Connexin Expression in Human Minor Salivary Glands: An Immunohistochemical Microscopy Study.

Connexins (Cxs) are transmembrane proteins involved in the formation of hemichannels and gap junctions (GJs). GJs are involved in various physiological functions, including secretion in glandular tissue. It has been demonstrated that Cx26, Cx32, and Cx43 are mainly expressed in glands, but no data are available in human salivary glands to date. The aim of our study was to investigate the presence and the localization of Cxs in human minor labial salivary glands. Immunofluorescence and immunoelectron microscopy were employed to evaluate the Cx26, Cx32, and Cx43 protein in human labial salivary gland biopsies (hLSGBs). RT-PCR was also used to detect their mRNA expression. Cx expression was found at both the mRNA and protein levels in all hLSGBs analysed. Cxs were observed at the level of the duct and acinar cells, as well as in myoepithelial cells. The localization of the three Cx types was very similar, suggesting colocalization of these Cxs in the same connexons. These results demonstrated the presence of Cxs in human salivary glands for the first time. Moreover, the few samples with primary Sjögren's Syndrome analysed only by immunofluorescence showed an alteration of the Cx expression, indicating that these proteins could be involved in salivary gland dysfunctions.

Serum, but Not Saliva, CXCL13 Levels Associate With Infiltrating CXCL13+ Cells in the Minor Salivary Gland Lesions and Other Histologic Parameters in Patients With Sjögren's Syndrome.

Recent studies suggest that elevated CXCL13 serum levels in patients with primary Sjögren's syndrome (pSS) associate with minor salivary gland (MSG) histologic features, disease severity, as well as high-risk status for non-Hodgkin lymphoma (NHL) development and NHL itself. In contrast, limited discriminative value of CXCL13 saliva levels has been reported. Prompt by these reports, we sought to validate the clinical utility of CXCL13 by investigating potential correlations of serum and saliva levels with MSG histopathologic [including CXCL13+-cell number, severity of infiltrates and germinal center (GC) formation], serologic and clinical parameters, as well as NHL. CXCL13 levels were evaluated in paired serum and saliva specimens of 45 pSS patients (15 with NHL; pSS-associated NHL: SSL), 11 sicca-controls (sicca-complaining individuals with negative MSG biopsy and negative autoantibody profile), 10 healthy individuals (healthy-controls) and 6 non-SS-NHLs. CXCL13+-cells were measured in paired MSG-tissues of 22 of pSS patients studied (including 7 SSLs) and all sicca-controls. CXCL13 serum levels were significantly increased in pSS and SSL patients compared to sicca- and healthy-controls and were positively correlated with the CXCL13+-cell number and biopsy focus-score. Serum CXCL13 was significantly higher in pSS patients with GCs, rheumatoid factor, hypocomplementemia, high disease activity, NHL and in high-risk patients for NHL development. CXCL13 saliva levels were significantly increased in SSL patients (compared to non-SS-NHLs), patients with GCs and in high-risk for NHL patients. Univariate analysis revealed that CXCL13 serum, but not saliva, levels were associated with lymphoma, an association that did not survive multivariate analysis. Conclusively, our findings confirm that serum, but not saliva, levels of CXCL13 are associated with histologic, serologic and clinical features indicative of more severe pSS.

Characterization of Lipids in Saliva, Tears and Minor Salivary Glands of Sjögren's Syndrome Patients Using an HPLC/MS-Based Approach.

The diagnostic work-up of primary Sjögren's syndrome (pSS) includes quantifying saliva and tear production, evaluation of autoantibodies in serum and histopathological analysis of minor salivary glands. Thus, the potential for further utilizing these fluids and tissues in the quest to find better diagnostic and therapeutic tools should be fully explored. Ten samples of saliva and tears from female patients diagnosed with pSS and ten samples of saliva and tears from healthy females were included for lipidomic analysis of tears and whole saliva using high-performance liquid chromatography coupled to time-of-flight mass spectrometry. In addition, lipidomic analysis was performed on minor salivary gland biopsies from three pSS and three non-SS females. We found significant differences in the lipidomic profiles of saliva and tears in pSS patients compared to healthy controls. Moreover, there were differences in individual lipid species in stimulated saliva that were comparable to those of glandular biopsies, representing an intriguing avenue for further research. We believe a comprehensive elucidation of the changes in lipid composition in saliva, tears and minor salivary glands in pSS patients may be the key to detecting pSS-related dry mouth and dry eyes at an early stage. The identified differences may illuminate the path towards future innovative diagnostic methodologies and treatment modalities for alleviating pSS-related sicca symptoms.

Diversity of mucins in labial glands of infants.

Mucins as highly glycosylated proteins comprise multiple functions like protection, homeostasis, immune defense, cell signaling. Various epithelial tissues including glandular structures express different specific mucin types. We investigated labial salivary glands in infants for the occurrence of MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, and MUC7 by immunohistochemistry. MUC1 and MUC4 were detected in serous and ductal glandular cells, partially intensified at the apical plasma membrane. MUC3 was found in ductal glandular cells and in myoepithelial cells. MUC5B exhibited a mosaic expression pattern in mucous glandular endpieces. MUC2 and MUC7 were abundant in serous acini. Glandular structures were negative for MUC5AC. A comprehensive study of specific mucins in labial salivary glands of infants was presented for the first time. As a representative of the minor salivary glands, labial glands are, due to their localization, directly exposed to environmental influences. The distribution of a broad spectrum of mucins in infantile labial glands indicates their importance early in human development to sustain oral health.

[Clinicopathologic and molecular genetic analysis of secretory carcinoma of salivary gland].

Objective: To investigate the clinicopathologic and molecular genetic features of secretory carcinoma of salivary gland (SCSG). Methods: Six cases of SCSG were collected from Zhejiang Provincial People's Hospital from January 2011 to March 2018. The clinical, histopathological and immunohistochemical features were analyzed and fluorescence in situ hybridization (FISH) was used to detect ETV6 gene rearrangement. Results: Four out of 6 tumors originated in the parotid gland and one of each in the minor salivary glands of soft palate and the buccal mucosa. Grossly, 4 cases were solid and 2 were partially cystic with maximum diameter ranging from 1.0 to 4.0 cm. Microscopically, 5 tumors showed typical features of low grade SCSG with tumor divided by thin fibrous septa into lobules composed of solid acinar, microcystic, follicular and papillary structures with abundant extracellular mucinous secretions. The tumor cells had cuolated or hobnail cytoplasm with low-grade nuclei and scarce mitoses. Perineural invasion was present in 1 case. The remaining tumor showed about 30% of the tumor areas with high-grade transformation characterized by proliferation of a distinct population of anaplastic cells arranged in irregular glandular, small nested and single cell patterns that were surrounded by desmoplastic stroma and invaded into surface mucosa with ulceration. Immunohistochemistry showed that all 6 tumors had diffuse and strong reactivities to S100 protein and cytokeratin 7, and 4 cases showed focal reactivity to gross cystic disease fluid protein 15 (GCDFP15), all were negative for discovered on gist 1 (DOG1), cytokeratin 20, p63 and calponin. High grade transformation cases were analysed, the high grade SCSG components showed a significantly increased Ki-67 index and cyclin D1 positive tumor cells compared to the conventional SCSG components. FISH analyses showed that 4 cases had ETV6 gene rearrangement. Eleven to seventy one months' follow-up showed no evidence of tumor recurrence nor metastasis. Conclusions: SCSG harbors characteristic genetic abnormalities with ETV6 gene rearrangement and typically shows a low grade morphology with occasionally, high grade transformation can be present.

Immunolocalization of Surfactant Proteins SP-A, SP-B, SP-C, and SP-D in Infantile Labial Glands and Mucosa.

Surfactant proteins in different glandular structures of the oral cavity display antimicrobial activity for protection of invading microorganisms. Moreover, they are involved in lowering liquid tension in fluids and facilitate secretion flows. Numerous investigations for studying the occurrence of surfactant proteins in glandular tissues were performed using different methods. In the oral cavity, minor salivary glands secrete saliva continuously for the maintenance of a healthy oral environment. For the first time, we could show that infantile labial glands show expression of the surfactant proteins (SP) SP-A, SP-B, SP-C, and SP-D in acinar cells and the duct system in different intensities. The stratified squamous epithelium of the oral mucosa revealed positive staining for SPs in various cell layers.

Predictive significance of CCL21 and CXCL13 levels in the minor salivary glands of patients with Sjögren's syndrome.

OBJECTIVES: To investigate whether CCL21 and CXCL13 expression levels in the minor salivary gland are associated with the laboratory and clinical manifestations of Sjögren's syndrome (SS). METHODS: Sociodemographic data on 106 SS patients were obtained and the glandular and extraglandular manifestations of the disease were documented. In addition, minor salivary gland biopsies were performed and the patients' laboratory findings were analysed. European League Against Rheumatism SS disease activity index (ESSDAI) values of SS disease activity at the time of biopsy and the SS disease damage index (SSDDI) values were also recorded. An immunohistochemical approach was used to semiquantitatively measure the CCL21 and CXCL13 expression in the minor salivary glands. RESULTS: The minor salivary glands of SS patients stained positively for CCL21 and CXCL13 in 46.2% (49/106) and 70.7% (75/106) of all cases, respectively. Higher-level expression of CCL21 and CXCL13 was associated with increases in ESR, IgG and rheumatoid factor levels, as well as anti-SS-A and -SS-B titers. A higher focus score and ESSDAI value at the time of biopsy were also associated with these chemokines. In patients with extraglandular manifestations of SS, the prevalence of lymphadenopathy increased with increasing CCL21 levels. CONCLUSIONS: The expression levels of CCL21 and CXCL13 within the lymphocytic infiltrates of SS patients were associated with several laboratory features of the disease as well as lymphadenopathy and the extent of clinical disease activity. CCL21 and CXCL13 levels can therefore serve as useful markers to predict the disease activity and prognosis of patients with SS.

MUC1/SEC and MUC1/Y overexpression is associated with inflammation in Sjögren's syndrome.

OBJECTIVES: To evaluate the expression and localization of MUC1/SEC and MUC1/Y isoforms in labial salivary glands (LSG) from Sjögren's syndrome patients (SS patients), as well as their in vitro expression induced by cytokines. SUBJECTS AND METHODS: Labial salivary gland from 27 primary SS patients and 22 non-SS sicca subjects were studied. Relative MUC1/SEC and MUC1/Y mRNA levels were determined by qPCR and protein levels by Western blotting. Induction of mucin mRNAs was assayed in vitro. Immunohistochemistry was used for localization. RESULTS: Relative MUC1/SEC and MUC1/Y mRNA and protein levels were significantly higher in LSG from SS patients. These mRNAs were induced by cytokines. MUC1/SEC and MUC1/Y were detected in acini apical region of control LSGs, and significant cytoplasmic accumulation was observed in acini of SS patients. MUC1/Y localized in acinar nuclei and cytoplasm of inflammatory cells of LSG from SS patients. A strong positive correlation was observed between cellular MUC1/SEC levels and glandular function determined by scintigraphy. CONCLUSIONS: We show for the first time that MUC1/SEC and MUC1/Y are expressed in LSG of both SS patients and non-SS sicca subjects. The observed overexpression and aberrant localization of MUC1/SEC and MUC1/Y and their induction by pro-inflammatory cytokines may favor the perpetuation of the inflammatory environment that disrupts the salivary glandular homeostasis in SS patients.

Saliva as a potential tool for diagnosis of dry mouth including Sjögren's syndrome.

OBJECTIVES: Recently, the use of saliva as a diagnostic tool has gained considerable attention because it is non-invasive and easy to perform repeatedly. In this study, we focused on soluble molecules in saliva to establish a new diagnostic method for xerostomia. MATERIALS AND METHODS: Saliva was obtained from 90 patients with Sjögren's syndrome (SS), 22 patients with xerostomia associated with neurogenic/neuropsychiatric disorders and drugs (XND), 30 patients with radiation-induced xerostomia (RX), and 36 healthy controls. Concentrations of helper T (Th) cytokines in saliva were measured by flow cytometric analysis. Concentrations of secretory IgA (SIgA) and chromogranin A (CgA) were measured by ELISA. RESULTS: Unstimulated and stimulated whole saliva from patients with SS, XND, and RX was significantly reduced compared with controls. Th1 and Th2 cytokines from SS patients were significantly higher than controls. Furthermore, Th2 cytokines were closely associated with strong lymphocytic accumulation in salivary glands from SS patients, while Th1 and Th17 cytokines were negatively associated. SIgA levels were not significantly different between all patient groups and controls. CgA levels from XND patients were significantly higher than controls. CONCLUSIONS: The measurement of cytokines, CgA, and SIgA in saliva is suggested to be useful for the diagnosis of xerostomia and also to reveal disease status.

Cholera toxin B subunit-binding and ganglioside GM1 immuno-expression are not necessarily correlated in human salivary glands.

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

Canalicular adenoma immunoprofile: a case report.

OBJECTIVES: To report a case of canalicular adenoma (CA) and discuss the use of immunohistochemistry to better address the diagnosis given some unusual characteristics in this patient. BACKGROUND: CA is an uncommon benign neoplasm that can develop in minor salivary gland duct tissues throughout the oral cavity. At histology, it shares several features with other salivary tumors. Immunohistochemistry can be useful in the differential diagnosis. MATERIALS AND METHODS: The clinical presentation consisted in a nodule on the left upper lip of an 85-year-old man. The patient's main complaint was upper denture instability secondary to soft tissue changes. The nodule was excised under local anesthesia and underwent histological and immunohistochemical examination to rule out any malignancy. RESULTS: Histological findings, cytokeratin positivity and the absence of any reactivity to specific markers of myoepithelial differentiation confirmed the epithelial nature of the lesion. CONCLUSION: The histological diagnosis of benign salivary tumors such as CA can be confirmed by immunohistochemistry.

Xerostomia in hereditary gelsolin amyloidosis.

Hereditary gelsolin amyloidosis (AGel amyloidosis) is a rare, dominantly inherited systemic disease with worldwide distribution, caused by c.654G > A or c.654G > T gelsolin gene mutation. The disease mainly manifests with late-onset dystrophy of the cornea, laxity of the skin and dysfunction of the cranial nerves whereas the oral manifestations have remained less-studied. To examine if AGel amyloidosis also affects salivary gland function, we studied 27 patients. In a questionnaire, 89% of them reported oral dryness, and 74% oral and ocular dryness. Unstimulated (UWS) and stimulated whole salivary flow (SWS) rates were measured, and salivary proteins were analyzed in the patients and controls. Hyposalivation according to UWS was detected in 67% of the patients, while decreased SWS occurred in 63% of the patients and 19% of the controls (p = 0.001). The secretion rates of salivary total protein and IgA were significantly lower in patients than controls. Histopathological analyses of labial salivary gland biopsies showed deposition of gelsolin amyloid, atrophy and inflammation. This study showed that AGel amyloidosis belongs to the differential diagnostic choices to be kept in mind in the patients presenting with xerostomia, low secretion rates of salivary total protein and IgA and/or deposition of amyloid in the minor salivary glands. AGel amyloidosis patients should be advised for efficient dental care.

Identification of (L)-3-hydroxykynurenine O-sulfate in the buccal gland secretion of the parasitic lamprey, Lethenteron japonicum.

Parasitic lampreys are known to secrete proteins having anticoagulant and vasodilator activities from the buccal glands during feeding on their host's blood. However, small molecules in the secretion have never been explored in detail. We examined the secretion of Japanese liver lamprey (Lethenteron japonicum) for small molecules and found an intensely fluorescent substance upon gel filtration. After purification by anion-exchange chromatography and reversed-phase HPLC, structure of the compound was determined to be L-3-hydroxykynurenine O-sulfate by NMR- and UV-spectrometry, complemented with enzymatic and chemical degradation. In vertebrates, the sulfate ester of 3-hydroxykynurenine is a compound that has been regarded as a urinary metabolite of tryptophan but not reported from normal tissues to date. Although the function of this molecule in the buccal glands remains to be elucidated, it is remarkable that the same substance was described in 1960s from two species of blood-sucking insects, Rhodnius prolixus and Triatoma infestans, suggesting its potential role in blood-feeding.

Deep sequencing of short RNAs reveals novel microRNAs in minor salivary glands of patients with Sjögren's syndrome.

OBJECTIVES: Sjögren's syndrome is a complex autoimmune disease of the salivary gland with an unknown etiology, so a thorough characterization of the transcriptome would facilitate our understanding of the disease. We use ultradeep sequencing of small RNAs from patients with Sjögren's syndrome and healthy volunteers, primarily to identify and discover novel miRNA sequences that may play a role in the disease. METHODS: Total RNA was isolated from minor salivary glands of healthy volunteers and patients with either high or low salivary flow and sequenced on the SOLiD platform. Prediction of mature miRNAs from the sequenced reads was carried out using miRanalyzer, and expression was validated using Taqman qPCR assays. RESULTS: We validated the presence of six previously unidentified miRNA sequences in patient samples and in several cell lines. One of the validated novel miRNAs shows promise as a biomarker for salivary function. CONCLUSION: Sequencing small RNAs in the salivary gland is largely unprecedented, but here, we show the feasibility of discovering novel miRNAs and disease biomarkers by sequencing the transcriptome.

Detection of AA-type amyloid protein in labial salivary glands.

OBJECTIVES: Among the diverse forms of amyloidosis, secondary type is the most frequent one. Diagnosis of amyloid deposition is based on the identification of the fibrillary protein amyloid by means of Congo Red (CR) or crystal violet (CV) stains, but these techniques do not differentiate between the different types of amyloid fibrils. The aim of this study was to identify by immunofluorescence (IF) AA amyloid a pathological fibrillar low-molecular-weight protein formed by cleavage of serum amyloid A (SAA) protein in labial salivary gland (LSG) biopsies from patients with secondary amyloidosis. STUDY DESIGN: 98 LSG were studied, 65 were from patients with secondary amyloidosis and 33 from subjects with chronic inflammatory diseases without evidence of this anomaly. All sections were stained with hematoxylin and eosin (H &E), CV, CR and IF using anti-AA antibodies. Positive and negative controls were used for all techniques. RESULTS: CV and CR demonstrated that the amyloid substance was found mainly distributed periductally (93.8%), followed by periacinar and perivascular locations (p <0.001); however, the IF demonstrated that amyloid AA substance predominates in the periacinar area (73.8%), followed by periductal and perivascular locations (p <0.001). IF has a sensitivity of 83%, 100% of specificity, 100% of predictive positive value and 75% of predictive negative value. CONCLUSIONS: The results of this study confirm the efficacy of the LSG biopsy as a highly reliable method for diagnosis of secondary amyloidosis.

Acinic cell carcinoma of minor salivary glands: a clinical and immunohistochemical study.

PURPOSE: Acinic cell carcinoma is a rare malignant tumor of salivary glands. The purpose of this study is to evaluate the clinical outcome of acinic cell carcinoma in a group of 11 patients, who were treated in our clinic, and to discuss the management as well as the immunohistochemical features and prognosis of this carcinoma. MATERIALS AND METHODS: The study included 11 patients with acinic cell carcinoma of the minor salivary glands who were treated in our clinic. The patients were 7 women and 4 men. The patients' age ranged from 46 to 83 years. The distribution of the primary sites was buccal mucosa (4) maxilla/maxillary sinus, etc, (2), hard palate (1), junction of soft/hard palate (1), lower lip (1), labio marginal sulcus (1), and vestibular sulcus and mandible (1). All patients were treated with surgery. Adjuvant radiotherapy was used in 3 patients. Immunohistochemical assay of expression of Ki-67, p53, EGFR, and c-erbB-2/neu markers was performed on specimens of all tumors. RESULTS: The mean follow-up range was 2 to 15 years. Of the 11 patients, 7 were alive (2, 3, 4, 5, and 15 years after the initial therapy). Two patients died of another cause free of the disease 9 and 10 years after the initial treatment, and 2 patients died of the disease (local recurrence, distant metastases 2 and 3 years later). Overexpression of immunohistochemical markers was evident for tumors with widespread metastases. CONCLUSIONS: Acinic cell carcinoma is a rare malignant tumor of the salivary glands, characterized by an indolent clinical course with the potential for both local recurrence and distant metastases. The immunohistochemical analysis of proliferation markers provides additional prognostic information for this tumor.

Electron microscopic immunogold localization of statherin in human minor salivary glands.

In this study, which supplements a recent article on the localization of statherin in human major salivary glands, we investigated the intracellular distribution of this peptide in minor salivary glands by immunogold cytochemistry at the electron microscopy level. In the lingual serous glands of von Ebner, gold particles were found in serous granules of all secreting cells, indicating that statherin is released through granule exocytosis. In buccal and labial glands, mostly composed of mucous tubuli, statherin reactivity was detected in the serous element, which represents only a small population of the glandular parenchyma. In these serous cells, however, statherin labeling was absent in secretory granules and restricted to small cytoplasmic vesicles near or partially fused with granules. Vesicle labeling could be related to the occurrence of an alternative secretory pathway for statherin in buccal and labial glands.

Ultrastructural localization of salivary mucins MUC5B and MUC7 in human labial glands.

As a result of their presence throughout the mouth in the submucosa or between muscle fibers, minor salivary glands secrete directly and continuously into the oral cavity, providing mucosal surfaces with highly glycosylated proteins that are active in bacterial aggregation and in oral tissue lubrication. In this study, we investigated the ultrastructural localization of the MUC5B and MUC7 mucins in human labial glands by means of a postembedding immunogold technique. Thin sections of normal human labial glands, obtained during surgery, were incubated with polyclonal antibodies to human salivary mucins MUC5B and MUC7, and then with gold-labeled secondary antibodies. Specific MUC5B reactivity was found in the secretory granules of mucous cells of all glands examined, and was associated with the luminal membrane of duct cells. MUC7 labeling was observed in the granules of both mucous and seromucous secretory cells of the glandular parenchyma. Quantitative analyses demonstrated that seromucous granules have higher immunogold labeling densities for MUC7 than mucous granules. Our immunohistochemical data extend the results of previous light microscopic studies of MUC5B and MUC7 localizations, pointing out the significant contribution of human labial glands in the secretion process of these two mucins.

Myoepithelial carcinoma of the retromolar area in a young female. A rare case report.

We present a case of a 34-year-old woman with myoepithelial carcinoma of the retromolar area. Myoepithilial carcinoma is a rare tumor of small salivary glands most usually located in the parotid gland. The major differential diagnosis of myoepithelioma is from pleomorphic adenoma. Little is known about the clinical and biological behavior and the prognosis of myoepithelial carcinoma and there is no consensus for its treatment. It is considered a low-grade malignancy; it sometimes shows aggressive behavior and may locally recur. Our patient was treated successfully with wide-local resection and remained free of disease for 6 months.