Search

Objective The goals of this study were to (i) establish a useful miniature pig (minipig) model for irradiation-induced oral mucositis and (ii) evaluate the effect of Tempol to prevent its development. Methods and Materials Minipigs were irradiated with 6 Gy for five consecutive days targeting the entire oral cavity. To prevent radiation damage, minipigs were treated with 30 mg kg−1 Tempol 10 min before irradiation (n = 4), while the radiation-alone group was similarly injected with saline (n = 4). Lesions were graded using an oral mucositis score and visual inspection every 3 days, and biopsy of multiple sites was performed at day 18. Weight and chest and abdominal circumferences were measured every 3 days. Results Lesions began about 12 days after the first irradiation fraction and healed about 30 days after irradiation. Epithelial thickness was calculated on the lingual and buccal mucosa on the 18th day after the first irradiation fraction. Tempol provided modest protection from ulceration after irradiation using this treatment strategy. Conclusions This study established a useful large animal model for irradiation-induced oral mucositis and showed modest beneficial effects of Tempol in limiting tissue damage. The latter finding may be potentially valuable in preventing oral mucositis in patients receiving irradiation for head and neck cancers.

We evaluated late effects of AdhAQP1 administration in five subjects in a clinical trial for radiation-induced salivary hypofunction (http://www.clinicaltrials.gov/ct/show/NCT00372320?order=). All were identified as initially responding to human aquaporin-1 (hAQP1) gene transfer (Baum et al, 2012). They were followed for 3-4 years after AdhAQP1 delivery to one parotid gland. At intervals we examined salivary flow, xerostomic symptoms, saliva composition, vector presence and efficacy in the targeted gland, clinical laboratory data, and adverse events. All displayed marked increases (71-500% above baseline) in parotid flow 3-4.7 years after treatment, with improved symptoms for ~ 2-3 years. There were some changes in [Na+] and [Cl−] consistent with elevated salivary flow, but no uniform changes in secretion of key parotid proteins. There were no clinically significant adverse events, nor consistent negative changes in laboratory parameters. One subject underwent a core needle biopsy of the targeted parotid gland 3.1 years post treatment and displayed evidence of hAQP1 protein in acinar, but not duct, cell membranes. All subjects responding to hAQP1 gene transfer initially had benefits for much longer times. First generation adenoviral vectors typically yield transit effects, but these data show beneficial effects can continue years after parotid gland delivery.

Head and neck irradiation (IR) during cancer treatment causes by-stander effects on the salivary glands leading to irreversible loss of saliva secretion. The mechanism underlying loss of fluid secretion is not understood and no adequate therapy is currently available. Delivery of an adenoviral vector encoding human aquaporin-1 (hAQP1) into the salivary glands of human subjects and animal models with radiation-induced salivary hypofunction leads to significant recovery of saliva secretion and symptomatic relief in subjects. To elucidate the mechanism underlying loss of salivary secretion and the basis for AdhAQP1-dependent recovery of salivary gland function we assessed submandibular gland function in control mice and mice 2 and 8 months after treatment with a single 15-Gy dose of IR (delivered to the salivary gland region). Salivary secretion and neurotransmitter-stimulated changes in acinar cell volume, an in vitro read-out for fluid secretion, were monitored. Consistent with the sustained 60% loss of fluid secretion following IR, a carbachol (CCh)-induced decrease in acinar cell volume from the glands of mice post IR was transient and attenuated as compared with that in cells from non-IR age-matched mice. The hAQP1 expression in non-IR mice induced no significant effect on salivary fluid secretion or CCh-stimulated cell volume changes, except in acinar cells from 8-month group where the initial rate of cell shrinkage was increased. Importantly, the expression of hAQP1 in the glands of mice post IR induced recovery of salivary fluid secretion and a volume decrease in acinar cells to levels similar to those in cells from non-IR mice. The initial rates of CCh-stimulated cell volume reduction in acinar cells from hAQP1-expressing glands post IR were similar to those from control cells. Altogether, the data suggest that expression of hAQP1 increases the water permeability of acinar cells, which underlies the recovery of fluid secretion in the salivary glands functionally compromised post IR.

Patients frequently experience a loss of salivary function following irradiation (IR) for the treatment of an oral cavity and oropharyngeal cancer. Herein, we tested if transfer of fibroblast growth factor-2 (FGF2) cDNA could limit salivary dysfunction after fractionated IR (7.5 or 9 Gy for 5 consecutive days to one parotid gland) in the miniature pig (minipig). Parotid salivary flow rates steadily decreased by 16 weeks post-IR, whereas blood flow in the targeted parotid gland began to decrease ~3 days after beginning IR. By 2 weeks, post-IR salivary blood flow was reduced by 50%, at which point it remained stable for the remainder of the study. The single preadministration of a hybrid serotype 5 adenoviral vector encoding FGF2 (AdLTR2EF1a-FGF2) resulted in the protection of parotid microvascular endothelial cells from IR damage and significantly limited the decline of parotid salivary flow. Our results suggest that a local treatment directed at protecting salivary gland endothelial cells may be beneficial for patients undergoing IR for oral cavity and oropharyngeal cancer.

Oral Diseases (2010) 17, 77–82 Objectives: Published studies of gene transfer to mouse salivary glands have not employed the parotid glands. Parotid glands are the likely target tissue for most clinical applications of salivary gene transfer. The purpose of the present study was to develop a convenient and reproducible method of retroductal gene transfer to mouse parotid glands. Methods: The volume for vector delivery was assessed by infusion of Toluidine Blue into Stensen’s ducts of Balb/c mice after direct intraoral cannulation. Recombinant, serotype 5 adenoviral vectors, encoding either firefly luciferase or human erythropoietin (hEpo), were constructed and then administered to parotid glands (107 vector particles/gland). Transgene expression in vivo was measured by enzyme activity (luciferase) or an enzyme-linked immunosorbent assay (hEpo). Vector biodistribution was measured by real-time quantitative (Q) PCR. Results: The chosen volume for mouse parotid vector delivery was 20 μL. Little vector was detected outside of the targeted glands, with both QPCR and luciferase assays. Transgene expression was readily detected in glands (luciferase, hEpo), and serum and saliva (hEpo). Most secreted hEpo was detected in saliva. Conclusion: These studies show that mouse parotid glands can be conveniently and reproducibly targeted for gene transfer, and should be useful for pre-clinical studies with many murine disease models.

Salivary glands are useful target organs for local and systemic gene therapeutics. For such applications, the regulation of transgene expression is important. Previous studies by us in murine submandibular glands showed that a rapamycin transcriptional regulation system in a single serotype 2, adeno-associated viral (AAV2) vector was effective for this purpose. This study evaluated if such a vector was similarly useful in rhesus macaque parotid glands.A recombinant AAV2 vector (AAV-TF-RhEpo-2.3w), encoding rhesus erythropoietin (RhEpo) and a rapamycin-inducible promoter, was constructed. The vector was administered to macaques at either of two doses [1.5 x 10(11) (low dose) or 1.5 x 10(12) (high dose) vector genomes] via cannulation of Stensen's duct. Animals were followed up for 12-14 weeks and treated at intervals with rapamycin (0.1 or 0.5 mg kg(-1)) to induce gene expression. Serum chemistry, hematology, and RhEpo levels were measured at interval.AAV-TF-RhEpo-2.3w administration led to low levels of rapamycin-inducible RhEpo expression in the serum of most macaques. In five animals, no significant changes were seen in serum chemistry and hematology values over the study. One macaque, however, developed pneumonia, became anemic and subsequently required euthanasia. After the onset of anemia, a single administration of rapamycin led to significant RhEpo production in this animal.Administration of AAV-TF-RhEpo-2.3w to macaque parotid glands was generally safe, but led only to low levels of serum RhEpo in healthy animals following rapamycin treatment.

Salivary glands are potentially useful target sites for multiple clinical applications of gene transfer. Previously, we have shown that serotype 2 adeno-associated viral (AAV2) vectors lead to stable gene transfer in the parotid glands of rhesus macaques. As AAV5 vectors result in considerably greater transgene expression in murine salivary glands than do AAV2 vectors, herein we have examined the use of AAV5 vectors in macaques at two different doses (n = 3 per group; 10(10) or 3 x 10(11) particles per gland). AAV5 vector delivery, as with AAV2 vectors, led to no untoward clinical, hematological or serum chemistry responses in macaques. The extent of AAV5-mediated expression of rhesus erythropoietin (RhEpo) was dose-dependent and similar to that seen with an AAV2 vector. However, unlike results with the AAV2 vector, AAV5 vector-mediated RhEpo expression was transient. Maximal expression peaked at day 56, was reduced by approximately 80% on day 84 and thereafter remained near background levels until day 182 (end of experiment). Quantitative PCR studies of high-dose vector biodistribution at this last time point showed much lower AAV5 copy numbers in the targeted parotid gland (approximately 1.7%) than found with the same AAV2 vector dose. Molecular analysis of the conformation of vector DNA indicated a markedly lower level of concatamerization for the AAV5 vector compared with that of a similar AAV2 vector. In addition, cellular immunological studies suggest that host response differences may occur with AAV2 and AAV5 vector delivery at this mucosal site. The aggregate data indicate that results with AAV5 vectors in murine salivary glands apparently do not extend to macaque glands.

Salivary glands (SGs) appear to be a useful target site for gene therapeutics. The ability to control transgene expression is essential for clinical application. Previously, in a proof-of-concept study, we have shown that the rapamycin-inducible transcriptional regulation system can regulate protein expression after adenoviral-mediated gene transfer to SGs. To evaluate the potential ability to utilize this regulatory system for long-term control of transgene expression in this tissue, we employed a 'third generation', single adenoassociated serotype 2 viral (AAV2) vector encoding human erythropoietin (hEPO) under the control of a rapamycin-inducible promoter. The vector, rAAV-TF2.3-hEPO (10(10) particles/animal), was delivered to mouse SGs. No detectable increase in serum hEPO or hematocrit levels was observed in the absence of rapamycin administration. However, rapamycin induced elevation of serum hEPO levels, as well as concomitant hematocrit changes, that were dose-dependent, completely reversible, and relatively stable over the course of this study (6 months), with no appreciable change in rapamycin responsiveness. Our results suggest that the rapamycin transcriptional regulation system delivered in a single AAV2 vector to SGs may be valuable for systemic protein replacement applications.

Transgene-encoded therapeutic secretory proteins can be efficiently secreted from salivary glands into saliva or the bloodstream after adenoviral (Ad)-mediated gene transfer. Since transgene expression from conventional vectors is typically unregulated, we evaluated the rapamycin-based dimerizer regulation system for control of transgene expression in, and consequent exocrine protein secreted from, rat salivary glands. We used human growth hormone (hGH) as a surrogate exocrine secretory protein. Two Ad vectors, Ad C4ZF3, encoding activation and DNA binding domain fusion polypeptides, and Ad Z12-I-GH-2, encoding hGH, were constructed and shown useful in vitro. Thereafter, both vectors were delivered into submandibular glands by retroductal infusion. After 24 h, rapamycin (0, 1, 3 or 10 mg/kg) was administered, and 20 h later hGH levels in saliva were determined. Salivary hGH levels were rapamycin concentration dependent. At a rapamycin dose of 10 mg/kg, total salivary hGH was 693+/-197 ng and the hGH concentration in saliva was 4.6+/-1.3 microg/ml. Over a 16-day experimental period, three separate administrations of rapamycin (3 mg/kg) induced distinct elevations of salivary hGH (approximately 100-200 ng total hGH) that were entirely rapamycin dependent. This study demonstrates for the first time pharmacological control of transgenic exocrine protein production and presence in saliva after salivary gland gene transfer, and the potential for its application to the management of oral, oropharyngeal and upper gastrointestinal tract disorders.

OBJECTIVE: To test the ability of two cationic lipoplexes, Vaxfectin and GAP-DLRIE/DOPE, to facilitate transfection and elicit immune responses from plasmid DNAs (pDNAs) after retrograde instillation into salivary glands. METHODS: Two pDNA expression vectors encoding either the influenza NP protein or human growth hormone (hGH) were complexed with the cationic lipid transfection reagents, GAP-DLRIE/DOPE or Vaxfectin, and delivered to the submandibular glands of rats. Samples from rats receiving the influenza NP protein pDNA and cationic lipoplexes were analyzed for anti-influenza NP-specific IgG1, IgG2a, and IgG2b in serum, and IgA in saliva, by an enzyme- linked immunosorbent assay (ELISA). Cytotoxic T-cell lymphocyte (CTL) assays were also performed. Transgene protein expression (hGH) was determined from extracts of submandibular glands of rats receiving hGH lipoplexes. RESULTS: Serum antibodies (IgG) against the NP protein developed and were highest in all rats vaccinated with GAP-DLRIE/DOPE or Vaxfectin. The major serum IgG subclass stimulated by this route of immunization was IgG2b, followed by IgG2a. CTL assay results showed statistically significantly higher percentage killing in the Vaxfectin group than controls (P pDNA/water), the difference with those receiving pDNA/GAP-DLRIE/DOPE (∼10-fold > pDNA/water) was not significant. CONCLUSIONS: Retrograde instillation of pDNA complexed with Vaxfectin into the salivary glands can stimulate cytotoxic and humoral responses to the influenza NP protein antigen. Optimization of the conditions required to stimulate humoral and secretory antibody formation may facilitate use of this tissue for specific clinical applications of pDNA immunization.

It is possible that, during primary saliva formation, aquaporins (AQPs) facilitate transcellular water flow across acinar cells to the lumina of salivary glands. In the rat submandibular gland (rSMG) AQP5 is localized in the apical membranes of acinar cells. The presence of a basolateral AQP in the same cell type has not been reported. We have therefore used immunofluorescence confocal microscopy to determine the subcellular localization of a newly discovered aquaporin, AQP8, in rSMG epithelial cells. The antibodies we used were made against the amino- or carboxyl-terminus (anti-rAQP8NT and anti-rAQP8CT, respectively) of an AQP8 cloned from rat pancreas and liver (rAQP8). Two lines of evidence suggest that both antibodies are suitable for immunolocalization studies. First, results of immunofluorescence confocal microscopy studies show that both antibodies bind to the plasma membranes of 293 cells infected with an adenovirus encoding rAQP8. Second, results of immunoblots of membranes from infected cells suggest that both antibodies bind to glycosylated and non-glycosylated forms of rAQP8. When tested in frozen sections of rSMG, we could not detect the binding of anti-rAQP8NT to any membranes. In contrast, anti-rAQP8CT binds to the basolateral membranes of acinar (but not ductal) epithelia, suggesting that rAQP8 resides in the basolateral membranes of acinar cells. Lack of anti-rAQPNT binding to basolateral membranes suggests that this epitope is not available in the membranes. Our evidence for the basolateral localization of rAQP8 in acinar cells, coupled with previous findings that AQP5 is localized apically in the same cells, raises the possibility that water crosses the acinar epithelium through these channels during primary saliva formation.

Adenoviral vectors effectively transfer genes to rat salivary glands. However, potent immune responses limit their use in vivo. Mice offer more opportunities than rats for the study of these immune processes. We first established conditions for infection of mouse salivary glands, with an adenoviral vector. The effects of time, viral dose, viral diluent buffer volume, and dexamethasone on expression of a transgene, luciferase, were determined by means of the recombinant vector AdCMVluc. Optimal luciferase expression was observed when the vector was suspended in 50 μL of buffer. This volume completely filled the gland parenchyma and slightly distended the capsule. Dexamethasone increased immediate transgene expression and reduced the acute inflammation one day following viral administration, but did not alter subsequent mononuclear inflammation or transgene expression 14 or 28 days later. An adenoviral vector encoding either antiinflammatory cytokine IL-4 or IL-10 was co-administered with AdCMVluc to increase transgene expression at 14 and 28 days. While this strategy did not extend the duration of luciferase expression, co-administration of AdCMVIL-10 with AdCMVluc almost completely eliminated the chronic inflammatory infiltrate in the glands after 28 days. This study demonstrates that adenoviral-mediated gene transfer to mouse submandibular glands is possible by intraductal cannulation and that reduction of either the acute or chronic inflammatory infiltrates was insufficient to increase long-term transgene expression in this tissue.

Sjogren's syndrome is a chronic systemic disease that primarily affects the salivary and lacrimal glands. The pathogenesis of Sjogren's syndrome is unknown. We hypothesize that reduced somatostatin activity is an important factor in promoting immune dysregulation in patients affected by Sjogren's syndrome. Somatostatin is a multifunctional peptide with potent immunomodulatory properties. Its effects include reduced lymphocytic activity, reduced gastric and intestinal secretions, activation of the hypothalamic-pituitary axis, and anti-inflammatory action, all opposite to the general presentation in Sjogren's syndrome. We suggest that the activity of somatostatin is low in patients affected by this disease, and this contributes significantly to the pathology observed.

Published studies of gene transfer to mouse salivary glands have not employed the parotid glands. Parotid glands are the likely target tissue for most clinical applications of salivary gene transfer. The purpose of the present study was to develop a convenient and reproducible method of retroductal gene transfer to mouse parotid glands.The volume for vector delivery was assessed by infusion of Toluidine Blue into Stensen's ducts of Balb/c mice after direct intraoral cannulation. Recombinant, serotype 5 adenoviral vectors, encoding either firefly luciferase or human erythropoietin (hEpo), were constructed and then administered to parotid glands (10(7) vector particles/gland). Transgene expression in vivo was measured by enzyme activity (luciferase) or an enzyme-linked immunosorbent assay (hEpo). Vector biodistribution was measured by real-time quantitative (Q) PCR.The chosen volume for mouse parotid vector delivery was 20μL. Little vector was detected outside of the targeted glands, with both QPCR and luciferase assays. Transgene expression was readily detected in glands (luciferase, hEpo), and serum and saliva (hEpo). Most secreted hEpo was detected in saliva.These studies show that mouse parotid glands can be conveniently and reproducibly targeted for gene transfer, and should be useful for pre-clinical studies with many murine disease models.

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.

The non-obese diabetic (NOD) mouse is not only a widely used model for diabetes mellitus type I, but also for the chronic autoimmune disease Sjögren's syndrome (SS), mainly affecting salivary and lacrimal glands. We studied the efficacy of local recombinant serotype 2 adeno-associated viral (rAAV2) vector transfer of immunomodulatory transgenes to alter the SS-like disease in NOD mice. Data collected over a 2-year period indicated a changing SS phenotype in these mice and this phenomenon was investigated.10(10) particles rAAV2LacZ/gland were delivered to both submandibular glands (SMGs) of NOD/LtJ mice at 8 weeks (before sialadenitis onset) of age. Salivary flow rates were determined at 8 weeks and time of killing. Blood glucose levels and body weights were measured weekly. After killing, saliva and SMGs were harvested. Analyses of salivary output, inflammatory infiltrates (focus score), SMG cytokine profile, body weight, and diabetes mellitus status were performed. Data from six different experimental studies over 2 years were analyzed and compared.Salivary flow rate, focus score, and SMG cytokines interleukin (IL)-2, IL-4, IL-6, IL-10, IL-12(p70), tumor necrosis factor-alpha and IFNgamma showed changes over time. There were no differences for body weight, diabetes mellitus prevalence, or blood glucose level of non-diabetic mice.This retrospective report is the first to describe longitudinal variability in the NOD mouse as a model for SS. We advise other investigators to continuously monitor the SS phenotype parameters and include appropriate controls when studying this disease in NOD mice.

OBJECTIVE: We examined the toxicity and biodistribution associated with a single administration of a first-generation, serotype 5, adenoviral vector encoding human growth hormone (hGH; AdCMVhGH) to a single rat submandibular gland in the presence of hydroxychloroquine (HCQ). Previously, we showed that hGH is primarily secreted into saliva (approximately ninefold serum level) when expressed as a transgene in salivary glands (e.g. Baumet al,1999),butadministrationofHCQsubstantially increases the hGH levels secreted into the bloodstream (Hoque et al, 2001). A potential application of this observation is for patients with adult hGH deficiency. METHODS: Six groups of male and female adult rats (n = 12 each) were studied, with zero to 1.5 · 10 11 particles of AdCMVhGH, ±HCQ, administered retroductally. Multiple clinical and pathological parameters, as well as vector tissue distribution, were assessed. RESULTS: All animals survived until the scheduled day of sacrifice, and essentially no untoward events were observed clinically or at gross necropsy. We observed no vector-related effects on clinical hematology evaluations and a single, transient significant change on clinical chemistry evaluations (increased serum globulin levels). Three days after AdCMVhGH administration, the vector distributed to all tissues analyzed with the exception of gonads and heart. By day 29, most organs, other than the targeted and contralateral submandibular glands, were negative for the presence of vector. On day 3, none of the animals tested positive for the presence of replication competent adenovirus in either their blood or saliva. CONCLUSION: Salivary gland delivery of AdCMVhGH ±HCQ appears associated with limited toxicity in rats. Oral Diseases (2006) 12, 137–144

Previous studies from our laboratory showed that therapeutic levels of transgene expression were obtained from mouse submandibular glands after retro-ductal administration of relatively low doses (109 particles/animal) of a recombinant serotype 2 adeno- associated viral vector (rAAV2) encoding human erythropoietin (hEPO). The recombinant hormone was preferentially secreted into the bloodstream rather than into saliva. Serum hEPO levels steadily increased for an 8|[ndash]|12 week period and remained relatively stable until the end of the experiment (54 weeks; Voutetakis et al, PNAS, 2004).

Therapeutic irradiation for head and neck cancer, and the autoimmune disease Sjogren's syndrome, lead to loss of salivary parenchyma. They are the two main causes of irreversible salivary gland hypofunction. Such patients cannot produce adequate levels of saliva, leading to considerable morbidity. We are working to develop an artificial salivary gland for such patients. A major problem in this endeavor has been the difficulty in obtaining a suitable autologous cellular component. This article describes a method of culturing and expanding primary salivary cells obtained from human submandibular glands (huSMGs) that is serum free and yields cells that are epithelial in nature. These include morphological (light and transmission electron microscopy [TEM]), protein expression (immunologically positive for ZO-1, claudin-1, and E-cadherin), and functional evidence. Under confocal microscopy, huSMG cells show polarization and appropriately localize tight junction proteins. TEM micrographs show an absence of dense core granules, but confirm the presence of tight and intermediate junctions and desmosomes between the cells. Functional assays showed that huSMG cells have high transepithelial electrical resistance and low rates of paracellular fluid movement. Additionally, huSMG cells show a normal karyotype without any morphological or numerical abnormalities, and most closely resemble striated and excretory duct cells in appearance. We conclude that this culture method for obtaining autologous human salivary cells should be useful in developing an artificial salivary gland.

To test the ability of two cationic lipoplexes, Vaxfectin and GAP-DLRIE/DOPE, to facilitate transfection and elicit immune responses from plasmid DNAs (pDNAs) after retrograde instillation into salivary glands.Two pDNA expression vectors encoding either the influenza NP protein or human growth hormone (hGH) were complexed with the cationic lipid transfection reagents, GAP-DLRIE/DOPE or Vaxfectin, and delivered to the submandibular glands of rats. Samples from rats receiving the influenza NP protein pDNA and cationic lipoplexes were analyzed for anti-influenza NP-specific IgG1, IgG2a, and IgG2b in serum, and IgA in saliva, by an enzyme- linked immunosorbent assay (ELISA). Cytotoxic T-cell lymphocyte (CTL) assays were also performed. Transgene protein expression (hGH) was determined from extracts of submandibular glands of rats receiving hGH lipoplexes.Serum antibodies (IgG) against the NP protein developed and were highest in all rats vaccinated with GAP-DLRIE/DOPE or Vaxfectin. The major serum IgG subclass stimulated by this route of immunization was IgG2b, followed by IgG2a. CTL assay results showed statistically significantly higher percentage killing in the Vaxfectin group than controls (P0.05). No rats developed IgA antibodies to NP protein in saliva. Animals receiving plasmid encoding hGH and either lipoplex expressed significantly higher amounts of hGH compared with those receiving the hGH plasmid and water. Although hGH expression was higher in the animals receiving pDNA/Vaxfectin (approximately 30-foldpDNA/water), the difference with those receiving pDNA/GAP-DLRIE/DOPE (approximately 10-foldpDNA/water) was not significant.Retrograde instillation of pDNA complexed with Vaxfectin into the salivary glands can stimulate cytotoxic and humoral responses to the influenza NP protein antigen. Optimization of the conditions required to stimulate humoral and secretory antibody formation may facilitate use of this tissue for specific clinical applications of pDNA immunization.

Saliva provides the principal protective milieu for teeth by modulating oral microbial ecosystems and reversing the initial phases of caries development. Patients with inadequate salivary function are at increased risk for dental decay. Therefore, it is likely that therapies that increase overall fluid output of these individuals will reverse early carious lesions. The most common causes of salivary dysfunction are medication usage, Sjögren's syndrome, and damage of salivary parenchyma during therapeutic irradiation. For patients with remaining functional acinar tissue, treatment with the parasypathomimetic secretogogues pilocarpine and Cevimeline may provide relief. However, these medications do not benefit all patients. The possibilities of using gene therapy and tissue engineering to develop treatments for those with severe salivary dysfunction are discussed.

The activated insulin-like growth factor-I receptor (IGF-IR) is implicated in mitogenesis, transformation, and anti-apoptosis. To investigate the role of the IGF-IR in protection from UV-mimetic-induced DNA damage, 4-nitroquinoline N-oxide (4-NQO) was used. In this study we show that the activation of the IGF-IR is capable of rescuing NWTb3 cells overexpressing normal IGF-IRs from 4-NQO-induced DNA damage as demonstrated by cellular proliferation assays. This action was specific for the IGF-IR since cells expressing dominant negative IGF-IRs were not rescued from 4-NQO UV-mimetic treatment. DNA damage induced by 4-NQO in NWTb3 cells was significantly decreased after IGF-IR activation as measured by comet assay. IGF-I was also able to overcome the cell cycle arrest, observed after 4-NQO treatment, thereby enhancing the ability of NWTb3 cells to enter S phase. Interestingly, the p38 mitogen-activated protein kinase pathway was shown to represent the main signaling pathway involved in the IGF-IR-mediated rescue of UV-like damaged cells. The ability of the IGF-IR to induce DNA repair was also demonstrated by infecting NWTb3 cells with UV-irradiated adenovirus. Activation of the IGF-IR resulted in enhanced beta-galactosidase reporter gene activity demonstrating repair of the damaged DNA. This study indicates a direct role of the IGF system in the rescue of damaged cells via DNA repair.

Sjögren's syndrome is a chronic systemic disease that primarily affects the salivary and lacrimal glands. The pathogenesis of Sjögren's syndrome is unknown. We hypothesize that reduced somatostatin activity is an important factor in promoting immune dysregulation in patients affected by Sjögren's syndrome. Somatostatin is a multifunctional peptide with potent immunomodulatory properties. Its effects include reduced lymphocytic activity, reduced gastric and intestinal secretions, activation of the hypothalamic-pituitary axis, and anti-inflammatory action, all opposite to the general presentation in Sjögren's syndrome. We suggest that the activity of somatostatin is low in patients affected by this disease, and this contributes significantly to the pathology observed.

The potential applications of gene transfer technology to all branches of medicine are increasing. It is quite likely that within the next 10-20 years surgical practice routinely will utilize gene transfer, at least adjunctively. The purpose of this review is to familiarize the oral and maxillofacial surgeon with this technology. Studies performed with salivary glands in animal models are presented as examples of proof of concept.

In this report, we have reviewed several of our recent studies using replication-deficient recombinant adenoviruses encoding either aquaporin 1 or 5. The aquaporins are a relatively recently described family of water channels that mediate osmotically-driven transmembrane water permeability. Recombinant adenoviruses are highly efficient gene transfer vectors and readily result in significant levels of transgene expression. These aggregate studies provide examples of how such "tools" can be used in a novel way to address problems of both clinical and biological significance.

This study examined the effect of gamma-irradiation (5 and 10 Gy) on the human submandibular cell line (HSG). Radiation treatment (5 Gy and 10 Gy) induced a dose-dependent decrease in cell proliferation, with a G2/M arrest of the cell cycle, and an increase in cell death (cells with2n DNA increased from 7% in control cells to 34% and 40% in 5 and 10 Gy irradiated cells, respectively). [Ca2+]i measurements demonstrated that the status of internal Ca2+ stores, and muscarinic receptor-mediated Ca2+ mobilization, in irradiated cells was comparable to that in non-irradiated cells. These data suggest that 1) irradiated HSG cells maintain normal physiology and 2) internal Ca2+ store depletion does not account for the decreased cell proliferation. To manipulate the radiation-induced cell cycle arrest, we examined the effect of the transcription factor E2F1, which has been shown to induce cell cycle progression in HSG cells (Lillibridge and O'Connell, 1997, J. Cell. Physiol., 1 72:343-350). The ability of irradiated HSG cells to express and appropriately route proteins was demonstrated by using adenovirus-mediated expression of beta-galactosidase, alpha1-antitrypsin, and aquaporin-1. Infection of HSG cells with an adenoviral vector encoding E2F1, either 12 h before or immediately following irradiation, but not post-irradiation, induced maintenance of cells in the S phase of the cell cycle, reduced the number of cells arrested at G2/M, and decreased the rate of appearance of cells with2n DNA. While the mechanism of irradiation-induced cell death has not yet been confirmed, these data suggest that expression of the E2F1 gene product in HSG cells can be a useful strategy to manipulate cell cycle events and reduce the initial loss of cells due to radiation.

The diagnosis and treatment of Sjögren's syndrome, which poses many severe complications, should be of critical interest to dentists, who are often the first practitioners to detect symptoms. Dentistry is an integral part of health care delivery for patients with this condition. Management of Sjögren's syndrome can be seen as a model for the expanded scope of dental care in the future.

We have previously suggested that although salivary glands function in an exocrine manner they might none the less offer a useful way to deliver therapeutic proteins systemically. As a direct functional test of this hypothesis, we constructed a recombinant adenovirus (AdCMVhGH) encoding human growth hormone (hGH) and then studied the biological action of hGH produced following transfer of the hGH gene to rat submandibular glands. At 48 h following infusion of AdCMVhGH into these glands via cannulation of the main excretory duct, serum levels of hGH were approximately 16 ng/ml and rat insulin-like growth factor-1 was elevated approximately 25%. Moreover, serum chemistry profiles of rats subjected to in vivo gene transfer displayed alterations in the BUN:creatinine ratio and triglyceride levels presumably reflecting the anabolic actions of the hGH. These results provide the first demonstration of systemic biological action from a transgene product secreted in an endocrine fashion from the salivary glands.

We have used a human salivary gland cell line (HSG) as a possible in vitro model to evaluate the effects of IFN-gamma on human salivary gland epithelium (Wu et al., 1994, 1996, 1997). In the present study, we examined the JAK-STAT signal-transduction pathway in IFN-gamma-treated HSG cells. We demonstrate that JAK2 and Stat1 are phosphorylated at tyrosine residues in a time- and concentration-dependent manner following exposure to IFN-gamma. In addition, we show that activation of this signalling pathway is decreased by the addition of a blocking antibody to the IFN-gamma receptor. The same maneuver is also able to reduce by approximately 50-70% the surface expression of two IFN-gamma-induced immunoregulatory molecules: HLA-DR and ICAM-1. These results demonstrate that the JAK2 and Stat1 signalling pathway is active in salivary-derived epithelial cells and may contribute to their immunopathologic destruction.

This study compared the localization of the alpha(v)beta3/5 integrins in the different cell types of the rat submandibular gland with the susceptibility of these cells to infection by an intraductally administered replication-deficient recombinant type 5 adenovirus coding for rat aquaporin 5 (termed AdrAQP5). We used a panel of antibodies and immunohistochemical procedures to determine the luminal membrane distribution of the integrins. The alpha(v)beta3/5 integrin subunits were present in luminal membranes of all ductal cell types and acini; however, the alpha(v) subunit was found to a lesser extent in the acini. After AdrAQP5 infection, the expression of AQP5 exhibited a similar, though not identical, cellular localization to that seen for alpha(v)beta3/5 integrins. AdrAQP5 infected all cell types in these glands, except excretory ducts, after intraductal administration, directing the transient expression of AQP5. In addition, the localization of alpha(v)beta3/5 integrins was examined in rabbit, monkey, and human submandibular and parotid glands. Although there were some interspecies differences, glands generally displayed the presence of these integrin subunits on luminal membranes of ductal cells and most acinar cells. These findings show that the presence of alpha(v)beta3/5 integrins on the luminal membranes of rat salivary epithelial cells is associated with the susceptibility of these cells to act as targets for adenoviral-mediated gene transfer. However, the abundance of alpha(v)beta3/5 integrin expression was not necessarily predictive of the extent of transgene expression in a particular cell type. Furthermore, because the salivary glands of rabbits, monkeys, and humans show a similar luminal membrane distribution of these integrins, it is likely that recombinant type 5 adenoviruses may be able to mediate in vivo gene transfer to salivary glands in these species.

Gelatinases have been shown to be regulated by many cytokines and growth factors, and have been implicated in the pathogenesis of certain autoimmune diseases via tissue destruction. High levels of several cytokines, including IFN-gamma and TNF-alpha, have been demonstrated in the salivary gland microenvironment of patients with Sjogren's syndrome (SS). How these cytokines may be contributing to the pathogenesis of this disease is not well understood. We hypothesized that IFN-gamma with or without (+/-) TNF-alpha could be playing a role in the pathogenesis of SS via the regulation of matrix metalloproteinase (MMP) levels. This study examined the role of IFN-gamma and (+) TNF-alpha in the regulation of the matrix metalloproteinases, MMP-2 (72 kD gelatinase A) and MMP-9 (92 kD gelatinase B). A human salivary gland cell line (HSG) has been used as a possible in vitro model to study the role of IFN-gamma + TNF-alpha in the pathogenesis of SS. The HSG cell line, in the presence of IFN +/- TNF-alpha, displays increased MMP-2 and MMP-9 gelatinolytic activity, protein and RNA levels. The increase in MMP activity was partially blocked with an antibody against the IFN-gamma receptor, and this was associated with a complete inhibition of the previously described IFN-gamma +/- TNF-alpha antiproliferative effect. However, incubation of IFN-gamma treated HSG cells with the synthetic MMP inhibitor BB94 did not alleviate this antiproliferative effect. In addition, we demonstrate that there are very high levels of MMP-9 in the saliva of patients with SS when compared to healthy control subjects. These data suggest that cytokines could be regulating MMP production by salivary epithelial cells and thus indicate a potential role for these cells in the pathogenesis of SS.

The mechanism of irradiation-induced hypofunction of the salivary glands is a process that is not fully understood. Here we examine the hypothesis that intracellular and redox-active ions of iron and copper, which are associated with the secretion granules, play a catalytic role in the irradiation-induced damage. Rats were subjected to head and neck irradiation (15 Gy X rays) and allowed to recover for 2 months. The function of the parotid and submandibular glands was then determined by pilocarpine-stimulated salivary secretion. A 45% decrease in the function of both glands was obtained when compared to nonirradiated controls. Treatment prior to irradiation (90 min) with cyclocytidine (200 mg/kg) led to a massive degranulation of the parotid gland and yielded nearly complete protection from radiation-induced damage. In contrast, pilocarpine stimulation prior to irradiation led to a marginal degranulation of the parotid gland and yielded only 13% protection. Neither agent caused degranulation of the submandibular gland mucous cells or yielded functional protection of this gland. Treatment with both agents yielded a marked increase in iron, copper and manganese levels in the parotid gland saliva. An analogous marked increase in the redox activity of iron and copper ions was recorded for the parotid saliva stimulated by pilocarpine and cyclocytidine. Pilocarpine-stimulated submandibular gland saliva contained metal levels similar to those of the parotid gland saliva. However, no redox activity and no increase in metal mobilization could be demonstrated in the submandibular gland saliva stimulated by both agents. The correlation between the patterns of gland degranulation, mobilization of redoxactive metals and the protection of gland function, for both parotid and submandibular glands, focuses attention on the catalytic roles played by transition metal ions in promoting free radical reactions, which likely participate in the process of injury to the tissue.

Increased levels of several cytokines, including interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), have been demonstrated in the salivary gland microenvironment of patients with Sjögren's syndrome (SS). How these cytokines may be contributing to the pathogenesis of the disease is not well understood. This study examined the role of IFN-gamma +/- TNF-alpha on cellular death in a cultured human salivary gland cell line (HSG). Cells treated long-term with IFN-gamma +/- TNF-alpha demonstrate a profound antiproliferative effect with a decrease in cell number to below that initially plated. Treatment of HSG cells with TNF-alpha alone did not have any significant effects on growth but did increase the expression of the IFN-gamma receptor. Cells labelled with propidium iodide and anti-digoxigenin dUTP/dATP were examined by flow cytometry to determine the percentage of cells exhibiting low DNA content and DNA strand breaks. The percentage of cells exhibiting subdiploid DNA and DNA strand breaks increased with increased time of exposure to the cytokines. The maximum percentage of cells exhibiting DNA degradation at 12 days was 58% for cells treated with IFN-gamma + TNF-alpha, 31% for IFN-gamma treated cells, and5% for TNF-alpha-treated and untreated cells. The cells with subdiploid (2n) DNA were subsequently demonstrated to represent two populations, both with evidence of increased DNA strand breaks but with differing light scatter characteristics. One population had features of cells undergoing necrosis, whereas the second population exhibited features of apoptosis. These findings were confirmed by transmission electron microscopy. Cells not exposed to cytokines did not exhibit significant evidence of either death process. We conclude that long-term exposure of a human salivary gland epithelial cell line to IFN-gamma +/- TNF-alpha leads to increased DNA degradation and subsequent cell death. This suggests a potential SS disease mechanism and implicates the role of the epithelial cell in this disease as an important area for future study.

Interferon-gamma (IFN-gamma) is a product of activated T-lymphocytes, and tumor necrosis factor-alpha (TNF-alpha) is a product of both lymphocytes and macrophages. These cell types are often present at sites of tissue damage secondary to chronic infection or autoimmune disease. The purpose of this study was to characterize the effects of TNF-alpha and IFN-gamma on a human submandibular gland epithelial cell line (HSG). IFN-gamma caused a concentration-dependent decrease in HSG cell growth (approximately 70% in 6 days). Conversely, TNF-alpha alone had little effect on the growth of these cells. When these cytokines were added in combination (20 units/ml TNF-alpha and 1,000 units/ml of IFN-gamma), there was a synergistic antiproliferative effect; no apparent cell growth was observed. The cytokine-induced antiproliferative effect was reversible. After the apparent cessation of cell growth for 3-6 days, removal of the cytokines permitted complete growth recovery. Further, cells that recovered and exhibited growth patterns that were similar to control cells remained susceptible to the antiproliferative effects of the cytokines. Flow cytometry revealed that the percentage of cells in G0/G1 with the combination of cytokines was significantly increased by 24 h. The antiproliferative effect of IFN-gamma alone and that of IFN-gamma and TNF-alpha in combination were blocked completely using an antibody to the IFN-gamma receptor. A hypothesized mechanism of tissue damage in autoimmune inflammatory disorders is via up-regulation of cell surface markers such as intercellular adhesion molecule type I (ICAM-1) and histocompatibility antigen HLA-DR which can exacerbate the inflammatory process. Treatment of HSG cells with IFN-gamma, with or without TNF-alpha, resulted in increased levels of ICAM-1 and the acquisition of HLA-DR expression. These aggregate data suggest that IFN-gamma alone can regulate the expression of cell surface markers involved in the inflammatory process as well as cause a potent yet reversible inhibition of HSG cell growth that is modulated by the presence of TNF-alpha.

Recently we employed two enantiomers of the muscarinic antagonist quinuclidinyl iodobenzilate (IQNB), and pharmacokinetic analyses, to define and quantitate nonspecific and specific binding to rat parotid gland muscarinic acetylcholine receptors (mAChRs) in vivo (Hiramatsu et al., 1993). The present studies were designed to utilize this same approach for evaluating mAChRs in three other morphologically different rat exocrine glands: the submandibular, sublingual and lacrimal glands. The metabolism and tissue distribution of the intravenously injected IQNB enantiomers were determined, and the resulting data were assessed in terms of their goodness of fit to several multicompartmental models. All three exocrine glands showed substantial nonspecific ligand distribution as measured with the receptor-inert enantiomer (SS)-IQNB. Nonspecific distribution represented 45, 21 and 36% of total ligand distribution in submandibular, sublingual and lacrimal glands, respectively, as measured with the receptor-active enantiomer (RR)-IQNB. The rank order of the binding potential, kinetically equivalent to Bmax/Kd, for (RR)-IQNB and these mAChRs was lacrimalsublingualsubmandibular glands (674 +/- 235575 +/- 109345 +/- 29). These results demonstrate that specific mAChRs in the exocrine glands can be measured in vivo with the (RR)-IQNB enantiomer and that despite some small quantitative differences, the distribution of (RR)- and (SS)-IQNB is similar in the three exocrine glands but is substantially different from that in brain and heart.

The purpose of this study was to examine the effects of different doses of X irradiation on the major rat salivary glands. The flow rates of the salivary glands were measured simultaneously in both parotid and submandibular glands of mature male Wistar rats at 3 and 40 days after head and neck irradiation with single doses of 2.5, 5, 7.5, 10, or 15 Gy. The parotid and submandibular glands were weighed at the time of saliva collection and total body weight was obtained weekly. Significant reductions in parotid salivary flow at 3 days and parotid and submandibular flow at 40 days were found. Diminished saliva output was dose-dependent and significantly reduced at radiation exposures of 7.5 Gy and greater. Submandibular function deteriorated between 3 and 40 days and the extent of hypofunction was comparable to the parotid gland at the latter time. Parotid and submandibular gland weights were reduced by irradiation in a dose-dependent manner at both 3 and 40 days. The effects were similar for both glands at the latter time. Total body weight was also reduced by the head and neck irradiation in a dose-dependent manner. There was significant mortality in the group receiving 15 Gy irradiation between 7 and 14 days after irradiation. The results demonstrate that parotid and submandibular glands may be affected comparably by equal doses of head and neck irradiation when examined at later times. In the period immediately after irradiation, there are significant differences in the responses of the major salivary glands.

Radiation-induced damage to salivary glands has been recognized for over 80 years. However, the mechanisms responsible for the destruction of gland parenchyma are still not known. The purpose of this study was to describe alterations in salivary function in the rat within the first 24 h after irradiation. Saliva was collected from the parotid and submandibular glands during radiation treatment and at four times (0.5, 1, 4, and 24 h) after 15 Gy X irradiation delivered to the head and neck. Total body weight, submandibular gland weight, and food and water intake were monitored and the total protein, sodium, and potassium content of the salivas was analyzed. The effects of radiation on salivary glands of the rat could be demonstrated by 24 h. Parotid salivary flow was reduced and the sodium concentration was significantly less than that in control animals. Submandibular gland weights declined markedly by 24 h. These effects appear to be influenced significantly by the animals' limited intake of food and water during this period, as well as the anesthesia administered.

It is not clear if aging distinctions can be made at the level of an organ or organism. The purpose of this study was to determine if a general definition of systemic aging, primary aging (influence of the passage of time), versus secondary aging (influence of extrinsic factors), can be used to discriminate the functional status of an individual organ system, the oral cavity. Thirty healthy, nonmedicated subjects (that is, those who exhibit primary aging) and 42 persons being treated for medical problems and taking prescription medications (that is, those who exhibit secondary aging), aged 75 to 96 years, from the oral physiology component of the Baltimore Longitudinal Study of Aging were evaluated. A standardized examination assessed gingival, periodontal, dental, and oral mucosal tissues. There were few substantive differences in oral health and function between primary and secondary aging subjects. Thus use of broad definitions of aging in an organism did not lead to meaningful predictions of the health or function of an individual organ system. Furthermore, the similarity in the oral condition between both groups studied here suggests substantial resiliency of the oral cavity during aging.

In general, considerable strength and much realism exist in the model as presented by Dr. McHugh. Progress in biological science is central to any scheme anticipating change in medicine, including dental and oral medicine. In my view, there are two weaknesses in his presentation: 1) a perhaps semantic difference with me over whether dentists should be trained as scientists or as individuals who can use science (fact and process); and 2) a substantive difference as to how to reinforce didactic, basic biomedical science in clinical training.

There is little known about the regulation of gene expression in rat parotid glands after exposure to ionizing radiation. The present studies investigate the effects of in vivo ionizing radiation, with subsequent stimulation of beta-adrenergic receptors by isoproterenol, on parotid gland function and on the expression of the early response genes, c-fos, c-jun, and jun B. Ionizing radiation diminished parotid gland weight and saliva output. Treatment of irradiated rats with isoproterenol increased the gland weight to levels similar to those in nonirradiated rats. However, such treatment had no effect on saliva output as indicated by measurements of parotid salivary flow rate. Irradiation alone increased the expression of c-fos, c-jun, and jun B. The combination of irradiation and isoproterenol had an additional effect on the levels of c-fos and jun B mRNAs and proteins particularly at earlier experimental times (1 to 8 h). Isoproterenol alone induced high levels of c-fos and jun B mRNA but not of c-jun mRNA. However, c-jun mRNA was induced markedly by radiation and 8 h of isoproterenol treatment, indicating a combined effect on c-jun gene expression. These observations suggest that the expression of the proto-oncogenes c-fos, c-jun, and jun B is probably regulated through differential signal transduction pathways which may be activated by these external stimuli and may be associated with functional changes induced in the rat parotid gland by ionizing radiation and by ionizing radiation and isoproterenol.

An HIV-1/ATH8-cell cytopathic system was used to characterize the previously reported anti-HIV-1 activity of human saliva. Inhibitory activity was demonstrated by monitoring viable cell counts, HIV-1 p24 core antigen, and reverse transcriptase levels. Nonfiltered whole saliva, sterilized by irradiation, protected the ATH8 cells from HIV-1 infection. When HIV-1/saliva mixtures were filtered following incubation, the quantity of virus was significantly less (approximately 50%) than in HIV-1/media-filtered controls, suggesting that salivary aggregation and/or agglutination may be involved in the inhibitory activity. However, a sufficient number of apparently morphologically intact viral particles were still present in the HIV-1/saliva filtrates to lead to infection. When saliva was filtered prior to incubation with HIV-1, these filtrates showed substantial inhibitory activity, although reduced compared with that of non-prefiltered saliva. We conclude that saliva likely has several means by which to inhibit HIV-1 infectivity.

We studied the effects of pilocarpine hydrochloride, a para-sympathomimetic agent, on major salivary gland output and subjective responses in 31 patients with salivary hypofunction. Pilocarpine hydrochloride (5-mg capsules, three times daily) was given for 5 months and a placebo was randomly assigned for 1 month in a double-blind fashion. Objective measurements of major salivary gland output, subjective impressions of oral moisture, treatment-related side effects, and a number of physiologic measures were assessed monthly. Pilocarpine significantly increased salivary output in 21 of the 31 patients. Subjective improvement in the feeling of oral dryness, speaking, chewing, and swallowing were reported by 27 individuals. Side effects, while common, generally were mild and tolerable. There were no significant alterations in cardiovascular or other physiologic measures. We conclude that pilocarpine is an effective and safe treatment for salivary gland hypofunction and xerostomia in selected patients. The increase in major gland output provides beneficial natural secretions and relief of oral dryness.

Saliva is important for the preservation and maintenance of oral health. It is unclear, however, how much saliva is required to maintain normal oral function. Major salivary gland flow rates, objective measurements of oral health, and subjective complaints of oral problems were assessed in different-aged, healthy persons. Results suggest that the comparison of major salivary gland flow rates of an individual with population standards to identify patients susceptible to the effects of salivary dysfunction is unreliable. Changes in salivary function over time are a more meaningful gauge of the impact of saliva on oral health. The clinician should monitor salivary production to identify patients with declining salivary gland output.