Chronic hyperglycemia predisposes to exaggerated inflammatory response and leukocyte dysfunction in Akita mice.

The role of polymorphonuclear neutrophils (PMN) in mediating diabetic tissue damage to the periodontium was investigated in a novel model of chronic hyperglycemia, the Akita mouse. Induction of acute peritoneal inflammation in wild-type (WT) and Akita mice resulted in exaggerated IL-6 response in Akita mice (2.9-fold increase over WT values) and a markedly increased chemokine response (KC, 2.6-fold; MCP-1, 2.6-fold; and MIP-1alpha, 4.4-fold increase over WT values). Chemotaxis to both fMLP and WKYMVm was significantly reduced in isolated Akita PMN compared with WT PMN as measured in a Boyden chamber. Superoxide release in contrast was significantly increased in Akita PMN as measured with cytochrome c reduction. Bone marrow-derived Akita PMN showed partial translocation of p47phox to the cell membrane without external stimulation, suggesting premature assembly of the superoxide-producing NADPH oxidase in hyperglycemia. In vivo studies revealed that ligature-induced periodontal bone loss is significantly greater in Akita mice compared with WT. Moreover, intravital microscopy of gingival vessels showed that leukocyte rolling and attachment to the vascular endothelium is enhanced in periodontal vessels of Akita mice. These results indicate that chronic hyperglycemia predisposes to exaggerated inflammatory response and primes leukocytes for marginalization and superoxide production but not for transmigration. Thus, leukocyte defects in hyperglycemia may contribute to periodontal tissue damage by impairing the innate immune response to periodontal pathogens as well as by increasing free radical load in the gingival microvasculature.

Two-hybrid analysis of human salivary mucin MUC7 interactions.

MUC7 is a low molecular weight monomeric mucin secreted by submandibular, sublingual and minor salivary glands. This mucin has been implicated in the non-immune host defense system in the oral cavity since it binds and agglutinates a variety of oral microbes. To investigate interactions between this mucin and other secretory salivary proteins, a submandibular gland prey library was screened with baits encoding the N- and C-terminal regions of MUC7 in the yeast two-hybrid system. The N-terminal region interacted with several secretory salivary proteins, whereas the C-terminal region did not. Interacting proteins included amylase, acidic proline-rich protein 2, basic proline-rich protein 3, lacrimal proline-rich protein 4, statherin and histatin 1. Formation of complexes between these proteins and the N-terminal region of MUC7 was confirmed in Far Western blotting experiments. Interactions between mucin and non-mucin proteins in saliva could protect complex partners from proteolysis, modulate the biological activity of complexed proteins or serve as a delivery system for distribution of secretory salivary proteins throughout the oral cavity.

Salivary micelles: identification of complexes containing MG2, sIgA, lactoferrin, amylase, glycosylated proline-rich protein and lysozyme.

Micelles represent macromolecular structures in saliva and the aim of this study was to identify salivary proteins that occur in these globular particles. Micelles were isolated from whole saliva (WS) collected from three individuals and analysed in different experiments. Samples were subjected to polyacrylamide gel electrophoreses, hydrolysed to determine their amino acid composition and total protein concentration, examined by scanning electron microscopy and examined on Western blots probed with a panel of antibodies directed against salivary proteins. On Coomassie Brilliant Blue stained gels, the banding pattern of whole saliva and micelles was similar but the intensity of bands was quite different. Amino acid analysis confirmed that the amino acid composition of micelles was distinct from that of whole saliva. Scanning electron microscopy showed that micelles exhibit a complex pattern consisting of individual particles or clusters of particles with different sizes and shapes. Micelles contain proteins with high (MG2 and secretory IgA), intermediate (lactoferrin, amylase and glycosylated proline-rich protein (PRP)) and low (lysozyme) molecular weight that were immuno-detected on blots probed with specific antibodies. Micelles represent particulate multicomponent structures in whole saliva that contain a subset of salivary proteins known to be important components of the innate immune system and are likely to play an important role in the maintenance of homeostasis in the oral environment.

Patterns of secretion of mucins and non-mucin glycoproteins in human submandibular/sublingual secretion.

The present investigation has characterised the influence of gustatory stimulation and duration of stimulation on the secretion pattern of salivary mucins MG1 and MG2 and non-mucin glycoproteins in submandibular/sublingual secretion (SMSL). Resting SMSL was collected for three 2 min intervals and stimulated SMSL was collected for ten 1 min intervals from six healthy subjects. Flow rates and total protein were significantly different under the two conditions. The secretion patterns of these proteins under resting and stimulated conditions was examined on periodic acid-Schiff reagent (PAS)-stained polyacrylamide gels using a Kodak Digital-Science Image Station. Image analyses revealed that the level of MG1 increased and the level of MG2 remained nearly the same after stimulation. Six other major glycoproteins (designated Band 1-6) were identified on the basis of their electrophoretic mobilities and immuno-reactivity on Western blots. After stimulation the intensity of Band 1 (lactoferrin and peroxidase) and Band 2 (amylase) decreased whereas the intensity of Band 3 (carbonic anhydrase), Band 4 (proline-rich glycoprotein) and Bands 5 and 6 (basic glycosylated proline-rich proteins) increased. These patterns probably reflect secretion from preformed vesicles since de novo synthesis would be unexpected within the time frame of these experiments. The variable patterns observed suggest that mucins and non-mucin glycoproteins in SMSL derive from different subsets of secretory vesicles, some of which may originate in mucous and others in serous acini, as well as in ductal cells. Quantification of mucins was performed by image analysis technology using purified MG1 and MG2 standards. Finally, the present investigation has shown that the secretory patterns of mucins and non-mucin glycoproteins from submandibular/sublingual glands are complex and represent an important aspect of salivary gland physiology.