Cystatin S-a candidate biomarker for severity of submandibular gland involvement in Sjögren's syndrome.

Objectives: Salivary cystatin S is a defence protein mainly produced by submandibular glands and involved in innate oral immunity. This study aimed to verify whether cystatin S was diversely expressed in different disease subsets of primary Sjogren's syndrome (pSS) patients, defined on the basis of salivary flow [unstimulated salivary flow rate (USFR)], minor salivary gland (MSG) focus score and submandibular gland ultrasonography abnormalities. We also evaluated miR-126 and miR-335-5p expression in MSG biopsies to verify whether an aberrant regulation of cystatin S at the glandular level may influence its salivary expression. Methods: Forty pSS patients and 20 sex- and age-matched healthy volunteers were included. Salivary cystatin S levels were assessed by western blot analysis using a stain-free technology. The expression of miR-126, miR-335-5p and cystatin S was assessed by quantitative PCR in 15 MSG biopsies differing for USFR and MSG focus score. Results: We found that salivary cystatin S was significantly decreased in pSS patients vs healthy volunteers ( P = 0.000), especially in those with hyposalivation. A positive correlation was observed between cystatin S and USFR ( r = 0.75, P = 0.01). Salivary cystatin S was also significantly reduced in patients with a submandibular gland ultrasonography score ⩾2. The expression levels of miR-126 and miR-335-5P increased in inverse proportion with USFR. The mRNA of cystatin S did not change significantly, suggesting post-transcriptional regulation. Conclusion: Cystatin S emerged as a promising biomarker for pSS, strongly correlated with glandular dysfunction. An upregulation of miR-126 and miR-335-5P might be implicated in its expression.

Epiregulin is critical for the acinar cell regeneration of the submandibular gland in a mouse duct ligation model.

Acinar cell regeneration from tubular structures has been reported to occur in duct-deligated salivary glands. However, the detailed process of acinar cell regeneration has not been clarified. We have developed a mouse duct ligation model to clarify the mechanisms underlying acinar cell regeneration, and we analyzed the epidermal growth factor receptor (EGFR) and epidermal growth factor (EGF) ligands using the model. We studied these ligands expressions in the course of acinar cell regeneration using immunohistochemistry and RT-PCR methods. In the duct-ligated portion of the submandibular gland (SMG) that underwent atrophy, newly formed acinar cells were observed arising from the tubular structures after the release of the duct obstruction. The constitutive expression of EGFR was observed by immunohistochemistry in both the duct-ligated and duct-deligated animals as well as in normal controls. The EGFR phosphorylation detected on the tubular structures after duct ligation paralleled the acinar cell regeneration. RT-PCR showed an increase in the epiregulin and heparin-binding EGF levels from day 0 to day 3 after the release of the duct obstruction. The EGF level was increased only after day 7. In vitro, cultured cells isolated from ligated SMGs proliferated and produced EGF ligands following the addition of epiregulin to the culture medium. These findings suggest that the tubular structures localized in an atrophic gland are the source of acinar cell regeneration of the salivary gland. The induction of EGF ligands, in particular epiregulin, may play an important role in acinar cell regeneration in this model.

Involvement of toll-like receptors in autoimmune sialoadenitis of the non-obese diabetic mouse.

The aim of this study was to characterize the expression of Toll-like receptors (TLRs) during the development of sialoadenitis in the non-obese diabetic mouse. Submandibular glands were dissected from non-obese diabetic mice at 4, 8, 10, 12, and 16 weeks of age. The mRNA expression levels of TLR1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, MyD88, and TRIF was quantified using real-time reverse transcription polymerase chain reaction. The mRNA expression levels in 4-week-old non-obese diabetic mice were used as controls. The expression levels of TLR1, 2, 4, and 9 were significantly higher at 8, 10, 12, and 16 weeks than the levels in the controls. The expression level of TLR3 was significantly higher at 16 weeks than in the controls. A group of mice were given drinking water containing 4.75% chloroquine starting at 4 weeks. Chloroquine caused a significant decrease in the expression of TLR1, 2, 3, 4, and 9 at 16 weeks compared with control mice who did not receive chloroquine. The areas of lymphocyte infiltration seen on serial sections of submandibular glands in the mice receiving chloroquine were significantly smaller than the areas of infiltration in control glands. Increased expression of Toll-like receptors may be involved in the development and/or progression of sialoadenitis in the non-obese diabetic mouse. Toll-like receptors may be a therapeutic target for autoimmune sialoadenitis.

Glycyrrhizic acid ameliorates submandibular gland oxidative stress, autophagy and vascular dysfunction in rat model of type 1 diabetes.

The burden of diabetes mellitus (DM) and associated complications is increasing worldwide, affecting many organ functionalities including submandibular glands (SMG). The present study aims to investigate the potential ameliorative effect of glycyrrhizic acid (GA) on diabetes-induced SMG damage. Experimental evaluation of GA treatment was conducted on a rat model of type I diabetes. Animals were assigned to three groups; control, diabetic and GA treated diabetic groups. After 8 weeks, the SMG was processed for assessment of oxidative stress markers, autophagy related proteins; LC3, Beclin-1 and P62, vascular regulator ET-1, aquaporins (AQPs 1.4 and 5), SIRT1 protein expressions in addition to LC3 and AQP5 mRNA expressions. Also, parenchymal structures of the SMG were examined. GA alleviated the diabetes-induced SMG damage via restoring the SMG levels of oxidative stress markers and ET-1 almost near to the normal levels most probably via regulation of SIRT1, AQPs and accordingly LC-3, P62 and Beclin-1levels. GA could be a promising candidate for the treatment of diabetes-induced SMG damage via regulating oxidative stress, autophagy and angiogenesis.

Switching to normal diet reverses kwashiorkor-induced salivary impairments via increased nitric oxide level and expression of aquaporin 5 in the submandibular glands of male Wistar rats.

Kwashiorkor, a form of malnutrition, has been shown to cause impaired salivary secretion. However, there is dearth of information on the mechanism that underlies this complication. Also, whether returning to normal diet after kwashiorkor will reverse these complications or not is yet to be discerned. Thus, this study aimed at assessing the mechanisms that underlie kwashiorkor-induced salivary impairments and to evaluate the effects of switching back to normal-diet on kwashiorkor-induced salivary impairments. Weaning rats were randomly divided into 3 groups (control group, kwashiorkor group (KG), re-fed kwashiorkor group (RKG)) of 7 rats each. The control group had standard rat chow while the KG and RKG were fed 2% protein diet for 6 weeks to induce kwashiorkor. The RKG had their diet changed to standard rat-chow for another 6 weeks. Blood and stimulated saliva samples were collected for the analysis of total protein, electrolytes, amylase, immunoglobulin A (IgA) secretion rate, leptin, and ghrelin. Tissue total protein, nitric oxide level, expressions of Na+/K+-ATPase, muscarinic (M3) receptor, and aquaporin 5 in the submandibular glands were also determined. Data were presented as means ± SEM and compared using ANOVA with Tukey's post hoc test. RKG showed improved salivary function evidenced by reduced salivary lag-time and potassium and increased flow rate, sodium, amylase, IgA secretion rate, leptin, submandibular nitric oxide level, and aquaporin 5 expression compared with KG. This study for the first time demonstrated that kwashiorkor caused significant reduction in salivary secretion through reduction of nitric oxide level and aquaporin 5 expression in submandibular salivary glands. Normal-diet re-feeding after kwashiorkor returned salivary secretion to normal.

Histological and histochemical observations on salivary microliths in ferret.

The fortuitous observation of salivary microliths in ferret was pursued in the present investigation. Major salivary glands obtained post-mortem from mature ferrets of either sex were examined with the use of histology and light microscopical histochemistry for calcium, protein, amino acids, mucosubstances and hydrolytic enzymes. Microliths were detected in most parotids, but were absent from submandibular and sublingual glands. The microliths were usually seen in lumens, and occasionally in parenchyma and interstices. They were variably stained for calcium, tryptophan, and neutral and acidic mucosubstances, similarly to acinar or ductal secretory granules. Unlike secretory granules, microliths showed autofluorescence, high levels of tyrosine and a low concentration of -SS- groups. Acid phosphatase and beta-glucuronidase reaction surrounded non-luminal microliths. The present data establish ferret as a new model for the investigation of salivary microliths and do not support the notion of microliths being almost absent from the parotid. Probably there is secretory inactivity in ferret parotid and this fosters the formation and accumulation of microliths containing calcium and disintegrated secretory material.

Radiation-induced progressive decrease in fluid secretion in rat submandibular glands is related to decreased acinar volume and not impaired calcium signaling.

The mechanism(s) of radiation-induced salivary gland dysfunction is poorly understood. In the present study, we have assessed the secretory function (muscarinic agonist-stimulated saliva flow, intracellular calcium mobilization, Na+/K+/2Cl- cotransport activity) in rat submandibular glands 12 months postirradiation (single dose, 10 Gy). The morphological status of glands from control and irradiated rats was also determined. Pilocarpine-stimulated salivary flow was decreased by 67% at 12 months (but not at 3 months) after irradiation. This was associated with a 47% decrease in the wet weight of the irradiated glands. Histological and morphometric analysis demonstrated that acinar cells were smaller and occupied relatively less volume and convoluted granular tubules were smaller but occupied the same relative volume, while intercalated and striated ducts maintained their size but occupied a greater relative volume in submandibular glands from irradiated compared to control animals. In addition, no inflammation or fibrosis was observed in the irradiated tissues. Carbachol- or thapsigargin-stimulated mobilization of Ca2+ was similar in dispersed submandibular gland cells from control and irradiated animals. Further, [Ca2+]i imaging of individual ducts and acini from control and irradiated groups showed, for the first time, that mobilization of Ca2+ in either cell type was not altered by the radiation treatment. The carbachol-stimulated, bumetanide-sensitive component of the Na+/K+/ 2Cl- cotransport activity was also similar in submandibular gland cells from control and irradiated animals. These data demonstrate that a single dose of gamma radiation induces a progressive loss of submandibular gland tissue and function. This loss of salivary flow is not due to chronic inflammation or fibrosis of the gland or an alteration in the neurotransmitter signaling mechanism in the acinar or ductal cells. The radiation-induced decrease in fluid secretion appears to be related to a change in either the water-handling capacity of the acini or the number of acinar cells in the gland.

Weddellite in submandibular gland calculus.

Scanning electron microscopy of a submandibular sialolith showed octahedral and dipyramidal crystals located at the surface layer of cortex. The crystal habits observed were consistent with those of a tetragonal crystal system. X-ray microdiffraction revealed the presence of weddellite. The origin of weddellite within the calculus remains to be determined.

Induction of C-reactive protein, serum amyloid P component, and kininogens in the submandibular and lacrimal glands of rats with experimentally induced inflammation.

The mRNAs for acute-phase proteins and kininogens were found to be increased in the submandibular gland (SMG) and extraorbital and intraorbital lacrimal gland (ELG and ILG) in response to experimentally induced inflammation in rats; i.e., 24 hours after subcutaneous injection of turpentine oil, mRNAs for C-reactive protein (CRP), serum amyloid P component (SAP), and H- and T-kininogens were induced in the SMG, ELG, and ILG of rats, whereas these mRNAs were not detected in the same tissues of normal control rats. The induction of mRNAs for these inflammatory proteins by turpentine oil was preceded by a transient increase in the level of mRNA for tumor necrosis factor-alpha (TNF-alpha) at 6 hours after subcutaneous injection of the oil. This was confirmed by injection of another inflammation inducer, lipopolysaccharide (LPS), which induced the TNF-alpha mRNA in the same way at 6 hours as turpentine oil did. The up-regulation of acute-phase proteins including kininogens in the SMG, ELG, and ILG suggest the existence of a strict defense system in the exocrine glands.

Sialolith crystals localized intraglandularly and in the Wharton's duct of the human submandibular gland: an X-ray diffraction analysis.

The exact cause of the formation of sialoliths is unknown. Detailed knowledge of pathogenesis of sialolithiasis and composition of sialoliths is necessary to define new therapeutic procedures. The crystalline components of 23 sialoliths of human submandibular gland were investigated by X-ray powder diffraction analysis. All of the sialoliths localized in the ducts in the submandibular gland consisted of hydroxylapatite. However, in the sialoliths in the Wharton's duct, hydroxylapatite as well as whitlockite and brushite could be found in all except one case. Whitlockite was observed more often in the nucleus of the sialoliths and it was a common co-phase along with hydroxylapatite. The nucleus in one sialolith consisted of brushite and the cortex showed a co-phase of hydroxylapatite and brushite. The occurrence of whitlockite in the sialoliths in Wharton's duct may be due to a higher concentration of calcium and phosphate in saliva in this duct.

Lipid composition of the matrix of human submandibular salivary gland stones.

The lyophilized stone matrix, prepared by exhaustive dialysis of pulverized stones against 10 per cent EDTA and water, yielded 9.6 +/- 0.9 mg per cent of lipids. Following fractionation of the lipid extract on silicic acid columns, 57 per cent of lipids were found in the neutral lipid fraction, 40 per cent in the glycolipid fraction and 3 per cent in the phospholipid fraction. Of the total neutral lipids, 77.3 per cent were represented by free fatty acids, 14.7 per cent by cholesterol, 4.1 per cent by triglycerides and 3.2 per cent by cholesterol esters. The glycolipid fraction was comprised of simple glycosphingolipids (11.3 per cent), mainly lactosyl- and glucosylceramides, and of neutral and sulphated glyceroglucolipids (88.7 per cent). The sulphated glyceroglucolipids consisted of tri- and tetraglucosyl glyceroglucolipids, whereas hexa- and octaglucosyl glyceroglucolipids were the predominant constituents of the neutral glyceroglucolipid fraction. The phospholipids exhibited a high content of sphingomyelin (22.3 per cent), phosphatidylcholine (12.1 per cent), phosphatidylethanolamine (11.4 per cent), phosphatidylserine (10.5 per cent) and lysophosphatidylcholine (13.3 per cent). The major fatty acids of the neutral lipid fraction were hexadecanoate, octadecanoate and octadecenoate. The glycolipids contained large quantities of hexadecanoate and tetracosanoate, while the hexadecanoate, octadecanoate and tetracosanoate were the principal fatty acids of the phospholipid fraction.

Transforming growth factor beta in chronic obstructive sialadenitis of human submandibular gland.

BACKGROUND: The exact pathomechanism of inflammation progress and fibrosis in chronic obstructive sialadenitis is unknown. The aim of the present study was to assess whether there is an association between transforming growth factor beta (TGF-beta) and fibrogenic process of chronic sialadenitis. METHODS: Tissue samples of 12 patients with chronic sialadenitis and 4 normal tissue samples of the submandibular gland were examined immunohistochemically for identification of TGF-beta. TGF-beta1 messenger RNA (mRNA) expression was analysed semiquantitatively using reverse transcription polymerase chain reaction and gel electrophoresis to correlate its expression levels with stages of the disease. RESULTS: TGF-beta positive cells could be found in the secretory duct system of all examined samples. However, an intense TGF-beta immunoreactivity was observed in inflamed salivary glands. With progress of disease TGF-beta1 mRNA expression increases significantly. CONCLUSION: Expression of TGF-beta in chronic sialadenitis and its apparent increase in advanced stages of the disease, suggests that this growth factor may play a role in glandular fibrosis.

Kuttner's tumor of salivary glands resembling marginal zone B-cell lymphoma of the MALT type: a histopathologic and immunohistochemical study of 7 cases.

Kuttner's tumor is a benign inflammatory process of the submandibular gland that presents as a hard mass mimicking a malignant neoplasm clinically. The histologic feature varies according to stage of evolution and severity of inflammation. We report here 7 cases of Kuttner's tumor that morphologically resemble primary salivary gland marginal zone B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT) type. Histologically, the lobular architecture was distorted and the septa showed sclerosis. There was a dense lymphoplasmacytoid infiltration with lymphoid follicle formation accompanied by loss of acini and ducts. In 4 cases, a few salivary gland ducts contained the lymphoid cells within the epithelium. However, a true lymphoepithelial lesion was observed in none of the 7 cases. Immunohistochemical study demonstrated a disrupted follicular dendritic cell network, which is a characteristic finding of follicular colonization of MALT-type lymphoma. In 6 cases, there were a few small foci of lymphocytes somewhat resembling centrocyte-like cells of MALT-type lymphoma. However, immunohistological study demonstrated the mixed nature of the cells resembling centrocyte-like cells. Moreover, the polytypic nature of B lymphocytes was demonstrated by immunohistochemistry and polymerase chain reaction.

Expression and localization of mRNA for epidermal growth factor and epidermal growth factor receptor in human cholesteatoma.

The roles of epidermal growth factor (EGF) and epidermal growth factor receptor (EGF-R) in the proliferation and progression of the epithelium of middle ear cholesteatoma were studied. An attempt was made to elucidate the site and degree of localization of the EGF mRNA and EGF-R mRNA in the epithelium of the cholesteatoma by means of the non-radioactive in situ hybridization method. Ten cholesteatoma specimens excised during operations and six normal skin specimens (control) collected from the external ear canal were used in this study. The signal of the EGF mRNA was slightly expressed in part of the basal cells in only one of the six control specimens, while the signal was strongly expressed along the basal cells of the cholesteatoma epithelium in five of the ten specimens. The signal of the EGF-R mRNA was observed along the basal cell layer in five of the six control specimens, while the signal was strongly expressed in all layers of the cholesteatoma epithelium in all ten specimens. The expression was especially marked in the basal cell layer. These findings suggest the possibilities that abnormal expression of the EGF-R mRNA in nearly entire epithelial layers of cholesteatoma is due to overexpression of EGF-R gene, and that there is a mechanism of epithelial basal cell proliferation through an autocrine regulatory system via EGF and EGF-R.

Ultrastructural and elemental analysis of sialoliths and their comparison with nephroliths.

AIM: Sialoliths are common in the submandibular gland and its duct system, although their exact cause of formation is still a matter of debate. The aims of this study were to: (a) analyze sialoliths ultrastructurally, and to determine the role of foreign bodies or organic materials in the formation of sialolith nuclei; and (b) compare nephroliths with sialoliths ultrastructurally. METHODS: Three sialoliths and two nephroliths were analyzed ultrastructurally by a scanning electron microscope and X-ray diffractometer. RESULTS: The main structures of the sialoliths were found to be hydroxyapatite crystals. No organic cores were observed in the central parts of the sialoliths. In nephroliths, calcium oxalate, calcium phosphate, and struvite crystals were found. The energy-dispersive X-ray microanalysis found that sialoliths and nephroliths were predominantly composed of elements comprising calcium, phosphorous, magnesium, sodium, chloride, silicon, iron, and potassium. CONCLUSION: Sialoliths in the submandibular salivary glands might form secondary to sialadenitis, but not via a luminal organic nidus.

Spectroscopic and thermal analysis of a submandibular sialolith of Wharton's duct resected using Nd:YAG laser.

A sialolith observed in the Wharton's duct of a male patient was resected using an Nd:YAG laser. This is the first report on the resection of sialolith using laser. The resected sample was analyzed for structural details using Fourier transform infrared (FTIR), FT-Raman, and fluorescence spectroscopic techniques. Other techniques like energy dispersive X-ray analysis, scanning electron microscopy, and thermal analysis were also used for the analysis of structural details. The major peaks of the vibrational spectra are observed to be due to the vibrations of the phosphate and hydroxyl groups of the inorganic part of the sample and the proteinaceous component of the organic part. The major elements in the sample are identified as calcium and phosphorous in the ratio 7:3. The fluorescence spectra recorded at excitation wavelengths 280, 325, and 410 nm showed emission maxima corresponding to the endogenous fluorescence of structural proteins and amino acids. The inorganic part of the sialolith remained stable even at temperatures up to 1,673 K. The spectroscopic studies indicated that the structure of the sialolith is similar to that of the dentine part of the human teeth. In situ disintegration of the sialolith involves very high temperature. High calcium and phosphorous content in the food may be attributed to one of the reasons for the formation of sialoliths.

Recovery of rat submandibular salivary gland function following removal of obstruction: a sialometrical and sialochemical study.

Functional recovery of the rat submandibular gland following ligation of the main excretory duct was examined. Rat submandibular glands were ligated for 1, 4 and 8 weeks using a micro-clip with a plastic tube. Micro-clips were removed and glands were allowed to recover for periods of 8, 16 and 24 weeks. Submandibular glands were stimulated with autonomimetic drugs (methacholine and isoprenaline) and salivas were collected from atrophic or de-ligated and contralateral control glands. Glands recovered almost full size (92% of control gland) following 24 weeks of de-ligation. Saliva volume secreted by ligated/de-ligated (RSM) and control (LSM) glands were similar with different doses of agonists. Protein output expressed per gram of tissue wet weight was similar from both ligated/de-ligated and control glands with all doses of agonist. Sodium and chloride levels were higher from de-ligated glands than contralateral control glands. Protein electrophoresis showed similar profiles of salivary proteins in all samples with some minor differences. Acinar cells in de-ligated glands showed a normal morphology, as indicated by light microscopy, whilst granular ductal cells were fewer and contained fewer secretory granules. Sodium potassium ATPase staining of striated ducts in de-ligated glands was similar to that of control glands. It can be concluded that rat submandibular glands can regenerate following severe atrophy and secrete normal amounts of saliva containing broadly a full profile of secretory proteins. In contrast to acinar cells, ductal cells appear not to recover full function.