Expression of mesenchymal stem cell marker CD90 on dermal sheath cells of the anagen hair follicle in canine species.

The dermal sheath (DS) of the hair follicle is comprised by fibroblast-like cells and extends along the follicular epithelium, from the bulb up to the infundibulum. From this structure, cells with stem characteristics were isolated: they have a mesenchymal origin and express CD90 protein, a typical marker of mesenchymal stem cells. It is not yet really clear in which region of hair follicle these cells are located but some experimental evidence suggests that dermal stem cells are localized prevalently in the lower part of the anagen hair follicle. As there are no data available regarding DS stem cells in dog species, we carried out a morphological analysis of the hair follicle DS and performed both an immunohistochemical and an immunocytochemical investigation to identify CD90+ cells. We immunohistochemically evidenced a clear and abundant positivity to CD90 protein in the DS cells located in the lower part of anagen hair follicle. The positive cells showed a typical fibroblast-like morphology. They were flat and elongated and inserted among bundles of collagen fibres. The whole structure formed a close and continuous sleeve around the anagen hair follicle. Our immunocytochemical study allowed us to localize CD90 protein at the cytoplasmic membrane level.

Apoptotic cell death in canine hair follicle.

Apoptotic cell death is an essential homeostatic mechanism involved in the control of cellular turnover in a variety of adult tissues. Cytoplasmic and nuclear condensation morphologically define this process whose biochemical hallmark is extensive DNA fragmentation into discrete oligonucleosomic units. Hair follicle growth and regression has been shown to be correlated with apoptosis in humans, mice, rats and guinea pigs. The present study was carried out to evaluate its implication in canine hair biology in order to define the spatio-temporal relationship between apoptosis and the hair cycle in dogs. As assessed by terminal deoxy-nucleotidyl transferase-mediated d-UTP nick-end-labelling (TUNEL) and by basic histological and ultrastructural assays, apoptotic cells appeared both in the growing and in the regressing follicle epithelium showing the well characterized morphological features described in the previous relevant literature.

Topographical localisation of glucidic residues and their variations in the canine zona pellucida during folliculogenesis.

In the present ultrastructural study, horseradish peroxidase-labelled lectins, in conjunction with antiperoxidase antibody and protein A-gold, were used to characterise and localise the oligosaccharide sequences of zona pellucida glycoproteins at different stages of follicular development in the canine ovary. Deacetylation and sialidase digestion were also performed before lectin cytochemistry. The zona pellucida of oocytes present in unilaminar primary follicles reacts with WGA- and RCA-I-lectins. The zona pellucida of oocytes present in bilaminar and trilaminar secondary follicles displays positivity to WGA, RCA-I, Con-A, UEA-I, and sialidase/SBA. This labelling pattern persists in the zona pellucida of oocytes present in antral tertiary follicles with the exception of WGA and RCA-I reactive sites which are differently distributed throughout the zona pellucida. The topographical distribution of these carbohydrates is not uniform throughout the zona pellucida, indicating the regionalization of oligosaccharide chains within three concentric bands of the zona matrix: an inner surface close to the oocyte plasma membrane, an intermediate portion and an outer layer in contact with the follicular cells. Our results demonstrated variations in the presence and distribution of the carbohydrate residues in the canine zona pellucida during different stages of follicular growth. We also observed the presence of vesicles in both the ooplasm and granulosa cells, showing a similar lectin binding pattern to that of the zona pellucida.

Histochemical localization of prostaglandin dehydrogenase activity for PGF-2 alpha in some bovine tissues.

Prostaglandin dehydrogenase activity is histochemically detected in various bovine tissues (kidney, liver, lung, parotid and naso-labial glands) using as substrate prostaglandin F-2 alpha. Kidney, liver and lung showed the highest intensity of the reaction, but parotid and naso-labial glands also displayed enzymatic activity at the level of the ductal cells.

Cytochemical identification of lymphocytes and other mononuclear cells in ovine and bovine hemal nodes.

Cytochemical and immunocytochemical studies were carried out with specific markers for B lymphocytes, dendritic reticulum cells (ATPase, 5'Nase, SIg) and T lymphocytes (ANAE, A.P.) in an attempt to identify the mononuclear cells present in bovine and ovine hemal nodes. The results show that primary nodes and mantle of secondary nodes are composed of B cells, whereas T cells are mainly localized in the interfollicular cords. Since such an arrangement resembles the picture in normal lymph nodes, but the direct contact between lymphatic tissue and blood is more reminiscent of the spleen, hemal nodes probably perform immunological functions similar to those of both normal lymph nodes and spleen.

Lectin histochemical study of bovine lingual glands.

Bovine lingual glands consist of mucous acini capped by demilunes. Information on the chemical structure of their secretory glycoconjugates was obtained by means of a battery of peroxidase-conjugated lectins with affinity for specific terminal sugars. Sialidase procedures followed by lectin staining were also used to visualize the sugar sequences. Stored secretions in mucous acinar cells contained fucose, N-acetylglucosamine, alpha and beta-N-acetylgalactosamine as terminal sugar residues and beta-galactose as penultimate sugar in a heterogeneous distribution. Demilunar cells failed to react with any of the lectins examined except that of Dolichos biflorus.

Histochemical study of lectin binding in the major salivary glands of adult fallow-deer (Dama dama L.).

The sugar residues in glycoconjugates present in the parotid and mandibular glands of the adult fallow-deer were detected and characterized by using a battery of eight different lectin-horseradish peroxidase conjugates. In some cases a treatment with sialidase preceded the lectin staining. Parotid secretory cells produced glycoconjugates with N-acetylgalactosamine, N-acetylglucosamine and mannose residues. Mucous acinar cells were the most reactive sites of the mandibular gland and contained conspicuous quantities of oligosaccharides with terminal sialic acid radicals. Galactosil-(beta 1-->3)N-acetylgalactosamine was the most abundant penultimate sugar linked to N-acetylneuraminic acid. Mandibular mucous cells also presented N-acetylglucosamine and sialylated components with the terminal dimer sialic acid-N-acetylgalactosamine. Demilunar cells contained glycoconjugates with fucose and mannose residues. The apical surface of duct cells was stained by all the lectins.

Carbohydrate histochemistry of lamb duodenum.

In order to elucidate the carbohydrate profile of the mucosa of lamb duodenum, conventional histochemical methods and a panel of 7 labelled lectins were used. In some cases, treatment with sialidase preceeded lectin staining. Carbohydrate histochemistry revealed the presence of sugar residues in the brush border of enterocytes, goblet cells and duodenal glands. All sites contained neutral and acid glycoconjugates. The presence of sulphomucins in goblet and duodenal gland cells was age-dependent. Enterocytes and duodenal gland cells contained abundant amounts of oligosaccharides with terminal sialic acid-galactosyl(beta1 --> 3)N-acetylgalactosamine, whereas goblet cells contained the penultimate N-acetylgalactosamine residue linked to sialic acid. These findings were not age-dependent, whereas scarce amounts of fucose were found in all sites especially in young animals. The findings obtained in the present study serve as a basis for future pathological studies in lamb and sheep.

Retrospective ultramicroscopic investigation on naturally cryptosporidial-infected commercial turkey poults.

The morphometric characteristics and the ultramicroscopic findings of Cryptosporidium spp. at various stages of their life cycle in the intestinal and bursal epithelial cells of naturally infected 30-day-old commercial turkeys are reported. Small, sporulated oocysts, observed in the small intestinal content after flotation, were identified as Cryptosporidium meleagridis on the basis of morphometric characteristics (round in shape and 4.5-5.0 microm in size) and the small intestinal localization. Light section examinations revealed the presence of the protozoon in multiple organs, but its prevalence was highest in the intestinal and bursal epithelial cells. Ultramicroscopic studies on ileum and bursal samples showed the presence of all the life cycle stages in the microvillar brush epithelial cells in both the organs examined. On the basis of the comparison of the morphology and the sizes of the microorganisms parasitizing the ileum and the bursa, hypotheses are considered on the possible species involved.

Immunogold study on lectin binding in the porcine zona pellucida and granulosa cells.

An ultrastructural localization of lectin receptors on the zona pellucida (ZP) of porcine antral oocytes and on the granulosa cells was performed using a panel of horseradish peroxidase-labelled lectins in conjunction with antiperoxidase antibody and protein A-gold. In some cases, lectin incubation was preceded by sialidase digestion. WGA-, Con-A-, UEA-I-, RCA-I-, PNA- and SBA-reactive sites were distributed differently in the porcine ZP. Sialidase digestion increased the positivity obtained with RCA-I and it was necessary to promote PNA and SBA reactivity. These results indicated that the ZP contained N-acetylglucosamine, a-mannose, a-fucose, b-Gal-(1-4)GlcNAc, b-Gal- (1-3)GalNAc, b-GalNAc and sialic acid residues. We also observed the presence of vesicles in both the ooplasm and granulosa cells, showing a similar lectin binding pattern to that of the ZP, thus suggesting that the oocyte and granulosa cells are the site of synthesis of ZP glucidic determinants.

Architecture of sheep haemal nodes.

Sheep haemal nodes are reported to be lymphatic organs within the blood circulation. In this study they were seen to be covered by a capsule of connective tissue, with a hilus in which some blood vessels were detectable. Below the capsule a subcapsular blood sinus and lymphatic nodules were present. A reticular meshwork extended through the interior; the interstices of the reticular meshwork contained a large number of free blood cells, many macrophages, lymphocytes and plasma cells. Blood collected in the subcapsular sinus and flowed through the reticular meshwork aided by the particular alignment of some of the reticular cells in channel-like passageways. Venules originated in the parenchyma and drained into efferent veins that left the organ at various points in the capsule. A large efferent blood vessel, apparently connected with the subcapsular sinus, was present in the hilus.

Glycoconjugates in the mandibular salivary gland of adult dogs revealed by lectin histochemistry.

The glycosidic residues in the mandibular glands of five adult dogs were studied by using seven different lectin-horseradish peroxidase conjugates. In some cases a treatment with sialidase preceded the lectin staining. The mucous acinar cells contained oligosaccharides with alpha- and beta-N-acetylgalactosamine, N-acetylglucosamine and fucose residues, whereas the demilunar cells contained glycoconjugates rich in sialic acid linked to the penultimate disaccharide galactosyl-(beta 1-->3) N-acetylgalactosamine.

Characterisation of sugar residues in glycoconjugates of pig mandibular gland by traditional and lectin histochemistry.

Sugar residues are important components of salivary gland secretion. Traditional histochemical methods and lectin histochemistry were used to characterise glycoconjugates present in the mandibular gland of normal adult pigs. Acinar cells contained abundant quantities of glycoconjugates with the terminal trisaccharide sialic acid - (alpha 2-->3, 6) galactosyl (beta 1-->3) N -acetylgalactosamine. Mandibular acinar cells also contained alpha and beta N -acetylgalactosamine and N -acetylglucosamine residues, whereas the demilunar cells contained glycoconjugates with fucose, mannose and N -acetylglucosamine residues. In the duct system a range of sugar residues were localised throughout the cell cytoplasm or limited to the apical surface. These results provide new knowledge concerning the structure of salivary glycoconjugates in normal adult pig and a basis for future pathological studies.

Detection of glycosidic residues in carpal glands of wild and domestic pig revealed by basic and lectin histochemistry.

Carpal glands are compound tubuloalveolar glands, located on the medial surface of the carpus. This study was carried out on samples from carpal glands of adult wild and domestic pigs of both sexes. We elucidated the glycosidic composition of carpal gland secretion in situ using traditional histochemical methods and lectin histochemistry. Some secretory cells exhibited an intense reaction with PAS in both wild and domestic pigs. Lectin histochemistry showed differences in the localization and composition of glycoconjugates secreted by carpal glands. A cytoplasmic positivity was revealed in the wild pig by the sequence sialidase-PNA and WGA, whereas in the domestic pig the reactivity was localized at the apical surface of some cells. LTA positive cells were found only in the carpal glands of the domestic pig.

Ultrastructure of bovine von Ebner's salivary glands.

Bovine von Ebner's glands were studied by electron microscopy. The gland consists of tubulo-alveolar adenomeres which open into an abbreviated duct system. The cells of the secretory acini show many morphological features typical of serous cells and contain numerous granules with a complex substructure. Short intercalated ducts connect the acini with excretory ducts which are lined with bistratified epithelium. The striated ducts are absent. The von Ebner's gland morphology was compared with that of the same gland in other species of mammals and with the ultrastructure of the major bovine salivary glands.

Complex carbohydrates occurring in the digestive apparatus of Umbrina cirrosa (L.) fry.

The glycosoaminoglycans in the digestive apparatus of immature fish have important biological functions and are involved in morphofunctional differentiation. The aim of this study was to investigate the glycoconjugate histochemistry in the different parts of the digestive apparatus (oesophagus, stomach, intestine) of Umbrina cirrosa (L.) fry using classical histochemical reactions (periodic acid-Schiff, Alcian blue pH 2.5, Alcian blue/periodic acid-Schiff, high iron diamine, low iron diamine) in conjunction with glycolytic digestions that degrade different classes of glycosoaminoglycans. No differences were observed in the reactivity to conventional histochemical staining of the oesophagus, stomach or intestine among 27-, 34- or 44-day-old fry. In the oesophagus, the mucopolysaccharides contained chondroitin sulphates B and A and/or C, heparan sulphate and chondroitin. In the stomach, only neutral glycoconjugates were revealed, whereas in the intestine there were only chondroitin sulphates. Some differences in the type and content of glycoconjugates were found in Umbrina cirrosa (L.) fry compared to those of adult subjects, probably related to different dietary habits and to changes in the environmental conditions.

A lectin histochemical study of the zygomatic salivary gland of adult dogs.

Seven lectins (PNA, DBA, SBA, UEA I, LTA, WGA and ConA), conjugated with horseradish peroxidase, were used to characterize the glycosidic residues in the zygomatic gland of adult dogs. In some cases (PNA and DBA), lectin staining was preceded by neuraminidase digestion. The acinar and tubular cells produced glycoconjugates with different sugar residues, presenting binding sits for all of the lectins used. The apical surfaces of the cells lining the intra- and interlobular ducts were also stained by all the lectins. In contrast, the demilunar cells only reacted with the Neu-PNA sequence and Con A.

Localization of glycoconjugates in dog parotid gland by lectin histochemistry.

Parotid glands from adult dogs were stained with a battery of seven horseradish peroxidase-conjugated lectins (PNA, UEA, LTA, DBA, SBA, WGA and ConA). In some cases (PNA and DBA) neuraminidase digestion was followed by lectin staining. Acinar cells contained conspicuous quantities of oligosaccharides with terminal sialic acid radicals. Galactosil-(beta 1-->3) N-acetylgalactosamine was the most abundant penultimate sugar linked to N-acetylneuraminic acid. Sialylated components having the terminal dimer sialic acid-N-acetylgalactosamine were found in the acinar cells. Secretory cells presented a heterogeneous distribution of glycoconjugates with terminal fucose and beta-N-acetylgalactosamine. Fucose, N-acetylglucosamine and alpha-N-acetylgalactosamine were present on the apical cytoplasm and surface of the striated and interlobular duct cells. This glycosidic composition was unaffected by extensive selective breeding. The role of abundant amounts of sialic acid radicals in the oral mucosa was considered.

Expression of mesenchymal stem cell marker CD90 on dermal sheath cells of the anagen hair follicle in canine species.

The dermal sheath (DS) of the hair follicle is comprised by fibroblast-like cells and extends along the follicular epithelium, from the bulb up to the infundibulum. From this structure, cells with stem characteristics were isolated: they have a mesenchymal origin and express CD90 protein, a typical marker of mesenchymal stem cells. It is not yet really clear in which region of hair follicle these cells are located but some experimental evidence suggests that dermal stem cells are localized prevalently in the lower part of the anagen hair follicle. As there are no data available regarding DS stem cells in dog species, we carried out a morphological analysis of the hair follicle DS and performed both an immunohistochemical and an immunocytochemical investigation to identify CD90+ cells. We immunohistochemically evidenced a clear and abundant positivity to CD90 protein in the DS cells located in the lower part of anagen hair follicle. The positive cells showed a typical fibroblast-like morphology. They were flat and elongated and inserted among bundles of collagen fibres.The whole structure formed a close and continuous sleeve around the anagen hair follicle. Our immunocytochemical study allowed us to localize CD90 protein at the cytoplasmic membrane level.

Immunohistochemical evaluation of intermediate filament nestin in dog hair follicles.

Hair follicles (HFs) are self-renewing structures that reconstitute themselves through the hair cycle. They maintain reservoirs of stem cells (SC) that are thought to reside in the bulge area, a region localized in the lowermost permanent portion of HFs. In mice and humans, HF bulge cells express nestin and present stem features as pluripotency. Nestin is a class VI intermediate filament protein; it was first described as a specific marker of CNS stem cells, but recent studies suggest that it may represent a more general stem cell marker (Wiese et al., 2004; Hoffman, 2006). Bulge cell characteristics have mainly been studied in mice and humans, but recently, a bulge-like region was identified also in dog HFs (Pascucci et al., 2006). In this work we investigate the presence and localization of nestin in dog HFs with the aim of evaluating its expression and to correlate it with the location of the bulge-like region. Immunostaining of skin samples collected from healthy dogs was performed by using a rabbit anti-nestin polyclonal antibody. The presence of a population of immunoreactive cells was revealed in the hair follicle middle region, at the arrector pili muscle insertion level. An immunohistochemical signal was detected only in primary hair follicles throughout the hair cycle. These observations led us to conclude that nestin positive cells are located in the bulge-like region of dog HFs and strengthen our hypothesis regarding the correlation between this region and the dog HF stem compartment.