Sox2 expression in breast tumours and activation in breast cancer stem cells.

The cancer stem cell (CSC) model does not imply that tumours are generated from transformed tissue stem cells. The target of transformation could be a tissue stem cell, a progenitor cell, or a differentiated cell that acquires self-renewal ability. The observation that induced pluripotency reprogramming and cancer are related has lead to the speculation that CSCs may arise through a reprogramming-like mechanism. Expression of pluripotency genes (Oct4, Nanog and Sox2) was tested in breast tumours by immunohistochemistry and it was found that Sox2 is expressed in early stage breast tumours. However, expression of Oct4 or Nanog was not found. Mammosphere formation in culture was used to reveal stem cell properties, where expression of Sox2, but not Oct4 or Nanog, was induced. Over-expression of Sox2 increased mammosphere formation, effect dependent on continuous Sox2 expression; furthermore, Sox2 knockdown prevented mammosphere formation and delayed tumour formation in xenograft tumour initiation models. Induction of Sox2 expression was achieved through activation of the distal enhancer of Sox2 promoter upon sphere formation, the same element that controls Sox2 transcription in pluripotent stem cells. These findings suggest that reactivation of Sox2 represents an early step in breast tumour initiation, explaining tumour heterogeneity by placing the tumour-initiating event in any cell along the axis of mammary differentiation.

Effects of VEGF administration or neutralization on the BBB of developing rat brain.

We investigated the effects of exogenous Vascular Endothelial Growth Factor VEGF combined with an enriched environment on BBB integrity after a minimal trauma induced during the first days of the critical visual period in rats, when peak levels of endogenous VEGF secretion are reached. VEGF was administered using osmotic mini-pumps placed in middle cortical layers of P18 Long-Evansrats. Tissue changes were evaluated using conventional histology. BBB integrity was shown by immunohistochemistry techniques for EBA and GluT-1. Mini-pump implantation produced a wider cavity in anti-VEGF infused rats. In VEGF-infused rats there was a damaged region around the cannula that was smaller in rats raised in an enriched environment (EE). The administration of VEGF induced a high concentration of plasma proteins in the neuropil around the point of cannula placement and a high inflammatory reaction. VEGF-infused rats raised in an EE showed a lower degree of extravasation and better tissue preservation. Anti-VEGF administration produced a lower protein expression profile and more widespread deterioration of tissue. Double immunofluorescence for EBA and GluT-1 showed that the administration of VEGF preserves the tissue, which remains present but not fully functional. In contrast, a combination of VEGF administration and an EE partially protects the functionally damaged tissue with a higher preservation of BBB integrity.

The role of eNOS in vascular permeability in ENU-induced gliomas.

Brain edema in gliomas is an epiphenomenon related to blood-brain-barrier (BBB) breakdown in which endothelial nitric oxide synthase (eNOS) plays a key role. When induced by vascular endothelial growth factor (VEGF), eNOS synthesizes nitric oxide that increases vascular permeability. We investigated the relationship between eNOS, VEGF and BBB dysfunction in experimental gliomas.Tumors were produced in Sprague-Dawley rats by transplacentary administration of Ethylnitrosourea (ENU). Immunoexpression of eNOS and VEGF(165) was studied to identify locations of vascular permeability. BBB permeability was evaluated using gadolinium and intravital dyes and BBB integrity by endothelial barrier antigen (EBA), glucose transporter-1 (GluT-1) and occludin immunostaining. Low grade gliomas displayed constitutive eNOS expression in endothelial cells and in VEGF-positive astrocytes surrounding vessels. Malignant gliomas overexpressed eNOS in aberrant vessels and displayed numerous adjacent reactive astrocytes positive for VEGF. Huge dilated vessels inside tumors and glomeruloid vessels on the periphery of the tumor showed strong immunopositivity for eNOS and a lack of occludin and EBA staining in several vascular sections. BBB dysfunction on these aberrant vessels caused increased permeability as shown by Gadolinium contrast enhancement and intravital dye extravasation.These findings support the central role of eNOS in intra- and peritumoral edema in ENU-induced gliomas.

Lectin histochemical localization of N- and O-linked oligosaccharides during the spermiogenesis of the urodele amphibian Pleurodeles waltl.

The aim of this work is the characterization of the glycoconjugates of the spermatids during the spermiogenesis of the testis of an urodele amphibian, Pleurodeles waltl, by means of lectins in combination with several chemical and enzymatic procedures, in order to establish the distribution of N- and O-linked oligosaccharides in these cells. The acrosome was the most relevant lectin-labeled structure. The O-linked oligosaccharides contained DBA- and SBA-positive GalNAc, AAA-positive Fuc and PNA-positive Gal beta1,3GalNAc. Sialic acid was scarcely observed, the Neu5Ac alpha2,3Gal beta1,4GlcNAc sequence was found in N-linked oligosaccharides. Additionally, N-linked oligosaccharides containing HPA-positive GalNAc and AAA-positive Fuc were found. Moreover, with some lectins the acrosome showed a variable composition of the oligosaccharides in the different steps of the sperm maturation. Some residues were found only in the early steps in maturating acrosome, while others were in the later steps, showing that acrosomal glycoconjugates are modified during acrosome development in spermiogenesis. The changes observed during acrosome maturation suggest the existence of a predetermined pattern of storage of the acrosome components and a progressive compression of them.

Lectin-gold localization of fucose residues in human gastric mucosa.

The oligosaccharides of the mucous gastric glycoproteins are involved in the protection of the gastric mucosa and are altered in different diseases. Therefore, it is important to know their composition in health, to better determine the alterations induced by the disease. Moreover, analysis of the molecular composition of the fundic gland cells has been previously used to obtain new insights into the origin of the different cell types. The aim of the present study was the localization in the subcellular structures of the fucose residues of the oligosaccharides in human fundic glands. For this, lectin cytochemical methods were used at the light and electron microscopic levels. They were combined with enzymatic and chemical treatments to characterize the nature of the oligosaccharide chains containing the fucose residues. The presence of this carbohydrate belonging to N- or O-linked oligosaccharides has been demonstrated in the secretory granules of the surface, gastric pit, mucous neck, and transitional cells of the fundic mucosa, and in the intracellular canaliculi and tubulovesicular system of the parietal cells. These fucose residues were added in the trans-Golgi regions to the elongating chains. Additional fucose linked to the innermmost N-acetylglucosamine of the N-linked oligosaccharides was found in the chief cells, being incorporated in the cis-Golgi. The findings in the transitional cells corroborate the origin of the chief cells from the mucous neck cells.

Secretion of fucosylated oligosaccharides related to the H antigen by human gastric cells.

Although the role of the blood group antigens in the gastrointestinal tract is not well understood, alterations in blood group-related antigens have been described in some pathological processes. Thus, the knowledge of their expression under normal conditions is of special interest. Those individuals expressing their ABO blood group in exocrine epithelia and secretions are called secretors. The aim of the present study was the localization of H antigen expression in the normal human gastric epithelial cells of non-O blood group individuals. For this, a monoclonal anti-H antibody was examined by immunocytochemical methods at both the light and electron microscopic levels. In combination with enzymatic and chemical treatments, the nature of the oligosaccharide chains containing the H antigen was characterized. The selected cases were four A secretors, three A nonsecretors, and three B non-secretors. The labeling of the anti-H antibody in the human stomach is described, irrespective of the blood group of the individuals. The staining was abolished when O-linked oligosaccharides were removed. Since commercially available anti-H antibodies usually also recognize other H-related antigens, the labeling of the antibody by H-related antigens cannot be dismissed. Our findings suggest the existence of H or H-related antigens in the O-linked oligosaccharides of the secretory granules of the surface, gastric pit, mucous neck, and transitional cells of the fundic mucosa, and in the intracellular canaliculi and tubulovesicular system of parietal cells. The H or H-related antigens were also localized in the apical membrane of all the cell types of the epithelial cells of the human fundic mucosa. The overall distribution of the H or H-related antigens in the stomach in non-O blood group individuals suggests the constitutive expression of an alpha(1,2)fucosyltransferase.

Analysis of the distribution of glycoconjugates in the digestive gland of the bivalve mollusc Mytilus galloprovincialis by conventional and lectin histochemistry.

We examined the distribution and pattern of reactivity of a panel of 16 lectins in the digestive gland of the bivalve mollusc Mytilus galloprovincialis at the light-microscopic level. Various chemical treatments were applied in combination with lectins to differentiate between N- and O-linked oligosaccharides. Several control reactions were carried out, including replacement of lectins by buffer and incubation with their specific competitive inhibitors. Some lectins reacted selectively with particular cell types, thus revealing a cell-specific glycoconjugate distribution pattern which is possibly related to the metabolic role of each cell type in the digestive gland. Glycoconjugates containing glucosamine, mannose, and sulfated galactose were associated with the endolysosomal system of digestive cells. These glycoconjugates were also found in small vesicles randomly distributed in the cytoplasm of adipogranular cells in the connective tissue. However galactosamine residues appeared to be associated mainly with basophilic cells. Fucose residues did not exhibit a cell-specific distribution and appeared in small amounts homogeneously distributed throughout the digestive gland tissue. Conventional histochemical reactions for carbohydrate detection revealed moderate amounts of periodic-acid-Schiff-positive, neutral carbohydrates widely distributed in digestive and connective tissues. Among the acid carbohydrates, most cell types contained complex sulfated carbohydrates, but not carboxylated ones; this agreed well with the complete lack of sialic acid residues in all cell types studied, as observed by lectin histochemistry.

N- and O-linked oligosaccharides in the secretory granules of rat Paneth cells: an ultrastructural cytochemical study.

Paneth cells are located at the base of the intestinal glands. The origin, composition, and function of these cells have not been well established. The sharing of a common pathway of development with the goblet cells has been suggested. The aim of the present study was to explore the cytochemical composition of rat Paneth cells and to discuss a possible developmental relationship between goblet and Paneth cells. Lectins (WGA, LTA, UEA-1, AAA, and HPA) were used as a precise tool for the ultrastructural localization of carbohydrates. Several procedures were performed in combination with lectin cytochemistry: beta-elimination, a reaction that specifically removes O-linked oligosaccharides (typical of mucin-type glycoproteins of goblet cells); and treatment with peptide N-glycosidase F, an enzyme that removes N-linked oligosaccharides from glycoproteins. Secretory granules of Paneth cells showed a biphasic nature composed of an electron-lucent peripheral halo containing O-linked oligosaccharides with GalNAc and GlcNAc residues and N-linked oligosaccharides with GlcNAc residues (only sparse Fuc residues were scarcely identified in O-linked oligosaccharides), and an electron-dense core containing N- and O-linked oligosaccharides with Fuc residues. Neither GlcNAc nor GalNAc was identified. The occurrence of O-linked oligosaccharides in the Paneth cells and the biphasic nature of the secretory granules, similar to that of transitional cells intermediate between mucous and serous cells of other tissues, favor the hypothesis of a common lineage for goblet and Paneth cells.