[Primary lung salivary gland-type duct carcinoma: a clinicopathological analysis of two cases and review of literature].

Objective: To investigate the clinicopathological features, diagnostic criteria and differential diagnosis of primary salivary gland-type duct carcinoma of lung(LSDC). Methods: Two patients with LSDC after surgical resection in Shanghai Pulmonary Hospital from 2020 to 2021 were included; their clinical parameters as well as pathological, immunohistochemical and molecular characteristics of the tumors were analyzed. The relevant literature was also reviewed. Results: Both patients were male, aged 49(case 1) and 64(case 2) years, respectively, and with a history of smoking. The chest computed tomography scan showed both lesions to be centrally located. Gross examination showed the maximum diameters were 16 mm and 35 mm, respectively. The histomorphology of LSDC resembled ductal carcinoma of breast, with intraductal islands of neoplastic cells, which also formed solid nests, papillary, micropapillary and cribriform structures. There was frequent accompanying comedo-like necrosis. The neoplasm cells were markedly heteromorphic, possessing large irregular nuclei with prominent nucleoli, abundant eosinophilic or clear cytoplasm, and mitotic figures were common. Both cases of LSDC were immunoreactive for CKpan, CK7, AR, HER2 staining was (2+) and were negative for TTF1, Napsin A, p40, GATA3, mammaglobin, GCDFP15, SOX10, PSA, P504S, ER, PR, vimentin, S-100, SMA, CK5/6 and p63. The tumor showed double-layer cell structure of the duct, and some basal cells/myoepithelial cells expressed p40 and CK5/6. Case 1 had no gene mutation while case 2 harbored TP53 and KMT2A gene mutation detected by next generation sequencing. Conclusions: LSDC is a very rare and highly aggressive salivary-type malignant tumor. The postoperative diagnosis mainly depends on histopathology and immunohistochemistry, attention should be paid to differential diagnosis to prevent missed diagnosis.

Localization of cystic fibrosis transmembrane conductance regulator signaling complexes in human salivary gland striated duct cells.

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

Activation of TLR9-dependent p38MAPK pathway in the pathogenesis of primary Sjögren's syndrome in NOD/Ltj mouse.

OBJECTIVE: The objective of this study was to investigate the potential role of Toll-like receptor 9-dependent p38 MAPK signaling pathway in the pathogenesis of primary Sjögren's syndrome (pSS) in NOD/Ltj mouse, aiming to identify an ideal target therapy model for human pSS. METHODS: NOD/Ltj mice were chosen as a model of pSS. The Toll-like receptor 9 and p-p38 MAPK double-positive peripheral blood mononuclear cells (PBMCs) of 4-, 5-, 8-, 10-, and 15-week-old NOD/Ltj mouse were analyzed by flow cytometry. The expressions of Toll-like receptor 9 and p-p38 MAPK in the submandibular gland (SMG) were also examined by immunohistochemistry. The change of stimulated salivary flow rate was dynamically measured, and the histopathology of SMG was evaluated by hematoxylin and eosin stain. RESULTS: The stimulated salivary flow rate in NOD/Ltj was reduced to 50-60% of the flow rate of control mice since the fifth week onwards. The Toll-like receptor 9 and p-p38 MAPK double-positive PBMCs in both groups increased gradually from 5 weeks, peaked at 8 weeks and then gradually decreased at 10 weeks, yet the percentage of Toll-like receptor 9 and p-p38MAPK double-positive PBMCs in 5-, 8-, and 10-week-old NOD/Ltj mouse was significantly increased compared with those in control subjects. After the 10th week onwards, there were no significant differences in the Toll-like receptor 9 and p-p38 MAPK double-positive PBMCs between NOD/Ltj mice and controls. Immunohistochemical staining showed that Toll-like receptor 9 was positive in the acinar epithelium cells and infiltrating lymphocytes in NOD/Ltj mice. p-p38 MAPK was detected in infiltrating lymphocytes and few ductal or acinar epithelium cells adjacent to infiltrating lymphocytes in NOD/Ltj mice. CONCLUSIONS: From the fifth week till the tenth week, Toll-like receptor 9 and p-p38 MAPK double-positive PBMCs were significantly increased in NOD/Ltj mice, accompanied with reduced stimulated salivary flow rate and Toll-like receptor 9 or p-p38 MAPK positive infiltrating lymphocytes observed in the SMG of NOD/Ltj mouse. Our results indicated that activation of Toll-like receptor 9-depended p38 MAPK signal pathway in PBMCs was an early event in pSS which made NOD/Ltj as an ideal therapy model to test the treatment effects of p38 MAPK or Toll-like receptor 9 inhibitors on pSS.

Expression and functional regulation of the nuclear receptors AHR, PXR, and CAR, and the transcription factor Nrf2 in rat parotid gland.

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

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.

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

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

Factors associated with fluoride concentrations in whole and parotid ductal saliva.

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

Cellular expression of Bcl-2 and Bax in atrophic submandibular glands of rats.

In submandibular gland atrophy, most acinar cells disappear by apoptosis, while many duct cells remain. The present study aimed to establish whether Bcl-2 and Bax, members of the Bcl-2 gene family, regulating the signalling pathway of apoptosis were involved in duct cell survival and acinar cell death in atrophic submandibular glands. The excretory duct of rat submandibular gland was doubly ligated with metal clips from 1 to 14 days to induce atrophy to the gland. The expressions of Bcl-2 and Bax in the atrophic submandibular gland were examined using immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). Immunohistochemically, Bcl-2 expression was identified in duct cells in the experimental glands at all time points. Some acinar cells showed Bax positivity 1 day after excretory duct ligation, and there were more Bax-positive acinar cells on days 3 and 5 when many apoptotic acinar cells were observed. Analysis by RT-PCR showed that the expression of mRNA for Bcl-2 became stronger as the glandular atrophy progressed and that Bax mRNA strongly expressed on days 1 and 3. These observations suggest that Bcl-2 inhibits duct cell apoptosis and Bax promotes apoptosis of acinar cells during atrophy of submandibular glands.

Aggressive invasive micropapillary salivary duct carcinoma of the parotid gland.

The presence of invasive micropapillary component has been reported to be associated with salivary duct carcinoma and poor outcomes. Herein is described a rare case of invasive micropapillary salivary duct carcinoma of the parotid gland in a 60-year-old man. The micropapillary component was approximately 70% of the area of the tumor. Squamous differentiation was focally seen adjacent to the micropapillary component. On immunohistochemistry the ordinary salivary duct carcinoma component was positive for gross cystic disease fluid protein-15 (GCDFP-15), androgen receptor (AR), and HER2/neu, whereas both micropapillary and squamous components were negative for GCDFP-15 and AR. Immunohistochemical staining for D2-40 highlighted the lymph vessel invasion of tumor cells. This patient developed metastases in the lymph nodes of the neck, and also in the liver, lung, and brain. The lymph nodes and liver metastases had both ordinary salivary duct carcinoma and micropapillary components. The patient died of tumor 11 months after the initial surgical operation. The results support that the presence of micropapillary component is associated with more aggressive behavior of salivary duct carcinoma. It is also important for pathologists to recognize that GCDFP-15 and AR expression can be reduced in micropapillary carcinoma in the differential diagnosis of metastatic tumor.

The implications of TrkA and MET aberrations in de novo salivary duct carcinoma.

Salivary duct carcinoma (SDC) is an aggressive carcinoma with poor prognosis. Although anti-HER2 therapy is a potential treatment option for HER2-positive SDC, other potential therapeutic targets are not known, in particular for HER2-negative cases. In this study, the recently identified receptors tyrosine kinases MET and tropomyosin-receptor kinase (Trk) were investigated as potential therapeutic targets. A total of 28 consecutive, surgically resected, de novo SDC cases were selected after evaluating histology and immunohistochemical expression of androgen receptor. Immunohistochemical expression of c-erb2, TrkA, TrkB, TrkC, and c-MET was analyzed, and the genetic status of the HER2 and MET genes was investigated through dual-color silver in situ hybridization. High expression of c-MET or Trk was defined as that above the median value. Among the 28 SDC cases, 64.3% (18/28) were HER2-positive. c-MET expression varied, with a median H-score of 65 (range, 0 to 200). Copy number gain and amplification of MET were noted in 57.1% (16/28) and 10.7% (3/28) of cases, respectively. TrkA was variably expressed, with a median H-score of 100 (range, 0 to250). High TrkA expression was significantly related to an inferior overall survival rate in HER2-negative SDC. High expression of TrkA and c-MET and MET copy number gain/amplification were frequent events in SDC, and high expression of TrkA revealed the tendency to be related to poor prognosis in HER2-negative SDC. TrkA and MET may be possible therapeutic targets in SDC, especially in HER2-negative SDC.

Differential expression of hormonal and growth factor receptors in salivary duct carcinomas: biologic significance and potential role in therapeutic stratification of patients.

Salivary duct carcinoma (SDC), a rare malignancy, manifests remarkable morphologic and biologic resemblance to high-grade mammary ductal carcinoma. We contend that, similar to mammary ductal carcinoma, hormones and growth factors may play a role in SDCs. Our aim was to determine the incidence and clinical significance of the expression of several hormone and growth factor receptors and evaluate their potential in therapeutic stratification of SDC patients in the largest cohort studied to date. Eighty-four archived tumor tissue blocks were analyzed immunohistochemically for expression of estrogen receptor-beta (ERbeta), androgen receptor (AR), and proline, glutamic acid, and leucine-rich protein-1 and growth factor receptors HER-2 and epidermal growth factor receptor. The results were correlated with available pathologic, demographic, and clinical data from 59 of 84 cases. Proline, glutamic acid, and leucine-rich protein-1, ERbeta, and AR were expressed individually in 94% (71/76), 73% (57/80), and 67% (56/84) of SDCs, respectively, and coexpressed in 45% (34/75). AR was expressed significantly more often in SDCs of men than in SDCs of women [79% (35/57) vs. 33% (9/27), P<0.001]. Epidermal growth factor receptor and HER-2 were overexpressed individually in 48% (40/83) and 25% (21/84), respectively, and co-overexpressed in 12% (10/83). Survival decreased significantly in patients with lymph node metastasis (P=0.002) and positive surgical margins (P=0.006). Lack of ERbeta expression correlated with increased local and regional recurrence (P=0.05 and P=0.002, respectively). Together, these results indicate that (a) ERbeta down-regulation is associated with adverse clinical features, (b) lymph node and surgical margin status are significant survival factors, and (c) the differential expression of these hormones and growth factor receptors may assist in triaging patients with SDC for novel therapies.

Mouse salivary glands and human beta-defensin-2 as a study model for antimicrobial gene therapy: technical considerations.

Transduction of salivary glands with antimicrobial peptide genes has great potential for oral infection control. Our ultimate goal is to introduce antimicrobial peptide genes into salivary glands that secrete these peptides into saliva to control bacterial/fungal infection in the oral cavity. However, an animal study model to test this potential has not been established. Therefore, we determined to test (i) whether the potent antimicrobial peptide human beta-defensin-2 (hBD-2) can be overexpressed in saliva after transduction of salivary glands and (ii) whether oral fungal infection can be developed in a NOD/SCID murine model. Lentiviral vector SIN18cPPTRhMLV bearing hBD-2 cDNA was introduced into SCID mouse submandibular glands via cannulation. Reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry or enzyme-linked immunosorbent assay (ELISA) were performed to detect hBD-2 expression in glands or in saliva. Candida albicans 613p was inoculated orally into SCID mice to establish oral candidiasis. Whilst expression of hBD-2 was detected in mouse salivary glands by RT-PCR and immunohistochemistry 1 day or 1 week following delivery of lentivirus, hBD-2 was not detected in saliva. There was recoverable C. albicans from the oral cavity and gastrointestinal tract 4 days to 4 weeks after infection, but there was no establishment of observable oral candidiasis in SCID mice under a stereomicroscope. Our data indicate that lentiviral vectors transduce mouse salivary glands, but not at a sufficient level to allow hBD-2 detection in saliva. Other vectors for gene transduction and additional treatment of SCID mice to establish oral candidiasis are needed in order to utilise mouse salivary glands to test antimicrobial gene therapy.

Primary salivary duct carcinoma of the lung, mucin-rich variant.

Primary salivary gland-type lung cancer is a heterogeneous group of neoplasms arising from the seromucinous glands of the respiratory tract. Histopathologically, they are identical to salivary gland neoplasms of the head and neck. While mucoepidermoid carcinoma and adenoid cystic carcinoma are overwhelmingly the most common subtypes found in the lung, reports of uncommon subtypes can be found in the literature. We report a case of a 73-year-old woman with primary lung salivary duct carcinoma, mucin-rich variant--an exceedingly rare subtype of an already rare malignant salivary-type neoplasm. One case of primary lung salivary duct carcinoma has been reported in the literature; however, the mucin-rich variant has never been described in the lung. Furthermore, the tumor in our case bears a rare BRAF G464V mutation. To our knowledge, this is the first reported case of a BRAF G464V mutation detected in a salivary duct carcinoma or any other salivary-type neoplasm.

Dual oxidases represent novel hydrogen peroxide sources supporting mucosal surface host defense.

Lactoperoxidase (LPO) is an enzyme with antimicrobial properties present in saliva, milk, tears, and airway secretions. Although the formation of microbicidal oxidants by LPO has been recognized for some time, the source of hydrogen peroxide (H2O2) for LPO-catalyzed reactions remains unknown. Reactive oxygen species produced by the phagocyte NADPH oxidase (phox) play a critical role in host defense against pathogens; however, analogous oxidant-generating systems in other tissues have not been associated with antimicrobial activity. Several homologues of gp91phox, the catalytic core of this enzyme, were described recently; dual oxidase (Duox)1/thyroid oxidase 1 and Duox2/thyroid oxidase 2 were identified in the thyroid gland and characterized as H2O2 donors for thyroxin biosynthesis. We examined Duox1 and Duox2 expression in secretory glands and on mucosal surfaces and give evidence for their presence and activity in salivary glands, rectum, trachea, and bronchium. Epithelial cells in salivary excretory ducts and rectal glands express Duox2, whereas tracheal and bronchial epithelial cells express Duox1. Furthermore, we detected Duox1-dependent H2O2 release by cultured human bronchial epithelial cells. Our observations suggest that Duox1 and Duox2 are novel H2O2 sources that can support LPO-mediated antimicrobial defense mechanisms on mucosal surfaces.

Expression of androgen receptor and prostatic specific markers in salivary duct carcinoma: an immunohistochemical analysis of 13 cases and review of the literature.

Salivary duct carcinoma (SDC) is an uncommon, pathologically distinct entity characterized by its morphologic resemblance to ductal carcinoma of the breast and highly aggressive behavior. Approximately two thirds of patients die within 4 years of initial diagnosis despite aggressive, combined surgical resection and radiotherapy. Review of the literature indicates that androgen receptor (AR), a marker frequently detected in prostatic carcinoma, is expressed in over 90% of SDCs, whereas two common breast carcinoma markers, estrogen and progesterone receptors (ER and PR), are expressed in only 1.3% and 6% of the tumors, respectively, by immunohistochemistry. This hormonal profile suggests that SDC, in contrast to its histiologic similarity to ductal carcinoma of the breast, is immunophenotypically more related to prostatic carcinoma. To substantiate this hypothesis, we performed immunohistochemical staining of 13 cases of SDC for the presence of AR and two prostatic markers, prostate specific antigen (PSA) and prostatic acid phosphatase (PAP). Our results showed multifocal, scattered, moderate immunostaining for PAP and diffuse, moderate immunostaining for PSA in seven (58.3%) and two (16.7%) cases, respectively. These results create a potential diagnostic challenge to surgical pathologists who are dealing with a metastatic adenocarcinoma of AR+/PSA+/-/PAP+/- phenotype, particularly in male patients of unknown primary. Metastatic salivary duct carcinoma should be given serious thought if clinical investigation fails to reveal a prostatic primary. The immunophenotypic homology that exists between SDC and prostatic carcinoma also suggests that antiandrogen therapy as used in the treatment of prostatic carcinoma might be beneficial in patients with metastatic SDC when all other conventional modalities fail.

Mucin-rich variant of salivary duct carcinoma: a clinicopathologic and immunohistochemical study of four cases.

Salivary duct carcinoma is a relatively uncommon aggressive neoplasm, typically found in the parotid glands of older men. The histologic appearance is that of an in situ and invasive high-grade adenocarcinoma, and it closely resembles ductal carcinoma of the breast. Several variants of the latter are very well known, but only papillary, sarcomatoid, and low-grade subtypes have so far been reported in salivary duct carcinoma. This study describes the clinicopathologic and immunohistochemical findings in four examples of an additional previously undescribed variant, rich in mucin. Each tumor showed areas of typical salivary duct carcinoma, but in addition there were lakes of epithelial mucin-containing malignant cells, i.e., mucinous (colloid) carcinoma. All four tumors expressed androgen receptors, cytokeratins, epithelial membrane antigen, gross cystic disease fluid protein-15, and carcinoembryonic antigen, but S-100 protein, other myoepithelial markers, and estrogen and progesterone receptors were negative. The mucin antigen profile showed positivity for MUC2, MUC5B, and MUC6 in all cases but only rare staining with MUC5AC and MUC7. Strong immunohistochemical overexpression of HER2/neu was demonstrated in one tumor, together with amplification by fluorescence in situ hybridization; another case was weakly positive with just one antiserum, but the remaining two tumors were completely negative. Small quantities of mucin have often been described in salivary duct carcinoma but not large extracellular mucinous lakes, which though prominent in the present series, were not as extensive as in mucinous adenocarcinoma. The relatively poor clinical outcome of the patients in our study mirrored that seen in usual-type salivary duct carcinoma and emphasizes the importance of differentiating mucin-rich salivary duct carcinoma from pure mucinous (colloid) adenocarcinoma, a tumor not fully defined, but possibly with a better prognosis.

Dopamine and serotonin receptors mediating contractions of the snail, Helix pomatia, salivary duct.

The combination of high performance liquid chromatography, bioassay and immunocytochemistry was applied to study the regulation of the salivary duct muscle of the snail, Helix pomatia. The major function of the duct appears to be to propel the saliva toward the buccal cavity during feeding. It has been established that serotonin and dopamine applied exogenously mimic the effect on the duct exerted by the stimulation of the salivary nerve. Immunohistochemistry revealed the presence of serotonin, but not dopaminergic nerve elements in the nerve and along the duct surface. However, both serotonin (14.9-15.5 pmol/mg) and dopamine (0.38-0.58 pmol/mg), as well as the synthesizing enzymes (tyrozine hydroxylase 0.28 pmol/mg tissue/h and DOPA 0.32 nmol synthesized DA/mg tissue/h) could regularly be assayed in the salivary duct by high performance liquid chromatography. When released following the stimulation of the salivary nerve, both monoamines were shown to interact with distinct membrane receptors. Dopamine elicited a sustained increase of the muscle tone in concentration-dependent manner (K(d)=1.5 microM). Mammalian D(1) receptor antagonist flupenthixol and fluphenazine attenuated, whereas the D(1) receptor agonist SKF-38393 mimicked the effect elicited by exogenous dopamine. Serotonin had a double effect on the salivary duct: a relaxing and a contracting one with different K(d) values 76 nM and 2.4 microM, respectively. 5-HT(2) receptor antagonist ritanserin and ketanserin attenuated the serotonin-induced relaxation. In contrast 5-HT(3) antagonist metoclopramide and MDL2222 decreased and 5-HT(3) receptor agonist 1-(m-chlorophenyl)-biguanide mimicked the serotonin-induced contraction, suggesting that serotonin exerted its action on two different receptor subtypes. The release of radiolabeled serotonin and dopamine upon nerve stimulation was found to be Ca-dependent. Furthermore, the increase in serotonin concentration induced a decrease of the potency of dopamine to elicit sustained contraction. These results provide evidence for the transmitter role of serotonin and dopamine in salivary duct. It is concluded that receptors reveal a pharmacological profile related to vertebrate D(1), 5-HT(2) and 5-HT(3) receptor subtypes. Moreover, it was found that the process of conveying the saliva is modulated by an interaction of dopamine and serotonin.

A morphogenetic concept of salivary duct regeneration and metaplasia.

The exact mechanisms of physiological regeneration and of metaplastic processes of the salivary duct have not been definitely established, although regeneration from a putative uncommitted stem cell population has long been favored. In the present study, double immunohistochemical labeling for Ki 67 and alpha-actin or different cytokeratin subtypes, respectively, made possible an exact localization and quantification of cellular proliferation in the regular salivary duct and in different types of metaplasia. Our data demonstrate a baseline proliferative capacity in all five cell types of the salivary duct. Luminal secretory cells of the acinus and intercalated duct regenerate independently from myoepithelial or basal cells. In contrast, the renewal of oxyphilic cells in the striated and excretory duct is maintained by proliferation and differentiation of basal cells. The great majority of metaplasias develops from uncommitted, Bcl-2 positive basal cells of striated/excretory ducts which possess an enormous capacity for pluridirectional morphogenetic differentiation. Despite this important role of basal cells, our findings demonstrate that all cell types principally have to be considered as potential progenitor cells for salivary gland tumors. The improved insight into regenerative and metaplastic processes of the salivary duct may contribute to a better understanding of the complex formal carcinogenesis.

Transplantation of cultured salivary gland cells into an atrophic salivary gland.

Patients with dry mouth have been treated with salivary substitutes and/or medications such as pilocarpine or cevimeline hydrochloride. These treatments temporarily relieve their symptoms and induce salivation from residual tissue. However, no treatment is available for the purpose of regenerating an atrophic gland. In this study, the feasibility of a cell transplantation therapy for the atrophic submandibular glands was investigated in rats. Further, the potential of cell differentiation into a useful phenotype was assessed by immunohistochemistry together with cell tracking with the fluorescent dye PKH 26. Rat submandibular glands were excised, and the salivary gland epithelial cells were cultured for 3 weeks with 3T3 cells as a feeder layer. Ductal ligation of the submandibular gland was employed to generate an atrophic gland. One week after the operation, the ligation was removed, and the cultured cells labeled with PKH 26 were injected into the atrophic submandibular glands. As a control, the cultured cells were also injected into normal submandibular glands. Two weeks after cell transplantation, the transplanted cells were detectable in both the experimental and control groups. The cells were clustered in the connective tissue between the lobules. Four weeks after transplantation, the labeled cells were detectable in the experimental group but not in the control group. In the atrophic glands, the scattered transplanted cells were observed over a broad area of the gland but localized mainly around the acini and ductal region. Immunostaining results showed a possible involvement of the transplanted cells in ductal regeneration, while neither myoepithelial nor acinar differentiations were observed within the 4 weeks since transplantation. This study demonstrated that cell transplantation to the salivary gland is feasible, and that the transplanted cells were selectively attracted to and remained in the damaged area without affecting normal tissue.

Expression of the antimicrobial peptide, human beta-defensin 1, in duct cells of minor salivary glands and detection in saliva.

The oral cavity is exposed to a variety of environmental insults. Salivary secretions play a critical role in maintaining oral health via innate host defense mechanisms and secretion of secretory IgA. Human beta-defensins (hBD) are antimicrobial peptides that are a component of the innate immune response; they are expressed in epithelia and are proposed to have a role in mucosal defense. hBD-1 mRNA is constitutively expressed in numerous mucosal tissues, including human gingiva and submandibular and parotid glands. Our objective was to detect the expression and localization of hBD-1 peptide in human salivary glands and in saliva. Minor salivary gland tissue was obtained from biopsies of patients with mucoceles (n = 20). hBD-1 peptide was detected by immunohistochemistry; expression was localized to the ductal cells and not the acinar cells of these glands. The peptide was located apically, toward the lumen in the duct cells. Further evaluation showed stronger hBD-1 expression in ducts with periductal inflammation, as indicated by the immunostaining of serial sections with anti-CD45 specific for B- and T-lymphocytes. Statistical analysis showed a strong correlation of hBD-1 staining and inflammation. Results of immunolocalization suggest that hBD-1 functions to protect salivary glands from retrograde infection, that expression of the peptide is enhanced in inflamed sites, and that post-transcriptional regulatory mechanisms may be involved in hBD-1 peptide expression. Western immunoblot analysis also detected hBD-1 peptide in unstimulated, whole, acidified saliva from normal volunteers. However, hBD-1 peptide associated with salivary mucin resulted in loss of the detection in a dot-immunoblot assay. Association of hBD-1 with salivary mucin may facilitate peptide distribution and adherence to oral surfaces and aid its function within the oral cavity.