Differential expression of glycans in the urothelial layers of horse urinary bladder.

BACKGROUND: Urothelium is a multilayer epithelium covering the inner surface of the urinary bladder that acts as a blood-urine barrier and is involved in maintaining the wellbeing of the whole organism. Glycans serve in the maturation and differentiation of cells and thus play a key role in the morphology and function of the multilayered epithelium. The aim of the present study was to examine the glycoprotein pattern of the horse urinary bladder urothelium by lectin histochemistry. METHODS: The study involved urinary bladders from four horse stallions. Tissue sections were stained with a panel of eleven lectins, in combination with saponification and sialidase digestion (Ks). RESULTS: Basal cells displayed high-mannose N-glycans (Con A), α2,6-linked sialic acid (SNA), and O-linked sialoglycans with sialic acids linked to Galßl,3GalNAc (T antigen) (KsPNA) and terminal N-acetylgalactosamine (Tn antigen) (KsSBA). The young intermediate cells expressed terminal N-acetylglucosamine (GlcNAc) (GSA II), galactose (GSA I-B4), T- and Tn antigens (PNA, SBA). The mature intermediate cells showed additional high-mannose N-glycans, O-linked sialoglycans (sialyl-T antigen, sialyl-Tn antigen), α2,6- and α2,3-linked sialic acid (MAL II), α1,2-linked fucose (UEA I), and GlcNAc (KsWGA). The latter residue marked the boundary with the overlying surface layer. Few Con A positive intermediate cells were seen to cross the entire urothelium thickness. The surface cells showed additional glycans such as T antigen and sialic acids linked to GalNAc binding DBA (KsDBA). Few surface cells contained α1,3-linked fucose (LTA), whereas some other cells displayed intraluminal secretion of mucin-type glycans terminating with GalNAcα1,3(LFucα1,2)Galß1,3/4GlcNAcß1 (DBA). The luminal surface expressed the most complex glycan pattern in the urothelium because only α1,3-linked fucose lacked among the demonstrated glycans. CONCLUSIONS: This study showed that the glycan pattern becomes more complex from the basal to surface layer of the urothelium and that surface cells could modify the composition of urine via the secretion of glycoproteins.

Ultrastructural characteristics of ovine bone marrow-derived mesenchymal stromal cells cultured with a silicon stabilized tricalcium phosphate bioceramic.

Bioceramics are being used in experimental bone engineering application in association with bone marrow derived mesenchymal stem cells (BM-MSCs) as a new therapeutic tool, but their effects on the ultrastructure of BM-MSCs are yet unknown. In this study we report the morphological features of ovine (o)BM-MSCs cultured with Skelite, a resorbable bioceramic based on silicon stabilized tricalcium phosphate (SiTCP), able to promote the repair of induced bone defect in sheep model. oBM-MSCs were isolated from the iliac crest, cultured until they reached near-confluence and incubated with SiTCP. After 48 hr the monolayers were highly damaged and only few cells adhered to the plastic. Thus, SiTCP was removed, and after washing the cells were cultured until they became confluent. Then, they were trypsinizated and processed for transmission electron microscopy (TEM) and RT-PCR analysis. RT-PCR displayed that oBM-MSCs express typical surface marker for MSCs. TEM revealed the presence of electron-lucent cells and electron-dense cells, both expressing the CD90 surface antigen. The prominent feature of electron-lucent cells was the concentration of cytoplasmic organelles around the nucleus as well as large surface blebs containing glycogen or profiles of endoplasmic reticulum. The dark cells had a multilocular appearance by the presence of peripheral vacuoles. Some dark cells contained endocytic vesicles, lysosomes, and glycogen aggregates. oBM-MSCs showed different types of specialized interconnections. The comparison with ultrastructural features of untreated oBM-MSCs suggests the light and dark cells are two distinct cell types which were differently affected by SiTCP bioceramic. Skelite cultured ovine BM-MSCs display electron-dense and electron-lucent cells which are differently affected by this bioceramic. This suggests that they could play a different role in bioceramic based therapy.

Ultrastructural study of cultured ovine bone marrow-derived mesenchymal stromal cells.

Ovine bone marrow-derived mesenchymal stromal cells (oBM-MSCs) represent a good animal model for cell-based therapy and tissue engineering. Despite their use as a new therapeutic tool for several clinical applications, the morphological features of oBM-MSCs are yet unknown. Therefore, in this study the ultrastructural phenotype of these cells was analysed by transmission electron microscopy (TEM). The oBM-MSCs were isolated from the iliac crest and cultured until they reached near-confluence. After trypsinization, they were processed to investigate their ultrastructural features as well as specific surface marker proteins by flow cytometry and immunogold electron microscopy. Flow cytometry displayed that all oBM-MSCs lacked expression of CD31, CD34, CD45, HLA-DR whereas they expressed CD44, CD58, HLAI and a minor subset of the cell population (12%) exhibited CD90. TEM revealed the presence of two morphologically distinct cell types: cuboidal electron-lucent cells and spindle-shaped electron-dense cells, both expressing the CD90 antigen. Most of the electron-lucent cells showed glycogen aggregates, dilated cisternae of RER, moderately developed Golgi complex, and secretory activity. The electron-dense cell type was constituted by two different cell-populations: type A cells with numerous endosomes, dense bodies, rod-shaped mitochondria and filopodia; type B cells with elongated mitochondria, thin pseudopodia and cytoplasmic connectivity with electron-lucent cells. These morphological findings could provide a useful support to identify "in situ" the cellular components involved in the cell-therapy when cultured oBM-MSCs are injected.

In situ characterization of glycans in the urothelium of donkey bladder: evidence of secretion of sialomucins.

The glycoprotein pattern was investigated by lectin histochemistry in the urothelium lining the urinary bladder of the donkey Equus asinus. Tissue sections were stained with a panel of twelve lectins, in combination with saponification and sialidase digestion (K-s). The urinary bladder urothelium has three distinct layers from the basal zone to the lumen consisting of basal, intermediate and superficial cells (umbrella cells). Cytoplasm of basal cells reacted with SNA, PNA, K-s-PNA, GSA I-B4 and Con A showing glycans ending with Neu5Acα2,6Gal/GalNAc, Neu5AcGalß1,3GalNAc, αGal and with terminal/internal αMan. The cytoplasm of umbrella cells displayed an increase of Neu5AcGalß1,3GalNAc and the appearance of Neu5AcGalß1,3GalNAc, Neu5acα2,3Galß1,4GlcNAc and Neu5AcGalNAc residues (MAL II, K-s-SBA and K-s-HPA staining). Scattered umbrella cells were characterized by glycans terminating with GalNAc binding DBA, SBA and HPA. The mucosa forms folds with a crypt-like appearance where the urothelium shows a different pattern of glycans. The bladder luminal surface stained with K-s-PNA, K-s-DBA, KOH-s-SBA, and K-s-HPA displaying a coating of sialoglycoproteins belonging to O-linked glycans (typical secretory moieties). These findings show that different glycosylation patterns exist along the donkey bladder urothelium, and different sub-populations of umbrella cells are present secreting the sialoglycans which constitute the protective gel layer lining the bladder.

Morphology and lectin-binding sites of pyloric caeca epithelium in normal and GnRH-treated Atlantic bluefin tuna (Thunnus thynnus) Linnaeus 1758.

Mucosal epithelium of pyloric caeca was studied in normal and in GnRH-treated Atlantic bluefin tuna Thunnus thynnus L., using morphological analysis, conventional and lectin glycohistochemistry. The lining epithelium consisted of columnar (absorptive) cells, goblet cells and intraepithelial leucocytes. The epithelium from normal animals was significantly taller than GnRH-treated samples. Conventional histochemistry displayed the same staining pattern in normal and hormone-treated specimens which showed a mixture of neutral and sulphated acidic glycoconjugates in the luminal surface and goblet cells, and neutral glycans in apical granules of enterocytes. Lectin histochemistry revealed a different glycoconjugate pattern in normal and GnRH-treated tunas. In normal specimens the luminal surface expressed sialoglycoconjugates which bound MAL II, SNA, KOH-sialidase-PNA, KOH-sialidase-SBA as well as asialoglycans stained with HPA, SBA, GSA I-B4 , LTA. N-linked glycans were highlighted by Con A and KOH-sialidase-WGA. In GnRH-treated tunas the luminal surface did not react with SNA, SBA and LTA. The columnar cells of normal tunas bound KOH-sialisase-PNA in the apical region, KOH-sialidase-PNA, KOH-sialidase-DBA, HPA, SBA, KOH-sialidase-SBA and KOH-sialidase-WGA in apical granules, GSA I-B4 and LTA in the supranuclear region. GnRH-treated specimens showed some columnar cells that stained with KOH-sialidase-WGA in the apical granules and with GSA I-B4 in the supranuclear region. The goblet cells of normal animals produced mucins positive to PNA, HPA, KOH-sialidase-DBA, SBA, GSA II. The latter three binding sites lacked in GnRH-treated tunas. The results suggest that the mucosal epithelium of Thunnus thynnus L. pyloric caeca expresses a complex glycan pattern that is affected by GnRH-treatment.

Pepsinogen and H,K-ATPase mediate acid secretion in gastric glands of Triturus carnifex (Amphibia, Caudata).

The gastric glands of Triturus carnifex (Amphibia, Caudata) have been examined by histochemical and immunohistochemical methods with particular regard to hydrochloric acid and pepsinogen secretion. Fundic glands consist of mucous neck cells, endocrine cells and oxynticopeptic cells producing both pepsinogen and hydrochloric acid. The neck cells showed an unexpected distribution pattern which was only observed in the oral fundus, and produced neutral mucins with glycosidic residues of GalNAc and Gal beta1,3GalNAc, and in this respect they differ from the neck cells of anuran amphibians. The secretion of pepsinogen and hydrochloric acid as demonstrated by immunolabelling with anti-H,K-ATPase and with anti-pepsinogen, respectively, seems not to vary significantly along the longitudinal axis of the stomach. The mechanism of gastric acid secretion seems to be mediated by an ATPase, having similar features to the mammalian gastric H,K-ATPase, and is localised in the luminal membrane and in the subapical cytoplasm of the oxynticopeptic cells. Unusually, the same cytoplasmic areas revealed binding specificity for the winged pea lectin (WPA) from Lotus tetragonolobus, even after beta elimination, indicating the presence of fucosyl residues in N-linked oligosaccharidic chains in glycoproteins of beta-H,K-ATPase subunits.