Detection of Epstein-Barr virus-associated antigens and DNA in salivary gland biopsies from patients with Sjogren's syndrome

Sjogren's syndrome (SS) is an autoimmune disorder characterized by lymphocytic infiltration of salivary and lacrimal glands. To determine whether Epstein-Barr virus (EBV) might play a role in the pathogenesis of this disorder, we used monoclonal antibodies and DNA probes to detect evidence of viral gene products and genomes in these patients' tissue biopsies and saliva. Cytoplasmic staining of epithelial cells (i.e., ductal and/or acinar cells) with monoclonal antibody against the EBV-encoded early antigen (EA-D) was noted in 8/14 salivary gland biopsies from SS patients. This antibody did not react with normal salivary glands or salivary gland tumors, nor with other tissues from SS patients. The reactive antigen in SS biopsies had a m.w. of 52,000 on the basis of immunoblotting experiments, similar to the EA-D antigen found in lymphoblastoid cells lytically infected with EBV. EBV DNA was detected in parotid biopsies from two SS patients in amounts ranging from 0.1 to 3 pg per 20 micrograms of cellular DNA. Southern blotting was used to demonstrate the reactivity with Bam V, Eco D, and Bam M probes. Parotid saliva samples from 8/20 SS patients contained EBV DNA detectable by slot blot hybridization. EBV DNA was not detected in saliva of age-matched controls, rheumatoid arthritis patients lacking sicca symptoms, or patients with benign parotid tumors. The presence of EBV in salivary gland and saliva samples was associated with clinically more severe SS, as manifested by extraglandular symptoms such as vasculitis, occurrence of "pseudolymphoma", and marked abnormalities in immunoglobulin levels. These results demonstrate an elevated content of EBV in salivary glands of SS patients, and suggest that this virus may play a role in pathogenesis.     

The structure of the termini of the Epstein-Barr virus as a marker of clonal cellular proliferation

The linear virion form of Epstein-Barr virus (EBV) DNA has variable numbers of direct tandem 500 bp repeats at each terminus. The terminal restriction endonuclease fragments and the fused terminal fragments in the intracellular episomal form are heterogeneous in size, and vary by increments of 500 bp. The structure of the termini of EBV in carcinomas of the nasopharynx and the parotid gland was compared with the EBV termini in monoclonal and polyclonal tissues or cell lines. A single band representing the EBV joined termini was detected in each of the carcinomas and in the monoclonal lymphoid proliferations. Polyclonal cell lines contained multiple forms of the joined termini. The detection of a homogeneous episomal population suggests that EBV-associated epithelial malignancies are clonal expansions of a single EBV-infected progenitor cell.     

Characterization of mouse salivary glands by water content and type

The thermoanalytical analysis was applied to samples of sublingual, submandibular and parotid glands from sexually mature mice of both sexes. Findings indicated that the three salivary glands show a behaviour of water release characteristic for each type of gland. Derivative thermogravimetry curves concerned with the sublingual and parotid glands belonging to male and female subjects exhibited overlapped results. As regards submandibular gland, instead, some differences emerged between subjects of different sex. Water content and types in sublingual, submandibular and parotid glands were discussed and related to the different morphological expression, histochemical reactivity and chemical composition of these organ tissues.     

High-risk human papillomavirus (HPV) in parotid lesions

Although several studies have reported that oropharyngeal infection with HPV may predispose to tumorigenesis, little is known about the etiological factors of salivary gland tumors and the presence of HPV. We studied 9 parotid lesions for HPV infection including an oncocytoma, an acinic cell carcinoma, a high-grade adenocarcinoma, a low-grade polymorphous adenocarcinoma, a Warthin's tumor and 2 pleomorphic adenomas, a lymphoepithelial cyst and a lipoma of the parotid gland. DNA was extracted from formalin-fixed and paraffin-embedded tissue sections. Solution PCR for HPV detection was performed using the GP5+/GP6+ primers, while HPV typing was carried out by multiplex PCR for HPV6, 11, 16, 18, and 33; positive samples were recorfirmed by PCR with specific primers for each type. Quantitative real-time PCR for the high-risk HPV genotypes 16, 18, 31, 33, 35, 52, 58 and 67 was also performed to quantitate the viral load. Finally, in situ PCR was employed with HPV16-specific primers by direct-detection method. Seven of the 9 parotid lesions were HPV positive while 6 of these 7 had been infected by HPV16 and/or HPV18 oncogenic types. High viral load of highrisk genotypes of HPV was found in the oncocytoma, in one of the pleomorphic adenomas, and in the Warthin's tumor. Finally, in situ PCR indicated that HPV16 amplification occurred in the salivary gland tumors. This is the first time that highrisk HPV genotypes are detected in these histological types of parotid lesions, suggesting the possible involvement of the virus in the disease.     

Cloning overlapping DNA fragments from the B95-8 strain of Epstein-Barr virus reveals a site of homology to the internal repetition

Overlapping, sheared DNA fragments from the B95-8 strain of Epstein-Barr virus were cloned in Charon 4A. Eleven recombinant phages plus one recombinant plasmid contained all of the sequences found in B95-8 virion DNA. Analysis of recombinant DNA molecules revealed a previously undetected site of homology to the internal repetition found in Epstein-Barr virus DNA. This site was adjacent to or at a site which was unstable when the recombinant DNA was propagated as phage DNA in procaryotic hosts.     

Increased expression of the enzyme beta 1-4-galactosyltransferase is associated with human parotid neoplasms

Human biopsy samples of parotid gland neoplasms were examined for the level of enzyme activity of the glycosyltransferase, beta 1-4-galactosyltransferase. An analysis of an adenoid cystic carcinoma, Warthin's tumor, mucoepidermoid carcinoma, and five pleomorphic adenomas all revealed elevated levels of enzyme activity. Evidence for plasma membrane beta 1-4-galactosyltransferase activity was provided by membrane fractionation as well as intact cell enzyme assays. On the other hand, the major protein of human saliva, salivary alpha-amylase, was substantially reduced in the same tissue compared with adjacent normal parotid gland tissue. The trichloroacetic acid-soluble proteins isolated from gland homogenates were also reduced in two of the carcinoma samples but increased in the pleomorphic adenomas. Additionally, the proliferation of these cells, in vitro, could be retarded by culturing in media containing the galactosyltransferase specific modifier protein, alpha-lactalbumin, or the nucleotide sugar, UDP-galactose.     

Human Salivary Micro-RNA in Patients with Parotid Salivary Gland Neoplasms

Background

Currently, clinical examination, ultrasound scanning (with or without fine needle aspiration cytology), preoperative CT-scan and MRI are available for the differential diagnosis of parotid gland swelling. A preliminary non-invasive salivary diagnostic tool may be helpful in the clinical decision making process. Altered salivary micro-RNA (miRNA) expression levels have been observed in saliva from patients with various cancers. Therefore, we investigated miRNA expression levels in saliva samples from patients with a parotid gland neoplasm using Human miRNA cards in comparison to controls.

Results

In the discovery phase, eight miRNAs were identified having different expression levels in patients compared to controls. In the validation phase, the differences in miRNA expression levels between patients and controls were confirmed for seven out of eight discovered miRNAs (p < 0.001). A combination of two miRNAs yielded a receiver-operator-characteristics curve with an AUC of 0.94 (95% CI: 0.87–1.00; sensitivity 91%; specificity 86%). Validation of discovered miRNAs in segregated collected parotid saliva revealed that expression of these miRNAs differ between whole saliva and parotid saliva.

Conclusions

A two miRNA combination can predict the presence of a parotid gland neoplasm. Furthermore, this study suggested that the identified, patient-specific, salivary miRNAs were not derived from the parotid gland itself.     

Precocious differentiation of mouse parotid glands and pancreas induced by hormones

Changes of alpha-amylase activity (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) in mouse parotid gland and pancreas were investigated during embryonic and postnatal development. Amylase activity in the parotid gland increased from around day 12 and reached the adult level on day 30. On the other hand, the activity in the pancreas increased during the last stage of gestation, decreased after birth, and then gradually increased from around day 15, reaching the adult level on day 35. Precocious differentiation of the parotid gland was induced by injections of hydrocortisone or thyroxine after birth, but these hormones did not have additive effects on the parotid gland. Injection of insulin had little effect when given alone, but suppressed the effects of the other two hormones on the gland. Only hydrocortisone increased the amylase activity in mouse pancreas during postnatal development, the other two hormones causing slight decrease in pancreatic amylase. Adrenalectomy and injection of hydrocortisone affected the parotid gland but not the pancreas of adult mice. These results suggest that hydrocortisone is involved in cytodifferentiations of the parotid gland and pancreas, and in maintenance of the parotid gland. Thyroxine may also be important in differentiation of the parotid gland.     

Effects of selective denervation and nerve growth factor on activity-mediated growth of rat parotid gland.

A dietary change from all liquid to solid food is followed by an average increase of 200% in [3H]thymidine uptake into the parotid gland of rat. However, removal of either the parasympathetic (Px) or the sympathetic (Sx) innervation to the parotid gland prior to the dietary change resulted in a partial inhibition of the increase; values for the parasympathectomized gland were 51% of those of the innervated gland, and values of the sympathectomized parotid gland were 42% of those of the innervated gland. Removal of both autonomic nerves caused a complete inhibition. Initiation of nerve growth factor (NGF) injection (1 microgram/kg body wt, two times daily for the 2 days of chow refeeding) at the time of chow refeeding had no effect on completely or partially denervated glands, and thymidine values for the denervated parotid gland of rats given NGF did not differ statistically from those of rats not given NGF. With parasympathectomy, sympathectomy, and complete denervation, weight of parotid gland was decreased from that of innervated glands, and administration of NGF had no effect on the denervation-induced decreases. The data show that both branches of the innervation to parotid gland must be intact to ensure a maximal increase in thymidine uptake with the dietary change from liquid to solid food. The level of the enzyme, beta 1-4 galactosyltransferase, involved in proliferation, also depended on the presence of intact nerves. Enzyme activity of innervated parotid gland showed an average increase of 200% with chow refeeding of rats previously on liquid diet, but with Px, the average increase was 51% of that of the innervated parotid, and with Sx, it was 41%. NGF administration did not cause any change in levels of this enzyme in any Px or PxSx parotid gland and only a small change in Sx parotid; it did increase levels of this enzyme in parotid of rats without submandibular-sublingual glands.     

Effects of nutritional conditions around weaning on carbonic anhydrase activity in the parotid gland and ruminal and abomasal epithelia of Holstein calves

Thirty-two male Holstein calves were used to investigate the effects of nutritional conditions around weaning and aging on carbonic anhydrase (CA) activity in the parotid gland and epithelium from the rumen and abomasum. We fed calf starter and lucerne hay as well as milk replacer (group N) or fed milk replacer either with (group S) or without (group M) administration of short-chain fatty acids (SCFA) through polypropylene tubing into the forestomach until 13 weeks of age. The diets were fed at 1000 hours and 1600 hours, and SCFA were administrated after milk replacer feeding at 1600 hours. Slaughter and tissue sampling were carried out between 1300 hours and 1430 hours at 1, 3, 7, 13, and 18 weeks of age. Tissue samples from five adult (1.5-2.0 years-old) Holstein steers were obtained from a local abattoir. In group N, CA activity in the parotid gland gradually and significantly increased toward the adult value, whilst in the epithelium from the rumen and abomasum, adult values were reached at 3 and 7 weeks of age, respectively. At 13 weeks, the activity for group N was significantly higher than that for the other two groups in the parotid gland, but there was no significant difference in the epithelium from the rumen and abomasum. The concentration of the carbonic isozyme VI in the parotid gland also changed with age but, in contrast to CA activity, had not reached adult levels by 13 weeks of age. In groups M and S, parotid saliva did not show any change toward an alkaline pH or toward a reciprocal change in the concentrations between Cl(-) and HCO(3)(-), even at 13 weeks of age. From these results we conclude that a concentrate-hay based diet around weaning has a crucial role in CA development in the parotid gland, but not in the epithelium of the rumen and abomasum.     

Precocious differentation of mouse parotid glands and pancreas induced by hormones

Changes of α-amylase activity (1,4-α-D-glucan glucanohydrolase, EC 3.2.1.1) in mouse parotid gland pancreas were investigated during embryonic and postnatal development. Amylase activity in the parotid gland increased from around day 12 and reached the adult level on day 30. On the other hand, the activity in the pancreas increased during the last stage of gestation, decreased after birth, and then gradually increased from around day 15, reaching the adult level on day 35. Precocious differentiation of the parotid gland was induceed by injections of hydrocortisone or thyroxine after birth, but these hormones did not have additive effects on the parotid gland. Injection of insulin had little effect when given alone, but suppressed the effects of the other two hormones on the gland. Only hydrocortisone increased the amylase activity in mouse pancreas during postnatal development, the other two hormones causing slight decrease in pancreatic amylase. Adrenalectomy and injection of hydrocortisone affected the parotid gland but not the pancreas of adult mice. These results suggest that hydrocortisone involved in cytodifferentiations of the parotid gland and pancreas, and in maintenance of the parotid gland. Thyroxine may also be important in differentiation of the parotid gland.     

High-resolution footprints of the DNA-binding domain of Epstein-Barr virus nuclear antigen 1.

The DNA-binding domain of Epstein-Barr virus nuclear antigen 1 was found by hydroxyl radical footprinting to protect backbone positions on one side of its DNA-binding site. The guanines contacted in the major groove by the DNA-binding domain of Epstein-Barr virus nuclear antigen 1 were identified by methylation protection. No difference was found in the interaction of the DNA-binding domain of Epstein-Barr virus nuclear antigen 1 with tandemly repeated and overlapping binding sites.     

Modulation of rat parotid cell alpha-adrenergic responsiveness at a step subsequent to receptor activation

Parotid gland acinar cells, prepared from 12- and 24-month-old rats, show decreased physiological responsiveness to alpha-adrenergic stimulation in vitro compared to cells from 3-month-old rats. K+ efflux, an index of water and electrolyte secretion, was approximately 35% lower with 12- and 24-month-old parotid cells. No loss of alpha-adrenergic receptors, their binding affinity for specific alpha-adrenergic ligands, or their relative subtype distribution, accompanied the diminished exocrine function. Conversely, a significant reduction in alpha-adrenergic-mediated phospholipid turnover in, and 45Ca2+ efflux from, parotid cells of older rats was observed. These changes in phospholipid metabolism and Ca2+ flux were parallel to changes seen in K+ efflux as judged by dose-response studies. When the alpha-adrenergic receptor was by-passed by using the Ca2+-ionophore A-23187 to elicit K+ efflux, young and old parotid cells were equally responsive. In aggregate the findings suggest that parotid gland cells from older rats display an altered alpha-adrenergic signal transduction mechanism at a site between the receptor and phospholipid turnover/Ca2+ mobilization.     

The safe face lift with bony anatomic landmarks to elevate the SMAS

The risk for facial nerve injury has been reported to be increased with the inclusion of superficial musculoaponeurotic system (SMAS) elevation as compared with a skin-only face lift. The facial nerve courses through the parotid gland. The SMAS is elevated superficial to the parotid gland. However, in elevating the SMAS anterior to the parotid gland, the facial nerve is at risk of injury where its branches emerge from the anterior edge of the parotid gland. The purpose of this study was to identify bony anatomic landmarks to predict the location of the anterior edge of the parotid gland to avoid injury to the facial nerve branches as they exit the parotid gland. The authors dissected 20 cadaver face halves to determine bony landmarks-the masseteric tuberosity and the inferior lateral orbital rim-to predict the location of the anterior parotid edge. Then they measured the anterior edge of the parotid gland in relation to the vector formed between these two bony landmarks. They identified and measured the most anterior portion of the parotid gland in relation to this vector. Then the most posterior aspect of the parotid gland in relation to this vector was measured. In the 20 dissections, the authors found the most anterior portion of the parotid gland to be 2.7 +/- 1.0 mm anterior to the vector from the inferior lateral orbital rim to the masseteric tuberosity. The most posterior part of the anterior edge of the parotid gland in relation to this vector was found to be 1.0 +/- 1.5 mm posterior to this vector. The parotid gland measured an average of 38.8 +/- 3.5 mm in width from the tragus to the anterior parotid edge. In elevating the SMAS with a face lift, the facial nerve branches can be predicted to exit the anterior edge of the parotid gland, which can be located 38.8 mm anterior to the tragus and near the vector from the inferior lateral orbital wall to the masseteric tuberosity.     

Squamous cell carcinoma of the parotid gland

Squamous cell carcinoma involving the parotid gland is an aggressive and rapidly advancing lesion which if not recognized and treated early will result in a high morbidity and mortality. We reviewed 30 patients with squamous cell carcinoma involving the parotid gland. Twenty-four patients had had previous epidermoid skin lesions in an area known to drain to the parotid gland and three resulted from direct extension into the gland from an overlying skin carcinoma, whereas only three were primary lesions of the gland. Patients who presented with involvement of the gland more than 4 months after excision of the skin lesion had a poor prognosis. Patients with epidermoid skin cancer in areas with a propensity to secondarily involve the parotid gland must be closely followed after treatment of the primary skin lesion.