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Intermittent fasting exerts a profound beneficial influence on a spectrum of diseases through various mechanisms including regulation of immune responses, elimination of senescent- and pathogenic cells and improvement of stem cell-based tissue regeneration in a disease- and tissue-dependent manner. Our previous study demonstrated that alternate-day fasting (ADF) led to alleviation of xerostomia and sialadenitis in non-obese diabetic (NOD) mice, a well-defined model of Sjögren's syndrome (SS). This present study delved into the previously unexplored impacts of ADF in this disease setting and revealed that ADF increases the proportion of salivary gland stem cells (SGSCs), defined as the EpCAM hi cell population among the lineage marker negative submandibular gland (SMG) cells. Furthermore, ADF downregulated the expression of p16 INK4a , a cellular senescence marker, which was concomitant with increased apoptosis and decreased expression and activity of NLRP3 inflammasomes in the SMGs, particularly in the SGSC-residing ductal compartments. RNA-sequencing analysis of purified SGSCs from NOD mice revealed that the significantly downregulated genes by ADF were mainly associated with sugar metabolism, amino acid biosynthetic process and MAPK signaling pathway, whereas the significantly upregulated genes related to fatty acid metabolic processes, among others. Collectively, these findings indicate that ADF increases the SGSC proportion, accompanied by a modulation of the SGSC property and a switch from sugar- to fatty acid-based metabolism. These findings lay the foundation for further investigation into the functionality of SGSCs influenced by ADF and shed light on the cellular and molecular mechanisms by which ADF exerts beneficial actions on salivary gland restoration in SS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The salivary glands are often damaged during head and neck cancer radiotherapy. This results in chronic dry mouth, which adversely affects quality of life and for which there is no long-term cure. Mouse models of salivary gland injury are routinely used in regenerative research. However, there is no clear consensus on the radiation regime required to cause injury. Here, we analysed three regimes of γ-irradiation of the submandibular salivary gland. Transcriptional analysis, immunofluorescence and flow cytometry was used to profile DNA damage, gland architecture and immune cell changes 3 days after single doses of 10 or 15 Gy or three doses of 5 Gy. Irrespective of the regime, radiation induced comparable levels of DNA damage, cell cycle arrest, loss of glandular architecture, increased pro-inflammatory cytokines and a reduction in tissue-resident macrophages, relative to those observed in non-irradiated submandibular glands. Given these data, coupled with the fact that repeated anaesthetic can negatively affect animal welfare and interfere with saliva secretion, we conclude that a single dose of 10 Gy irradiation is the most refined method of inducing acute salivary gland injury in a mouse model., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Background: Modernization has made individuals prefer processed and cooked foods (soft food), but this eating habit may have negative effects on the oral cavity. However, laboratory animals fed with soft diet are commonly used in an attempt to clarify this issue, and various oral tissues, including the salivary glands have been examined. In this review, we summarize the findings of previous studies concerning the responses of salivary glands to daily intake of soft diet., Highlight: The weight of the parotid glands decreased in rodents fed with soft diet (liquid or powder). In atrophic parotid glands, acinar cell shrinkage is histologically observed and the DNA content is reduced, showing that the atrophy is caused by a decrease in the size and number of acinar cells. Immunohistochemical examinations showed that the decrease in the acinar cell number was induced by suppression of acinar cell proliferation and acceleration of apoptosis. The atrophic parotid glands recovered following a change from soft to pellet diet. Other salivary glands, such as the submandibular, sublingual, and palatine glands, responded only slightly to the soft diet feeding., Conclusion: Accumulated research data showed that a soft diet negatively affects the parotid glands much more than other salivary glands and that atrophic parotid glands are able to recover by switching to a hard diet. Therefore, it should be emphasized that good eating habits are important for not only digestion but also the health of oral tissues, including the salivary glands., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (Copyright © 2022 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.)

Plasmacytoid dendritic cells (pDCs) produce type I interferons (IFNs) and promote pathogenesis of multiple autoimmune diseases. Autoimmune Sjögren's syndrome (SS) primarily affects salivary and lacrimal glands, causing their inflammation, destruction and dysfunction. pDCs and type I IFN activity are elevated in salivary glands of SS patients, and this study seeks to elucidate the in vivo actions of pDCs in SS pathogenesis using the non-obese diabetic (NOD) mouse model. We confirmed the type I IFN-dependency of SS development in female NOD mice and elevation of pDC-type I IFN in their submandibular glands (SMGs). We administered a pDC-depleting anti-BST2/CD317 antibody to female NOD mice from 4 to 7 weeks of age at the early stage of SS, and assessed SS pathologies at age 10 weeks, the time of disease onset. Depletion of pDCs impeded the development of SMG inflammation and secretory dysfunction. It drastically reduced the amount of type I IFN mRNA and the number of total leukocytes, and T- and B lymphocytes in SMGs. Gene expression analyses showed that pDC depletion markedly diminished SMG expression of IL-7, BAFF, TNF-α, IFN-γ, CXCL9, CXCL11, CD40, CD40L, Lt-α, Lt-β and NOS2. Hence, pDCs critically contribute to the development and onset of SS-like salivary gland exocrinopathy., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Diabetes mellitus (DM) is one of the major metabolic diseases. Xerostomia and salivary gland dysfunction are of its common oral complications. Exosomes, as a new therapeutic potential containing nucleic acids, proteins and lipids, act as effective vehicles for target molecules delivery. Accordingly, their therapeutic use is gaining much interest. Therefore, this work aimed to assess the therapeutic efficacy of salivary exosomes in ameliorating DM and combating xerostomia as a complication of salivary gland dysfunction in diabetic rats. In the current study, salivary exosomes were injected intravenously to rats of group II (Salivary Exo-treated group) one week after diabetes induction. Group I (Diabetic group) was left untreated. Blood sugar level was checked weekly. Water intake, salivary flow rate, salivary amylase and serum nitric oxide were assessed before and after diabetes induction and at the end of the study. After 5 weeks from the beginning of the study, salivary gland tissues were dissected and examined histologically and ultrastructurally. Gene expression of the inflammatory markers NFκB/p65 and TNFα was assessed by polymerase chain reaction. The results showed that salivary exosomes reduced blood glucose levels and enhanced salivary glands' function. This was indicated by a decrease in water intake, salivary amylase and serum nitric oxide in addition to an increase in salivary flow rate. This was confirmed histologically, ultrastructurally and via downregulation of NFκB/p65 and TNFα gene expression. Our results concluded that salivary exosomes could be considered as a novel cell free based therapy in treatment of xerostomia and salivary gland dysfunction in DM.

Objective: Persistent (≥2 months) major salivary gland enlargement in primary SS (pSS) patients is a well-known sign of possible involvement by B cell lymphoma. The study aimed to evaluate the diagnostic accuracy and safety of US-guided core needle biopsy (CNB) of major salivary glands compared with open surgical biopsy., Methods: Prospective pSS patients (cases) with clinically persistent salivary gland enlargement underwent US-guided CNB and were compared with retrospective pSS patients (controls) submitted to open surgical biopsy. The features analysed were pre-biopsy clinical and laboratory findings, adequacy of the material for histology and diagnostic-rendered and biopsy-related complications (reported by the patient with a questionnaire and clinically verified)., Results: Thirteen cases underwent US-guided CNB: in nine, biopsy was performed on the parotid gland and in four it was performed on the submandibular gland. Sufficient material was obtained for pathological diagnosis in all samples. The final diagnoses were 5 (38.5%) B cell lymphoma, 1 (7.7%) lymphoepithelial sialadenitis, 4 (30.7%) other sialadenitis (granulomatous consistent with sarcoidosis, IgG4-related disease, chronic sclerosing, diffuse chronic) and 3/13 (23.1%) miscellaneous lesions. Thirteen controls underwent open surgical biopsy of the parotid. In one, inadequate material was obtained, while in 12 (92.3%) the pathologic diagnoses were 4 (33.3%) B cell lymphoma, 2 (16.7%) lymphoepithelial sialadenitis, 4 (33.3%) uncertain lymphoproliferative lesions and 2 (16.7%) miscellaneous lesions. Six cases (46.1%) reported six transient complications and 12/13 (92.3%) controls had 2 persistent and 14 transient complications., Conclusion: US-guided CNB represents a novel, clinically relevant and safe approach for the management of pSS patients with parotid or submandibular persistent enlargement., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Aims: Radiotherapy has become a basic treatment modality for head and neck cancer. However, radiotherapy results in inevitable side effects, particularly radiation sialadenitis, that significantly impairs quality of life. A previous study indicated that nerve growth factor (NGF) has a radio-protective effect, but the mechanism was not determined in salivary glands. In this study, we explored the functional role and mechanism regarding how NGF protects salivary glands against IR-induced damage., Main Methods: Human salivary gland (HSG) cells and C57BL/6 mice were selected to establish an IR-induced salivary gland damage model in vitro and in vivo. Recombinant NGF protein and NGF siRNA and over-expression plasmids were applied to manipulate NGF expression in vitro. AAV-NGF was retrogradely perfused into the submandibular gland (SMG) through the SMG duct to manipulate NGF expression in vitro. Small-molecule inhibitors and siRNAs were applied to inhibit AKT and JNK. Western blotting, quantitative PCR, flow cytometry and histology assays were performed to analyse the functional role and mechanism of NGF., Key Findings: Our study demonstrated that NGF expression was upregulated following radiotherapy both in human HSG cells and mouse SMG tissues. NGF could reduce IR-induced HSG cell apoptosis, and AAV-mediated gene therapy could restore the salivary flow rate and protect the salivary gland against IR-induced apoptosis in vivo. Mechanistically, NGF protects salivary glands from IR-induced apoptosis by de-phosphorylating JNK kinase rather than promoting AKT phosphorylation., Significance: The current study findings indicated that the modulation of the NGF pathway might prevent IR-induced salivary hypo-function., (Copyright © 2020. Published by Elsevier Inc.)

Background: Juvenile Sjögren's Syndrome (jSS) is a rare phenomenon that may appear as primary jSS or associated with mixed connective tissue disease (MCTD) and other autoimmune diseases as secondary jSS. With currently no standard diagnostic procedures available, jSS in MCTD seems to be underdiagnosed. We intended to describe and identify similar distinct salivary gland ultrasound (SGUS) findings in a cohort of primary and secondary jSS patients, focusing on sicca like symptoms and glandular pain/swelling in the patients'history., Methods: We present a single-center study with chart data collection. B-mode examinations of salivary glands were obtained with a linear high-frequency transducer and evaluated using the scoring-system of Hocevar. Inclusion criteria were: (i) primary or secondary jSS and/or (ii) diagnosis of MCTD and additionally (iii) any presence of sicca like symptoms or glandular pain/swelling., Results: Twenty five patients with primary (pjSS) and secondary jSS (sjSS) were included in the study (n = 25, 21 female, 4 male), with a median age of 15.3 years at the time of first visit and a mean disease duration of 4.9 years. Pathologic SGUS findings were observed in 24 of 25 patients, with inhomogeneous parenchymal appearances with hypoechoic lesions present in 96% of patients. At least one submandibular gland was affected in 88.5% of the whole group, and all patients in the MCTD-group. Twenty of twenty five patients were scanned and scored on a second visit. Pre-malignancies or mucosa-associated lymphoid tissue (MALT) were detected in biopsies of three patients (Hocevar scoring of 40, 33, and 28)., Conclusion: SGUS in patients with pjSS and sjSS is a helpful first-line tool to detect and score salivary gland involvement, in particular when keratoconjunctivitis sicca, xerostomia, or glandular swelling occurs. Juvenile MCTD patients have a significant risk of developing secondary jSS. We propose SGUS as a method in the diagnostic workup and screening for inflammatory changes. Further studies have to determine the predictive value of SGUS for follow up.

Purpose: To explore the role of salivary fat fraction (FF) values in evaluating patients with primary Sjögren's syndrome (pSS)., Material and Method: A total of 28 patients with pSS, ten patients with suspected pSS, and 28 volunteers were enrolled. The FF values of the parotid and submandibular glands were generated from mDIXON Quant. A one-way analysis of variance was used to compare the FF values among the groups. A receiver-operating characteristic analysis was applied to assess the diagnostic performance of the parotid and submandibular FF values in distinguishing patients with pSS from non-pSS subjects. In particular, we focused on distinguishing patients with grade 0 pSS from those with suspected pSS., Results: The parotid and submandibular mean FF value of patients with pSS was significantly higher than that of healthy volunteers. The submandibular mean FF value of patients with pSS was higher than those suspected ones. Submandibular FF value performed better than parotid FF value in differentiating patients with pSS from those non-pSS subjects (area under the curve [AUC] = 0.927 vs. 0.734), patients with pSS from patients with suspected pSS (AUC = 0.907 vs. 0.725). This value also performed better at distinguishing patients with grade 0 pSS from those patients with suspected pSS (AUC = 0.925 vs. 0.783)., Conclusions: The salivary gland FF value, especially the submandibular gland FF value, distinguished patients with pSS from those patients with suspected pSS and healthy volunteers., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: This study aimed to analyze the effects of comprehensive protection of bilateral parotid glands (PG-T), contralateral submandibular gland (cSMG), and accessory salivary glands in the oral cavity (OC) by helical tomotherapy for head-and-neck cancer patients., Methods: Totally 175 patients with histologically confirmed head-and-neck cancer treated with helical tomotherapy were recruited. The doses delivered to PG-T, cSMG, and OC were constrained to be as low as possible in treatment planning. The saliva flow rates and xerostomia questionnaire were evaluated. Correlation between xerostomia and other clinical factors were assessed using univariate and multivariate models. The impact of salivary gland dose on locoregional (LR) recurrence was assessed by Cox analysis. ROC curve was used to determine the threshold of mean dose for each gland., Results: The median follow-up was 25 (19-36) months. The OC mean dose, PG-T mean dose, cSMG mean dose, age, clinical stage (II and III versus IV), and both unstimulated and stimulated saliva flow rates were significantly correlated with xerostomia. The OC mean dose, cSMG mean dose, age, and clinical stage were predictors of xerostomia after adjusting PG-T mean dose, and unstimulated and stimulated saliva flow rates. Xerostomia was significantly decreased when the mean doses of PG-T, cSMG, and OC were kept below 29.12Gy, 29.29Gy, and 31.44Gy, respectively. At 18 months after radiation therapy, early LR recurrence rate was only 4%., Conclusion: Comprehensive protection of salivary glands minimized xerostomia in head-and-neck cancer patients treated by helical tomotherapy, without increasing early LR recurrence risk., Competing Interests: The authors declare that they have no conflicts of interest.

Family with sequence similarity 20‑member C (FAM20C), a recently characterized Golgi kinase, performs numerous biological functions by phosphorylating more than 100 secreted proteins. However, the role of FAM20C in the salivary glands remains undefined. The present study demonstrated that FAM20C is mainly located in the cytoplasm of duct epithelial cells in the salivary glands. Fam20cf/f; Mmtv‑Cre mice were created in which Fam20c was inactivated in the salivary gland cells and observed that the number of ducts and the ductal cross‑sectional area increased significantly, while the number of acinar cells was reduced. The granular convoluted tubules (GCTs) exhibited an accumulation of aberrant secretory granules, along with a reduced expression and altered distribution patterns of β nerve growth factor, α‑amylase and bone morphogenetic protein (BMP) 4. This abnormality suggested that the GCT cells were immature and exhibited defects in developmental and secretory functions. In accordance with the morphological alterations and the reduced number of acinar cells, FAM20C deficiency in the salivary glands significantly decreased the salivary flow rate. The Na+, Cl- and K+ concentrations in the saliva were all significantly increased due to dysfunction of the ducts. Furthermore, Fam20c deficiency significantly increased BMP2 and BMP7 expression, decreased BMP4 expression, and attenuated the canonical and noncanonical BMP signaling pathways in the salivary glands. Collectively, the results of the present study demonstrate that FAM20C is a key regulator of acinar and duct structure and duct maturation and provide a novel avenue for investigating novel therapeutic targets for oral diseases including xerostomia.

Xerostomia is a common consequence of radiotherapy in head and neck cancer. The objective was to compare the regional radiation dose distribution in patients that developed xerostomia within 6 months of radiotherapy and those recovered from xerostomia within 18 months post-radiotherapy. We developed a feature generation pipeline to extract dose volume histogram features from geometrically defined ipsilateral/contralateral parotid glands, submandibular glands, and oral cavity surrogates for each patient. Permutation tests with multiple comparisons were performed to assess the dose difference between injury vs. non-injury and recovery vs. non-recovery. Ridge logistic regression models were applied to predict injury and recovery using clinical features along with dose features (D10-D90) of the subvolumes extracted from oral cavity and salivary gland contours + 3 mm peripheral shell. Model performances were assessed by the area under the receiver operating characteristic curve (AUC) using nested cross-validation. We found that different regional dose/volume metrics patterns exist for injury vs. recovery. Compared to injury, recovery has increased importance to the subvolumes receiving lower dose. Within the subvolumes, injury tends to have increased importance towards D10 from D90. This suggests that different threshold for xerostomia injury and recovery. Injury is induced by the subvolumes receiving higher dose, and the ability to recover can be preserved by further reducing the dose to subvolumes receiving lower dose.

Objective: The aim of this study was to determine the effects of prolonged hyperoxia on salivary glands and salivary secretion in mice., Design: Male C57BL/6 J mice were kept in a 75% oxygen chamber (hyperoxia group) or a 21% oxygen chamber for 5 days. We measured the secretion volume, protein concentration, and amylase activity of saliva after the injection of pilocarpine. In addition, we evaluated the histological changes induced in the submandibular glands using hematoxylin and eosin and Alcian blue staining and assessed apoptotic changes using the TdT-mediated dUTP nick end labeling (TUNEL) assay. We also compared the submandibular gland expression levels of heme oxygenase-1 (HO-1), superoxide dismutase (SOD)-1, and SOD-2 using the real-time polymerase chain reaction., Results: In the hyperoxia group, salivary secretion was significantly inhibited at 5 and 10 min after the injection of pilocarpine, and the total salivary secretion volume was significantly decreased. The salivary protein concentration and amylase activity were also significantly higher in the hyperoxia group. In the histological examinations, enlargement of the mucous acini and the accumulation of mucins were observed in the submandibular region in the hyperoxia group, and the number of TUNEL-positive cells was also significantly increased in the hyperoxia group. Moreover, the expression levels of HO-1, SOD-1, and SOD-2 were significantly higher in the hyperoxia group., Conclusion: Our results suggest that hyperoxia reduces salivary secretion, and oxidative stress reactions might be involved in this., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Lipoxin receptor (ALX)/N-formyl peptide receptor (FPR)-2 is a G-protein-coupled receptor that has multiple binding partners, including the endogenous lipid mediators resolvin D1, lipoxin A 4 , and the Ca 2+ -dependent phospholipid-binding protein annexin A1. Previous studies have demonstrated that resolvin D1 activates ALX/Fpr2 to resolve salivary gland inflammation in the NOD/ShiLtJ mouse model of Sjögren syndrome. Moreover, mice lacking the ALX/Fpr2 display an exacerbated salivary gland inflammation in response to lipopolysaccharide. Additionally, activation of ALX/Fpr2 has been shown to be important for regulating antibody production in B cells. These previous studies indicate that ALX/Fpr2 promotes resolution of salivary gland inflammation while modulating adaptive immunity, suggesting the need for investigation of the role of ALX/Fpr2 in regulating antibody production and secretory function in mouse salivary glands. Our results indicate that aging female knockout mice lacking ALX/Fpr2 display a significant reduction in saliva flow rates and weight loss, an increased expression of autoimmune-associated genes, an up-regulation of autoantibody production, and increased CD20-positive B-cell population. Although not all effects were noted among the male knockout mice, the results nonetheless indicate that ALX/Fpr2 is clearly involved in the adaptive immunity and secretory function in salivary glands, with further investigation warranted to determine the cause(s) of these between-sex differences., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.

Background: Salivary gland (SG) injurious agents are all translated into loss of salivation (xerostomia). An association has been established between activation of innate immunity and SG injury and dysfunction. However, it remains unclear how the secretory epithelia respond by halting saliva production., Methods: C57BL/6 submandibular glands (SMGs) were acutely challenged using a single dose of the innate immune stimulant: polyinosinic-polycytidylic acid (poly (I:C)). Secretory capacity of the infected SMGs was substantiated by assessing the flow rate in response to pilocarpine stimulation. Depletion of the acute inflammatory cells was achieved by pre-treating mice with RB6-8C5 depletion antibody. Flow cytometry, histology and immunohistochemistry were conducted to verify the immune cell depletion. Epithelial expression of saliva-driving molecules: muscarinic 3 receptor (M3R), aquaporin 5 water channel (AQP5), Na-K-CL-Cotransporter 1 (NKCC1) and transmembrane member 16A (TMEM16A), was characterized using RT-qPCR and immunohistochemistry. Tight junction (TJ) protein; zonula occludens (ZO-1) and basement membrane (BM) protein; and laminin were assessed by immunohistochemistry., Results: Innate immune challenge prompted dysfunction in the exocrine SGs. Dysregulated gene and protein expression of molecules that drive saliva secretion was substantiated. Aberrant expression of TJ and BM proteins followed innate immune activation. Hyposalivation in the current model was independent of myeloperoxidase (MPO)-positive, acute inflammatory cells., Conclusions: In this study, we developed a novel injury model of the SGs, featuring acute secretory dysfunction and immediate structural disruptions. Our results ruled out the injurious role of aggressively infiltrating inflammatory cells., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: We analyzed the diagnostic performance of the MR imaging findings of the parotid, submandibular, and sublingual glands to discriminate between patients with and without Sjögren's syndrome., Methods: We retrospectively analyzed the correlation between the MR imaging and histopathological findings obtained from 69 patients with clinically suspected Sjögren's syndrome. We evaluated the heterogeneous signal intensity distribution on T1- and T2-weighted images, the multiple high-signal-intensity spots on MR sialograms, and the volume of the parotid, submandibular, and sublingual salivary glands., Results: The multiple high-signal-intensity spots in the parotid gland showed the highest sensitivity and diagnostic accuracy (82% and 83%, respectively). In addition, the multiple high-signal-intensity spots and the heterogeneous signal intensity distribution in the submandibular gland showed high specificity (100% and 88%, respectively). The volume of the submandibular gland, but not that of the parotid or sublingual gland, was smaller in patients with Sjögren's syndrome., Conclusions: The presence of multiple high-signal-intensity spots on an MR sialogram in the parotid gland should be considered the best diagnostic indicator for Sjögren's syndrome. The presence of spots, heterogeneity, and the change to smaller volumes in the submandibular gland were also helpful because of their high specificity, particularly in advanced cases., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Radiotherapy is commonly used in patients with oral cavity and pharyngeal cancers, usually resulting in irreversible salivary hypofunction. Currently management of radiation damage to salivary glands still remains a great challenge. Recent studies show that activation of mammalian target of rapamycin (mTOR) occurs in salivary gland lesions, making it possible to apply mTOR inhibitor for treatment. Our results indicate inhibition of mTOR by rapamycin significantly alleviated irradiation-induced salivary hypofunction by restoring 46% salivary flow rate and protecting histological structures in swine. Furthermore, rapamycin protected human submandibular gland cell line (HSG) from irradiation-induced cell depletion and loss of cell proliferation capacity. These findings lay the foundation for a new clinical application of rapamycin to prevent irradiation-induced salivary hypofunction.

A total of 136 subjects (51 SS patients, 35 sicca syndrome patients without SS, and 50 healthy volunteers) were enrolled in this study. The mean SWV value for salivary glands of SS patients was statistically higher than that of controls (2.81 ± 0.66 m/s versus 1.85 ± 0.28 m/s for parotid glands and 2.29 ± 0.34 m/s versus 1.82 ± 0.25 m/s for submandibular glands, resp.). Combining SWV values of parotid and submandibular glands gives a sensitivity of 88.2% (95% CI: 76.1-95.6%) and specificity of 96.0% (95% CI: 86.3-99.5%) at the cutoff point of 2.19 m/s, with an AUROC of 0.954 (95% CI: 0.893-0.986). In addition, combining SGUS score and SWV value yields a sensitivity of 98.0% (95% CI: 89.6-100%), specificity of 90.0% (95% CI: 78.2-96.7%), and AUROC of 0.962 (95% CI: 0.904-0.990). Classification tree considering the sequential use of SGUS score and SWV value achieved 92.1% accuracy for diagnosis of SS. Similarly, the ROC curve of combined SGUS scores and SWV values yields an AUROC of 0.954 (95% CI: 0.885-0.987), sensitivity of 97.1% (95% CI: 85.1-99.9%), and specificity of 92.2% (95% CI: 81.1-97.8%) for separating sicca syndrome patients (without SS) from SS patients. Combining SGUS and VTQ provides a promising tool for diagnosis of SS., Competing Interests: The authors have no conflict of interests to disclose for this study.

Sjögren's syndrome (SS) is a chronic inflammatory autoimmune disorder that causes secretory dysfunction of the salivary glands leading to dry mouth. Previous studies reported that tight junction (TJ) proteins are down-regulated and lose polarity in human minor salivary glands with SS, suggesting that TJ structure is compromised in SS patients. In this paper, we utilized the NOD/ShiLtJ mouse with the main goal of evaluating this model for future TJ research. We found that the organization of apical proteins in areas proximal and distal to lymphocytic infiltration remained intact in mouse and human salivary glands with SS. These areas looked comparable to control glands (i.e., with no lymphocytic infiltration). TJ staining was absent in areas of lymphocytic infiltration coinciding with the loss of salivary epithelium. Gene expression studies show that most TJs are not significantly altered in 20-week-old NOD/ShiLtJ mice as compared with age-matched C57BL/6 controls. Protein expression studies revealed that the TJ proteins, zonula occludens-1 (ZO-1), occludin, claudin-12, as well as E-cadherin, do not significantly change in NOD/ShiLtJ mice. Our results suggest that ZO-1, occludin and E-cadherin are not altered in areas without lymphocytic infiltration. However, future studies will be necessary to test the functional aspect of these results., (© The Author(s) 2014.)

Glucocorticoids (GCs) induce insulin resistance (IR), a condition known to alter oral homeostasis. This study investigated the effects of long-term dexamethasone administration on morphofunctional aspects of salivary glands. Male Wistar rats received daily injections of dexamethasone [0.1 mg/kg body weight (b.w.), intraperitoneally] for 10 days (DEX), whereas control rats received saline. Subsequently, glycaemia, insulinaemia, insulin secretion and salivary flow were analysed. The parotid and submandibular glands were collected for histomorphometric evaluation and Western blot experiments. The DEX rats were found to be normoglycaemic, hyperinsulinaemic, insulin resistant and glucose intolerant (P < 0.05). DEX rat islets secreted more insulin in response to glucose (P < 0.05). DEX rats had significant reductions in the masses of the parotid (29%) and submandibular (16%) glands (P < 0.05) that was associated with reduced salivary flux rate. The hypotrophy in both glands observed in the DEX group was associated with marked reduction in the volume of the acinar cells in these glands of 50% and 26% respectively (P < 0.05). The total number of acinar cells was increased in the submandibular glands of the DEX rats (P < 0.05) but not in the parotid glands. The levels of proteins related to insulin and survival signalling in both glands did not differ between the groups. In conclusion, the long-term administration of dexamethasone caused IR, which was associated with significant reductions in both mass and flux rate of the salivary glands. The parotid and submandibular glands exhibited reduced acinar cell volume; however, the submandibular glands displayed acinar hyperplasia, indicating a gland-specific response to GCs. Our data emphasize that GC-based therapies and insulin-resistant states have a negative impact on salivary gland homeostasis., (© 2014 The Authors. International Journal of Experimental Pathology © 2014 International Journal of Experimental Pathology.)

Background: Protein-tyrosine sulfation is a post-translational modification of an unknown number of secreted and membrane proteins mediated by two known Golgi tyrosylprotein sulfotransferases (TPST-1 and TPST-2). We reported that Tpst2-/- mice have mild-moderate primary hypothyroidism, whereas Tpst1-/- mice are euthyroid. While using magnetic resonance imaging (MRI) to look at the thyroid gland we noticed that the salivary glands in Tpst2-/- mice appeared smaller than in wild type mice. This prompted a detailed analysis to compare salivary gland structure and function in wild type, Tpst1-/-, and Tpst2 -/- mice., Methodology/principal Findings: Quantitative MRI imaging documented that salivary glands in Tpst2-/- females were (≈) 30% smaller than wild type or Tpst1-/- mice and that the granular convoluted tubules in Tpst2-/- submandibular glands were less prominent and were almost completely devoid of exocrine secretory granules compared to glands from wild type or Tpst1-/- mice. In addition, pilocarpine-induced salivary flow and salivary α-amylase activity in Tpst2-/- mice of both sexes was substantially lower than in wild type and Tpst1-/- mice. Anti-sulfotyrosine Western blots of salivary gland extracts and saliva showed no differences between wild type, Tpst1-/-, and Tpst2-/- mice, suggesting that the salivary gland hypofunction is due to factor(s) extrinsic to the salivary glands. Finally, we found that all indicators of hypothyroidism (serum T4, body weight) and salivary gland hypofunction (salivary flow, salivary α-amylase activity, histological changes) were restored to normal or near normal by thyroid hormone supplementation., Conclusions/significance: Our findings conclusively demonstrate that low body weight and salivary gland hypofunction in Tpst2-/- mice is due solely to primary hypothyroidism.

Xerostomia as a result of salivary gland damage is a permanent and debilitating side effect of radiotherapy for head and neck cancers. Effective treatments for protecting, or restoring, salivary gland function are not available. Here we report that irradiation treatment leads to activation of the calcium-permeable channel, transient potential melastatin-like 2 (TRPM2), via stimulation of poly-ADP-ribose polymerase. Importantly, irradiation induced an irreversible loss of salivary gland fluid secretion in TRPM2+/+ mice while a transient loss was seen in TRPM2-/- mice with >60% recovery by 30 days after irradiation. Treatment of TRPM2+/+ mice with the free radical scavenger Tempol or the PARP1 inhibitor 3-aminobenzamide attenuated irradiation-induced activation of TRPM2 and induced significant recovery of salivary fluid secretion. Furthermore, TPL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) induced complete recovery of function in irradiated TRPM2-/- mice. These novel data demonstrate that TRPM2 is activated by irradiation, via PARP1 activation, and contributes to irreversible loss of salivary gland function.

A 23-year-old man was referred to our Emergency Department due to an acute-onset hemoptysis and the associated bilateral swelling of all of his major salivary glands. The elevated levels of systemic inflammatory markers with hypereosinophilia and the concurrent presence of multiple lung infiltrates made it difficult to perform a differential diagnosis, as these symptoms can also be indicative of infectious, autoimmune or hematologic disorders. A histological examination of the patient's left submandibular gland revealed strong clues as to the final diagnosis of eosinophilic granulomatosis with polyangiitis with an atypical clinical presentation, thus allowing for the administration of early and successful conservative therapy. The outcome of our case suggests that systemic vasculitis represents a rare but possible cause of acute bilateral sialadenitis in young patients.

Objectives: The purpose of this prospective study was to characterize the MR relaxometric features of the major salivary glands in patients with sickle cell disease (SCD)., Methods: 15 patients with SCD (aged 19.8-43.6 years) and 12 controls were imaged with the mixed turbo-spin echo pulse sequence. The major salivary glands were manually segmented and T1, T2 and secular T2 relaxometry histograms were modelled with Gaussian functions., Results: Shortened T1 relaxation times were seen solely in the submandibular glands of patients with SCD (747.5±54.8 ms vs 807.1±38.3 ms, p<0.001). Slight T2 and secular T2 shortening were seen in the parotid gland; however, this difference was not significant (p=0.07). The sublingual gland showed no changes under MR relaxometry. There was no difference in glandular volumes, and no correlation was demonstrated between history of blood transfusion and salivary gland relaxometry., Conclusions: Patients with SCD exhibited changes in quantitative MRI T1 relaxometry histograms of the submandibular glands.

Purpose: Patients treated with radiotherapy for head-and-neck cancer invariably suffer its deleterious side effect, xerostomia. Salivary hypofunction ensuing from the irreversible destruction of glands is the most common and debilitating oral complication affecting patients undergoing regional radiotherapy. Given that the current management of xerostomia is palliative and ineffective, efforts are now directed toward preventive measures to preserve gland function. The human homolog of Tousled protein, TLK1B, facilitates chromatin remodeling at DNA repair sites and improves cell survival against ionizing radiation (IR). Therefore, we wanted to determine whether a direct transfer of TLK1B protein to rat salivary glands could protect against IR-induced salivary hypofunction., Methods: The cell-permeable TAT-TLK1B fusion protein was generated. Rat acinar cell line and rat salivary glands were pretreated with TAT peptide or TAT-TLK1B before IR. The acinar cell survival in vitro and salivary function in vivo were assessed after radiation., Results: We demonstrated that rat acinar cells transduced with TAT-TLK1B were more resistant to radiation (D₀ = 4.13 ± 1.0 Gy; α/β = 0 Gy) compared with cells transduced with the TAT peptide (D₀ = 4.91 ± 1.0 Gy; α/β = 20.2 Gy). Correspondingly, retroductal instillation of TAT-TLK1B in rat submandibular glands better preserved salivary flow after IR (89%) compared with animals pretreated with Opti-MEM or TAT peptide (31% and 39%, respectively; p < 0.01)., Conclusions: The results demonstrate that a direct transfer of TLK1B protein to the salivary glands effectively attenuates radiation-mediated gland dysfunction. Prophylactic TLK1B-protein therapy could benefit patients undergoing radiotherapy for head-and-neck cancer., (Published by Elsevier Inc.)

The aim of this study is to evaluate the diagnostic value of quantifying salivary gland scintigraphy in correlation to the labial biopsy findings of Sjögren's syndrome (SS). Thirty patients suspected of having SS referred to our clinic for salivary gland scintigraphy were included to this study. All patients underwent salivary gland biopsy as well. The severity of histopathologic changes was graded according to the Chisholm and Mason scoring system. Dynamic scintigraphy was performed and region of interests (ROI) were drawn. Time activity curves for salivary glands were generated. Count rates of maximum, minimum activity after lemon juice stimuli, and last minute activities of parotid and submandibular glands were obtained. On the basis of this ROI counts, excretion fraction (EF%) was calculated for all salivary glands. The mean EF% for normal parotid gland and pathologic parotid gland was 54.5 ± 13.9 and 45.8 ± 18.42, respectively, while it was 46.7 ± 11.7 for the normal submandibular gland and 29.3 ± 18.8 for the pathologic submandibular gland. With progression in histopathologic grades from 0 to 4, the EF decreased in all salivary glands. Decreased EF in the salivary glands is correlated with the SS, and salivary gland scintigraphy is a sensitive and valid method for evaluation of the function of the salivary glands.

Background: This study focuses on the potential of ReGeneraTing Agent OTR4120 (RGTA-OTR4120) to treat radiation-induced damage of salivary glands. RGTAs are biopolymers designed to mimic the effects of heparan sulphate, thereby stimulation tissue repair and regeneration., Methods: C3H mice were irradiated with a single dose of 15 Gy in the head and neck region. RGTA-OTR4120 was injected 24 h after radiotherapy, followed by weekly injections. At 2, 6 and 10 weeks after radiotherapy, salivary flow rates were measured and animals were sacrificed to obtain parotid and submandibular glands for histology. Periodic acid Schiff stain was performed to visualize mucins that are produced by acinar cells. Amylase and total protein content were measured in saliva samples., Results: Salivary flow rates were increased at 2 weeks, but not at 6 and 10 weeks after radiotherapy with RGTA-OTR4120 administration, compared to irradiated controls. Two and 10 weeks after radiotherapy, the mucin production activity of acinar cells was increased under influence of RGTA administration. RGTA-OTR4120 did not influence amylase or total protein secretion., Conclusion: RGTA-OTR4120 administration has a positive effect on salivary flow rates in irradiated mice on the short term. The effect was absent 10 weeks after radiotherapy, while at that time point, mucin producing activity of acinar cells was elevated by RGTA-OTR4120 administration. Given these results and the advantages of RGTA use in irradiated patients, further investigation on the potential of this drug to treat radiation-induced salivary gland damage, alone or in combination with other drugs, such as amifostine, is suggested., (© 2012 John Wiley & Sons A/S.)

Purpose: Many head and neck cancer survivors treated with radiotherapy suffer from permanent impairment of their salivary gland function, for which few effective prevention or treatment options are available. This study explored the potential of transient activation of Wnt/β-catenin signaling in preventing radiation damage to salivary glands in a preclinical model., Methods and Materials: Wnt reporter transgenic mice were exposed to 15 Gy single-dose radiation in the head and neck area to evaluate the effects of radiation on Wnt activity in salivary glands. Transient Wnt1 overexpression in basal epithelia was induced in inducible Wnt1 transgenic mice before together with, after, or without local radiation, and then saliva flow rate, histology, apoptosis, proliferation, stem cell activity, and mRNA expression were evaluated., Results: Radiation damage did not significantly affect activity of Wnt/β-catenin pathway as physical damage did. Transient expression of Wnt1 in basal epithelia significantly activated the Wnt/β-catenin pathway in submandibular glands of male mice but not in those of females. Concurrent transient activation of the Wnt pathway prevented chronic salivary gland dysfunction following radiation by suppressing apoptosis and preserving functional salivary stem/progenitor cells. In contrast, Wnt activation 3 days before or after irradiation did not show significant beneficial effects, mainly due to failure to inhibit acute apoptosis after radiation. Excessive Wnt activation before radiation failed to inhibit apoptosis, likely due to extensive induction of mitosis and up-regulation of proapoptosis gene PUMA while that after radiation might miss the critical treatment window., Conclusion: These results suggest that concurrent transient activation of the Wnt/β-catenin pathway could prevent radiation-induced salivary gland dysfunction., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The interaction between proteins and cell receptors is related to tissue homeostasis such as in salivary glands. In this respect, alterations in hormone levels caused by hyperglycaemic conditions may interfere with this interaction, intensifying the damage caused by diabetes mellitus. Hormone replacement therapy is an option to reverse this damage, but doubts still exist regarding the efficacy of this procedure. The objective of this study was to evaluate the effect of oestrogen replacement therapy combined with insulin treatment on the expression of oestrogen (ER-alpha) and insulin receptors (INS-R) in the salivary glands of spontaneously diabetic mice. Twenty-five mice were divided into five group of 5 animals each: group I (NOD diabetic), group II (NOD diabetic treated with insulin), group III (NOD diabetic treated with oestrogen), group IV (NOD diabetic treated with insulin and oestrogen), and group V (control BALB/c mice). Group II received insulin, group III received oestrogen, and group IV received insulin plus oestrogen administered daily for 20 days. Groups I and V received saline for the same period of time to simulate treatment. Glucose and oestrogen levels were monitored during the experimental period and salivary gland samples were collected at the end of the experiment for fluorescence microscopy analysis of ER-alpha and INS-R. Animals receiving oestrogen replacement therapy plus insulin showed regulation of the expression of oestrogen and insulin receptors. Oestrogen treatment alone contributed to the recovery of these cell receptors. These results indicate that oestrogen replacement therapy alone, and especially when combined with insulin, is important for the recovery of the interaction between functional proteins and their receptors, thus contributing to the reestablishment of tissues damaged by the hyperglycaemic condition., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Salivary gland function decreases after radiation doses of 39 Gy or higher. Currently, submandibular glands are not routinely spared. We implemented a technique for sparing contralateral submandibular glands (CLSM) during contralateral elective neck irradiation without compromising PTV coverage., Methods: Volumetric modulated arc therapy (RapidArc™) plans were applied in 31 patients with stage II-IV HNC without contralateral neck metastases, all of whom received elective treatment to contralateral nodal levels II-IV. Group 1 consisted of 21 patients undergoing concurrent chemo-radiotherapy, with elective nodal doses of 57.75 Gy (PTVelect) and 70 Gy to tumor and pathological nodes (PTVboost) in 7 weeks. Group 2 consisted of 10 patients treated with radiotherapy to 54.45 Gy to PTVelect and 70 Gy to PTVboost in 6 weeks. All clinical plans spared the CLSM using individually adapted constraints. For each patient, a second plan was retrospectively generated without CLSM constraints ('non-sparing plan')., Results: PTV coverage was similar for both plans, with 98.7% of PTVelect and 99.2% of PTVboost receiving ≥95% of the prescription dose. The mean CLSM dose in group 1 was 33.2 Gy for clinical plans, versus 50.6 Gy in 'non-sparing plans' (p < 0.001). In group 2, mean CLSM dose was 34.4 Gy for clinical plans, and 46.8 Gy for non-sparing plans (p = 0.002)., Conclusions: Elective radiotherapy to contralateral nodal levels II-IV using RapidArc consistently limited CLSM doses well below 39 Gy, without compromising PTV-coverage. Future studies will reveal if this extent of dose reduction can reduce patient symptoms., (© 2011 Doornaert et al; licensee BioMed Central Ltd.)

Purpose: Salivary glands are significantly affected when head and neck cancer patients are treated by radiation. We evaluated the effect of human keratinocyte growth factor (hKGF) gene transfer to murine salivary glands on the prevention of radiation-induced salivary hypofunction., Experimental Design: A hybrid serotype 5 adenoviral vector encoding hKGF (AdLTR(2)EF1α-hKGF) was constructed. Female C3H mice, 8 weeks old, were irradiated by single (15 Gy) or fractionated (6 Gy for 5 days) doses to induce salivary hypofunction. AdLTR(2)EF1α-hKGF or AdControl was administered (10(8) - 10(10) particles per gland) to both submandibular glands (SG) by retrograde ductal instillation before irradiation (IR). Salivary flow was measured following pilocarpine stimulation. Human KGF levels were measured by ELISA. SG cell proliferation was measured with bromodeoxyuridine labeling. Endothelial and progenitor or stem cells in SGs were measured by flow cytometry. The effect of SG hKGF production on squamous cell carcinoma (SCC VII) tumor growth was assessed., Results: In 3 separate single-dose IR experiments, salivary flow rates of mice administered the AdLTR(2)EF1α-hKGF vector were not significantly different from nonirradiated control mice (P > 0.05). Similarly, in 3 separate fractionated IR experiments, the hKGF-expressing vector prevented salivary hypofunction dramatically. Transgenic hKGF protein was found at high levels in serum and SG extracts. AdLTR(2)EF1α-hKGF-treated mice showed increased cell proliferation and numbers of endothelial cells, compared with mice treated with AdControl. hKGF gene transfer had no effect on SCC VII tumor growth ± radiation., Conclusions: hKGF gene transfer prevents salivary hypofunction caused by either single or fractionated radiation dosing in mice. The findings suggest a potential clinical application., (©2011 AACR.)

Objective: To analyse and compare the expression of Palate, Lung, and Nasal Epithelium Clone (PLUNC) proteins in salivary glands from patients with and without AIDS (control group) using autopsy material., Methods: We analysed the expression of PLUNCs using immunohistochemistry in parotid (n = 45), submandibular (n = 47) and sublingual gland (n = 37) samples of AIDS patients [30 with normal histology, 21 with mycobacteriosis, 14 with cytomegalovirus (CMV) infection, 30 with chronic non-specific sialadenitis, and 30 HIV-negative controls. In situ hybridization (ISH) for SPLUNC 2 in the HIV-negative group was performed., Results:   SPLUNC 1 expression was detected in the mucous acini of submandibular and sublingual glands, and SPLUNC 2 were seen in the serous cells. LPLUNC 1 expression was only positive in the salivary ducts. There was a higher expression of SPLUNC 2 in AIDS patients with CMV infection and mycobacteriosis when compared with all other groups. The intensity of staining for SPLUNC 2 was greater around the lesions than the peripheral ones. ISH for SPLUNC 2 showed perinuclear positivity in the serous cells in all HIV-negative cases., Conclusions:   SPLUNC 1 and LPLUNC 1 proteins were similarly expressed in the salivary glands of AIDS patients and non-HIV patients. CMV infection and mycobacteriosis increase SPLUNC 2 expression in serous cells in the salivary gland of AIDS patients., (© 2010 John Wiley & Sons A/S.)

We used forebrain-specific conditional presenilin 1 (PS1) and presenilin 2 (PS2) double knockout mice (dKO mice), which exhibit neurodegenerative disease-like symptoms, including inflammation of the brain and periphery, to investigate whether periodontal and salivary tissues display alterations. Mandibles were dissected for alveolar bone height analysis. Maxillae were fixed and decalcified for histological observation and osteoclast detection. Submandibular glands were fixed for histological observation. The submandibular gland and the gingiva of the mandibular incisor teeth were used to assay inflammatory mediators. At 9 months, the number of osteoclasts had significantly increased in the periodontal ligament and the periodontal tissues exhibited obvious histomorphological abnormalities in the dKO mice compared to the control mice at the same age. Alveolar bone loss in dKO mice increased with age. The salivary tissues in dKO mice exhibited obvious age-dependent histomorphological abnormalities. The levels of the inflammatory mediators IL-1β, TNF-α, and GM-CSF in the submandibular gland and gingiva also increased in an age-dependent manner. These findings suggest that inflammation in the dKO brain could expand to the periphery, including the oral tissue, which could ultimately induce abnormalities in the periodontal and salivary tissues.

We determined mRNA levels of bone morphogenetic protein 7 (BMP7), a growth and differentiation factor belonging to the transforming growth factor-beta superfamily, in the salivary glands of mice with streptozotocin (200 mg/kg, i.p.)-induced diabetes. We also examined the effects of BMP7 on secretion of saliva and degenerative change in salivary glands in diabetic mice. In normal mice, BMP7 mRNA levels were high in the submandibular gland and low in the parotid gland, while in diabetic mice, levels were significantly decreased in the parotid gland, but not in the submandibular gland. No significant difference was observed in mRNA levels of BMP receptors between normal and diabetic mice. In diabetic mice, pilocarpine (4 mg/kg, i.p.)-stimulated salivary secretion showed a remarkable decrease in both parotid and submandibular gland, although degree of reduction was smaller in the latter. Notable degeneration with vacuolation and atrophy was also found in parotid gland, whereas degeneration of submandibular gland was slight. Administration of BMP7 (50 and 100 microg/kg, i.v.) in diabetic mice induced a significant increase in salivary secretion, with rate of recovery higher in parotid gland than in submandibular gland. In diabetic mice, BMP7 also exhibited a powerful protective effect in degenerated salivary gland, especially in parotid gland. These results suggest that BMP7 acts to prevent diabetic damage in salivary gland, and that its cytoprotective effect is closely correlated with mRNA levels in tissue.

The present study was performed for determining the optimal timing of MR sialography by use of the Japanese pickled plum (umeboshi) for promoting secretion by the salivary glands. MR sialography was performed in four healthy male volunteers. The four volunteers were examined before and 10 min after stimulation with umeboshi. On the next examination, three volunteers were examined before and after umeboshi stimulation every 1 min up to 5 min to allow assessment of the temporal changes in duct visualization. Dilatation of the salivary gland ducts and improvement of the visualization of the ducts were obtained after stimulation with umeboshi. The difference in the dilatation of the parotid duct was statistically significant. In the temporal study, the salivary gland ducts were shown to be dilated at 2 min after stimulation. As a result, 2 min after stimulation is the optimal timing for MR sialography by use of umeboshi as a stimulator of salivary secretion.

We have used a recently described model in which a ras oncogene is expressed in cytokeratin 5 (K5)-expressing cells on doxycycline administration to explore the effects of this oncogene in salivary glands of adult mice. Inducible expression of a mutated K-ras gene under the control of the K5 promoter led to the development of hyperplastic and dysplastic epithelial lesions and carcinomas, with an incidence of 100% and a minimum latency of a week. All major salivary glands were affected, as well as a set of previously undescribed buccal accessory salivary glands located on the apex of the masseter muscle, close to the oral angle. The tumors appear to arise from the cytokeratin 5-positive basal cell compartment. Myoepithelial cells participated in the hyperplasias but not in carcinomas, because the tumors are negative for smooth muscle actin. Carcinomas did not accumulate immunoreactive p53 but are positive for p63, as assayed by immunohistochemistry using an antibody against the N terminus of DeltaN p63, a splice variant of p63 that can inhibit p53 transcriptional activity. In this study, we provide evidence that the ras oncogene, targeted to a specifically sensitive cell compartment within the salivary glands, can trigger a series of event that are sufficient for full carcinogenesis.

Severe xerostomia is a common late radiation consequence, which occurs after irradiation of head and neck malignancies. The aim of the present study was to analyze apoptosis and proliferation and their relationship during the late post-irradiation phase. C57BL/6 mice were locally irradiated in head and neck region with a single dose of 7.5 or 15 Gy and their submandibular glands were collected at 40 and 90 days after irradiation. To identify apoptotic cells, the TUNEL method was employed and immunohistochemistry with proliferating cell nuclear antigen (PCNA) was used for detecting proliferation. Histological changes at day 40 were mild in contrast to day 90 when glands of irradiated mice showed severe atrophy, vacuolization and mononuclear infiltration. Acinar cells, granular and intercalated duct cells of mice irradiated with 7.5 and 15 Gy expressed higher apoptotic index than cells of non-irradiated, control glands at both examined time points. At 40 days, a higher proliferation index in granular and intercalated duct cells was detected only in group irradiated with 7.5 Gy. At 90 days, proliferation index for all cell types in both irradiated groups was similar to the controls. According to our results, the imbalance between apoptosis and proliferation caused by X-irradiation may be the reason for gland impairment during the late post-irradiation phase.

The spontaneous non-obese diabetic (NOD) mouse model of Sjögren's syndrome provides a valuable tool to study the onset and progression of both the autoimmune response and secretory dysfunction. Our purpose was to analyse the temporal decline of salivary secretion in NOD mice in relation to the autoimmune response and alterations in various signalling pathways involved in saliva secretion within each salivary gland. A progressive loss of nitric oxide synthase activity in submandibular and parotid glands started at 12 weeks of age and paralleled the decline in salivary secretion. This defect was associated with a lower response to vasoactive intestinal peptide in salivary flow rate, cAMP and nitric oxide/cGMP production. No signs of mononuclear infiltrates or local cytokine production were detectable in salivary glands in the time period studied (10-16 weeks of age). Our data support a disease model for sialadenitis in NOD mice in which the early stages are characterized by defective neurotransmitter-mediated signalling in major salivary glands that precedes the autoimmune response.

Objectives: To evaluate ultrasonography (US) of salivary glands in primary Sjögren's syndrome (SS) and to compare US with parotid magnetic resonance (MR) imaging and MR sialography., Methods: US examination of parotid, submandibular and sublingual glands was performed on 27 patients with primary SS, 27 healthy controls and 27 symptomatic controls without SS. The results were compared with parotid MR imaging and MR sialography and the clinical features of the patients., Results: Salivary gland abnormalities, parenchymal inhomogeneity or adipose degeneration, were visualized in 21 (78%) SS patients, in one healthy control and in two symptomatic controls by US. Eighteen (67%) patients had changes in the parotid and submandibular glands and 8 (30%) changes in the sublingual glands. In the comparison, MR sialography was found to be the most sensitive method (96%), followed by MR imaging (81%) and US (78%), in detecting glandular changes. The specificity of US was 94%. The US and MR results were related to anti-Ro/SSA positivity but not to saliva secretion. The focus scores were related only to parotid MR imaging findings., Conclusions: US, MR imaging and MR sialography with modern technology have reached such a good accuracy in visualizing glandular structural changes that they are promising alternatives to the conventional invasive examinations in the diagnostics of SS.

Human fractalkine (CX3CL1), a delta-chemokine, is implicated in the mediation of multiple cell functions. In addition to serving as a chemotactic factor for mononuclear cell subtypes, membrane-bound fractalkine may promote viral infection by interacting with virions that encode putative fractalkine-binding proteins. Fractalkine expression in normal epithelial tissues studied to date is either constitutive or is upregulated with inflammation. In salivary glands, the expression of fractalkine is unknown. Moreover, salivary glands are a major site for the persistent and productive infection by human herpesvirus (HHV)-7, which encodes two putative fractalkine-binding gene products, U12 and U51. Surprisingly, the cellular distribution of HHV-7 in major salivary glands has not been explored. We therefore determined by immunohistochemistry the cellular localization of fractalkine in three different salivary glands: parotid, submandibular, and labial glands. Fractalkine expression was highly variable, ranging from high to undetectable levels. We further examined the association of fractalkine with inflammatory cell infiltration or HHV-7 infection of salivary epithelial cells. Inflammatory cells were always adjacent to epithelial cells expressing fractalkine, consistent with a function of fractalkine in inflammatory cell recruitment and/or retention in salivary glands. In contrast, HHV-7-infected epithelial cells did not always express fractalkine, suggesting that fractalkine may not be an absolute requirement for viral entry.

Introduction: The aim of this study was to correlate structural, histomorphological damage of the salivary gland with scintigraphic findings during fractioned radiotherapy., Methods: The head and neck area of 27 WAG/RijH rats was irradiated with (60)Co-gamma-rays (60 Gy/30f/6 weeks). A port-system was implanted and (99m)Tc-pertechnetat applied at different stages of irradiation (0, 16, 30, 46, 60 Gy and 6 months post irradiation)., Results: After the application of 16 Gy an intra- and extra-cellular oedema developed in the salivary glands. The progressive vacuolisation (30 Gy) passed over into lipomatosis (46 Gy) and necrosis (60 Gy) in the parotid and mandibular glands. Six months after irradiation treatment, the chronic histomorphological damage corresponded to stage II according to Seifert. The corresponding loss in gland function was 13% (16 Gy); 26% (30 Gy); 57% (46 Gy); 75% (60 Gy) and 66.5% (6 months post irradiation)., Conclusion: This animal model demonstrates the correlation between histomorphological and scintigraphic findings.

This study explored the feasibility of developing an animal model for radiation-induced salivary gland injury with a radiation protocol identical to current clinical practice. Three male Hanford minipigs were subjected to fractionated daily irradiation with a total dose of 70 Gy; structural and functional measures were compared with those of a control group of minipigs. We found that irradiated submandibular and parotid glands were one-third to one-half the gross size of control glands. Whereas no pathologic changes were noted in control glands, irradiated glands consistently demonstrated significant parenchymal loss with extensive acinar atrophy and interstitial fibrosis, enlarged nuclei in remaining acinar cells, and ductal dilatation and proliferation. Stimulated salivary flow was reduced by 81% in irradiated animals compared with preirradiation flow (P <.001); salivary flow in the control group increased by 30% during the same period (P <.001). The observed radiation-induced structural and functional salivary gland changes are comparable in every respect to those observed following irradiation of human salivary glands.

In 1896 Kuttner reported four cases which he described as induration of the submandibular gland. Histologically, they showed chronic inflammation and fibrosis. Sporadic cases of this entity that have come to be known as Kuttner's tumor or chronic sclerosing sialadenitis of the submandibular gland and have been reported throughout the 20th century. This inflammatory tumor has been under-recognized, and awareness of its importance and probable immunologic background have recently become evident. We report an unusual case of chronic sclerosing sialadenitis, affecting both parotid glands, both submandibular glands, and minor salivary glands of the oral cavity, and explore the immunohistochemical profile of this entity., (Copyright 2003, Elsevier Science (USA). All rights reserved.)

Clinical studies show that amifostine can reduce xerostomia and mucositis during radiotherapy of head and neck cancers. The aim of this study was to evaluate the radioprotective potency of amifostine with respect to late toxicity of salivary glands of rats. The head-neck-area of 8 male WAG/RijH rats (295 +/- 7 g) were irradiated with 60Co-gamma-rays (60 Gy/30 f/6 weeks). Amifostine (250 mg/m2 body surface) was applied via a venous port 15 min before each irradiation. Rats of a control group were irradiated with the same schedule with equal volumes of physiological saline. The morphological and sialoscintigraphical findings clearly demonstrate that amifostine has a remarkable cytoprotective effect on the late toxicity of irradiated salivary glands.

A variable response following ductal ligation of feline salivary glands corresponds to the human condition but contrasts with a predictable atrophy in obstructed salivary glands of rodents popularly used as a model for human salivary problems. The present investigation is concerned with a possible reason for the variable response, namely the preservation of the innervation. Ducts of feline submandibular and sublingual salivary glands were ligated with or without the inclusion of the chorda tympani. Inclusion led to a delayed initial response followed by progressive atrophy until the parenchyma was extremely atrophic, whereas avoidance of the chorda led to the variable response in which variable numbers of acini of a similar form to normal persisted. The results establish the atrophic effect of inclusion of the chorda tympani in ductal ligation and indicate the caution that should be exercised in the extrapolation of the rodent model to the human condition.

The salivary gland is considered to be a reservoir of many growth factors in rodents. In humans, the epidermal growth factor, basic fibroblast growth factor, and insulin and insulin-like growth factor family have also been detected in this gland, but their physiological role remains unclear. In this study, we focused on bFGF, which is a well-known mitogen for various types of cells, and is present in the salivary gland as well as in saliva. The roles of bFGF in the salivary gland were investigated by three different procedures. First, the effects of bFGF on the salivary gland cells were investigated with a monolayer culture of normal submandibular gland cells. The effects of different concentrations of bFGF on the second passage of these cultured cells were examined. In both human and rat cultured submandibular gland cells, bFGF accelerated the cell proliferation at a concentration of 100 ng/mL or higher. Next, an atrophic model of the rat submandibular gland was used to examine the ability of bFGF to accelerate tissue repair. Two weeks after ductal ligation, the ligature was removed, and various amounts of bFGF, isoproterenol, or saline were administered via a retrograde duct instillation. Both isoproterenol and bFGF increased acinar and ductal cell proliferation significantly. To determine the role of bFGF in saliva, we investigated its effect on the healing process of oral mucosal defects. Four-millimeter mucosal defects were made to the depth of the periosteum in the rat palate under anesthesia. bFGF or vehicle alone was applied once only at the time of surgery as a suspension. At days 3, 5, and 7 in the bFGF group, significant increases in the degree of re-epithelialization were found in treated groups. These results indicate that its action as a mitogen stimulus is the major effect of bFGF on salivary gland cells and mucosal epithelium.

Recent reports have alluded to various tissue effects secondary to fine-needle aspiration (FNA), particularly infarction observed in resected salivary gland masses, precluding accurate histologic diagnosis. Our experience with the use of 25-gauge needles indicates otherwise. We retrospectively reviewed 94 resected salivary gland masses previously sampled by FNA, looking for infarction, hemorrhage, needle track tumor seeding, and fibrosis. We assessed the significance of these complications and their impact on the histologic diagnosis. The median interval from FNA to excision was 25 days. Variable degrees of infarction and hemorrhage were present in 7 cases (7%) and 9 cases (10%), respectively. Infarction ranged from 5% to 80% (average, 20%), while hemorrhage averaged less than 20% of the material on the tissue sections. Significant infarction was present in acinic cell carcinomas (3/7), but histologic diagnosis was not compromised, and tissue alterations were absent. We conclude that FNA of salivary gland lesions using 25-gauge needles is safe and does not significantly alter the histologic diagnosis. The tissue effects observed did not preclude accurate diagnostic interpretation in any case.