A potential role of an intracellular signaling defect in neutrophil functional abnormalities and promotion of tissue damage in patients with localized juvenile periodontitis.

Localized juvenile periodontitis is a destructive form of periodontal inflammatory disease which has its onset at puberty. The etiopathology of the disease is still unclear but neutrophils have been suggested to play a major role both in the production and development of the disorder. About 70% of the patients with localized juvenile periodontitis exhibit neutrophil functional abnormalities, such as decreased chemotaxis and phagocytosis. Interestingly, it has been frequently reported that the same hypoactive cells show an enhanced respiratory burst response and increased adhesion. Several possible mechanisms explaining neutrophil anomalies in localized juvenile periodontitis have been proposed. These include the presence of soluble serum factors capable of modulating neutrophil function, altered cell-surface receptor expression and/or function, and a change in the post-receptor signaling events. Recently, a growing evidence has accumulated showing that the diacylglycerol metabolism could be altered in neutrophils from patients with localized juvenile periodontitis. This change, which may be due to a defect in a major diacylglycerol metabolizing enzyme, diacylglycerol kinase, results in enhanced accumulation of diacylglycerol in activated cells. Because diacylglycerol is an endogenous activator of protein kinase C, the increased and prolonged generation of diacylglycerol could lead to abnormal pattern of protein kinase C-regulated neutrophil functions, explaining the parallel hypo- and hyperactivities.

Increased adhesion of peripheral blood neutrophils from patients with localized juvenile periodontitis.

Adhesion of peripheral blood neutrophils from 5 patients with localized juvenile periodontitis (LJP) and age- and gender-matched healthy controls was measured using a semi-automated 96-well microtiter plate assay method. Both unstimulated and formyl-methionyl-leucyl-phenylalanine (FMLP, 10-1000 nM)-stimulated neutrophils from LJP patients showed in general higher adhesion than did their controls. After 15-60 min incubation with 100 and 1000 nM FMLP the numbers of adherent cells were significantly (p < 0.05), 2.1-2.6-fold higher in LJP patients than in controls. Neutrophils from these LJP patients showed also enhanced respiratory burst activity in response to unopsonized zymosan stimulation. To test whether a decrease in intracellular diacylglycerol (DAG) kinase activity could account for the increased neutrophil adhesion of LJP patients normal neutrophils were treated with R59949 (10 microM), a DAG-kinase inhibitor. Both unstimulated and FMLP-stimulated normal neutrophils showed significantly (p < 0.05) enhanced adhesion after R59949-treatment. Taken together, our data indicate that neutrophils from the 5 LJP patients investigated here exhibit 2 parallel hyperactivities, namely increased adhesion and enhanced production of reactive oxygen species. Furthermore, our present and previous (Hurttia et al., J Periodont Res 1997; 32: 401-407) results suggest that the observed neutrophil functional abnormalities in some LJP patients may be associated with decreased cellular DAG-kinase activity. It is proposed that the hyperadherent and -active neutrophils may promote the development of LJP by causing tissue damage in the periodontium.

Evidence of an association between functional abnormalities and defective diacylglycerol kinase activity in peripheral blood neutrophils from patients with localized juvenile periodontitis.

Peripheral neutrophils from patients with localized juvenile periodontitis (LJP) show functional abnormalities, such as impaired locomotion and enhanced respiratory burst activity. A defect in intracellular signalling mechanism has been proposed to be responsible for some changes, but direct evidence is lacking. In this study we have determined the activity of diacylglycerol (DAG) kinase, an enzyme controlling the DAG/protein kinase C (PKC) pathway, in crude cytosolic and membrane fractions of neutrophils from 5L JP patients and age and gender-matched normal individuals. No difference was observed in the DAG kinase activity in subcellular fractions from unstimulated cells between the 2 groups. When normal neutrophils were stimulated with N formyl-methionyl-leucyl-phenylalanine (FMLP), the enzyme activity was markedly increased in both subcellular fractions. In contrast, neutrophils from 3 of the 5 LJP patients tested completely failed to rise the DAG kinase activity upon chemoattractant stimulation. These data indicate that in some LJP patients the neutrophil DAG kinase may be defective. To examine whether a decrease in DAG kinase activity could account for some neutrophil abnormalities seen in LJP, normal neutrophils were treated with R59022, a DAG kinase inhibitor, that has been shown to reduce DAG kinase activity in human neutrophils. Upon stimulation with FMLP, R59022-treated normal neutrophils showed significantly reduced chemotactic response and enhanced respiratory burst activity, two typical functional abnormalities featured by LJP cells. It is concluded that a defect in DAG kinase may cause, through an abnormal accumulation of the endogenous PKC activator DAG some of the functional changes observed in neutrophils from LJP patients.

Subcellular localization of diacylglycerol kinase activity in stimulated and unstimulated human peripheral blood lymphocytes and neutrophils.

The diacylglycerol (DAG) kinase activity was determined in crude cytosol and membrane fractions in human peripheral blood lymphocytes and neutrophils. In neutrophils the basal lipid kinase activity was identical in the two subcellular fractions whereas in lymphocytes the basal enzyme activity was 1.6-fold higher in the membrane fraction. In general, the DAG kinase activity in lymphocyte fractions was 10- to 20-fold higher than in neutrophil fractions. When lymphocytes were stimulated with phytohaemagglutinin for 4 hours, a significant decrease in the DAG kinase activity in the membrane fraction was detected. By contrast, a 30-min activation with N-formyl-methionyl-leucyl-phenylalanine markedly increased the lipid kinase activity in both subcellular fractions in neutrophils. This activity was partially inhibited by the compound R59022. These results suggest that the DAG kinase is widely distributed in human leukocytes, but the enzyme's activity and regulation may vary in different leukocyte types.

Oral fluid elastase as an indicator of periodontal health.

The study was designed to find out whether oral elastase activity could be used as a simple biochemical indicator of periodontal health. Both stimulated whole saliva and water rinse samples were collected from subjects with different degrees of adult periodontitis, gingivitis or healthy periodontium. In both sample types, elastase was mostly bound to insoluble fraction and preferred valine containing synthetic substrate, similar to neutrophil elastase. The elastase measurement required very little manipulation or time and its reproducibility was found to be good. The elastase levels were found to be negligible in edentulous subjects and usually very low in subjects with healthy periodontium. In about 85% of periodontitis cases having at least 1 deep periodontal pocket ( > or = 6 mm), clearly elevated elastases levels were detected in both the saliva and r rinse samples. In advanced periodontitis cases, the colour reaction took place in 0.5 to 2 h. In localized periodontitis cases, 2- to 18-h incubations were required for positive reaction. There was a good correlation between the elastase activity and the number of deep periodontal pockets and the average community periodontal index of the subjects. Elastase activity was not a good indicator of gingivitis. About 45% of gingivitis cases were positive with the elastase test, and the enzyme values were not significantly increased in experimental gingivitis. In a longitudinal study on advanced periodontitis cases, elastase levels dropped dramatically as a result of clinically successful therapy, close to the values of healthy subjects. The oral elastase test could serve as a valuable adjunct in periodontal screening and assessment of treatment efficacy.

Diacylglycerol in peripheral blood neutrophils from patients with localized juvenile periodontitis.

Neutrophils from patients with localized juvenile periodontitis (LJP) show several functional abnormalities. Recently, it has become increasingly apparent that the reason for these changes lies in part at the post receptor level of cellular metabolism. In this study we have analyzed intracellular diacylglycerol (DAG), a second messenger and an endogenous activator of protein kinase C, in unstimulated and agonist-stimulated neutrophils, from five LJP patients showing a chemotaxis defect and matched normal individuals. No difference was observed in the basal cellular DAG between the two groups. In neutrophils from LJP patients the DAG levels increased by 67% and 111% from the basal level following stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP) and unopsonized zymosan particles, respectively, while in control cells the mean increases were 36% and 65%, respectively. Incubation with serum-opsonized zymosan particles produced an identical rise in DAG in both groups. These data indicate that the stimulation of receptors for FMLP and unopsonized zymosan may produce an enhanced accumulation of DAG in neutrophils from LJP patients. In addition to DAG mass analysis, we determined the effect of R59022, a DAG-kinase inhibitor, on zymosan-stimulated luminol-amplified chemiluminescence (CL) of neutrophils. In control cells R59022 significantly enhanced unopsonized zymosan induced CL, but it had no effect on cells from LJP patients, suggesting a possible change in the regulation of DAG-kinase in LJP.

Increased respiratory burst activity is associated with normal expression of IgG-Fc-receptors and complement receptors in peripheral neutrophils from patients with juvenile periodontitis.

The respiratory burst activity in peripheral neutrophils from nine patients with localized juvenile periodontitis (LJP) and age- and sex-matched healthy controls was studied by measuring the intensity of luminol-enhanced chemiluminescence (CL) induced by unopsonized and three differently opsonized zymosan particles, formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol myristate acetate (PMA). The neutrophils from LJP patients showed in general more intense CL with all activators than did their controls. Particularly, the CL response induced by unopsonized zymosan particles and FMLP were significantly higher (p < 0.05 and 0.001). When comparisons were made between female LJP patients (n = 6) and matched controls, also serum-opsonized and IgG-opsonized zymosan particles produced CL was significantly increased (p < 0.05). In order to determine whether the elevated CL responses to zymosan particles were due to altered levels of the interacting receptors on neutrophil surface, an immunofluorescence analysis of the expression of IgG-Fc-receptors (FcR) and complement receptors (CR) was performed with flow cytometry. No significant difference in the expression of FcRII, FcRIII, CR1 and CR3 was detected in LJP group compared to controls. Since the elevated CL responses can not be explained by changes in receptor numbers it is hypothesized that the increased respiratory burst activity in LJP may be caused by altered post receptor signalling pathway.

Immunohistochemical detection of androgen receptors in human oral mucosa.

Oral mucosal biopsies from 11 healthy volunteers, 7 women and 4 men, were analysed for the localization of androgen, oestrogen and progesterone receptors. The samples were dissected as quickly as possible and immediately frozen in liquid nitrogen to be stored at -70 degrees C. Only androgen receptors could be detected by the methods used. These were mainly located in the nuclei of basal epithelial cells, and to some extent in the nuclei of fibroblasts and endothelial cells. Failure in the localization of oestrogen and progesterone receptors might have been due either to low numbers of receptors or to the insensitivity of antibodies used. The method is thus suitable only for detecting androgen receptors.

Altered tissue metabolism of progesterone in pregnancy gingivitis and granuloma.

The metabolism of progesterone may play a special role in the gingival physiology. The lower the metabolism the higher its hormonal activity in the tissue. In healthy human gingiva, progesterone is metabolized only partially and is therefore in an active form. In the present study, gingival samples from pregnancy gingivitis (n = 1) and granulomas (n = 4) were studied histologically and biochemically. All samples were homogenized and then incubated with [4-14C]-progesterone and NADPH for 2 h at pH 7.4 and 37 degrees C. The metabolites were separated and characterized with column, solvent and thin-layer chromatographies as well as radioautography and quantified with liquid scintillation counting. The results showed low metabolism of progesterone, indicating active hormonal function as in healthy gingiva. It is suggested that progesterone functions as an immunosuppressant in the gingival tissues of pregnant women, preventing the rapid acute-type of inflammatory reaction against plaque, but allowing an increased chronic-type of tissue reaction, resulting clinically in an exaggerated appearance of inflammation.

Vascular and cellular responses to guanidino propyl piperazine CK0569A, a new anti-plaque agent.

Guanidino propyl piperazine CK0569A (abbreviation piperazine) used as a 0.1% mouthrinse has been shown to be effective against plaque bacteria. In addition to its efficacy as an antibacterial agent, its safety should be taken into consideration. In this study the effect of piperazine on the microcirculation of the hamster cheek pouch was studied. The effect of this drug was also investigated on rat leukocytes in vivo and on macrophages and erythrocytes in vitro. Piperazine caused a distinct retardation in the microcirculation of the hamster cheeck pouch even at a low concentration (0.01%), but the effect was abolished in 5 min. The number of leukocytes decreased markedly in the experimentally induced inflammatory exudate in the presence of 0.05% piperazine as compared to the controls. The macrophages in vitro released their lysosomal and cytoplasmic enzymes and lost their viability. The erythrocytes in vitro were destroyed in the presence of 0.005% piperazine as well. Based on these results it can be suggested that 0.005-0.05% piperazine under experimental conditions causes changes that indicate loss of cell viability.

Role of granulation tissue and fibroblasts in gingival testosterone metabolism in the rat in vitro.

The metabolism of testosterone by experimental granulation tissue, fibroblasts and the oral mucosa of rats of both sexes was studied. The experimental granulation tissue was produced by implanting viscose-cellulose sponges beneath the dorsal skin of female and male rats for 21 days. The granuloma capsules, fibroblasts in the sponges and the oral mucosae were homogenated. Mitochondrial, microsomal and soluble fractions were incubated with [4-14C]testosterone and NADPH for 30 min at pH 7.4 and 37 degrees C. The metabolites were identified with column and TLC and radioautography and quantified with liquid scintillation counting. The experimental granulation tissue and fibroblasts of both sexes showed less activity in metabolizing testosterone than the gingival tissue. The tissues were shown to contain 3 alpha-, 3 beta- and 17 beta-hydroxysteroid dehydrogenase and 5 alpha- and 5 beta-steroid hydrogenase activities. The activities of the enzymes in the oral mucosae were higher than in the experimental granulation tissue and fibroblasts.

Biochemical, microbiologic, and clinical comparisons between two dentifrices that contain different mixtures of sugar alcohols.

It has been customary to think that in a dentifrice only a few of its ingredients would be active and have clinically significant effects on dental caries, oral hygiene, and the levels of caries-inducive microorganisms or harmful plaque metabolic products. Therefore, most of the emphasis has been placed on the type of fluorine compounds, abrasives, or similar dentifrice ingredients. This study shows that such common dentifrice components as the humectants, which contribute to the texture, rheologic characteristics, and shelf life of the product, also may affect the type of dental plaque grown on the tooth surfaces between toothbrushings or during long-term neglect of toothbrushing or of oral hygiene. Commonly used humectants include sorbitol, a sugar alcohol of the hexitol type, which is used often in sugarless candies. This study showed that when sorbitol in a dentifrice was replaced by xylitol, a sugar alcohol of the pentitol type, the dental plaque of human subjects contained more ammonia and significantly less bacterial polysaccharides. It is accepted generally that ammonia neutralizes plaque acids and that bacterial polysaccharides are involved in promoting caries. Xylitol-containing dentifrice also reduced the saliva levels of S mutans. The results further indicated that if sorbitol and xylitol could be compared in a short-term dentifrice study that relied on subjective and coarse plaque determinations only, no differences between those dentifrices would be found necessarily. To demonstrate the differences between the experimental dentifrices used in this study, it was necessary to analyze specific plaque components and the salivary levels of S mutans.(ABSTRACT TRUNCATED AT 250 WORDS)