for growth and differentiation of mixed epithelial and connective tissues in organ culture.

The effect on tissue differentiation and growth in vitro of certain of the factors implicated in collagen synthesis (ascorbic acid, alpha-ketoglutarate and oxygen) and the influence of hydrocortisone was studied using organ cultures of fetal mouse mandible as a mixed epithelial and connective tissue system. Using serum-free Waymouth's MB 752/1 chemically-defined medium, addition of high levels of ascorbic acid (300mug per ml), hydrocortisone (1mug per ml) and oxygen (95%) enhanced differentiation in a number of tissues, in particular skin and appendages, tooth germs and bone, while osteoid and dentine production were noticeable promoted. It is suggested that an essential aspect of media design for organ culture involves the incorporaation of collagen-promoting factors to the in vitro enviornment particularly with regard to the controlling role implicated for collagen in a variety of biological processess.

Quantitative evaluation of autoradiographs by X-ray spectroscopy.

Measurements of mass of developed silver in autoradiograms have been made using X-ray microanalysis. This procedure has been applied to a scanning electron microscopic study of protease-induced cell stimulation in 3H-thymidine-labelled bladder tissues in vitro. Differences in silver concentrations over individual nuclei were demonstrated by X-ray analysis and quantitative data was obtained showing an increased rate of 3H-thymidine incorporation in protease-treated cultures. The initial evidence indicates that X-ray microanalysis could provide a potentially useful quantitative procedure for autoradiography.

Epithelial-stromal interactions in the adult bladder: urothelial growth, differentiation, and maturation on culture facsimiles of bladder stroma.

Analysis of the responsiveness of isolated adult urothelium to a series of different stromal cell-extracellular matrix combinations demonstrated the capacity of stromal cells to induce and maintain normal patterns of urothelial growth, differentiation, and maturation in vitro. By incorporating embryonic mesenchymal derived (Swiss 3T3) cells into type I collagen matrices, simplified three-dimensional tissue-like facsimiles of bladder stroma were derived. When recombined with sheets of isolated urothelium these facsimiles could approximately reproduce the capacity of natural stromal tissue to support the expression of normal urothelial tissue specific characteristics. In contrast cocultures between urothelia and monolayers of 3T3 cells, applied to the surface of planar collagen substrata could only permit urothelial cell attachment but not growth or differentiation whereas lethally irradiated 3T3 (feeder) cells, under similar experimental conditions, could support the maintenance of an immature or incompletely differentiated urothelium. Conditioned medium elaborated by cultured 3T3 cells could not stimulate further differentiation in urothelia cultured alone on planar collagen substrata. These studies indicate that a significant portion of the regulatory capacity of the stroma in stromal-urothelial interactions can be accounted for by the activities of a closely applied population of stromal cells, provided the cells are viable and presented to the urothelium in a three-dimensional context in combination with collagen. The capacity of embryonic mesenchymal cells to express properties appropriate to the development of a multilayered terminally differentiated urothelium suggests that normal interactions between adult urothelium and stroma are of limited specificity with the urothelium requiring an essential input of permissive signals only.

Gastrointestinal uptake and translocation of microparticles in the streptozotocin-diabetic rat.

Uptake and translocation of particulates across the mucosal barrier of the gastrointestinal (GI) tract is now generally recognised but the effect of pathophysiologically induced changes on this process is less well established. This study evaluated the effect of diabetes mellitus on GI absorption of particles, comparing particle localisation and particle loading in different microanatomical sites of the primary organ (small intestine) and possible particle translocation pathways to selected secondary organs (mesenteric lymph nodes, liver, spleen) in normal and streptozotocin-induced diabetic animals. Fluorescent polystyrene latex particles (approximately 2 microns diameter) were fed orally to young adult Sprague-Dawley rats and quantitative bulk tissue and morphological techniques used to chart particle transit across the small intestine to secondary organs 0.5 h postadministration. In the normal animal, epifluorescence and confocal laser scanning microscopy provided confirmatory evidence for particle absorption within the primary organ and transport to other sites in the body. By contrast, in the diabetic animal, particle translocation and peripheral distribution were reduced with approximately 30% decrease in particle loading in the epithelial/nonepithelial tissue compartments. This could be a consequence of gastric retention and altered intestinal motility and permeability which are known to be associated with diabetes.

Urothelium-specific antibody and lectin surface mapping of bladder urothelium.

Coupled ligand-colloidal gold complexes were found to provide a convenient approach for the localization of scanning electron microscopy of cell surface membrane antigens and lectin-binding sites on bladder urothelium and for the immunocytochemical identification of urothelial cell populations at different stages of differentiation. The ligands used to prove the membrane were a urothelium-specific rabbit antibody raised to a urothelial membrane-associated antigen (UMA), and two lectins: Concanavalin A (Con A) and peanut agglutinin (PNA). A complex luminal surface distribution pattern was demonstrated by the UMA antigen related to the stage of urothelial cell maturation and differentiation. UMA could be detected on the surface of immature and early differentiating intermediate cells, but was absent from the late differentiation stage, becoming re-expressed as the cells matured and was found in greatest abundance on the terminally differentiated superficial cells. It was absent on cells in benign hyperplasia of the urothelium. Cellular and regional differences in lectin binding to the urothelial cell surface was suggested with Con A receptors localized uniformly over the superficial cells, and PNA receptors confined to linear arrays or occasional clusters over the apical surface but evenly dispersed over the lateral surface of these cells.

A methodological basis for SEM autoradiography: biosynthesis and radioligand binding.

A method is described for scanning electron microscope (SEM) autoradiography whereby preservation of high resolution cell surface details is retained together with degelatination of the emulsion without gross loss or redistribution of silver grains. This method should provide a convenient medium-sized marker for SEM (using secondary, backscattered electron and X-ray imaging) topographic studies of biosynthesized molecules, and of cell surface receptors and antigens, using indirect or direct labelling procedures with radio-labelled ligands.

Colloidal gold--a powerful tool in scanning electron microscope immunocytochemistry: an overview of bioapplications.

Colloidal gold may be conjugated to a wide variety of macromolecules, provides a versatile system for immunocytochemical studies by various types of microscopy (light and fluorescent microscopy, scanning (SEM) and transmission (TEM) electron microscopy), and is significantly contributing to the development of SEM immunocytochemistry as a routine analytical procedure. A comprehensive overview has been compiled of the literature on SEM bioapplications of colloidal gold. This is illustrated through a selected series of studies focussing on a) cell surface receptor-ligand interactions; b) expression of cell surface lectin-binding sites; c) surface distribution of extracellular matrix components; and d) visualization of gold-labelled cytoskeletal elements with emphasis on the use of backscattered electron imaging as a powerful analytical adjunct in the development of SEM immunocytochemistry.

A review of the colloidal gold marker system.

Colloidal gold can be used as a particulate marker for the detection and localization of target molecules by various modes of microscopy (light and fluorescent microscopy, scanning and transmission electron microscopy) using both direct and indirect labeling approaches. Several techniques are available for the preparation of gold markers in a size range of 5nm to 150nm, their mean size and shape characteristics and absorption spectra varying with particle size. Under appropriate conditions, colloidal gold will bind macromolecules by non-covalent electrostatic adsorption with little change in the specific activity of the bound macromolecule. This interaction is influenced by a number of factors including ionic concentration, pH conditions (in correlation with the protein pI values) and protein stabilizing levels. Presence of reactive protein on probes can be demonstrated and quantitated by direct and indirect radioactive binding assays and agglutination assays. These assays provide convenient procedures for characterizing stability, and behaviour in storage, of gold probes. Stability of gold probes under conditons where competing proteins are present, under freeze-thaw cycles and under SEM preparation conditions have been evaluated in this paper. Some of the basic procedures in the application of gold probes to cell labeling are briefly discussed together with certain limitations of the colloidal gold marker system. A bibliography of gold probe cell labeling studies is included.