Diabetes reduces statherin in human parotid: immunogold study and comparison with submandibular gland.

BACKGROUND AND OBJECTIVE: Alteration of salivary gland secretion is one of the consequences of diabetes. In a recent study on the submandibular gland of diabetic subjects, we found changed expression of statherin, a salivary protein of fundamental importance in preserving tooth integrity, whose reduction was related with the high incidence of oral diseases in patients with diabetes. The goal of this report is to extend the study to human parotid gland and to compare the effects of diabetes on statherin expression with those previously described in submandibular gland. MATERIALS AND METHODS: Fragments of parotid glands obtained from diabetic and non-diabetic patients were fixed, dehydrated, embedded in Epon Resin and processed for the immunogold histochemistry. The staining density was expressed as number of gold particles per µm(2) and statistically evaluated. RESULTS AND CONCLUSIONS: In all samples, statherin reactivity was specifically localized in secretory granules of acinar cells. The statistical analysis showed that labelling density was significantly lower in diabetic than in non-diabetic parotid glands and that diabetes affects protein expression at identical extent in parotid and submandibular glands. The results strengthen the hypothesis that a reduced statherin secretion may be responsible for the higher incidence of oral disorders in diabetic subjects.

Reduced statherin reactivity of human submandibular gland in diabetes.

BACKGROUND AND OBJECTIVES: Statherin is a salivary protein involved in the formation of enamel pellicle and in regulation of calcium homeostasis. Diabetes and other pathologies affect both salivary flow and protein secretion by salivary glands, causing increased susceptibility to mucosal infections, tooth demineralization, and caries. The purpose of this study was to compare the statherin expression in submandibular glands of healthy and diabetic subjects. MATERIALS AND METHODS: Fragments of submandibular glands obtained from diabetic and non diabetic patients were fixed, dehydrated, embedded in Epon Resin and processed for the immunogold histochemistry. The results were statistically evaluated. RESULTS: Specific statherin labeling was demonstrated in secretory granules of acinar cells in both diabetic and normal samples. The staining was much more intense in the latter compared to those of diabetics. The labeling density was quantified by evaluating the number and spatial distribution of gold particles within the granules. The number of gold particles was significantly lower in glands from diabetics than in control glands. CONCLUSIONS: The results obtained suggest that a reduced statherin secretion by salivary glands might be partly responsible for a less effective protection of the oral tissues, resulting in an higher incidence of caries and oral infections associated with diabetes.

Electron microscopic detection of statherin in secretory granules of human major salivary glands.

In order to increase current knowledge regarding statherin secretion into the oral cavity, ultrastructural localization of this peptide was investigated in human salivary glands by using a post-embedding immunogold staining technique. Statherin reactivity was found inside the granules of serous cells of parotid and submandibular glands. In parotid granules immunostaining was preferentially present in the less electron-dense region, whereas in submandibular serous granules the reactivity was uniform and the dense core always stained. By contrast, none or weak reactivity was observed in serous cells of major sublingual glands. These findings reveal for the first time the subcellular localization of statherin by electron transmission microscopy and confirm that of the three major types of salivary glands, the parotid and submandibular glands are the greatest source of salivary statherin. Moreover, they suggest that more than one packaging mechanism may be involved in the storage of statherin within serous granules of salivary glands.

Immunocytochemical localization of Lewis blood group antigens in human salivary glands.

We demonstrated the immunohistochemical distribution of Le-a and Le-b blood group antigens in human major and minor salivary glands at the ultrastructural level by applying a post-embedding immunogold staining method. In secretors' glands, a faint Le-a reactivity was found only in mucous droplets, whereas Le-b antigen was intensely stained in secretory granules of most mucous cells, in those of intercalated duct cells, in the pale granular matrix of some serous cells, and, when osmication was omitted, in cytoplasmatic vesicles and cell surfaces of striated ducts. In the submandibular gland of a non-secretor, Le-a antigen was considerably stained in mucous droplets, whereas Le-b reactivity was restricted to the striated duct cells. These results indicate that the secretor status affects the secretion of Lewis antigens by mucous, serous, and intercalated duct cells but not the presence of Le-b as a surface antigen in striated duct cells.

Subcellular localization of blood group substances ABH in human salivary glands.

We investigated the subcellular localization of ABH antigens in human submandibular, sublingual, and buccal glands by applying a post-embedding immunogold method using monoclonal antibodies specific for A, B, and H antigens. In most glands the immunoreactivity was usually restricted to mucous cells, in which only secretory granules and sometimes Golgi cisternae were specifically labeled. A and B antigens were demonstrated only in the glands of type A, B, and AB subjects, while H antigen was visualized in glands from individuals of all blood types. Moreover, differences were observed in the relative distribution of ABH antigens, depending on the type of gland.

Human meibomian glands: a histochemical study for androgen metabolic enzymes.

Human meibomian glands were treated for the histochemical demonstration of several enzymatic activities. The 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD), 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) appeared intensely reactive in the differentiating excretory cells, and weakly reactive in the basal cells and in the epithelial cells of the proximal portion of the ducts. The results indicate that meibomian glands can metabolize androgens by the reductive pathway, characteristic of target tissues. The finding of an intense reactivity for the enzymes glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) is also discussed.

3D-structure of cells of human salivary glands as seen by SEM.

To demonstrate by SEM the topography and cytoarchitecture of the different parenchymal components of human salivary glands, we have employed a number of techniques that allow either the exposure of internal and lateral cell surfaces or, following the removal of connective tissue, the visualization of endpieces, ducts, and myoepithelial cells. Serous glands consist of indented acini attached to the ducts in a grape-like fashion, whereas mucous and mixed glands are made up of smooth tubuli. Myoepithelial cells (mecs), which are abundant on the surfaces of acini, tubuli, and intercalated ducts, are sparse on striated ducts. They are star-shaped on acini, striated ducts, and most of the tubuli. Spindle-shaped mecs are seen, instead, on intercalated ducts and, occasionally, on mucous and mixed tubuli as well. Cells of striated ducts split into a number of large basal portions whose surface is covered by long laminated processes responsible for the striations seen with TEM. Excretory ducts are lined by small cup-shaped basal cells and by tall cylindrical cells, which are completely covered by short processes oriented at random. When observed from below, after removal of the basal lamina, the basal surfaces of cells of excretory ducts exhibit polygonal areas delimited by short reliefs. Those of striated ducts show, instead, long laminar processes arranged radially. Results presented here are discussed and put in relationship to the mechanism of saliva production.

Immunocytochemical localization of blood group ABH antigens in the human parotid gland.

The aim of this study was to determine whether the parotid gland, like other salivary glands, produces blood group antigens. A post-embedding immunogold staining (IGS) method for ABH antigens was applied to osmicated and nonosmicated, routinely prepared specimens of normal human parotid glands. A and B antigens were not detected, whereas H antigen was found in acini and ducts of glands from A, B, and O subjects. In osmicated samples, the only sites of H labeling were secretory granules of a few acinar cells and those of most intercalated duct cells. In both cell types, only the pale matrix of the granules was stained. In non-osmicated samples, secretory granules showed the same distribution pattern of H, although gold particles were more abundant, and basolateral membranes of acinar and striated duct cells and the apical vesicles of striated duct cells were also labeled. Our results suggest that small amounts of H antigen are secreted by the parotid gland.

Diffuse lymphoid tissue associated with the human bulbourethral gland. An immunohistologic characterization.

This study investigates the presence and distribution of immunocompetent cells in bulbourethral glands obtained from four multiorgan transplant donors. Monoclonal antibodies reacting with B (Leu 12+) and T (Leu 4+) cells, suppressor-cytotoxic cells (Leu 2+), helper-inducer (Leu 3a+) and natural killer (Leu7+) phenotypes, monocyte-macrophages, (LeuM3+), and cells expressing interleukin-2 receptor and HLA-DR antigen were tested in all specimens using an indirect immunoperoxidase staining procedure. T lymphocytes were estimated to represent 10% of the mucosal cell population. Almost all intraepithelial lymphocytes were suppressor-cytotoxic (CD8+) cells. The results demonstrate the presence of a defined distribution of immunocompetent cells in these sex accessory glands. Their role in combatting infections or other chronic genitourinary diseases is still undefined.

An immunohistochemical study of the distribution of ABH antigens in human submandibular glands at the light and electron microscopic levels.

The distribution of blood group antigens ABH in submandibular glands was studied at light and electron microscopy levels by applying ImmunoGold Silver Staining (IGSS) and post-embedding ImmunoGold (IGS) methods, respectively. In IGSS treated samples, a cytoplasmic and a surface form of antigen localization were discernible in the glandular parenchyma. The former was restricted to most mucous cells and to scattered serous cells: A and B antigens were demonstrated in mucous cells of A and B type glands, while H antigen appeared in most mucous and occasional serous elements regardless of the blood type of donors. The latter appeared as a strong H reactivity on cell surfaces of serous acini and ducts regardless of the patient blood type. The IGS method was applied both on non-osmicated samples embedded in LR White resin and on osmicated, Epon embedded samples. In non-osmicated tissues, antigen labelling was revealed in secretory granules and cell surfaces. Positive secretory granules were found in most mucous cells and occasional serous, intercalated, and striated duct cells. A and B antigens weakly reacted in mucous cells of A and B type glands, respectively, while strong H reactivity was seen in mucous, serous, intercalated and striated duct cells of glands of all types. Surfaces labelled with H antigen were found on both lumenal and basolateral membranes of striated ducts in glands of all types. IGS method applied on osmicated, Epon embedded samples, selectively revealed blood group antigens in secretory granules of serous cells but not in the apical vesicles of striated ductal cells. Cell surfaces were completely unreactive.

Ultrastructural localization of blood group antigens in human exocrine pancreas by immunogold labelling.

The cytochemical distribution of ABH and Lewis antigens was investigated in human pancreas by means of a post-embedding immunogold staining method which permitted antigens associated with secretory materials to be distinguished from those bound to cell surfaces. H and Le-b antigens were found in secretory granules of acinar and interlobular duct cells. In the form of surface-associated antigens, H and Le-b were found in acini and interlobular ducts, Le-a in intralobular ducts. In ductal cells, the same antigens detected in cell surfaces were also found in small cytoplasmic vesicles. It is suggested that by the emptying of these vesicles blood group antigens may be released both into the lumen and into the interstitial spaces.

A granular cell in the proximal intercalated duct of human parotid and submandibular glands.

In human parotid and submandibular gland unusual granulated cells are observed at the acinar-intercalated duct junction. These cells, which show a well developed Golgi apparatus, greatly differ from the typical elements of the intercalated ducts mainly due to the presence of abundant secretory granules of unknown nature. A complex substructure distinguishes these granules from those of conventional ductal cells and from those of acinar cells as well. In addition, some differences exist in the morphology of the granules observed in parotid with respect to those of submandibular gland.

Fine structure of excretory ducts of human salivary glands.

Excretory ducts of human major salivary glands are lined by an epithelium consisting of principal cells and by a discontinuous row of basal cells. The principal cells are tall and columnar with mitochondria, large lipofuscin granules and a central nucleus. Just beneath the plasmalemma bordering the lumen, their cytoplasm contains a number of small granules and vesicles similar to those observed in cells of striated ducts. Both in TEM and SEM, these cells also show large apical protrusions devoid of cytoplasmic organelles that may represent a kind of apocrine secretion. The cytoarchitecture of the principal cells seems to be at variance with that of cells of striated ducts. First, the cell body remains unique and does not split into major basal processes. Second, these cells usually lack the long laminated basal folds, housing vertically aligned mitochondria, that are typical of striated ducts. Instead, below the smooth area occupied by the junctional complexes, the lateral cell surfaces are completely covered by a great number of short irregular processes. These organelle-free folds are apparently involved in the mechanism of ion transport since, at their level, there is a strong reactivity for the transporting enzyme K(+)-pNPPase. The basal cells, which are small and cuboidal, have a dense and filamentous cytoplasm. Their functional role is still uncertain.

Immunocytochemical investigation of the subcellular distribution of some secretory products in human salivary glands.

The subcellular distribution of epidermal growth factor (EGF) and of Lewis antigens was demonstrated by means of a post-embedding immunogold cytochemical method in human parotid and submandibular gland. Acinar secretory granules were reactive for EGF and difucosylated Lewis antigens, cell surfaces of acini and striated ducts reacted only for difucosylated antigens; a great number of cytoplasmic vesicles in acinar and ductal cells were reactive for all Lewis antigens and EGF. These results suggest that substances which enter saliva are released not only through granule exocytosis by acinar cells, but also via other routes: the vesicular system revealed in both acinar and ductal cells could play a fundamental role in driving some products both towards the lumen and towards basolateral cell surfaces and intercellular spaces.

Ultrastructural localization of blood group antigens in human salivary glands.

The subcellular distribution of several blood group antigens was studied in human major and minor salivary glands by means of a postembedding immunogold staining method. In each gland, antigens were detected as secretory products and as cell surface components. A, B, H and Lewis (a,b,x,y) antigens were found in mucous cells depending on the ABO and secretor status; reactivities were confined to the secretory granules. In serous and ductal cells, both the secretory granules and cell membranes were reactive, but only for H, Le-b and Le-y irrespective of the ABO and secretor status.

Localisation of epidermal growth factor receptor in mucous cells of human salivary glands.

EGFR activation has been related to an increase in synthesis and secretion of mucins in epithelial cells, so that the use of EGFR tyrosine kinase inhibitors has been proposed in the therapy of mucin hypersecretory diseases. In this paper, we describe the ultrastructural localisation of EGFR in the mucous elements of human major and minor salivary glands and relate it to mucin distribution. A post-embedding immunogold staining method has been applied to normal surgical samples of human submandibular, sublingual, and labial glands, using a mouse monoclonal antibody specific for the intracellular domain of human EGFR. In mucous cells of all the glands examined, specific reactivity was detected in the cytoplasmic basolateral portions and near the mucous droplets, but not on cell surfaces. Since this pattern of labelling must be related to the internalisation process of the ligand-GFR complex, our results support the hypothesis that EGFR activation takes place in mucous cells and affects mucin production in human salivary glands.

Ultrastructural localization of epidermal growth factor receptor in human parotid gland.

The epidermal growth factor receptor (EGFR) is widely distributed in several organs in which, following interaction with its ligand, it can affect development and differentiation. The aim of this study was to define the distribution of EGFR in human parotid gland by means of a post-embedding immunogold staining method. Normal human parotid glands obtained at surgery were routinely prepared for electron microscopy. Semithin and ultrathin sections were treated for immunocytochemistry using a mouse monoclonal antibody specific for EGFR and a goat anti-mouse gold conjugated secondary antiserum. At the light microscope level, EGFR reactivity was revealed by a specific dark staining in both acinar and ductal cells. At the electron microscope level, EGFR was strongly stained in the cytoplasmic compartments and occasionally labeled on cell surfaces. In acinar cells, it appeared to be associated with small vesicles of uncertain nature that were scattered among the secretory granules. EGFR-positive vesicles were also observed in the ductal cells, with the most intense labeling being localized in striated ducts. Since cytoplasmic vesicles were previously found to be EGF-positive, these results may be due to the presence of the EGF-EGFR complex that is internalized after binding of EGF to the surface EGFR.

Ultrastructural localization of glycodelin oligosaccharides Le-x and Le-y in human seminal vesicles by immunogold staining.

Histo-blood group antigens Le-x and Le-y are oligosaccharidic terminals that characterize many glycoproteins in the human tissues. In seminal plasma, they are expressed as part of the so-called glycodelin S, which is suggested to regulate sperm capacitation/decapacitation. It has recently been demonstrated that the core protein of glycodelin S is secreted by seminal vesicles. Here we show that epithelial cells of human seminal vesicles also release the Le-x and Le-y antigens. The presence of these substances in secretory material was revealed by means of an immunogold staining method in normal surgical samples. The results suggest that glycodelin S is secreted by seminal vesicles in its finished glycosylated form. Moreover, antigen reactivity was also revealed associated with plasma membranes.

Subcellular localization of epidermal growth factor receptor in human submandibular gland.

The subcellular distribution of the epidermal growth factor receptor (EGFr) was demonstrated in the normal human submandibular gland by means of immunogold cytochemistry. EGFr labelling appeared in both acinar and ductal cells, where strong immunoreactivity was associated with a tubulovesicular system near the basolateral surfaces. In addition, groups of reactive vesicles were highlighted among secretory granules of both serous and mucous cells and at the apex of ductal cells. Basolateral vesicles were interpreted as being a result of EGFr internalization after activation by an exogenous ligand, although the functional meaning of those located apically remains unclear.

Subcellular localization of epidermal growth factor in human parotid gland.

The intracellular distribution of epidermal growth factor was investigated in human parotid gland by immunogold cytochemistry at the electron-microscopy level. Epidermal growth factor immunoreactivity was demonstrated in both acini and ducts. In acinar cells, secretory granules appeared moderately stained, clearly indicating that parotid gland contributes to salivary epidermal growth factor through granule exocytosis. In ductal cells, gold particles were found to decorate numerous cytoplasmic vesicles, particularly abundant in striated duct cells. Since epidermal growth factor reactive vesicles were seen not only at the cellular apex, but nearby lateral plasma membranes as well, it leads to the hypothesis that epidermal growth factor may be discharged both apically into the saliva, and basally into the interstitium.