Bertil Kinnby

Plasminogen binding by oral streptococci from dental plaque and inflammatory lesions.

Plasminogen binding by bacteria is a virulence factor important for the entry and dissemination of bacteria in the body. A wide variety of bacteria bind plasminogen, including both organisms causing disease and components of the normal oral flora. The purpose of this study was to examine the characteristics of plasminogen binding by six clinical isolates of oral streptococci from both dental plaque and inflammatory lesions. All the strains bound plasminogen with approximately the same affinity, and binding was specific and lysine-dependent as evidenced by its inhibition by epsilon-aminocaproic acid. All of the test strains were capable of activating bound plasminogen to plasmin without the addition of a plasminogen activator, and subsequent analysis revealed the presence of streptokinase in all strains. However, the streptococci exhibited fibrinolytic activity only in the presence of plasminogen and this could be inhibited by the addition of epsilon-aminocaproic acid. SDS-PAGE and 2D gel electrophoresis coupled with plasminogen ligand blotting showed that only a subset of the total proteins (2-15) were involved in the binding of plasminogen. Partial identification of the binding proteins revealed that four glycolytic enzymes, enolase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate mutase, were predominant in binding plasminogen. The binding of plasminogen by bacteria from pus did not differ from that of the strains from supragingival plaque. The findings illustrate how apparently innocuous commensal bacteria are capable of utilizing a mechanism that is generally regarded as being of importance to pathogenicity and suggest an additional role of plasminogen binding.

Localization of plasminogen activators and plasminogen-activator inhibitors in human gingival tissues demonstrated by immunohistochemistry and in situ hybridization

The plasminogen-activating system plays an important part in tissue proteolysis in physiological as well as pathological processes. Plasminogen activators u-PA (urokinase) and t-PA (tissue) as well as the inhibitors PAI-1 and PAI-2 are present in gingival crevicular fluid in concentrations significantly greater than in plasma. This fact, and the finding that the concentrations of t-PA and PAI-2 are higher in areas with gingival inflammation, indicate local production of these components. The present study describes, by means of in situ hybridization and immunohistochemistry, the localization of the plasminogen activators and their inhibitors in gingival tissues from patients undergoing periodontal surgery. t-PA mRNA and t-PA antigen were primarily found in the epithelial tissues, predominantly in the sulcular and junctional regions, although occasionally in the oral epithelium and in blood vessels of the connective tissue. u-PA and u-PA-receptor signals were seen in single cells within the junctional and sulcular epithelia and adjacent to blood vessels close to the junctional epithelium, but rarely in the oral epithelium. Similar to t-PA, the predominant location of PAI-2 mRNA was the gingival epithelia. In the junctional and sulcular epithelia, PAI-2 mRNA was seen throughout the thickness, while in the oral epithelium the strongest signals were seen in stratum granulosum and stratum spinosum. PAI-1 mRNA was invariably found in the connective tissue associated with blood vessels. The present study confirms earlier indications of local production of plasminogen activators and their inhibitors in gingival tissues. In addition, the results demonstrate that t-PA and PAI-2 in these patients are produced predominantly in the epithelial tissues. Furthermore, the presence of t-PA and PAI-2 seems to be most pronounced in the areas likely to be subjected to bacterial assault.

Mucins MUC5B and MUC7 in minor salivary gland secretion of children and adults

OBJECTIVE The study was designed to investigate the relative amount of MUC5B and MUC7 in minor salivary glands in children and adults, in order to test the hypothesis that secretion of salivary mucins changes between childhood and adulthood. METHODS Ninety individuals in the age-groups 3-year-olds, 14-year-olds, and young adults 20-25 year-olds were recruited. Sialopapers were applied on the labial and the buccal mucosa and then placed in the Periotron 8,000 (Proflow ) for calculation of the amount of saliva. The assessment of MUC5B and MUC7 was carried out in an ELISA using the LUM5B-2 and the LUM7-1 antiserum, respectively. RESULTS MUC5B and MUC7 were detected in the labial minor gland saliva in all age groups. In buccal gland saliva, only a few individuals in each age group showed detectable amounts of the mucins. In the labial area, a significantly lower level of MUC7 was noted in 3-year-olds compared with adults. CONCLUSION The results indicate a site-dependent difference in minor gland mucin secretion and an age-related difference in the labial gland secretion of MUC7.

Increasing expression of tissue plasminogen activator and plasminogen activator inhibitor type 2 in dog gingival tissues with progressive inflammation

Urokinase and tissue-type plasminogen activators (u--PA and t--PA) are serine proteases that convert plasminogen into plasmin, which degrades matrix proteins and activates metalloproteinases. The PAs are balanced by specific inhibitors (PAI--1 and PAI--2). Local production of t--PA and PAI--2 was recently demonstrated in human gingival tissues. The aim now was to investigate the production and localization of t--PA and PAI--2 in gingival tissues from dogs in three well-defined periodontal conditions; clinically healthy gingiva, chronic gingivitis and an initial stage of ligature-induced loss of attachment. At the start of the experiment the gingiva showed clear signs of inflammation. Clinically healthy gingiva were obtained after 21 days period of intense oral hygiene. Attachment loss was induced by placing rubber ligatures around the neck of some teeth. Biopsies were taken from areas representing the different conditions and prepared for in situ hybridization and immunohistochemistry. In clinically healthy gingiva both t--PA mRNA and antigen were expressed in a thin outer layer of the sulcular and junctional epithelia. No t--PA signals or staining were seen in connective tissue. Both mRNA signaling and immunostaining for t--PA were stronger in chronic gingivitis. In areas with loss of attachment, t--PA mRNA as well as antigen were found in the sulcular and junctional epithelia to a similar degree as in gingivitis. Occasionally the connective tissue was involved, especially in connection with vessels. PAI--2 mRNA was seen in a thin outer layer of the sulcular and junctional epithelia in clinically healthy gingiva, but no signals were seen in connective tissue. PAI--2 antigen was found primarily in the outer layer of the sulcular and junctional epithelia. Some cells in the connective tissue were stained. In gingivitis, PAI--2 signals were mainly found in the same locations, but more intense and extending towards the connective tissue. Immunostaining was seen in the outer half of the sulcular and junctional epithelia as well as in the upper part of the connective tissue, close to the sulcular epithelium. In sites with loss of attachment, PAI--2 mRNA was found throughout the sulcular and junctional epithelia, as was the antigen, which stained intensely. No PAI--2 mRNA was seen in connective tissue; the antigen was found scattered, especially near vessels. This study shows that the expression of both t--PA and PAI--2 increases with experimental gingival inflammation in the dog, and furthermore, the two techniques demonstrate a strong correlation between the topographical distribution of the site of protein synthesis and the tissue location of the antigens for both t--PA and PAI--2. The distribution correlates well with previous findings in humans.

The localization of the relaxed form of plasminogen activator inhibitor type 2 in human gingival tissues.

Abstract. The plasminogen activating system is important in extracellular proteolysis. Plasmin degrades tissues and activates proteases. Plasminogen activators (tissue type; t-PA and urokinase type; u-PA) and plasminogen activator inhibitors (PAI-1, PAI-2) are found in high concentrations in gingival crevicular fluid (GCF). Previous findings indicate the significance of PAI-2 in gingival inflammation. When PAI-2 inhibits a plasminogen activator its conformation relaxes and neoepitopes can be detected with a monoclonal antibody (#2H5). Our aim was to study if and where in the gingival region PAI-2 has acted as an inhibitor. Methodological studies were performed on GCF with western blotting. Frozen sections of human gingiva were studied immunohistochemically. The methodological studies showed that our antibody #2H5 selectively detects relaxed low molecular weight non-glycosylated PAI-2. Total PAI-2 and relaxed PAI-2 were found in all gingival epithelia with a honeycomb-like staining. Relaxed PAI-2 showed the most pronounced staining in the cell layers near the surface of the epithelium and no staining in the suprabasal layers, while total PAI-2 was found throughout the epithelium, often more pronounced suprabasally. The results showed that PAI-2 indeed has acted as an inhibitor of a protease in gingival tissues, primarily in the epithelia. The results also suggest primarily an intracellular localization and thus the interaction of PAI-2 with a protein other than t-PA.

Glycoprotein 340 and sialic acid in minor-gland and whole saliva of children, adolescents, and adults.

Glycoprotein 340 (gp-340) is a bacterial-binding glycoprotein found in major-gland and minor-gland saliva. Sialic acid, a common terminal structure of salivary glycoproteins, interacts with microorganisms and host ligands, as well as with free radicals. This study investigated the contents of gp-340 and sialic acid in minor-gland saliva and whole saliva of children (3 yr of age), adolescents (14 yr of age), and adults (20-25 yr of age). Labial-gland saliva and buccal-gland saliva were collected on filter paper, and unstimulated whole saliva was collected by draining into a tube. The relative amount of gp-340 and sialic acid was determined by ELISA and by enzyme-linked lectin assay (ELLA), respectively. In minor-gland saliva, no statistically significant differences in gp-340 and sialic acid were seen between the age-groups. Among adults, significantly lower amounts of gp-340 and sialic acid were seen in labial saliva compared with buccal saliva. In whole saliva, the amount of gp-340 was significantly lower among adults compared with children. No differences between genders were seen. Stable content of gp-340 and sialic acid in minor-gland saliva across the age-groups, and a higher content of gp-340 in the whole saliva of the youngest age-group (3-yr-olds) compared with the adult group, may reflect that those components are vital innate factors of immunity in childrens saliva.

Bacterial profiles and proteolytic activity in peri-implantitis versus healthy sites

Peri-implantitis is a biofilm-induced destructive inflammatory process that, over time, results in loss of supporting bone around an osseointegrated dental implant. Biofilms at peri-implantitis sites have been reported to be dominated by Gram-negative anaerobic rods with a proteolytic metabolism such as, Fusobacterium, Porphyromonas, Prevotella and Tannerella, as well as anaerobic Gram-positive cocci. In this study, we hypothesized that protease activity is instrumental in driving bone destruction and we therefore compared the microbial composition and level of protease activity in samples of peri-implant biofluid (PIBF) from 25 healthy subjects (H group) and 25 subjects with peri-implantitis (PI group). Microbial composition was investigated using culture techniques and protease activity was determined using a FITC-labelled casein substrate. The microbial composition was highly variable in subjects both in the H and PI groups but one prominent difference was the prevalence of Porphyromonas/Prevotella and anaerobic Gram positive cocci which was significantly higher in the PI than in the H group. A subgroup of subjects with peri-implantitis displayed a high level of protease activity in the PIBF compared to healthy subjects. However, this activity could not be related to the presence of specific bacterial species. We propose that a high level of protease activity may be a predictive factor for disease progression in peri-implantitis. Further longitudinal studies are however required to determine whether assessment of protease activity could serve as a useful method to identify patients at risk for progressive tissue destruction.

Effect of fluoride and chlorhexidine digluconate mouthrinses on plaque biofilms

Objective : To develop a model in which to investigate the architecture of plaque biofilms formed on enamel surfaces in vivo and to compare the effects of anti-microbial agents of relevance for caries on biofilm vitality. Materials and Methodology : Enamel discs mounted on healing abutments in the pre-molar region were worn by three subjects for 7 days. Control discs were removed before subjects rinsed with 0.1% chlorhexidine digluconate (CHX) or 0.2% sodium fluoride (NaF) for 1 minute. Biofilms were stained with Baclight Live/Dead and z-stacks of images created using confocal scanning laser micoscopy. The levels of vital and dead/damaged bacteria in the biofilms, assessed as the proportion of green and red pixels respectively, were analysed using ImageTrak® software. Results : The subjects showed individual differences in biofilm architecture. The thickness of the biofilms varied from 28-96µm although cell density was always the greatest in the middle layers. In control biofilms, the overall levels of vitality were high (71-98%) especially in the area closest to the enamel interface. Rinsing with either CHX or NaF caused a similar reduction in overall vitality. CHX exerted an effect throughout the biofilm, particularly on the surface of cell clusters whereas NaF caused cell damage/death mainly in the middle to lower biofilm layers. Conclusion : We describe a model that allows the formation of mature, undisturbed oral biofilms on human enamel surfaces in vivo and show that CHX and NaF have a similar effect on overall vitality but differ in their sites of action.

PAI-2/SerpinB2 inhibits proteolytic activity in a P. gingivalis-dominated multispecies bacterial consortium

OBJECTIVE The aim of this study was to investigate the ability of the serine protease inhibitor plasminogen activator inhibitor type 2 (PAI-2/Serpin B2) to inhibit proteases produced by a multispecies bacterial consortium in vitro. BACKGROUND Gingival and periodontal inflammation is associated with an increased flow of protein-rich gingival fluid. This nutritional change in the microenvironment favors bacteria with a proteolytic phenotype, triggering inflammation and associated tissue breakdown. PAI-2 is produced by macrophages and keratinocytes and is present in very high concentrations in gingival crevicular fluid; the highest level in the body. DESIGN A multispecies bacterial consortium comprising nine bacterial strains, resembling the conditions in a periodontal pocket, was grown planktonically and as a biofilm. After seven days PAI-2 was added to the consortium and the proteolytic activity was assayed with fluorogenic protease substrates; FITC-labeled casein to detect global protease activity, fluorescent H-Gly-Pro-AMC for serine protease activity and fluorescent BIKKAM-10 for Porphyromonas gingivalis-associated protease activity. Protease activity associated with biofilm cells was examined by confocal scanning laser microscopy. RESULTS PAI-2 inhibited proteolytic activity of the bacterial consortium, as seen by decreased fluorescence of all substrates. PAI-2 specifically inhibited P. gingivalis proteolytic activity. CONCLUSION To our knowledge, this is the first time that PAI-2 has been shown to inhibit bacterial proteases. Given the high concentration of PAI-2 in the gingival region, our results indicate that PAI-2 might play a role for the integrity of the epithelial barrier.

Modeling the development of proteolytic phenotypes in multi-species oral biofilms

ABSTRACT In chronic periodontitis, subgingival biofilms induce an inflammatory response leading to periodontal tissue destruction which may cause tooth loss. The response includes exudation of fluid (GCF) from the gingival pocket, giving rise to a protein-rich micro-environment in the biofilm. We hypothesize that proteolytic activity in biofilms is a virulence factor contributing to sustained inflammation. To study the influence of GCF on proteolytic activity in a periodontitis-associated biofilm, a multi-species consortium (Porphyromonas gingivalis, Fusobacterium nucleatum, Parvimonas micra and Streptococcus constellatus) was grown in 10% equine serum (to model GCF) or BHI (control) for 2-5 days. Cell-associated and secreted proteolytic activity was investigated with zymography, fluorometry or fluorescent substrates in combination with fluorescence in situ hybridization (FISH). After 2 days, streptococci dominated the consortium in BHI whereas in serum diversity was maintained over 5 days. The serum consortium also developed proteolytic activity, which was absent in BHI. Zymography revealed an array of secreted proteases. FISH revealed proteolytic activity associated with the cell surface of P. gingivalis and F. nucleatum but not P. micra or S. constellatus. Thus, serum favored survival of P. gingivalis and F. nucleatum as well as production of proteases that can act as virulence factors in chronic periodontitis.