Differential immunohistochemical expression of type I collagen and matrix metalloproteinase 2 among major salivary glands of young and geriatric mice

Abstract Objective This study aimed to demonstrate the immunohistochemical changes associated with MMP-2 and type 1 collagen separately for the first time in the major salivary glands (the parotid, submaxillary, and sublingual glands) that occur with aging in mice. Material and Methods Fourteen Balb/c white mice (50-80 g) were used in this study. The animals were divided into two equal groups. Group I consisted of young animals (2-month-old) (n=7) and Group II consisted of older animals (18-month-old) (n=7). After routine histological follow-ups, Hematoxylin-eosin (H&E), Masson's Trichrome staining and immunohistochemical staining was performed for type I collagen and MMP-2. Results We observed that there were age-related decreases in the number of acinar cells, increase in eosinophilic zymogen granules in cells, collagen accumulation in fibrotic areas and dilatation in interlobular ducts. Also, while type I collagen and MMP-2 immunoreactivity were moderate in the salivary glands of the young mice, they were high in the salivary glands of the old mice (p=0.001). In the H-score assessment, MMP-2 immunoreactivity was lower at a significant level in young mice than in old mice (p=0.001). Conclusions This study showed that anatomical, physiological and morphological abnormalities occur in all three major salivary glands as a natural consequence of aging.

mRNA expression and localization of LPS-induced ß-defensin isoforms in rat salivary glands.

ß-defensins are small, cationic peptides with broad-spectrum antimicrobial activity that are produced by mucosal epithelia. However, little is known about the expression of ß-defensins in the major salivary glands. The purpose of this study was to characterize expression of rat ß-defensin-1 (RBD-1) and -2 (RBD-2) mRNA within the major salivary glands together with the effect of injection of intraductal lipopolysaccharide (LPS) on that expression. ß-defensin mRNA expression was quantitated by RT-PCR in salivary gland tissues and salivary acinar and striated duct cells collected by laser captured microdissection. RBD-1 and -2 were expressed in the parotid gland, the submandibular gland, and the sublingual gland. ß-defensins were expressed in both the acinar and striated duct cells of the major salivary glands. Intraductal injection of LPS increased expression of RBD-1 and -2 mRNA, which peaked at 12 hrs. These results suggest that salivary cells (acinar and striated duct cells) have the potential to produce ß-defensins.

Patterns and variability in electrophoretic polypeptide profiles of human saliva in a healthy population.

Electrophoretic polypeptide profiles of normal human saliva differ markedly between different reports. Since both methodological variations and polymorphism may explain these differences, in this study we aimed to establish whether or not the salivary electrophoretic polypeptide profiles of subjects from a healthy population share discrete molecular features. To this end, parotid, submandibular/sublingual and whole salivas were collected separately from each of 40 young and 34 elderly clinically healthy adults and processed for SDS-polyacrylamide gel electrophoresis and Coomassie blue staining. Each type of glandular saliva displayed a different group of invariant (i.e. present in every subject) electrophoretic polypeptide bands while whole saliva showed a profile that reflected mostly the combined contribution of the major salivary glands. Some minor variant (i.e. absent in some subjects) bands were identified in each type of saliva. Regarding those interindividual variations, no age- or sex-dependence was appreciated. Altogether, these results demonstrate the occurrence of distinctive electrophoretic polypeptide patterns, in addition to some minor variations, for each type of normal saliva, thus providing a background for further populational studies on salivary polypeptide profiles.

Immunolocalization of PTHrP in the sublingual glands of three mammals.

The present study deals with immunohistochemical localization of PTHrP in sublingual glands of white mouse, bank vole, and common vole. PTHrP immunoreactivity was observed in epithelial cells of striated, interlobular and main excretory ducts of the salivary glands in all the three animal species tested. However, we found no positive reaction for PTHrP in epithelial cells of the intercalated ducts. In striated duct cells, the reaction intensity was species-dependent. In bank vole and common vole, the reaction was very strong, while in white mouse very weak. In the remaining segments of excretory ducts (interlobular and main excretory duct) we found no species-related differences in the reaction intensity or character. Myoepithelial cells surrounding ducts and mucous tubules with serous demilunes in sublingual glands were also PTHrP-negative in all the three animal species tested.

Proliferation and distribution of myoepithelial cells during atrophy of the rat sublingual gland.

BACKGROUND: The present study was aimed to determine the proliferation and distribution of myoepithelial cells during atrophy of rat sublingual glands. METHODS: The excretory duct of the right sublingual gland of rats was doubly ligated with metal clips to induce atrophy in the gland. The atrophic sublingual glands were taken from 1 to 28 days after duct ligation and examined with single immunohistochemistry for actin as a marker of myoepithelial cells and with immunohistochemical double staining for actin and proliferating cell nuclear antigen (PCNA) as a marker of proliferating cells. RESULTS: In unligated sublingual glands, myoepithelial cells embraced acini and intercalated ducts, but not striated and interlobular excretory ducts. In the early stages of atrophy, myoepithelial cells surrounded small ducts but not large ones. However, in the later stages of atrophy, myoepithelial cells were also observed at the periphery of the large ducts. The immunohistochemical double staining showed that there were PCNA-positive myoepithelial cells in the normal as well as in the atrophic sublingual glands. However, the PCNA labeling indices of myoepithelial cells were low in the unligated and atrophic sublingual glands, and there were no statistically significant differences in these labeling indices. CONCLUSION: The observations suggest that the distribution of myoepithelial cells change during atrophy of rat sublingual glands and that myoepithelial cells have low proliferative activity in both the normal and atrophic condition of rat sublingual glands.

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.

[Assessment of peripheral blood perfusion during open heart surgery with sublingual PCO2 measured by ISFET-PCO2 sensor].

Sublingual tissue PCO2 (PSLCO2) was continuously monitored with an ISFET-based PCO2 sensor during and after the open-heart surgery under cardiopulmonary bypass (CPB) in order to study the effect of CPB on the peripheral blood perfusion. In addition, PSLCO2 monitoring was carried out in several cases of off-pump CABG. In the cases of open-heart surgery with CPB, PSLCO2 increased from 35.0 +/- 5.6 mmHg at the induction of anesthesia to the maximum value of 55.7 +/- 6.0 mmHg during CPB. After declamping of the aorta, PSLCO2 decreased gradually to 49.0 +/- 4.0 mm Hg 6 hr after the admission to ICU. The value of arterial lactate as another index of peripheral blood perfusion also increased gradually after the start of CPB, reaching to the maximum value of 8.8 +/- 1.1 mmol.l-1 just after being admitted into ICU. In the case of off-pump CABG, PSLCO2 and arterial lactate showed a slight increase during the later part of the surgery, but the change was not so significant as in the case of open-heart surgery under CPB. Through this study, typical changing pattern of PSLCO2 during the open-heart surgery was recognized. The change of PSLCO2 always preceded that of arterial lactate. We also experienced one case in which early stage of hypoperfusion was detected through the monitoring of PSLCO2. These results suggest clinical advantages of PSLCO2 monitoring.

Salivary gland nucleotide receptors: evidence for functional expression of both P2X and P2Y subtypes.

A growing body of information now supports the suggestion that P2 receptors for extracellular nucleotides (primarily ATP) have a role in regulating salivary gland function. There is solid pharmacological and molecular evidence for the presence of P2X ligand-gated ion channel nucleotide receptors (P2X4 and P2X7/P2Z). More recently, our group and others have obtained evidence that multiple P2Y G protein-coupled nucleotide receptors (P2Y1 and P2Y2) are also expressed. Our studies have focused on defining the conditions under which P2Y receptors are expressed, the functional consequences of their activation, and the importance of co-expression of P2X and P2Y receptors. Functional and molecular approaches have been used to identify the P2 subtypes in salivary glands and in salivary cell lines. Assays include measurement of changes in [Ca2+]i, changes in transcellular short circuit current in monolayers, and RT-PCR to assess changes in receptor mRNA levels. The main observations are: (1) P2Y1 receptor activity is present in the submandibular gland (SMG) of immature rats but decreases over the first four weeks following birth, although mRNA levels remain relatively constant; (2) P2Y2 receptors are present in the cell lines and are up-regulated during short-term culture of normal parotid, sublingual, and SMG cells and following ligation of the main excretory duct of SMG; and (3) the P2X subtypes, P2X4 and P2X7, and the P2Y subtypes, P2Y1 and P2Y2, are co-expressed in salivary glands and salivary cell lines, and exhibit distinct basolateral versus apical localization in polarized cell monolayers as well as discrete patterns of intracellular signaling.

In vitro studies of the regulation of mucous gland secretion.

Our laboratory is concerned with elucidation of mechanisms regulating the exocrine secretion of mucin glycoproteins by mucous cells of salivary mucous glands. As a model system for our studies we use short-term cultures of acinar structures isolated from rat sublingual glands. Recent results are discussed from an on-going study of cross-talk between the two primary signaling pathways regulating exocrine secretion from isolated sublingual acini: the muscarinic cholinergic and vasoactive intestinal peptide (VIP) pathways. The combination of muscarinic agonist (carbachol) and VIP elicits a secretion equivalent to about 150% of the additive sum of the secretory responses to each agonist alone. This synergistic secretory response is only observed at submaximal concentrations of carbachol. VIP thus serves to decrease the EC50 for carbachol nearly three-fold. Results are thus far consistent with the hypothesis that a sustained rise in the intracellular concentration of calcium ions induced by VIP accounts for synergistic secretion and the heightened sensitivity to carbachol.

Confocal and electron microscopy to characterize sialoglycoconjugates in mouse sublingual gland acinar cells.

Double lectin labeling for confocal microscopy and lectin-protein A-gold binding for electron microscopy were applied to the mouse sublingual gland in order to study surface and cytoplasmic sialoglycoconjugates. For this purpose, serially cut sections were submitted to sialidase followed by incubation with lectins recognizing usually acceptor sugars for terminal sialic acids. At the electron microscope level, the residues subtended to sialic acid were individually identified on adjacent sections by an indirect technique of labeling, whereas with confocal microscopy the above sugars were simultaneously visualized on the same section by a double staining method using fluorescein isothiocyanate (FITC)- and tetramethylrhodamine isothiocyanate (TRITC)-conjugated lectins. Acinar cells were found to contain the terminal sequence sialic acid-beta-galactose in abundance while the sequence sialic acid-alpha-N-acetylgalactosamine appeared to be present in modest amounts. Both sialoglycoconjugates were homogeneously codistributed inside acinar cells. The combination with a saponification method also allowed the occurrence of C4 acetylated sialic acids linked to beta-galactose to be discovered, at the electron microscope level, on acinar cell secretory products.

Molecular cloning of a novel high molecular weight mucin (MG1) from human sublingual gland.

A human sublingual gland cDNA library was screened with a polyclonal antiserum against deglycosylated MG1 and a positive clone, pSM2-1, was isolated which codes for 196 amino acids in the carboxyl-terminal region of this mucin. This region is cysteine-rich and contains a C2-like domain upstream of the extreme carboxyl-terminal domain in which the arrangement of cysteines is nearly identical to that in human von Willebrand factor, human intestinal mucin MUC2, human tracheobronchial mucin MUC5 and porcine and bovine submaxillary gland mucins. Northern analyses with pSM2-1 showed MG1 transcripts are abundant in sublingual gland and barely detectable in submandibular gland. This study provides the first primary sequence data on human salivary mucin MG1 and the significance of the results is discussed with respect to the biosynthesis and differential expression of MG1 in human salivary glands.

Simple mucin-type carbohydrate antigens in major salivary glands.

Simple mucin-type carbohydrate antigens Tn, sialosyl-Tn and T are often markers of neoplastic transformation and have very limited expression in normal tissues. We performed an immunohistological study of simple mucin-type carbohydrate antigens, including H and A variants, with well-defined monoclonal antibodies (MAb) on frozen and paraffin-embedded normal salivary gland tissue from 22 parotid, 14 submandibular, six sublingual, and 13 labial glands to elucidate the simple mucin-type glycosylation pattern in relation to cyto- and histodifferentiation. The investigated carbohydrate structures were predominantly observed in the cell cytoplasm, most often in the supranuclear area, suggesting localization to the Golgi region, whereas ductal contents were unstained. Mucous acinar cells expressed Tn, sialosyl-Tn, and H and A antigens, regardless of glandular location. Serous acinar cells, on the other hand, expressed A, H, and inconstantly sialosyl-T, Tn, and sialosyl-Tn antigens in major salivary glands, whereas serous cells of minor (labial) salivary glands expressed H exclusively, Tn and sialosyl-T antigens inconstantly, but never sialosyl-Tn and A antigens. The difference may be related to a more simple cytodifferentiation of serous cells of minor (labial) salivary glands as compared with major salivary glands. Duct cells in major salivary glands expressed A, H, and inconstantly T, sialosyl-T, and Tn antigens, whereas minor (labial) salivary glands ducts exclusively expressed H, T and sialosyl-T antigens, differences that may be related to dissimilarities in the duct system. Myoepithelial cells and basal cells exclusively expressed T and sialosyl-T antigens, which may prove useful in studies of salivary gland tumors, since these cells are known to play a key role in the histological characteristics of some salivary gland tumors. The results indicate a similar glycosylation pattern in the different major salivary glands, whereas minor (labial) salivary gland differ slightly in serous and duct cells. The limited and exclusive intracellular expression of the immature Tn, sialosyl-Tn, and T antigens indicates that these structures may be of value as markers of salivary gland tumors.

Developmental studies on vasoactive intestinal peptide, substance P and calcitonin gene-related peptide in salivary glands of postnatal rats.

The development of vasoactive intestinal peptide, substance P and calcitonin gene-related peptide in parotid, submandibular and sublingual glands of the male rat was followed by immunochemistry and immunocytochemistry. The total amounts of these peptides increased in surges during the first 8 weeks of the animal's life; one within 2-4 weeks and the other beginning 1-2 weeks later. Nerve fibres containing these peptides were present at birth showing a pattern of distribution similar to that in adults. During the first 4 weeks the nerve fibres increased in number.

Large-scale purification and characterization of the major phosphoproteins and mucins of human submandibular-sublingual saliva.

The major components of human submandibular-sublingual saliva (HSMSL) are mucins, amylases, cystatins, proline-rich proteins and statherin. Structure-function studies of these molecules have been hampered by the small amounts of purified materials that can be isolated from human secretions. The present study describes an integrated purification protocol for the large-scale preparation of many of these molecules. To dissociate partially heterotypic complexes among salivary molecules, HSMSL was initially fractionated into four pools by gel filtration with 6 M-guanidine hydrochloride. Subsequent fractionation of these four pools by gel-filtration and ion-exchange chromatography resulted in the purification of high- and low-Mr mucins, neutral and acidic cystatins, acidic and basic proline-rich proteins and statherin. Many variants or isoforms of these salivary molecules have been identified and biochemically characterized. Biochemical studies indicated that the low-Mr mucin exists as two isoforms which vary in their sialic acid to fucose ratios. Three isoforms of acidic cystatin S were characterized which differ in their phosphate content. Two isoforms of a basic proline-rich peptide were identified; the smaller peptide was a truncated form missing the first seven amino acids.

Transferrin receptor expression in oral tumors.

Transferrin receptor expression in oral tumors was examined by staining with monoclonal antibody against the human transferrin receptor. The cells with positive reaction were recognized in the basal and parabasal layers of the normal epithelium. The staining was found in all the malignant tumors but not in the benign tumors. These results suggest that the immunohistochemical analysis of the transferrin receptor is useful for the diagnosis of oral malignant tumor in addition to the clinical and pathological examinations.

Estudo histoquímico das glândulas salivares linguais posteriores do Coendu Villosus (Mammalia, Rodentia) / Histochemical study of the posterior lingual salivary glands of Coendu Villosus (Mammalia, Rodentia)

Os autores estudaram com métodos histoquímicos, a natureza do material elaborado pelas glândulas linguais posteriores de Coendu Villosus. Com base nos resultados obtidos, foi possível concluir que: 1) o produto de secreção das glândulas de Weber contém uma sulfosialomucina, fato que permite classificar essas glândulas como sendo do tipo mucoso; 2) o produto de secreção das glândulas de Von Ebner contém uma glico-proteína, tipo sero-mucoso