A replication-deficient, recombinant adenovirus encoding human aquaporin-1 (hAQP1), the archetypal water channel, was constructed. This virus, AdhAQP1, directed hAQP1 expression in several epithelial cell lines in vitro. In polarized MDCK cell monolayers, hAQP1 was localized in the apical and basolateral plasma membranes. Fluid movement across monolayers infected by AdhAQP1 in response to an osmotic gradient was Ϸ4-fold that seen with uninfected monolayers or monolayers infected by a control virus. When AdhAQP1 was administered to rat submandibular glands by retrograde ductal instillation, significant hAQP1 expression was observed by Western blot analysis in crude plasma membranes and by immunohistochemical staining in both acinar and ductal cells. Three or four months after exposure to a single radiation dose (17.5 or 21 Gy, respectively), AdhAQP1 administration to rat submandibular glands led to a two-to threefold increase in salivary secretion compared with secretion from glands administered a control virus. These results suggest that hAQP1 gene transfer may have potential as an unique approach for the treatment of postradiation salivary hypofunction.

The trp gene family has been proposed to encode the store-operated Ca 2؉ influx (SOC) channel(s

Adult mesenchymal stem cells have the proclivity to differentiate along multiple lineages giving rise to new bone, cartilage, muscle, or fat. Collagen, a normal constituent of bone, provides strength and structural stability and is therefore a potential candidate for use as a substrate on which to engineer bone and cartilage from their respective mesenchymal-derived precursors. In this study, a collagen- glycosaminoglycan scaffold was used to provide a suitable three-dimensional (3-D) environment on which to culture adult rat mesenchymal stem cells and induce differentiation along the osteogenic and chondrogenic lineages. The results demonstrate that adult rat mesenchymal stem cells can undergo osteogenesis when grown on the collagen-glycosaminoglycan scaffold and stimulated with osteogenic factors (dexamethasone, ascorbic acid, beta-glycerophosphate), as evaluated by the temporal induction of the bone-specific proteins, collagen I and osteocalcin, and subsequent matrix mineralization. The osteogenic factors were coupled to activation of the extracellular-regulated protein kinase (ERK), and this kinase was found to play a role in the osteogenic process. As well as supporting osteogenesis, when the cell-seeded scaffold was exposed to chondrogenic factors (dexamethasone and TGF-1beta), collagen II immunoreactivity was increased, providing evidence that the scaffold can also provide a suitable 3-D environment that supports chondrogenesis.

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Although replication-deficient adenoviruses can efficiently transfer genes to the salivary glands, the current vectors precipitate an immediate, transient decrease in salivary function. To study the cause of this salivary hypofunction, 10(6)-10(10) plaque-forming units (pfu) of the vector AdCMV beta gal were delivered by retrograde ductal infusion to the submandibular glands (SMGs) of rats. Microscopic analysis of infected glands showed a dose-related, rapidly developing inflammatory response, which at the highest amount of virus was characterized by a predominantly neutrophil-containing infiltrate, focal necrosis, and edema. Moreover, the glands of nude rats developed similar morphologic changes to those of immunocompetent rats. After 3 days, the volume of stimulated saliva secreted from SMGs receiving AdCMV beta gal (6.75 x 10(9) pfu) was approximately 20% that of controls. UV-inactivated virus caused a similar decrease in saliva output. We evaluated to what extent the anti-inflammatory glucocorticoid, dexamethasone, could suppress inflammation and preserve salivary function. Three days after infusion with a high dose of AdCMV beta gal (6.75 x 10(9) pfu), the glands from dexamethasone-treated animals showed markedly less inflammation and no necrosis. Furthermore, there was no significant difference in the average amount of saliva secreted from the infected glands (105 +/- 17 microliters) compared to the control glands (123 +/- 18 microliters). In addition, dexamethasone extended the expression of beta-galactosidase in the SMGs. These results suggest that the adenovirus-mediated acute inflammation in rat SMG is responsible for diminished gland function and transgene expression. Furthermore, we demonstrate a useful role for glucocorticoids in controlling acute inflammation during experimental gene transfer with current adenovirus vectors.

The purpose of this study was to elucidate some of the factors on which accurate shade taking depends. Custom shade tabs (0.5, 1.0 and 1.5 mm porcelain thickness) were fabricated from selected Vita and Shofu porcelains. Four main factors were investigated, namely, the difference between the two types of porcelain used, the effect of light quality, the effect of porcelain thickness and the experience of the observer. The chi-square test for independence at a probability level of P <0.05 was used to analyse the results. The results indicated that in ideal light there was no difference between the two porcelains (P=0.58). The experienced observers proved better than the novice observers in ideal light conditions (P=0.003). Thickness was also significant in the overall results (P=0.0001), in that thicker samples gave more accurate results. The results indicate that in adverse light, there was an overall difference between the two porcelains (P=0.046), but no difference between the experienced and novice observers. The thickness made no difference to the experienced or the novice observer in adverse light. These results indicate that the most influential factor on shade taking was the light quality (P <0.0001); better results were obtained overall for the ideal light situation. In ideal light thicker samples gave better results (P=0.0001).

Gene transfer to the salivary glands holds the potential for the therapy of salivary gland disorders and for delivery of therapeutic proteins to the mouth and upper gastrointestinal tract. Administration of the recombinant adenovirus vectors Ad.RSV beta gal [coding for the intracellular protein beta-galactosidase (beta-Gal)] and Ad alpha 1AT [coding for human alpha 1-antitrypsin (alpha 1-AT), a secreted protein] to salivary gland cell lines in vitro demonstrated exogenous gene expression. Retrograde ductal injection of the Ad.RSV beta gal vector to rat salivary glands in vivo resulted in beta-Gal expression in acinar and ductal cells. Exposure of submandibular glands in vivo to Ad alpha 1AT resulted in expression of alpha 1-AT mRNA transcripts, de novo synthesis of alpha 1-AT, and secretion in the saliva. To evaluate the feasibility of adenovirus-mediated gene transfer to human glands, human minor salivary glands were infected ex vivo with Ad.RSV beta gal, and implanted into severe combined immunodeficient mice. Evaluation of the human tissue demonstrated beta-Gal activity. These observations demonstrate that adenovirus vectors are capable of direct delivery of genes to the salivary glands, suggesting a variety of possible gene therapy applications.

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