Minor salivary gland immunohistology in the diagnosis of primary Sjögren's syndrome

Background:  Focal lymphocytic infiltrates of minor salivary glands are considered target-organ related signs of Sjögren's syndrome. The percentages of plasma cells expressing IgA, IgG and IgM in minor salivary gland biopsies have also been suggested as useful in establishing a diagnosis of Sjögren's syndrome, and this study aimed at evaluating this method.
Methods:  All biopsies from patients under investigation for Sjögren's syndrome ( n  = 210) at our department during 4 years were analyzed for IgA, IgG and IgM producing cells by immunohistochemistry, and related to Sjögren classification parameters.
Results:  A focus score ≥1 was observed in 67/210 patients and the frequency of IgA producing cells was <70% in 42/210 patients. Sufficient clinical data for classification of disease were available for 57/210 patients. Patients were classified as having primary Sjögren's syndrome (pSS) ( n  = 9), secondary Sjögren's syndrome (sSS) ( n  = 12) or non-Sjögren's syndrome (non-SS) ( n  = 36). IgA expressing cells were significantly decreased ( P  < 0.01) and IgG expressing cells significantly increased ( P  < 0.02) in patients with pSS compared to non-SS. Also, increased numbers of salivary gland IgG producing plasma cells correlated with increased IgG serum levels ( P  < 0.001). However, there was no significant difference between sSS and non-SS with regard to IgA, IgG or IgM expressing cells in the glands.
Conclusions:  Our results support previous reports indicating the relevance of quantitative evaluation of Ig isotype expression in plasma cells in the clinical investigation of Sjögren's syndrome and further indicate a difference in plasma cell populations between pSS and sSS.     

pSS患者唇腺细胞中Bcl-2的表达及其与细胞色素C的关系

目的：探讨原发性干燥综合征（pSS）患者唇腺细胞凋亡的发生机制。方法：收集临床患者，将其分为pSS组和非干燥综合征组。采用免疫组化的方法检测两组患者唇腺细胞Bcl一2蛋白的表达及含量；制备唇腺组织匀浆及提取线粒体，检测CytC的含量。使用SPSSl4．0软件对两组数据进行统计学分析。结果：pSS患者唇腺细胞中Bc1-2蛋白的表达和线粒体CytC的含量显著低于非干燥综合征组；pSS患者唇腺细胞胞浆CytC含量显著高于非干燥综合征组。结论：pSS的发病机制与Bcl-2蛋白及CytC诱导的细胞凋亡有关。     

Salivary secretion, mucin concentrations and candida carriage in HIV-infected patients

Objectives:  To test whether the submandibular/sublingual (SMSL) salivary secretion, mucin concentration and candida carriage status were altered in human immunodeficiency virus-positive (HIV+) patients.
Subjects and methods:  SMSL saliva collected from 48 HIV-infected and 31 HIV-negative men were analyzed for flow rates, total protein and mucin concentrations. Salivary cultures were performed for Candida assessment.
Results:  The salivary flow rate and protein secretion of the HIV+ patients was 37% and 32% less than that of the controls ( P  <   0.0001, P  =   0.0087). The mucin concentrations (MG1 and MG2) were higher in the HIV+ subjects compared with controls ( P  =   0.0186, P  =   0.0014); however, the mucin secretions were not different. The frequency of Candida -positive cultures was higher in the HIV+ subjects than in the controls (61.4% vs 24.1%, P  =   0.0018). In the HIV-infected group, the unstimulated SMSL flow rates were lower in Candida -positive than in Candida -negative patients ( P  =   0.0158).
Conclusion:  The salivary secretion of the SMSL glands was reduced in HIV infection. Although the mucin concentration increased in HIV+ subjects, mucin secretion was not altered. Highly active antiviral therapy had no effect on salivary function. We found an association between the level of candida carriage and salivary flow rate in HIV-infected patients.     

Different proteomic protein patterns in saliva of Sjögren's syndrome patients

Objective:  To investigate the salivary protein profile in patients with Sjögren's syndrome (SS), and healthy control subjects.
Materials and methods:  Unstimulated whole saliva samples were collected from 16 age-matched females; eight healthy subjects and eight patients diagnosed with SS (six primary SS, one incomplete SS and one primary SS associated with B cell lymphoma). Proteins were extracted and separated individually by 2D sodium dodecyl sulphate–polyacrylamide gel electrophoresis. Selected protein spots of interest were analysed by electrospray ionization – tandem mass spectrometry. Obtained data were searched against the Swiss-Prot and NCBI non-redundant protein databases using Mascot software.
Results:  Two groups of patterns of protein expression were observed in the eight SS patients: a major group (six patients) with significant expression differences from the healthy subjects and the second group (two patients) with a pattern similar to the eight healthy subjects.
Conclusion:  In this preliminary study, protein expression differences were found between SS patients and healthy subjects. Individual analysis of SS patients exhibited two patterns of protein expression with no direct relation to the clinical, serological or histological severity of disease. This study emphasizes the difficulty of the present proteomic knowledge to diagnose and monitor the sequel of SS development.     

Follicular dendritic cells confirm lymphoid organization in the minor salivary glands of primary Sjögren’s syndrome

Background: Sjögren’s syndrome (SS) is an autoimmune chronic inflammatory disorder affecting the salivary and lacrimal glands. The aim of this study was to explore immunophenotypic features of chronic inflammatory reactions in the minor salivary glands in patients with primary SS (pSS). Methods: Formalin‐fixed, paraffin‐embedded labial minor salivary gland tissue sections from randomly selected patients with pSS (n = 60) were investigated for the expression of CD21, CD23, CD35 and IgD by immunohistochemistry. Results: Based on the distribution and staining pattern of CD21, CD23, CD35 and IgD in lymphoid aggregates, several stages of chronic inflammatory reactions were observed. In 12/60 (20%) patients, lymphoid infiltrates with germinal centre (GC)‐like features such as extensive networks of CD21‐, CD23‐ and CD35‐positive cells were observed in the minor salivary gland tissue. Smaller networks and /or focal infiltrates with scattered CD21⁺, CD23⁺ and CD35⁺ cells were observed in the remaining 48/60 (80 %) cases. When dividing patients according to the presence (GC+) or the absence (GC?) of GC in the minor salivary glands, the mean focus score was significantly higher in the GC+ patients (P < 0.05). Double staining of the minor salivary glands revealed focal infiltrates with follicular dentritic cell networks and B cells resembling normal GCs in tonsillar tissue. Conclusion: A particular cellular profile was demonstrated in a sub‐group of patients with pSS and could be linked to serological aberrations. These findings warrant further prospective studies.     

Effect of HAART on salivary gland function in the Women's Interagency HIV Study (WIHS)

Objective:  To determine the impact of highly active antiretroviral therapy (HAART) on salivary gland function in human immunodeficiency virus (HIV) positive women from the Women's Interagency HIV Study (WIHS).
Design:  Longitudinal cohort study.
Subjects and methods:  A total of 668 HIV positive women from the WIHS cohort with an initial and at least one follow-up oral sub-study visit contributed 5358 visits. Salivary gland function was assessed based on a dry mouth questionnaire, whole unstimulated and stimulated salivary flow rates, salivary gland enlargement or tenderness and lack of saliva on palpation of the major salivary glands.
Main outcome measures:  Changes in unstimulated and stimulated flow rates at any given visit from that of the immediate prior visit (continuous variables). The development of self-reported dry mouth (present/absent), enlargement or tenderness of salivary glands (present/absent), and absence of secretion on palpation of the salivary glands were binary outcomes (yes/no).
Results:  Protease Inhibitor (PI) based HAART was a significant risk factor for developing decreased unstimulated ( P  =   0.01) and stimulated ( P  =   0.0004) salivary flow rates as well as salivary gland enlargement ( P  =   0.006) as compared with non-PI based HAART.
Conclusions:  PI-based HAART therapy is a significant risk factor for developing reduced salivary flow rates and salivary gland enlargement in HIV positive patients.     

Expression of two drug-metabolizing cytochrome P450-enzymes in human salivary glands

Objective:  The oral cavity is constantly lubricated by saliva and even small amounts of xenobiotics and / or their metabolites in the saliva may affect the oral mucosa. Our aim was therefore to clarify if xenobiotic metabolizing enzymes CYP1A2 and CYP3A4 are expressed in salivary glands.
Methods:  Formalin-fixed paraffin-embedded specimens from parotid (10), submandibular (7) and labial (10) salivary glands were examined immunohistochemically and by in situ hybridization for expression of CYP1A2 and CYP3A4 protein and mRNA.
Results:  CYP1A2 and CYP3A4 protein and mRNA were detected in ductal and seromucous / serous acinar cells in all gland types although to a varying degree and intensity. Mucous acinar cells were positive to a lesser extent.
Conclusion:  The results indicate a xenobiotic metabolizing capability of salivary glands. This may have implications for development of oral mucosal disease as a result of mucosal exposure to metabolites originating from internal sources (blood) as well as from saliva.     

Salivary profile and oxidative stress in children and adolescents with cystic fibrosis

J Oral Pathol Med (2010) 39 : 16–21
Background:  Cystic fibrosis (CF) is characterized by altered exocrine secretions; however, no comprehensive compositional profile of CF serous and mucous saliva secretions has been published.
Design:  We analyzed salivary flow rate and composition, and oxidative stress-related parameters, comparing CF patients with non-CF bronchiectasis patients and the healthy controls.
Results:  Median salivary magnesium concentration and lactate dehydrogenase activity were significantly lower in CF patients than in the healthy controls. Salivary total protein concentration was 45% higher in CF patients than in non-CF bronchiectasis patients. CF patients showed 8% lower levels of peroxidase compared with non-CF bronchiectasis. Salivary total antioxidant status, superoxide dismutase and uric acid values in the CF group were higher by 15%, 35% and 31%, respectively, than in both control groups.
Conclusions:  Cystic fibrosis patients demonstrated altered salivary profile, especially in antioxidant enzymatic and molecular activity, possibly resulting from the oral cavity's ongoing inflammatory and oxidative process. Free radical mechanisms may be involved in CF pathogenesis.     

Initial comparison of proteomic profiles of whole unstimulated saliva obtained from generalized aggressive periodontitis patients and healthy control subjects

Background and Objective:  Salivary proteomics technology can be used to evaluate the disease progession of periodontitis and the systemic screening of proteomes of saliva from subjects with aggressive periodontitis has not been available. The objective of this preliminary study was to compare the proteomic profile of whole unstimulated saliva of subjects with generalized aggressive periodontitis (GAgP) with that of healthy volunteers to identify proteins, the levels of which were significantly altered between the two groups.
Material and Methods:  Whole unstimulated saliva was obtained from five subjects with GAgP and five healthy subjects, and proteins were separated using two-dimensional gel electrophoresis. Proteins, the levels of which were significantly different between the two groups, were identified by computer image analyses and subsequent electrospray ionization tandem mass spectrometry.
Results:  Eleven proteins that exhibited a different level in the GAgP group vs. the control group were identified. Compared with whole saliva of healthy control subjects, the levels of serum albumin, immunoglobulin (Ig) γ2 chain C region, Ig α2 chain C region, vitamin D-binding protein, salivary α-amylase and zinc-α2 glycoprotein were increased in whole unstimulated saliva of GAgP subjects, while those of lactotransferrin, elongation factor 2, 14-3-3 sigma, short palate, lung and nasal epithelium carcinoma-associated protein 2 precursor and carbonic anhydrase 6 were decreased.
Conclusion:  Comparison of the proteomic profile of whole unstimulated saliva of GAgP subjects with that of healthy control subjects revealed at least 11 differential proteins. The approach applied herein might be helpful to aid understanding of the etiology of GAgP.     

Acute salivary gland hypofunction in the duct ligation model in the absence of inflammation

Objective:  The commonly associated aetiology of salivary gland inflammation and salivary hypofunction has led to the widely held belief that inflammation causes salivary gland hypofunction. Indeed, our own recent study seemed to support this contention. Here, we tested the hypothesis that, in an acute duct ligation model, eliminating inflammation the submandibular gland would recover normal function.
Materials and methods:  Ligation of the rat submandibular gland excretory duct for 24 h was used to induce inflammation and salivary gland hypofunction. A group of duct ligated rats was compared with a second group given dexamethasone, on the day of duct ligation. Twenty-four hours later salivary gland function was assessed and salivary glands were collected.
Results:  Histology and myeloperoxidase activity assay revealed a profound decrease in inflammatory cell infiltration of ligated glands from rats given dexamethasone, compared with ligated glands in the absence of dexamethasone. Salivary flow rate evoked by methacholine was decreased ( P  < 0.01) by approximately 56% (ligated vs control, 79 ± 9  μ l min⁻¹ g⁻¹ vs 177 ± 11  μ l min⁻¹ g⁻¹) and salivary flow from ligated dexamethasone-treated and ligated glands was similar.
Conclusion:  Despite eliminating the inflammatory reaction in the ligated gland, salivary hypofunction was not reversed, suggesting that other mechanisms must be at work in the ligation-induced salivary hypofunction.     

Activation of innate immune responses through Toll-like receptor 3 causes a rapid loss of salivary gland function

Background:  Recent studies have demonstrated the expression of Toll-like receptor 3 (TLR3) in salivary glands and epithelial cell lines derived from Sjögren's syndrome (SS) patients. As viral infections are considered to be a trigger for SS, in this study we investigated whether in vivo engagement of TLR3 affects salivary gland function.
Methods:  Female New Zealand Black/WF1 mice were repeatedly injected with polyinosinic:polycytidylic acid [poly(I:C)]. TLR3 expression within submandibular glands was studied using immunohistochemistry. RNA levels of inflammatory cytokines in the submandibular glands were determined by real time polymerase chain reaction. Pilocarpine induced saliva volume was used as an index of glandular function.
Results:  Immunohistochemical analysis of submandibular glands showed TLR3 expression in epithelium of serous and mucous acini, granular convoluted tubules, and ducts. Poly(I:C) treatment rapidly up-regulated the mRNA levels of type I interferon (IFN) and inflammatory cytokines in the submandibular glands. One week after treatment, the saliva volumes in poly(I:C) treated mice were significantly reduced in comparison with the phosphate-buffered saline (PBS) treated mice. Hematoxylin and eosin staining showed that salivary gland histology was normal and lymphocytic foci were not detected. Glandular function recovered after poly(I:C) treatment was stopped.
Conclusions:  Our results demonstrate that engagement of TLR3 within the salivary glands results in a rapid loss of glandular function. This phenomenon is associated with the production of type I IFN and inflammatory cytokines in the salivary glands. Restoration of glandular function suggests that for viral etiology of SS, a chronic infection of salivary glands might be necessary.     

Transient TWEAK overexpression leads to a general salivary epithelial cell proliferation

Objectives:  Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine that has pro-apoptotic, pro-angiogenic and pro-inflammatory effects. In liver, TWEAK leads to proliferation of progenitor oval cells, but not of mature hepatocytes. This study evaluated the hypothesis that TWEAK overexpression in salivary glands would lead to the proliferation of a salivary progenitor cell.
Methods:  A recombinant, serotype 5 adenoviral vector encoding human TWEAK, AdhTWEAK, was constructed, initially tested in vitro , and then administered to male Balb/c mice via cannulation of Wharton's duct. TWEAK expression in vivo was monitored as protein secreted into saliva and serum by enzyme-linked immunosorbent assays. Salivary cell proliferation was monitored by proliferating cell nuclear antigen staining and apoptosis was monitored using TUNEL staining.
Results:  AdhTWEAK administration led to a dose-dependent, transient TWEAK protein expression, detected primarily in saliva. Salivary epithelial cell proliferation was generalized, peaking on ∼days 2 and 3. TWEAK expression had no detectable effect on apoptosis of salivary epithelial cells.
Conclusion:  Transient overexpression of TWEAK in murine salivary glands leads to a general proliferation of epithelial cells vs a selective stimulation of a salivary progenitor cell.     

Probiotic bacteria affect the composition of salivary pellicle and streptococcal adhesion in vitro

Introduction:  The use of probiotic bacteria is increasing worldwide and at least some of them can transiently colonize the oral cavity. Several studies have shown that probiotic bacteria, which are often thought of in relation only to intestinal health, can also affect the oral ecology, but the mechanisms for this are largely unknown. The aim of this study was to investigate in vitro if the probiotic bacteria used in commercial products affect the protein composition of the salivary pellicle and the adherence of other oral bacteria.
Methods:  Salivary pellicle on hydroxyapatite and the adhesion of two oral streptococci, Streptococcus mutans and Streptococcus gordonii , were used as a model.
Results:  Probiotic bacteria that bound to saliva-coated hydroxyapatite reduced the adhesion of S. mutans but the inhibitory effect on the adherence of S. gordonii was weaker. Salivary pellicle protein composition was modified by all the strains tested. The modifications in the pellicle affected the adherence of S. mutans but not of S. gordonii . Two of the proteins missing from the pellicles made of saliva-treated with the probiotic bacteria were identified as salivary agglutinin gp340 and salivary peroxidase. All bacterial strains bound salivary agglutinin gp340. The ability of the probiotic bacteria to degrade peroxidase was demonstrated with purified bovine lactoperoxidase and two of the probiotic strains.
Conclusion:  This in vitro study showed that probiotic strains used in commercial products may affect the oral ecology by specifically preventing the adherence of other bacteria and by modifying the protein composition of the salivary pellicle.     

Morphometric analysis of CD34-positive vessels in salivary gland adenoid cystic and mucoepidermoid carcinomas

Background:  Carcinomas of the salivary glands are uncommon and morphologically a diverse group of malignancies. To evaluate the prognostic value of CD34 immunostaining of the vessels in adenoid cystic carcinoma (AdCC) and mucoepidermoid carcinoma (MEC), an automated image analysis method was used.
Method:  In a nationwide study, covering salivary gland cancer (SGC) patients in Finland 1991–1996, 37 AdCC and 18 MEC patients (M 25, F 30, age 25–90, mean 63) were included. In addition to clinical characteristics the size, shape, staining intensity and vessel density in CD34 immunostained histologic samples were measured.
Results:  Altogether 4433 vessels were measured from AdCC and 2615 from MEC tumor. Of the total tumor vessels measured, 2651 were from patients who deceased with disease (Group I) and 4397 were from specimens derived from those who did not die of disease (Group II) during the 10-year follow-up. The staining intensity was significantly higher in MEC than in AdCC tumor ( P  = 0.0005). In MEC, the Group I patients had a higher staining intensity among high-grade patients compared with patients with low grade disease, whereas the tumors in Group II had a lower staining intensity among the high-grade compared with the low grade tumors ( P  = 0.018). A higher vessel density was found in patients with MEC in group II compared with group I ( P  = 0.017).
Conclusions:  The staining intensity of CD34 positive vessels in MEC was higher than in AdCC. In MEC, higher staining intensity of vessels in high-grade tumors and lower vessel density in all MEC patients, predicted poor survival.     

Innervation pattern and Ca2+ signalling in labial salivary glands of healthy individuals and patients with primary Sjögren's syndrome (pSS)

Abstract: We have characterised the innervation pattern and intracellular Ca²⁺-signalling in labial salivary glands (LSG) of 16 patients with primary Sjögren's syndrome (pSS) and 27 healthy controls. Numerous immunoreactive nerve fibers (IRF) containing vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) were found around acini, ducts and blood vessels. Substance P (SP)-, neuropeptide Y-, tyrosine hydroxylase- and nitric oxide synthase-IRF were mainly surrounding ducts and blood vessels. The majority of pSS patients had inflamed LSG and the presence of focal lymphocytic infiltrates (FI) were more frequent and pronounced as compared with healthy controls. In areas with normal or diffusely inflamed LSG tissue, pSS patients demonstrated the same distribution of IRF as healthy controls with similar histology. However, IRF were absent in central areas of FI both in pSS and age-matched healthy controls. Although all pSS patients had hyposalivation, stimulation with acetylcholine, norepinephrine, phenylephrine, isoproterenol, VIP, PACAP, SP, adenosine 5'-triphosphate and uridine 5'-triphosphate induced the same increase in the intracellular free Ca²⁺ concentration in LSG acini from both pSS patients and healthy controls, indicating the presence of functional receptor systems in vitro .     

A clinico-pathologic study of 311 intra-oral salivary gland tumors in Thais

Background:  There have been several epidemiologic studies on intra-oral salivary gland tumors in several countries, but little is known of these tumors in Thailand.
Objectives:  To determine the relative frequency and distribution of various types of intra-oral salivary gland tumors in the Thai population.
Methods:  The files of the Department of Oral Pathology, Faculty of Dentistry, Chulalongkorn University, from 1969 to 2007 were searched for intra-oral salivary gland tumors. Histopathologic slides were reviewed and reclassified according to the 2005 WHO Classification of Head and Neck Tumors. The age, gender, race, and anatomical distribution of the tumors were collected from the patients' records.
Results:  Of the 16,358 accessioned cases, 311 cases (1.90%) were diagnosed as intra-oral salivary gland tumors. One hundred and forty-seven cases (47.27%) were benign tumors, while 164 cases (52.73%) were malignant tumors. The mean age of the patient ± SD = 41.57 ± 16.65 years. Females outnumbered male patients by a ratio of M:F = 1:1.38. Almost all except one patient were Thais. Pleomorphic adenoma was the most common intra-oral salivary gland tumor. The majority of cases occurred at the palate.
Conclusions:  Pleomorphic adenoma is the most common intra-oral salivary gland tumor and the most common benign intra-oral salivary gland tumor, while mucoepidermoid carcinoma is the most common malignant intra-oral salivary gland tumor. Intra-oral salivary gland tumors in Thailand elicit similar trend as in previous studies, with only minor differences such as the ranking of some tumors, the higher incidence of intra-bony location, and the lower incidence of polymorphous low-grade adenocarcinoma.     

Enamel defects and salivary methylmalonate in methylmalonic acidemia

Introduction and objective:  To characterize enamel defects in patients with methylmalonic acidemia (MMA) and cobalamin (cbl) metabolic disorders and to examine salivary methylmalonate levels in MMA.
Subjects and methods:  Teeth from patients ( n  = 32) were evaluated for enamel defects and compared with age- and gender-matched controls ( n  = 55). Complementation class ( mut , cblA , cblB and cblC ) and serum methylmalonate levels were examined. Primary teeth from two patients were examined by light and scanning electron microscopy and salivary methylmalonate levels from two patients were analyzed.
Results:  Enamel defects were significantly more prevalent per tooth in the affected group than the control group, across complementation types ( P  <   0.0001). The mut MMA subgroup had a significantly higher prevalence per individual of severe enamel defects than controls ( P  =   0.021), and those with enamel defects exhibited higher serum methylmalonate levels than those without ( P  = 0.017). Salivary methylmalonate levels were extremely elevated and were significantly higher than controls ( P  =   0.002). Primary teeth were free of enamel defects except for two cblC patients who exhibited severe enamel hypoplasia. One primary tooth from a cblC patient manifested markedly altered crystal microstructure.
Conclusion:  Enamel anomalies represent a phenotypic manifestation of MMA and cbl metabolic disorders. These findings suggest an association between enamel developmental pathology and disordered metabolism.     

Peroxiredoxin I is overexpressed in oncocytic lesions of salivary glands

Background:  Oncocytic lesions, particularly frequent in the salivary glands, are characterized by cells with an atypical accumulation of mitochondria. This accumulation has been recognized as a compensatory mechanism to intrinsic functional defects of these organelles, resulting in energy production impairment and increased generation of reactive oxygen species (ROS), including hydrogen peroxide (H₂O₂). Peroxiredoxin I (Prx I) is a H₂O₂ scavenging protein and the expression of its yeast homolog was reported to be influenced by mitochondrial function.
Methods:  In this study, we evaluated Prx I expression in oncocytic lesions of salivary glands by immunohistochemistry.
Results:  Our results showed that Prx I is overexpressed in oncocytes regardless of the salivary gland lesion where they appear.
Conclusions:  These results suggest that Prx I expression in oncocytes is related to its ability to decompose mitochondrial-derived H₂O₂ and that it could provide to the cells a protective role in an environment that, by continuously producing potential DNA-damaging ROS, predisposes to genome instability and cellular transformation.