Alfredo Aguirre

Artificial Salivas: Present and Future:

Modern technology has allowed us to understand better the functions of saliva and now provides a rationale for developing: (1) diagnostic reagents for monitoring oral and systemic health status and (2) replacement therapies for individuals with salivary dysfunctions. Several areas of dental research are directed at augmenting or enhancing both the quality and quantity of saliva for individuals with dry mouth. An “intrinsic” approach is being explored which utilizes medications such as pilocarpine and bromhexine to stimulate the salivary glands to produce more saliva. An “extrinsic” approach proposes to use topically applied artificial saliva. Studies in our laboratory have been directed toward developing artificial salivas which incorporate many of the protective features of “native” saliva. An ideal artificial saliva should be “long-lasting”, provide lubrication, inhibit colonization of microflora responsible for dental caries and gingivitis, and coat the oral soft tissues for protection against environmental insult and desiccation. Studies are currently under way to determine the structural requirements of salivary molecules responsible for these protective functions. Composite salivary molecules consisting of multiple biologically active or “functional domains” could then be designed and synthesized based upon primary sequence and conformational analyses, computer-assisted structural predictions, and in vitro testing. These supersalivary substances could then be used as saliva substitutes for targeting to selected oral surfaces to promote mineralization, hydration, and/or regulate microbial-mediated disease.

Lubrication and viscosity features of human saliva and commercially available saliva substitutes

The lubricating features and viscosity of human saliva and five commercially available saliva substitutes were compared. The results indicate that little correlation exists between these parameters. Saliva substitutes based on carboxymethylcellulose do not appear to lubricate biocompatible hard interfaces well and, therefore, might not protect against the rapid attrition observed in xerostomic individuals. In contrast, a mucin-based substitute proved to be a better lubricant with values comparable to whole human saliva.

Immunochemical quantitation of α-amylase and secretory IgA in parotid saliva from people of various ages

The alpha-amylase (128 subjects) and secretory IgA (118 subjects) concentration in stimulated parotid saliva of healthy individuals aged 23-84 years, was determined. They were divided into three age groups: I, 23-39; II, 40-59; and III, 60-84 years old. The concentrations (microgram/ml) of alpha-amylase for group I = 803.6; II = 648.0; and III = 652.4; the concentrations for secretory IgA for group I = 96.2; II = 101.8; and III = 97.7. The Kruskal-Wallis test revealed no significant differences between groups for alpha-amylase or secretory IgA.

In vitro characterization of human salivary lubrication

The friction coefficients for parotid and submandibular-sublingual salivas of 7 subjects were measured. At the load and speeds used, the lubricatory properties of the secretions followed the McKee-Petroffs curve. The friction coefficients for both secretions approximated those reported for boundary and thin film lubrication.

Lubrication of selected salivary molecules and artificial salivas

The lubrication regime displayed by human salivas (parotid and submandibular-sublingual), purified salivary molecules (the mucins MG1 and MG2 and α-amylases), and selected artificial salivas (Oracare D, Saliva Substitute, and Orthana) was assessed in vitro using a friction-testing device. Thin-film (boundary) lubrication was observed for all of the salivary samples and two of the artificial salivas examined. Oracare D, a glycerol-based artificial saliva, was the exception since it lubricated by a thick-film (hydrodynamic) regime. On a molar basis, the best lubricants of the purified salivary molecules were MG1 > MG2 ≈ nonglycosylated α-amylases ≈ glycosylated α-amylases.

Ectopic thyroid tissue in the submandibular region

This report describes an unusual location of ectopic thyroid gland tissue. A growth in the left submandibular area was surgically excised, and the microscopic examination of the specimen revealed thyroid tissue with colloid goiter. Because this entity cannot be clinically distinguished from a salivary gland tumor, ectopic thyroid tissue should be considered in the differential diagnosis of swellings involving the submandibular area.

Chronic ulcerative stomatitis: clinical, histopathologic, and immunopathologic findings.

Chronic ulcerative stomatitis (CUS) is a mucocutaneous disease primarily involving mucosal surfaces, but occasionally may involve the skin. Clinically, CUS patients exhibit erosive or ulcerative lesions of the oral mucosa that resemble erosive oral lichen planus. Direct immunofluorescence (DIF) studies of mucosal or skin biopsies reveal a unique pattern of IgG immunoglobulin bound to nuclei of keratinocytes of the basal and lower one third cell layers, the stratified epithelial specific (SES) antinuclear antibody (ANA) pattern. Patient sera also exhibit circulating SES-ANA reactions on indirect immunofluorescence (IIF) using an esophagus substrate. We report the clinical and immunopathologic findings of 3 cases of CUS and demonstrate autoantibody recognition of the CUS antigen on Western blot. An important reason to distinguish CUS from other oral ulcerative conditions is that it may be refractory to standard treatments with topical corticosteroids, and favorable clinical responses may be achieved with hydroxychloroquine pharmacotherapy.

Immunochemistry and immunogenicity of low molecular weight human salivary mucin

The purposes of this study were to examine the immunogenicity of the low molecular weight human salivary mucin (MG2) and determine its distribution within major and minor human salivary glands. Anti-MG2 sera were produced in Balb/c mice by a variety of immunization schedules. Chromatographically or electrophoretically purified MG2 and partially purified mucin chromatographic fractions exposed to mild denaturing conditions were not immunogenic. Only MG2 without prior exposure to urea or guanidine was able to elicit an immune response. A murine anti-MG2 monoclonal antibody (clone 1/F9) was produced and its monospecificity confirmed by immuno-dot blotting and SDS-PAGE Western transfer. Clone 1/F9 (IgG1; kappa) was of moderate affinity and was directed to a Pronase- and TPCK trypsin-sensitive but periodate-resistant epitope which was not blood group- or sialic acid-specific. Immunocytochemical studies of frozen tissue sections with clone 1/F9 using both indirect and direct methods revealed that MG2 was more heterogeneously distributed within submandibular than labial glands and was not found in parotid or palatine glands. The use of a polyclonal rabbit anti-MG2 reagent in either frozen or paraffin-embedded tissues gave the same immunocytochemical results as those obtained with the monoclonal antibody.

Levels of salivary cystatins in periodontally healthy and diseased older adults

Cystatins are cysteine protease inhibitors present in a variety of tissues and body fluids, including saliva. One possible function of these molecules may be to modulate tissue destruction in periodontal diseases. To investigate the potential role of salivary cystatins in these events, the levels of cystatins in saliva from periodontally healthy and diseased individuals were measured by enzyme-linked immunosorbent assay. Flow rates and total protein content were determined in all the samples collected, while cysteine protease inhibitory activity was assessed in submandibular-sublingual secretions. Statistical analysis showed no significant differences in the levels and activity of salivary cystatins in periodontally healthy and diseased individuals. These findings suggest that comparing the levels of cystatins in glandular salivas may not be a suitable indicator of periodontal disease status.

ALX/FPR2 receptor for RvD1 is expressed and functional in salivary glands

Sjögrens syndrome (SS) is an autoimmune disorder characterized by chronic inflammation and destruction of salivary and lacrimal glands, leading to dry mouth, dry eyes, and the presence of anti-nuclear antibodies. Despite modern advances, the current therapies for SS have no permanent benefit. A potential treatment could involve the use of resolvins, which are highly potent endogenous lipid mediators that are synthesized during the resolution of inflammation to restore tissue homeostasis. Our previous studies indicate that ALX/FPR2, the receptor for RvD1, is expressed and active in the rat parotid cell line Par-C10. Specifically, activation of ALX/FPR2 with RvD1 blocked inflammatory signals caused by TNF-α and enhanced salivary epithelial integrity. The goal of this study was to investigate RvD1 receptor expression and signaling pathways in primary salivary cells. Additionally, we determined the role of the aspirin-triggered 17R analog (AT-RvD1, a more chemically stable RvD1 epimeric form) in prevention of TNF-α-mediated salivary inflammation in mouse submandibular glands (mSMG). Our results indicate that ALX/FPR2 is expressed in mSMG and is able to elicit intracellular Ca2+ responses and phosphorylation of Erk1/2, as well as Akt. Given that these signaling pathways are linked to cell survival, we investigated whether AT-RvD1 was able to prevent programmed cell death in mSMG. Specifically, we determined that AT-RvD1 prevented TNF-α-mediated caspase-3 activation. Finally, we show that ALX/FPR2 is expressed in human minor salivary glands with and without SS, indicating the potential therapeutic use of AT-RvD1 for this condition.