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Salivary glands are the principal organs responsible for secreting saliva in the oral cavity. Tumors, trauma, inflammation, and other factors can cause functional or structural damage to the glands, leading to reduced saliva secretion. In this study, we innovatively prepared a acinar-mimetic silk fibroin-collagen-astragalus polysaccharide (SCA) scaffold using low-temperature three-dimensional (3D) printing and freeze-drying techniques. We evaluated the material properties and cell compatibility of the scaffold in vitro and implanted it into the damaged parotid glands (PG) of rats to assess its efficacy in tissue reconstruction and functional repair. The results demonstrated that the SCA scaffold featured a porous structure resembling natural acini, providing an environment conducive to cell growth and orderly aggregation. It exhibited excellent porosity, water absorption, mechanical properties, and biocompatibility, fulfilling the requirements for tissue engineering scaffolds. In vitro, the scaffold facilitated adhesion, proliferation, orderly polarization, and spherical aggregation of PG cells. In vivo, the SCA scaffold effectively recruited GECs locally, forming gland-like acinar structures that matured gradually, promoting the regeneration of damaged PGs. The SCA scaffold developed in this study supports tissue reconstruction and functional repair of damaged PGs, making it a promising implant material for salivary gland regeneration., Competing Interests: Declaration of competing interest I have nothing to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Salivary gland tumors are highly variable in clinical presentation and histology. The World Health Organization (WHO) classifies 22 types of malignant and 11 types of benign tumors of the salivary glands. Diagnosis of salivary gland tumors is based on imaging (ultrasound, magnetic resonance imaging) and fine-needle aspiration biopsy, but the final diagnosis is based on histopathological examination of the removed tumor tissue. In this pilot study, we are testing a new approach to identifying peptide biomarkers in saliva that can be used to diagnose salivary gland tumors. The research material for the peptidomic studies was extracts from washings of neoplastic tissues and healthy tissues (control samples). At the same time, saliva samples from patients and healthy individuals were analyzed. The comparison of the peptidome composition of tissue extracts and saliva samples may allow the identification of potential peptide markers of salivary gland tumors in patients' saliva. The peptidome compositions extracted from 18 tumor and 18 healthy tissue samples, patients' saliva samples (11 samples), and healthy saliva samples (8 samples) were analyzed by LC-MS tandem mass spectrometry. A group of 109 peptides was identified that were present only in the tumor tissue extracts and in the patients' saliva samples. Some of the identified peptides were derived from proteins previously suggested as potential biomarkers of salivary gland tumors (ANXA1, BPIFA2, FGB, GAPDH, HSPB1, IGHG1, VIM) or tumors of other tissues or organs (SERPINA1, APOA2, CSTB, GSTP1, S100A8, S100A9, TPI1). Unfortunately, none of the identified peptides were present in all samples analyzed. This may be due to the high heterogeneity of this type of cancer. The surprising result was that extracts from tumor tissue did not contain peptides derived from salivary gland-specific proteins (STATH, SMR3B, HTN1, HTN3). These results could suggest that the developing tumor suppresses the production of proteins that are essential components of saliva.

Background: Mammary analogue secretory carcinoma (MASC) is characterized by similar histologic, immunohistochemical, and molecular features with breast secretory carcinoma. MASC usually occurs in adults. Case report: A 4-year-old boy presented with a right infra-auricular mass. Features of the tumor include solid, tubular, and papillary growth patterns, with homogenous eosinophilic secretions inside microcystic structures. Immunohistochemical stains showed strong, diffuse staining for CK7, S100, pan-TRK protein. P63 was positive in a peripheral pattern. Fluorescence in situ hybridization (FISH) analysis showed the characteristic ETV6-NTRK3 gene fusion. Conclusion: Typical histological, immunohistochemical, and molecular features are present in MASC occurring early in childhood.

Background: Saliva has been studied as a better indicator of disorders and diseases than blood. Specifically, the salivary glucose level is considered to be an indicator of diabetes mellitus (DM). However, saliva collection methods can affect the salivary glucose level, thereby affecting the correlation between salivary glucose and blood glucose. Therefore, this study aims to identify an ideal saliva collection method and to use this method to determine the population and individual correlations between salivary glucose and blood glucose levels in DM patients and healthy controls. Finally, an analysis of the stability of the individual correlations is conducted., Methods: This study included 40 age-matched DM patients and 40 healthy controls. In the fasting state, saliva was collected using six saliva collection methods, venous blood was collected simultaneously from each study participant, and both samples were analyzed at the same time using glucose oxidase peroxidase. A total of 20 DM patients and 20 healthy controls were arbitrarily selected from the above participants for one week of daily testing. The correlations between salivary glucose and blood glucose before and after breakfast were analyzed. Finally, 10 DM patients and 10 healthy controls were arbitrarily selected for one month of daily testing to analyze the stability of individual correlations., Results: Salivary glucose levels were higher in DM patients than healthy controls for the six saliva collection methods. Compared with unstimulated saliva, stimulated saliva had decreased glucose level and increased salivary flow. In addition, unstimulated parotid salivary glucose was most correlated with blood glucose level (R 2 = 0.9153), and the ROC curve area was 0.9316, which could accurately distinguish DM patients. Finally, it was found that the correlations between salivary glucose and blood glucose in different DM patients were quite different. The average correlation before breakfast was 0.83, and the average correlation after breakfast was 0.77. The coefficient of variation of the correlation coefficient before breakfast within 1 month was less than 5%., Conclusion: Unstimulated parotid salivary glucose level is the highest and is most correlated with blood glucose level, which can be accurately used to distinguish DM patients. Meanwhile, the correlation between salivary glucose and blood glucose was found to be relatively high and stable before breakfast. In general, the unstimulated parotid salivary glucose before breakfast presents an ideal saliva collecting method with which to replace blood-glucose use to detect DM, which provides a reference for the prediction of DM.

Amphibians produce defensive chemicals which provide protection against both predators and infections. Within species, populations can differ considerably in the composition and amount of these chemical defenses. Studying intraspecific variation in toxins and linking it to environmental variables may help us to identify the selective drivers of toxin evolution, such as predation pressure and infection risk. Recently, there has been a renewed interest in the unique toxins produced by salamanders from the genus Salamandra: the samandarines. Despite this attention, intraspecific variation has largely been ignored within Salamandra-species. The aim of this study was to investigate whether geographic variation in profiles of samandarines exists, by sampling 4 populations of Salamandra atra over its range in the Dinaric Alps. In addition, we preliminary explored whether potential variation could be explained by predation (counting the number of snake species) and infection risk (cultivation and genomic analyses of collected soil samples). Salamanders from the 4 populations differed in toxin composition and in the size of their poison glands, although not in overall toxin quantity. Nor predation nor infection risk could explain this variation, as populations barely differed in these variables. Sampling over a much broader geographic range, using better estimators for predation and infection risk, will contribute to an improved understanding of how environment may shape variation in chemical defenses. Nevertheless, as the 4 populations of S. atra did differ in their toxin profiles, we propose that this species provides an interesting opportunity for further ecological and evolutionary studies on amphibian toxins., (© 2020 International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.)

Toads of the genus Bufo synthesize and accumulate bufadienolides (BDs) in their parotoid glands. BDs are cardiotonic steroids that play an important role in defense against the toads' predators. Three bufonid taxa occur in mainland Japan, Bufo japonicus formosus, B. j. japonicus, and B. torrenticola. The chemical structures of BDs isolated from B. j. formosus were studied several decades ago, but there is no further information on the toxic components of Japanese toads and their metabolism. In this study, we analyzed BDs of toads from throughout Japan and compared the BD profiles by liquid chromatography/mass spectrometry (LC/MS) and hierarchical cluster analysis (HCA). We observed BDs in three taxa of Japanese toads, and identified five of the most common BDs by nuclear magnetic resonance (NMR) analyses. Of the five BDs, only bufalin was detected in all individuals. HCA of individual BD profiles divided the three taxa into five primary clusters and several subclusters. This result indicates that BD profiles differ both among and within the taxa. The clustering pattern of BDs is generally concordant with a phylogenetic tree reconstructed from the mitochondrial cytochrome b gene of Japanese toads. Our results suggest that the BDs of Japanese toads have diversified not in response to specific selective pressures, but simply due to population structuring over evolutionary time.

The biological activity of Rhinella icterica parotoid secretion (RIPS) and some of its chromatographic fractions (RI18, RI19, RI23, and RI24) was evaluated in the current study. Mass spectrometry of these fractions indicated the presence of sarmentogenin, argentinogenin, (5 β ,12 β )-12,14-dihydroxy-11-oxobufa-3,20,22-trienolide, marinobufagin, bufogenin B, 11α,19-dihydroxy-telocinobufagin, bufotalin, monohydroxylbufotalin, 19-oxo-cinobufagin, 3α,12 β ,25,26-tetrahydroxy-7-oxo-5 β -cholestane-26- O -sulfate, and cinobufagin-3-hemisuberate that were identified as alkaloid and steroid compounds, in addition to marinoic acid and N -methyl-5-hydroxy-tryptamine. In chick brain slices, all fractions caused a slight decrease in cell viability, as also seen with the highest concentration of RIPS tested. In chick biventer cervicis neuromuscular preparations, RIPS and all four fractions significantly inhibited junctional acetylcholinesterase (AChE) activity. In this preparation, only fraction RI23 completely mimicked the pharmacological profile of RIPS, which included a transient facilitation in the amplitude of muscle twitches followed by progressive and complete neuromuscular blockade. Mass spectrometric analysis showed that RI23 consisted predominantly of bufogenins, a class of steroidal compounds known for their cardiotonic activity mediated by a digoxin- or ouabain-like action and the blockade of voltage-dependent L-type calcium channels. These findings indicate that the pharmacological activities of RI23 (and RIPS) are probably mediated by: (1) inhibition of AChE activity that increases the junctional content of Ach; (2) inhibition of neuronal Na + /K + -ATPase, leading to facilitation followed by neuromuscular blockade; and (3) blockade of voltage-dependent Ca 2+ channels, leading to stabilization of the motor endplate membrane.

Marinobufagenin is a bufadienolide compound detected mainly in skin and parotoid gland secretions of Rhinella marina (L.) toad. Bufadienolides regulate the Na + /K + -ATPase pump by inhibiting the cardiotonic steroid dependent-site and act as cardiac inotropes with vasoconstrictive properties. Marinobufagenin and other bufadienolides, such as telocinobufagin and bufalin, are thought to be found endogenously in mammals in salt-sensitive hypertensive states such as essential hypertension, congestive heart-failure, and preeclampsia. The role of marinobufagenin as antimicrobial agent and its cytotoxic potential have also been recognized. The particular interest around marinobufagenin prompts us to consider the Rhinella marina toad venom as a possible source for molecules with pharmacological and/or diagnostic potential. In this article, two different approaches of extraction and purification of marinobufagenin from Rhinella marina (L.) venom are studied: (i) Preparative thin-layer chromatography combined to mass spectrometry and/or ultraviolet detection and (ii) solid-phase extraction coupled with fractionation on high-performance liquid chromatography. Different chromatographic conditions are tested for each approach. The solid-phase extraction combined with high-performance liquid chromatography fractionation approach was preferred as it offered a greater yield, was less time-consuming and allowed us to selectively isolate marinobufagenin. Both protocols aim to provide efficient and convenient methods for toad venom extraction, based on an easily automatable and systematized strategy., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The parotoid gland of bufonids is characterized as a specialized integument region, formed by different gland types. The secretion elaborated by the largest glandular alveoli has been related to animal chemical defense and is constituted by granular protein content, associated with a basophilic and alcianophilic material with features of glycoconjugates. This study aimed to identify and characterize the glycoconjugates in the secretion of the largest granular gland of the parotoid gland of Rinella icterica by histochemical and immunohistochemical techniques at light microscopy, biochemical methods, and nuclear magnetic resonance spectroscopy. Our results showed that the glycoconjugate content contains a mixture of chondroitin‑6‑sulfate (C6S) and chondroitin-non-sulfate (C0S). Thus, chondroitin sulfate probably plays an important role in gland physiology, probably protecting the protein content while inside the secretory portion., (Copyright © 2018. Published by Elsevier B.V.)

Radio-ligand therapy (RLT) with 177 Lu-PSMA-617 is a promising option for patients with metastatic castration-resistant prostate-cancer (mCRPC). A prospective phase-II study (EUDRACT/RSO,2016-002732-32) on mCRPC is ongoing at IRST (Meldola, Italy). A total of 9 patients (median age: 68 y, range: 53⁻85) were enrolled for dosimetry evaluation of parotid glands (PGs), kidneys, red marrow (RM) and whole body (WB). Folic polyglutamate tablets were orally administered as PGs protectors and 500 mL of a 10% mannitol solution was intravenously infused to reduce kidney uptake. The whole body planar image (WBI) and blood samples were acquired at different times post infusion (1 h, 16⁻24 h, 36⁻48 h and 120 h). Dose calculation was performed with MIRD formalism (OLINDA/EXM software). The median effective half-life was 33.0 h (range: 25.6⁻60.7) for PGs, 31.4 h (12.2⁻80.6) for kidneys, 8.2 h (2.5⁻14.7) for RM and 40.1 h (31.6⁻79.7) for WB. The median doses were 0.48 mGy/MBq (range: 0.33⁻2.63) for PGs, 0.70 mGy/MBq (0.26⁻1.07) for kidneys, 0.044 mGy/MBq (0.023⁻0.067) for RM and 0.04 mGy/MBq (0.02⁻0.11) for WB. A comparison with previously published dosimetric data was performed and a significant difference was found for PGs while no significant difference was observed for the kidneys. For PGs, the possibility of reducing uptake by administering glutamate tablets during RLT seems feasible while further research is warranted for a more focused evaluation of the reduction in kidney uptake.

Human basic proline-rich proteins and basic glycosylated proline-rich proteins, encoded by the polymorphic PRB1-4 genes and expressed only in parotid glands, are the most complex family of adult salivary proteins. The family includes 11 parent peptides/proteins and more than 6 parent glycosylated proteins, but a high number of proteoforms with rather similar structures derive from polymorphisms and post-translational modifications. 55 new components of the family were characterized by top-down liquid chromatography-mass spectrometry and tandem-mass platforms, bringing the total number of proteoforms to 109. The new components comprise the three variants P-H S 1 → A, P-Ko P 36 → S, and P-Ko A 41 → S and several of their naturally occurring proteolytic fragments. The paper represents an updated reference for the peptides included in the heterogeneous family of proteins encoded by PRB1/PRB4. MS data are available via ProteomeXchange with the identifier PXD009813.

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.

There is considerable potential for the use of ion mobility mass spectrometry in structural glycobiology due in large part to the gas-phase separation attributes not typically observed by orthogonal methods. Here, we evaluate the capability of traveling wave ion mobility combined with negative ion collision-induced dissociation to provide structural information on N-linked glycans containing multiple fucose residues forming the Lewis x and Lewis y epitopes. These epitopes are involved in processes such as cell-cell recognition and are important as cancer biomarkers. Specific information that could be obtained from the intact N-glycans by negative ion CID included the general topology of the glycan such as the presence or absence of a bisecting GlcNAc residue and the branching pattern of the triantennary glycans. Information on the location of the fucose residues was also readily obtainable from ions specific to each antenna. Some isobaric fragment ions produced prior to ion mobility could subsequently be separated and, in some cases, provided additional valuable structural information that was missing from the CID spectra alone. Graphical abstract ᅟ.

Previous studies have reported that the umami taste of monosodium l-glutamate (MSG) and salty-smelling odors (e.g., soy sauce, bacon, sardines) enhance the perception of saltiness. This study aimed to investigate the neural basis of the enhancement of saltiness in human participants using functional near-infrared spectroscopy (fNIRS). University students who had passed a taste panel test participated in this study. Sodium chloride solutions were presented with or without either 0.10% MSG or the odor of soy sauce. The participants were asked to drink a cup of the stimulus and to evaluate only saltiness intensity in Experiment 1, as well as other sensory qualities in Experiment 2, and temporal brain activity was measured using fNIRS. In Experiment 3, the participants were asked to evaluate saltiness intensity using the time-intensity (TI) method, and the response of the parotid salivary glands was measured using fNIRS. The fNIRS data showed that the added MSG and soy sauce enhanced the hemodynamic response in temporal brain regions, including the frontal operculum, but no effect on the hemodynamic salivary responses was detected. These results indicate that the perceived enhancement of saltiness occurs in the brain region that is involved in central gustatory processing. Furthermore, the results of the sensory evaluations suggest that enhancement of saltiness by the addition of MSG is mainly based on fusion of the salty-like property of MSG and saltiness of NaCl, whereas enhancement by the addition of soy sauce odor is mainly based on modulation of the temporal dynamics of saltiness perception.

Objective: To compare the recently proposed Milan System for Reporting Salivary Gland Cytopathology (MSRSGC) with the four-tiered reporting system (FTRS) followed at our institute., Methods: Parotid gland fine-needle aspirates reported over a period of 5 years were analysed. These aspirates had been placed into 4 categories according to the FTRS: unsatisfactory (UNS), no evidence of malignancy/negative (NEG), inconclusive for malignancy (INC), and diagnostic for malignancy/positive (POS). Aspirates with follow-up histopathology were then categorized according to the MSRSGC as follows: non-diagnostic, non-neoplastic, atypia of unde-termined significance (AUS), neoplasm, suspicious for malignancy, and malignant. The risk of malignancy (ROM) was calculated., Results: A total of 893 parotid region aspirates were evaluated and histopathology was available for 190 cases (21%). ROM in MSRSGC groups, namely non-diagnostic, non-neoplastic, AUS, neoplasm, suspicious for malignant neoplasm, and malignant, was 44, 8, 0, 12, 81 and 100%, respectively. ROM in FTRS groups, namely UNS, NEG, INC, and POS, was 45, 13, 67 and 100%, respectively., Conclusions: MSRGC and FTRS are comparable with respect to the ROM across groups. Compared to FTRS, the further subcategorisation of the non-malignant group, the use of specific nomenclature, and the reproducibility of MSRGC provide proper risk stratification, thereby guiding better management and resulting in improved patient care., (© 2018 S. Karger AG, Basel.)

In this study, we investigated the effect of bufalin on the human ether-à-go-go-related gene (hERG) K + channels using the perforated patch recording technique. We measured a half-maximal inhibitory concentration (IC 50 ) of 24.83 μM and maximal inhibitory effect of 39.45 ± 1.14% with bufalin. These findings suggest that bufalin is a potent hERG K + channel blocker and may provide a new way for understanding Chan Su-induced arrhythmia., (© 2017 Société Française de Pharmacologie et de Thérapeutique.)

Saliva is an easily collected biological fluid with potentially important diagnostic value. While gel electrophoresis is generally used for salivary analysis, we employed the capillary isoelectric focusing technique to allow for a rapid, automated mode of electrophoresis. Capillary isoelectric focusing coupled with UV whole column imaging detection (iCIEF) was used to develop a robust protocol to characterize salivary α-amylase collected from various glands. Notably, three sample preparation methods were examined: ultrafiltration, gel-filtration, and starch affinity interaction with salivary amylase. Salivary α-amylase separated into two major peaks before sample treatment; while both filtration methods and starch affinity interaction of salivary amylase enhanced the resolution of isozymes, desalting with gel-filtration displayed the best recovery and the highest resolution of isozymes. Good agreement existed between the observed isoelectric points and the values reported in the literature. In addition, a high level of precision was apparent, and the relative standard deviation for replicates was less than 0.5% for pIs (peak positions) and below 10% for peak area. Furthermore, saliva secreted from the parotid gland proved to have a higher amylase content compared to either secretions from the submandibular/sublingual complex, or whole saliva, as well as amylase enhancement under stimulation. The results suggest that the iCIEF technique can be used to accurately resolve and quantitate amylase isozymes in a rapid and automated fashion, and that gel-filtration should be applied to saliva samples beforehand to allow for optimal purification and characterization., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We used isobaric mass tagging (iTRAQ) and lectin affinity capture mass spectrometry (MS)-based workflows for global analyses of parotid saliva (PS) and whole saliva (WS) samples obtained from patients diagnosed with primary Sjögren's Syndrome (pSS) who were enrolled in the Sjögren's International Collaborative Clinical Alliance (SICCA) as compared with two control groups. The iTRAQ analyses revealed up- and down-regulation of numerous proteins that could be involved in the disease process (e.g., histones) or attempts to mitigate the ensuing damage (e.g., bactericidal/permeability increasing fold containing family (BPIF) members). An immunoblot approach applied to independent sample sets confirmed the pSS associated up-regulation of β2-microglobulin (in PS) and down-regulation of carbonic anhydrase VI (in WS) and BPIFB2 (in PS). Beyond the proteome, we profiled the N-glycosites of pSS and control samples. They were enriched for glycopeptides using lectins Aleuria aurantia and wheat germ agglutinin, which recognize fucose and sialic acid/N-acetyl glucosamine, respectively. MS analyses showed that pSS is associated with increased N-glycosylation of numerous salivary glycoproteins in PS and WS. The observed alterations of the salivary proteome and N-glycome could be used as pSS biomarkers enabling easier and earlier detection of this syndrome while lending potential new insights into the disease process.

The localization of dopamine stores and the expression and localization of dopamine (DAT) and vesicular monoamine transporters (VMAT) type-1 and -2 and of dopamine D1-like and D2-like receptor subtypes were investigated in rat submandibular, sublingual, and parotid salivary glands by HPLC with electrochemical detection, as well as immunochemical and immunohistochemical techniques. Male Wistar rats of 2 mo of age were used. The highest dopamine levels were measured in the parotid gland, followed by the submandibular and sublingual glands. Western blot analysis revealed DAT, VMAT-1, VMAT-2, and dopamine receptors immunoreactivity in membrane preparations obtained from the three glands investigated. Immunostaining for dopamine and transporters was developed within striated ducts. Salivary glands processed for dopamine receptors immunohistochemistry developed an immunoreaction primarily in striated and excretory ducts. In the submandibular gland, acinar cells displayed strong immunoreactivity for the D2 receptor, while cells of the convoluted granular tubules were negative for both D1-like and D2-like receptors. Parotid glands acinar cells displayed the highest immunoreactivity for both D1 and D2 receptors compared with other salivary glands. The above localization of dopamine and dopaminergic markers investigated did not correspond closely with neuron-specific enolase (NSE) localization. This indicates that at least in part, catecholamine stores and dopaminergic markers are independent from glandular innervation. These findings suggest that rat major salivary glands express a dopaminergic system probably involved in salivary secretion. The stronger immunoreactivity for dopamine transporters and receptors in striated duct cells suggests that the dopaminergic system could regulate not only quality, but also volume and ionic concentration of saliva., (Copyright © 2015 the American Physiological Society.)

Most of the lymphoproliferative diseases involving the salivary glands represent indolent non-Hodgkin B-cell lymphoma (marginal zone lymphoma) related to chronic autoimmune sialadenitis (Sjögren disease). Other types of non-Hodgkin lymphomas involve the salivary glands less frequently. On rare occasions, classical Hodgkin lymphoma (CHL) and nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) present initially as a primary salivary gland mass. We analyzed a series of CHL (n=3) and NLPHL (n=6) presenting initially as parotid gland tumors concerning their pattern (parenchymal vs. intraparotid lymph node) and the presence of salivary inclusions and epithelial proliferations within the lymphoma infiltrate. The pattern of infiltration was determined on hematoxylin and eosin-stained slides assisted by immunostaining for pancytokeratin to highlight lobular salivary gland parenchyma. Patients included 6 male and 3 female individuals with a mean age of 62 years (range, 36 to 88 y). Lymphoma was localized within intraparotid lymph nodes in 8 cases and was limited to salivary parenchyma in 1 case. Parenchymal involvement in nodal-based cases was scored as absent (3) or minimal (5). Salivary inclusions (acini and ductules) within affected lymph nodes were noted in 6 cases (4/5 NLPHLs and 2/3 CHLs). In 3/6 NLPHL cases, salivary inclusions showed variable proliferative changes ranging from prominent lymphoepithelial lesions to cystic and oncocytic (Warthin-like) epithelial changes. Scanty small lymphoepithelial lesions were seen in 1 of the 3 CHL cases. One NLPHL in the intraparotid lymph node was accompanied by prominent lymphoepithelial sialadenitis in the absence of clinical signs of Sjögren disease. This study highlights that a majority of parotid gland Hodgkin lymphomas arise within intraparotid lymph nodes. Frequent entrapment and proliferation of salivary ducts and acini within the lymphoma infiltrate might mimic a variety of benign lymphoepithelial mass-forming lesions (nonsebaceous lymphadenoma, Warthin tumor, and autoimmune sialadenitis). Pancytokeratin stain is helpful for reliable assessment of the background architecture.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-dependent protein kinase (PKA)-regulated Cl(-) channel, crucial for epithelial cell regulation of salt and water transport. Previous studies showed that ezrin, an actin binding and A-kinase anchoring protein (AKAP), facilitates association of PKA with CFTR. We used immunohistochemistry and immunogold transmission electron microscopy to localize CFTR, ezrin, and PKA type II regulatory (RII) and catalytic (C) subunits in striated duct cells of human parotid and submandibular glands. Immunohistochemistry localized the four proteins mainly to the apical membrane and the apical cytoplasm of striated duct cells. In acinar cells, ezrin localized to the luminal membrane, and PKA RII subunits were present in secretory granules, as previously described. Immunogold labeling showed that CFTR and PKA RII and C subunits were localized to the luminal membrane and associated with apical granules and vesicles of striated duct cells. Ezrin was present along the luminal membrane, on microvilli and along the junctional complexes between cells. Double labeling showed specific protein associations with apical granules and vesicles and along the luminal membrane. Ezrin, CFTR, and PKA RII and C subunits are co-localized in striated duct cells, suggesting the presence of signaling complexes that serve to regulate CFTR activity., (© 2015 Eur J Oral Sci.)

Objective: To determine and compare the presence and in situ localization of the glycosphingolipid ganglioside GM1 in human salivary glands using the biomarkers for GM1: cholera toxin and antibodies against GM1., Materials and Methods: Immunohistochemical analyses were performed on sections of adult human submandibular, parotid and palatinal glands using cholera toxin sub-unit B and two polyclonal antibodies against ganglioside GM1 as biomarkers., Results: Immunofluorescence microscopy showed that the toxin and antibodies were co-localized in some acini but not in others. The cholera toxin mainly reacted with the cell membranes of the mucous acini in the submandibular gland, while incubation with the antibody against GM1 gave rise to a staining of the cytoplasm. The cytoplasm in some secretory acinar cells in the parotid gland was stained by the cholera toxin, whereas only small spots on the plasma membranes reacted with anti-GM1. The plasma membranes in the parotid excretory ducts appeared to react to anti-GM1, but not to cholera toxin., Conclusions: Cholera toxin induces the expression of ion channels and carriers in the small intestine and increases the production of secretory mucins. Although their mutual immunohistochemical localization may differ, both cholera toxin and ganglioside GM1 are present in the mucin-producing acini from salivary glands. This could point to a relationship between ganglioside expression and production of salivary mucins.

Nuclear receptors and transcription factors regulate the functions of many genes involved in cellular physiology and pathology (e.g. tumorigenesis and autoimmune diseases). The present study was performed to define the expression and the regulation of aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), constitutive androstane receptor (CAR), and nuclear factor E2-related factor 2 (Nrf2) in the rat parotid gland. Constitutive expression, as well as expression after stimulation with specific inducers for AhR [2,3,7,8-tetrachloro-dibenzylo-p-dioxin (TCDD)], Nrf2(oltipraz), PXR (dexamethasone), and CAR (phenobarbital), was evaluated using the quantitative PCR. Cellular localization of the nuclear receptors and the transcription factor was visualized by immunohistochemical staining. The study revealed constitutive expression of AhR as well as Nrf2, and their induction by TCDD andoltipraz, respectively. Immunohistochemical analysis revealed constitutive, predominantly cytoplasmic, expression of the AhR receptor, especially in interlobular striated duct cells, with nuclear shift upon exposure to TCDD. Inducible expression of Nfr2 was found mainly in the cytoplasm of intralobular striated duct cells. Constitutive expression of PXR and CAR was not found. Bearing in mind the involvement of AhR and Nrf2 in the regulation of many genes, it seems that these factors may play also a role in salivary gland physiology and pathology.

This study was designed to investigate whether extracorporeal shock wave lithotripsy (ESWL) exposure to parotid gland produces an oxidative stress in parotid glands of rats. Twelve male Wistar-albino rats, 6 months of age with an average body weight of 250-300 g, were divided randomly into two groups, each consisting of six rats. The animals in the first group did not receive any treatment and served as control. The left parotid glands of animals in group 2 (ESWL treated) received a thousand 18 kV shock waves after anesthetizing the rats with 50 mg/kg of ketamine. The animals in both groups were killed 72 hours after the ESWL treatment, and the parotid glands were harvested for the determination of lipid peroxidation product malondialdehyde (MDA), antioxidant glutathione (GSH) levels and the activities of antioxidant enzymes such as superoxide dismutase (SOD), GSH-Px and catalase (CAT). It was found that MDA level increased in parotid glands of rats after the ESWL treatment. The SOD, GSH-Px and CAT enzyme activities, and the level of antioxidant GSH decreased in parotid gland of rats after the ESWL treatment. It was concluded that short-term ESWL treatment caused an increase in the free radical production and a decrease in the antioxidant enzyme activity in parotid glands of ESWL-treated rats., (© The Author(s) 2012.)

Sclerosing polycystic adenosis (SPA) of salivary glands is a tumorous lesion of salivary glands, with clinical presentation of a slow-growing mass characterized by a combination of histological features, some of which are reminiscent of mammary fibrocystic disease. SPA is mostly unifocal, but rarely may be multifocal and/or bilateral. Recurrences have been reported in up to 19% of cases. Although originally considered pseudoneoplastic, the occurrence of "dysplasia" and carcinoma in situ of ductal epithelium, and recent evidence of clonality suggest a possible neoplastic nature. Herein we describe, for the first time, two cases of SPA in two sisters (7 and 33 years old). The younger patient experienced multiple recurrences. This is the first report of familial occurrence of SPA, suggesting a possible genetic background., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

In this study we investigated the differences in the properties of pellicles formed from stimulated parotid saliva (PS), which contains little or no mucin; and stimulated whole mouth saliva (WMS), which contains mainly two types of mucin: MUC5B and MUC7. By contacting WMS and PS with quartz-crystal microbalance with dissipation monitoring (QCM-D) and dual polarisation interferometer (DPI) hydroxyapatite (the main component of enamel) coated sensors, we observed the formation and structure of the respective salivary pellicles. As this was the first time that DPI hydroxyapatite sensors have been used to measure salivary pellicle adsorption; the techniques combined allowed us to measure the hydrated mass, dry mass, thickness and viscoelastic properties of the pellicle; but also to record the density of the PS and WMS formed pellicles. Subsequently, the PS pellicle was shown to form a denser layer than WMS pellicle; which would suggest that the proteins present in PS are also responsible for forming the dense basal layer of the acquired enamel pellicle. Whereas proteins present in the WMS are more likely to help form the softer outer layer of the pellicle. The data presented help to further define the mechanisms leading to the multi-layered structure of the salivary pellicle and demonstrate that salivary composition has an important effect on the structural properties of the adsorbed pellicle., (Copyright © 2013 Elsevier B.V. All rights reserved.)

β-defensins are small, cationic peptides with broad-spectrum antimicrobial activity that are produced by mucosal epithelia. However, little is known about the expression of β-defensins in the major salivary glands. The purpose of this study was to characterize expression of rat β-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. β-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. β-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 β-defensins.

In the membrane fraction of mouse parotid gland (PG), the protein level of aquaporin 5 (AQP5), a member of the water channel family, was increased by injection (ip) of isoproterenol (IPR), a β-adrenergic agonist, at 1 h, and stayed at high levels until 6 h; this change occurred simultaneously as amylase secretion. The AQP5 level then decreased and returned toward the original level at 12-48 h. After IPR injection, the AQP5 mRNA gradually increased and reached a maximum at 24 h. The facts suggest a rapid appearance of AQP5 at plasma membrane by IPR and subsequent degradation/metabolism by activation of proteolytic systems. Pretreatment of animals with two calpain inhibitors, N-Ac-Leu-Leu-methininal (ALLM) and calpeptin, as well as a protein synthesis inhibitor, cycloheximide (CHX), significantly suppressed the IPR-induced AQP5 degradation in the PG membrane fraction; such suppression was not observed by two proteasome inhibitors, MG132 and lactacystin, or the lysosome denaturant chloroquine, although most of these inhibitors increased AQP5 protein levels in unstimulated mice. The AQP5 protein was also degraded by μ-calpain in vitro. Furthermore, we demonstrated that μ-calpain was colocalized with AQP5 in the acinar cells by immunohistochemistry, and its activity in the PG was increased at 6 h after IPR injection. These results suggest that the calpain system was responsible for IPR-induced AQP5 degradation in the parotid gland and that such a system was coupled to the secretory-restoration cycle of amylase in the PG.

The hormone melatonin influences oral health through a variety of actions, such as anti-inflammatory, anti-oxidant, immunomodulatory and antitumour. Many of these melatonin functions are mediated by a family of membrane receptors expressed in the oral epithelium and salivary glands. Using immunoblotting and immunohistochemistry, recent studies have shown that the melatonin membrane receptors, MT1 and MT2, are present in rat and human salivary glands. To date, no investigation has dealt with the ultrastructural distribution of the melatonin receptors. This was the aim of the present study, using the immunogold method applied to the human parotid gland. Reactivity to MT1 and, with less intensity, to MT2 appeared in the secretory granules of acinar cells and in the cytoplasmic vesicles of both acinar and ductal cells. Plasma membranes were also stained, albeit slightly. The peculiar intracytoplasmic distribution of these receptors may indicate that there is an uptake/transport system for melatonin from the circulation into the saliva., (© 2013 Anatomical Society.)

Nuclear receptors and transcription factors coordinate expression of many genes, and regulation of their expression determines cellular response to various endo- and exogenous factors. There is paucity of data regarding expression of nuclear receptors and factors in salivary glands. In the present study, a focus was placed on human parotid gland expression of aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR, NR1I2), constitutive androstane receptor (CAR, NR1I3) and nuclear factor E2-related factor 2 (Nrf2). Parotid salivary tissue was obtained from patients undergoing the gland dissection. Quantitative real-time PCR aimmunohistochemical staining were used for expression studies. The highest mRNA expression was documented for NFE2L2 coding for Nrf2. Lower expression was seen in the case of AHR gene coding for AhR. PXR was constitutively present at very low level and CAR expression was below the limit of quantification. Immunohistochemical evaluation of the parotid gland specimens revealed cytoplasmic Nrf2 expression in striated duct cells as well as within myoepithelial cells. Acinar cells were mostly negative for Nrf2. Expression of AhR was found within the cytoplasm in striated duct cells. Acinar and myoepithelial cells were negative for AhR. Having in mind their role in regulating function of many enzymes and transmembrane transporters, expression of these factors seem play a role in salivary gland physiology, pathology as well as drug transport and metabolism.

Eight Merino sheep (49.4 ± 4.23 kg BW) and 8 Alpine goats (53.2 ± 2.51 kg BW) were used to study the effect of ingestion of quebracho tannins on salivation. Four sheep and 4 goats were individually fed a daily allotment of 20 g DM of alfalfa hay/kg BW (Control). Another 4 sheep and 4 goats were also given 20 g DM of alfalfa hay/kg BW supplemented with 50 g of quebracho/kg DM (Tannin) for a period of 64 d. The saliva secretion from the left parotid gland was collected by insertion of a polyvinyl chloride catheter into the parotid duct and the amount of parotid saliva produced recorded over three 48-h periods on d 1 and 2 (P1), d 31 and 32 (P2), and d 61 and 62 (P3) after the tannin feeding was initiated. The total amount of saliva produced was estimated from rumen water kinetics determined on d 4, d 34, and d 64 of the experiment. Experimental design was completely randomized, with repeated measures on each experimental unit, performing separate analysis for sheep and goats. Parotid saliva production was not affected by the sampling period in either animal species receiving the Control diet. Corresponding values for sheep were 2.04, 2.12, and 2.27 L/d (P = 0.89) and for goats 1.65, 1.79, and 1.86 L/d (P = 0.95). Sheep fed the Tannin diet produced 55, 73, and 107% of the amount of saliva recorded in sheep fed the Control diet on P1, P2, or P3, respectively. Corresponding values in goats were 88, 130, and 134% on P1, P2, or P3, respectively. Estimated total saliva production was not affected (P = 0.50 for sheep and P = 0.97 for goats) by the ingestion of quebracho. There was no difference (P > 0.10) in osmotic pressure, P, Mg, Ca, urea, and protein concentrations in parotid saliva. There were, however, differences in Na and K concentrations in response to the ingestion of quebracho tannins, with Na concentrations increasing (P = 0.05) and K concentrations decreasing (P = 0.04) in sheep saliva and pH increasing (P = 0.05) in goat saliva. In conclusion, the inclusion of quebracho at 50 g/kg DM for 64 d does not appear to alter saliva production in sheep and goats.

Ruminant nutrition relies upon the symbiotic relationship that exists with microbial populations in the rumen. Urea transported across the ruminal epithelia and secreted by the salivary glands is a key source of nitrogen for microbial growth in the rumen. As ruminal urea transport can be mediated by specific UT-B urea transporters, this study investigated whether UT-B urea transporters were also present in the bovine salivary gland. Western blotting experiments detected only small amounts of UT-B protein in whole-cell lysate from the bovine parotid gland. In contrast, strong 32 to 34 and 40 kDa UT-B proteins were detected in parotid plasma membrane-enriched protein, showing the importance of using enriched samples. These signals were also detected in rumen and correspond to bovine UT-B1 and UT-B2 urea transporters, respectively. Further immunolocalization studies identified that these proteins were located in the ductal system of the parotid gland. This study, therefore, confirmed the presence of UT-B urea transporter protein in the bovine parotid salivary gland., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The diagnosis of primary Sjögren's syndrome (pSS) is difficult due to the lack of specific laboratory and clinical tests. As an initial step for the global discovery of changes in the abundance of parotid salivary proteins in pSS, a pooled sample was compared to that from healthy control subjects by multidimensional protein identification technology (MudPIT). A total of 1246 proteins were identified by MudPIT. The abundance of 477 of these proteins did not change, 529 were only detected in either the pSS or HC sample, while 206 of these proteins were significantly upregulated ≥ twofold and 34 were downregulated ≤ 0.5. Ingenuity Pathway Analyses of differentially expressed proteins identified by MudPIT resulted in the identification of 100 significant pathways. The same samples were quantified in parallel using RP MS. Fifty-eight of 71 proteins identified by RP overlapped with MudPIT results. Five proteins were further analyzed by targeted label-free quantification to confirm the similar relative differential expression observed by RP and MudPIT approaches. The present study supports the use of MS for global discovery and validation of marker proteins for improved and early diagnosis of pSS., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Although cell proliferation is related to tumour aggressiveness and prognosis, there are few studies describing the expression of proliferative markers in salivary gland cancer. Our aim was to assess the long-term prognostic value of the proliferating cell nuclear antigen (PCNA) in a large group of histologically different salivary gland cancers. We analysed the expression of PCNA in 159 patients with parotid gland cancer by means of immunohistochemistry. The mean follow-up time was 56.6 months. A high expression of PCNA showed a significant correlation to the patients' pathological lymph node stage (p = 0.004). A high PCNA expression significantly indicated a poor 5-year disease-free (p = 0.046) and overall survival rate (p = 0.018). The PCNA expression was the only prognostic factor for a worse 5-year disease-free and overall survival in acinic cell carcinomas (p = 0.004, p = 0.022). The correlation between PCNA expression and survival probabilities of salivary gland cancer might make proliferation markers helpful tools in patient follow-up, prognosis and targeted therapy in salivary gland cancer in future.

Human zymogen granule protein 16 (ZG16p) contains a Jacalin-like lectin domain, although its glycan-binding properties are not fully understood. Here, we screened the glycan-binding specificity of ZG16p by recently developed glycoconjugate microarray. ZG16p appeared to exhibit selective binding to α- and β-linked mannose-polyacrylamide-biotin probes. In more quantitative analysis using frontal affinity chromatography, dissociation constants to two types of polyvalent mannose, i.e. high-density mannose and yeast mannan, were determined to be 1.3 and 1.7 µM, respectively. Mutation of the evolutionarily conserved amino acid Asp151, which is involved in sugar binding among the Jacalin-related lectins (JRLs), abolished binding activity to mannose. By immunohistochemical staining, ZG16p was specifically detected in mucus-secreting cells of the digestive system such as serosanguineous acinar cells of the parotid gland, acinar cells of the pancreas and goblet cells of the intestine. Finally, we showed that ZG16p recognizes pathogenic Candida and Malassezia species in a polyvalent mannose-dependent manner. We propose that ZG16p is a novel member of mannose-specific JRLs, which recognizes pathogenic fungi through non-self polyvalent mannose in the digestive system.

Aquaporin-5 (AQP5), a water channel, plays key roles in salivary secretion. The novel phosphorylation of AQP5 was investigated by using human salivary gland (HSG) cells and mouse salivary glands. In the HSG cells stably transfected with a wild-type mouse AQP5 construct, a protein band immunoreactive with antibody against phosphorylated PKA substrate was detected in the AQP5 immunoprecipitated sample, and its intensity was enhanced by short-term treatment of the cells with 8-bromo-cAMP, forskolin, or phorbol 12-myristate 13-acetate, but not by that with A23187 calcium ionophore. Such enhancement was inhibited in the presence of H-89, a PKA inhibitor. An AQP5 mutant (AQP5-T259A) expressed by transfection of HSG cells was not recognized by anti-phosphorylated PKA substrate antibody, even when the cells were stimulated with the protein kinase activators. Immunoblotting and immunofluorescence studies using a specific antibody detecting AQP5 phosphorylated at its Thr259 demonstrated that AQP5 was rapidly and transiently phosphorylated at the apical membrane of acinar cells in the submandibular and parotid glands after administration of isoproterenol, but not pilocarpine. Furthermore, both AQP5 and AQP5-T259A were constitutively localized at the plasma membrane in HSG cells under the resting and forskolin-stimulated conditions. These results suggest that AQP5 is phosphorylated at its Thr259 by PKA through cAMP, but not Ca(2+), signaling pathways, and that this phosphorylation does not contribute to AQP5 trafficking in the salivary gland cells.

Objective: Apart from its role as an inhibitory neurotransmitter, γ-aminobutyric acid is also thought to regulate various stages of cell proliferation and differentiation in the brain and periphery. The present study aimed to assess the levels of γ-aminobutyric acid and its biochemical precursor glutamic acid (glutamate) in benign parotid tumours and in unstimulated parotid saliva., Method: Unstimulated parotid saliva was collected bilaterally, using the swab method, in 20 patients with unilateral pleomorphic adenoma or Warthin's tumour. Samples of tumour and adjacent salivary tissue were collected during tumour resection., Results: Concentrations of γ-aminobutyric acid and glutamate, but not aspartate, were significantly higher in the tumour tissue than in the non-tumour tissue. There was no significant difference in salivary concentrations of γ-aminobutyric acid, glutamate or aspartate, comparing the involved and non-involved side., Conclusion: The present results provide preliminary evidence that γ-aminobutyric acid may be involved in the growth of benign parotid tumours.

Previously (Shan et al, 2005), we reported that adenoviral vector-mediated transfer of the human aquaporin-1 (hAQP1) cDNA to minipig parotid glands following irradiation (IR) transiently restored salivary flow to near normal levels. This study evaluated a serotype 2, adeno-associated viral (AAV2) vector for extended correction of IR (single dose; 20 Gy)-induced, parotid salivary hypofunction in minipigs. At 16 weeks following the IR parotid salivary flow decreased by 85-90%. AAV2hAQP1 administration at week 17 transduced only duct cells and resulted in a dose-dependent increase in salivary flow to approximately 35% of pre-IR levels (to approximately 1 ml per 10 min) after 8 weeks (peak response). Administration of a control AAV2 vector or saline was without effect. Little change was observed in clinical chemistry and hematology values after AAV2hAQP1 delivery. Vector-treated animals generated high anti-AAV2 neutralizing antibody titers by week 4 (approximately 1:1600) and significant elevations in salivary (approximately 15%), but not serum, granulocyte macrophage colony-stimulating factor levels. Following vector administration, salivary [Na(+)] was dramatically increased, from approximately 10 to approximately 55 mM (at 4 weeks) and finally to 39 mM (8 weeks). The findings demonstrate that localized delivery of AAV2hAQP1 to IR-damaged parotid glands leads to increased fluid secretion from surviving duct cells, and may be useful in providing extended relief of salivary hypofunction in previously irradiated patients.

Background/aims: There are still uncertainties regarding the use of whole and parotid ductal saliva as indicators of chronic exposure to fluoride. This study evaluated the effect of water fluoride concentration, age, gender, geographical area and localization (urban/rural) on fluoride concentrations in whole and ductal saliva., Methods: Subjects (n = 300) aged 3-7, 14-20, 30-40 and 50-60 years, from five communities (A-E) with different fluoride concentrations in the drinking water, participated in the study. Two samples of drinking water and parotid and whole saliva were collected for each subject and were analyzed for fluoride using appropriate electrode techniques., Results: Mean water F concentrations (±SE, mg/l, n = 60) were 0.09 ± 0.01, 0.15 ± 0.01, 0.66 ± 0.01, 0.72 ± 0.02, and 1.68 ± 0.08 for A-E, respectively. Mean F concentrations (±SE, mg/l, n = 15) ranged between 0.014 ± 0.002 (A, 3-7 years) and 0.297 ± 0.057 (D, 14-20 years) for whole saliva and 0.009 ± 0.001 (C, 30-40 years) and 0.284 ± 0.038 (E, 50-60 years) for parotid saliva. Results of multivariate linear regression analysis showed that geographical area and water fluoride concentration exerted the strongest influence in whole and ductal saliva F concentrations, respectively., Conclusion: Therefore, parotid ductal saliva seems to be a more appropriate biomarker of fluoride exposure, and factors like age and localization should also be considered when using this biomarker., (Copyright © 2011 S. Karger AG, Basel.)

We investigated the histology and carbohydrate content of the parotid and mandibular glands of the barking deer (Muntiacus muntjak). Three adult males were used. Paraffin wax sections of the glands were stained with haematoxylin and eosin (HE), alcian blue (AB), pH 2.5 and periodic acid Schiff (PAS). The acinar cells of the parotid gland were serous, whereas those of the mandibular gland were of the mixed type. The acini of the mandibular gland comprised serous and mucous cells with the mucous type predominating. AB and PAS staining showed high concentrations of acidic and neutral carbohydrates in the mucous cells, but not in the serous cells of the mandibular gland. These carbohydrates were also found in moderate-to-high concentrations in the secreted material in the mandibular duct lumen. However, these carbohydrates were not found in acinar cells of the parotid gland or in the serous cells of the mandibular gland. Thus, carbohydrates in the saliva of the barking deer appear to be produced mainly by the mucous cells of the mandibular glands., (© 2010 Blackwell Verlag GmbH.)

This study describes the identification and structural characterization of Sus scrofa statherin. HPLC-electrospray ionization mass spectrometry analysis on pig parotid secretory granule extracts evidenced a peptide with a molecular mass value of 5381.1 +/- 0.6 Da and its truncated form, devoid of the C-terminal Ala residue, with a molecular mass value of 5310.1 +/- 0.6 Da. The complete sequence of pig statherin gene was determined by sequencing the full-length cDNA obtained by rapid amplification of cDNA ends. The gene is 549 base pairs long and contains an open reading frame of 185 nucleotides, encoding a 42-amino acid secretory polypeptide with a signal peptide of 19 residues. This sequence presents some typical features of the four statherins characterized till now, showing the highest degree of amino acid identity with bovine (57%) and human statherin (39%). Pig statherin is mono-phoshorylated on Ser-3, while primate statherins already characterized are di-phosphorylated on Ser-2 and Ser-3. This difference, probably connected to the Asp-4 --> Glu substitution, suggests the involvement of the Golgi-casein kinase, which strictly recognizes the SX(E/pS) consensus sequence., ((c) 2010 European Peptide Society and John Wiley & Sons, Ltd.)

In the mature rat parotid gland, myoepithelial cells (MEC) invest intercalated ducts, but not acini. During postnatal development, however, these cells differentiate around both intercalated ducts and acini, then translocate to only intercalated ducts during weaning. Previously, we found that thyroxine (T(4)) accelerates translocation of cells with small secretory granules from acini into intercalated ducts and the number of apoptotic cells increased tremendously with high doses. We present here additional analysis of the effects of T(4) on developing rat parotid gland, namely, the distribution of MEC and the proliferation of parenchymal cells. Beginning at age four days, pups were given daily subcutaneous injections of low, medium, and high doses of T(4) or vehicle or no injection. At ages 4, 7, 10, and 15 days, glands were excised and processed for light microscopy. Sections were double-immunostained with antibodies against proliferating cell nuclear antigen (PCNA) and actin, and counterstained with hematoxylin. Proliferative activity was assessed via PCNA histochemistry and MEC were identified using actin histochemistry. MEC in the T(4) groups invested mostly acini at 15 days in vehicle/normal glands and mostly intercalated ducts after 10 days in the T(4) groups. The proliferative activity of acinar cells and MEC in vehicle/normal glands declined progressively with age and T(4) increased the rate of this decline in the MEC in a dose-dependent manner. We conclude that T(4) accelerates the translocation of MEC from acini to intercalated ducts and that an important mechanism is the more rapid decline in the proliferative activity of MEC than in acinar cells in the T(4) groups. Some of the decline in the proliferative activity of all cells in the high and medium dose T(4) groups after seven days may have been due to dose-related thyroxine toxicity.

Human saliva is a protein-rich, easily accessible source of potential local and systemic biomarkers to monitor changes that occur under pathological conditions; however, little is known about the changes in abundance associated with normal aging. In this study, we performed a comprehensive proteomic profiling of pooled saliva collected from the parotid glands of healthy female subjects, divided into two age groups 1 and 2 (20-30 and 55-65 years old, respectively). Hydrophobic charge interaction chromatography was used to separate high- from low-abundance proteins prior to characterization of the parotid saliva using multidimensional protein identification technology (MudPIT). Collectively, 532 proteins were identified in the two age groups. Of these proteins, 266 were identified exclusively in one age group, while 266 proteins were common to both groups. The majority of the proteins identified in the two age groups belonged to the defense and immune response category. Of note, several defense related proteins (e.g., lysozyme, lactoferrin and histatin-1) were significantly more abundant in group 2 as determined by G-test. Selected representative mass spectrometric findings were validated by Western blot analysis. Our study reports the first quantitative analysis of differentially regulated proteins in ductal saliva collected from young and older female subjects. This study supports the use of high-throughput proteomics as a robust discovery tool. Such results provide a foundation for future studies to identify specific salivary proteins which may be linked to age-related diseases specific to women.

Two peptides (MW 1211.7 and 928.5 Da) were detected by RP-HPLC-ESI-MS analysis of parotid saliva secreted upon continuous parasympathetic stimulation. The peptide with the higher mass (PSPFr-A) corresponded to the N-terminal dodecapeptide (Fragment 1-12) of rat parotid secretory protein (PSP), while the peptide with the lower mass (PSPFr-B) corresponded to the 4-12 fragment of the same protein. During stimulation, the PSPFr-A secretion increased, while the PSPFr-B secretion decreased (HPLC-ESI-MS). In the presence of cycloheximide, PSPFr-A was not demonstrated, while the PSPFr-B secretion decreased. In the presence of aprotinin, the PSPFr-B secretion was almost abolished, while the PSPFr-A secretion increased to higher levels than those observed in the absence of the inhibitor. In vitro perfusion, with artificial solution, of stimulated rat parotid glands excluded that the fragments were derived from the circulation. Neither peptide occurred in enriched granule preparations from unstimulated glands. The results suggest that at least two pathways--granular and vesicular--are responsible for the generation of the two peptides. PSPFr-A is the first cleavage product in both pathways. PRPFr-B is probably generated from granular PSPFr-A only and, at the end of the granule mediated pathway, by the action of an enzyme of the serine protease class.

The present investigation is an attempt to determine the occurrence, elemental composition and formation of microliths in the parotid of ferret. Parotids from four normal ferrets were examined by electron microscopy and X-ray microanalysis. Crystalline microliths were found in phagosomes of acinar cells, which occasionally contained secretory material, and in phagosomes situated between mitochondria of striated ductal cells. Crystalline microliths and microliths that consisted of granular material either without crystals or mixed with a component of crystals were found in lumina, where they were often associated with cellular debris. The crystals contained calcium and phosphorus. Phagy and stagnation related to pockets of inefficient secretory activity have been previously found to be features of the parotid of ferret. Thus, possibly persistent degradation of redundant cellular material, particularly secretory granules, in phagosomes results in accumulation of calcium and leads to calcified microliths, whereas consolidation of stagnant debris extracellularly does not involve such accumulation and leads to non-calcified or mixed microliths.

Diabetes mellitus (DM) is a widespread disease with high morbidity and health care costs. An experimental animal model was employed, using morphological and biochemical methods, to investigate the effects of DM on the expression and compartmentation of salivary gland proteins. The distribution of proline-rich proteins (PRP), submandibular mucin (Muc10) and the regulatory (RI and RII) subunits of cyclic AMP-dependent protein kinase type I and type II was determined in the parotid and submandibular (SMG) glands of rats treated with streptozotocin. Quantitative immunocytochemistry of secretory granules in diabetic glands revealed decreases of 30% for PRP in both the parotid and SMG, and a 40% decrease in Muc10 in the SMG. Immunogold labelling showed that RII decreased in nuclei and the cytoplasm in diabetic acinar cells while labelling of secretory granules was similar in control and diabetic parotid. Electrophoresis and Western blotting of tissue extracts of two secretory proteins showed that the response to DM and insulin treatment was gland specific: PRP showed little change in the SMG, but decreased in the parotid in DM and was partially restored after insulin treatment. Photoaffinity labelling showed only RI present in the SMG and mainly RII in the parotid. The results of this and previous studies demonstrating highly specific changes in salivary protein expression indicate that the oral environment is significantly altered by DM, and that oral tissues and their function can be compromised. These findings may provide a basis for future studies to develop tests using saliva for diabetic status or progression in humans.

It has been demonstrated that parotid glands of rats infected with Trypanosoma cruzi present severe histological alterations; changes include reduction in density and volume of the acini and duct systems and an increase in connective tissue. We evaluated the association between morphological changes in parotid glands, circulating testosterone levels and epidermal growth factor receptor (EGF-R) expression in experimental Chagas disease in rats. Animals at 18 days of infection (acute phase) showed a significant decrease in body weight, serum testosterone levels and EGF-R expression in the parotid gland compared with a control group. Since decreases in body weight could lead to a reduction in circulating testosterone concentration, we believe that the reduction in EGF-R expression in parotid glands of infected rats is due to alterations in testosterone levels and atrophy of parotid glands is caused by changes in EGF-R expression. Additionally, at 50 days (chronic phase) of infection parotid glands showed a normal histological aspect likely due to the normalization of the body weight. These findings suggest that the testosterone-EGF-R axis is involved in the histological changes.

Several studies have shown the antidiabetic properties of sodium tungstate. In this study, we evaluated some biochemical parameters of the parotid salivary gland of streptozotocin-induced diabetic rats treated with sodium tungstate solution (2 mg/ml). The studied groups were: untreated control (UC), treated control (TC), untreated diabetic (UD), and treated diabetic (TD). After 2 and 6 weeks of treatment, parotid gland was removed and total protein and sialic acid (free and total) concentration and amylase and peroxidase activities were determined. Data were compared by variance analysis and Tukey test (p < 0.05). The sodium tungstate treatment modestly decreased the glycemia of streptozotocin-induced diabetic rats. At week 2 of the study, parotid gland of diabetic rats presented a reduction of total protein concentration (55%) and an increase of amylase (120%) and peroxidase (160%) activities, free (150%) and total (170%) sialic acid concentration. No alteration in the evaluated parameters at week 6 of the study was observed. Sodium tungstate presented no significant effect in parotid gland. Our results suggest that diabetes causes initial modification in biochemical composition of parotid. However, this gland showed a recovery capacity after 6 week of the experimental time. Sodium tungstate has no effect in peripheral tissues, such as salivary glands.

Objectives: This study aimed to clarify the significance of cluster of differentiation 9 glycoprotein gene expression in human parotid gland tumours., Methods: We retrospectively analysed immunohistochemical staining for cluster of differentiation 9 glycoprotein in parotid gland tumours., Results: Cluster of differentiation 9 glycoprotein was consistently detected in the normal parotid gland. Regarding benign parotid gland tumours, cluster of differentiation 9 glycoprotein was present in 13 of 18 pleomorphic adenomas, in all Warthin tumours tested (21/21) and in all cases of basal cell adenoma tested (four of four). In contrast, positive staining for cluster of differentiation 9 glycoprotein was less often observed in malignant parotid tumours. Cluster of differentiation 9 glycoprotein was present in 11 of 14 mucoepidermoid carcinomas, in two of five acinic cell carcinomas and in two of five adenoid cystic carcinomas., Conclusions: There was a statistically significantly reduced expression of cluster of differentiation 9 glycoprotein in malignant parotid gland tumours, compared with benign parotid gland tumours (p < 0.05). These results suggest that a low level of cluster of differentiation 9 glycoprotein expression in parotid gland tumours may be associated with malignancy.

Amelanotic malignant melanoma in the oral region is extremely rare and has not previously been reported in the parotid gland. This present case report describes an amelanotic malignant melanoma in the parotid gland, with no other primary lesion detectable. The medical history, pathology and immunohistochemical analysis of the case are described and the relevant literature is reviewed in order to help in the recognition of this uncommon tumour. Amelanotic malignant melanoma is a melanoma subtype with little or no pigmentation and, because of this lack of pigmentation and its wide-ranging clinical appearance, it often defies clinical diagnosis. A high level of vigilance is, therefore, necessary in diagnosing such a tumour in the parotid gland. Where it is suspected, positive expression of S-100 protein and human melanoma black 45(HMB45) using immunohistochemical analysis can be considered reliable methods of confirming diagnosis.