Immune responses to porphyromonas gingivalis infection suppress systemic inflammatory response in experimental murine model.

Periodontitis is a localized infectious disease caused by periodontopathic bacteria such as Porphyromonas gingivalis (P. gingivalis), and the severity correlates to significance of immune responses. Recently, it has been reported that periodontitis is associated with the development of systemic disease such as diabetes and atherosclerosis because of increasing invasion of oral pathogens to the circulation. However, the association between local and systemic infectious responses is still unclear. In the present study, we examined the differences of biological responses in animals with or without bacterial infection. After Balb/c mice were infected subcutaneously with live P. gingivalis W83, serum, skin and liver were collected according to experimental protocol. The skin and liver tissues were observed pathologically by haematoxylin-eosin staining, and serum IL-6 levels were measured using ELISA method. Throughout the experimental period, conditions of the mice were observed continuously. As expected, severe infiltration of leukocytes were observed at inflamed skin corresponding to the number of bacterial challenges. Although no inflammatory appearance of skin was observed, serum IL-6 levels were increased dramatically (P <0.01, Student's t-test) and liver tissues were injured in the mice without bacterial challenge. Interestingly, although severe inflammatory appearance of the skin was observed, serum IL-6 levels were not increased and no inflammatory responses were observed in the liver of the 3-times bacterially challenged group. Importantly, immunoglobulin G against P. gingivalis W83 was detected in the blood of mice with 3-times bacterial challenge corresponding to improvement of weight loss and survival. In conclusion, although multiple infections develop severe localized inflammation, the immune system should be sufficient to protect the systemic inflammatory responses.

FGF-2 stimulates periodontal regeneration: results of a multi-center randomized clinical trial.

The efficacy of the local application of recombinant human fibroblast growth factor-2 (FGF-2) in periodontal regeneration has been investigated. In this study, a randomized, double-blind, placebo-controlled clinical trial was conducted in 253 adult patients with periodontitis. Modified Widman periodontal surgery was performed, during which 200 µL of the investigational formulation containing 0% (vehicle alone), 0.2%, 0.3%, or 0.4% FGF-2 was administered to 2- or 3-walled vertical bone defects. Each dose of FGF-2 showed significant superiority over vehicle alone (p < 0.01) for the percentage of bone fill at 36 wks after administration, and the percentage peaked in the 0.3% FGF-2 group. No significant differences among groups were observed in clinical attachment regained, scoring approximately 2 mm. No clinical safety problems, including an abnormal increase in alveolar bone or ankylosis, were identified. These results strongly suggest that topical application of FGF-2 can be efficacious in the regeneration of human periodontal tissue that has been destroyed by periodontitis.

In situ ATR-FTIR study of oxygen reduction at the Pt/Nafion interface.

We have developed a new half-membrane-electrode assembly (MEA)-type cell that allows us to conduct attenuated total reflectance-Fourier transform infrared (ATR-FTIR) measurements at the Pt/Nafion interface under humidified N(2)/O(2) atmosphere. The cell consists of a gas-diffusion type anode placed on a carbon separator with a gas flow field, a Pt film cathode deposited chemically on an Si ATR prism and a Nafion NRE(R)211 electrolyte sandwiched between them. The construction allows the control of the atmosphere at the cathode by those at the anode via the electrolyte of 20-microm thickness. An infrared absorption band was observed at 1400-1403 cm(-1) under humidified oxygen atmosphere in close association with the appearance of ORR current. Its absence under N(2) atmosphere and insensitivity to the change from H(2)O to D(2)O humidification led us to ascribe the band to the O-O vibration of the adsorbed oxygen molecule O(2)(ads). The band intensity increased with increasing ORR current but decreased significantly in the limiting current region. However, the stability of the species at potentials as high as 1.1 V vs. the reversible hydrogen electrode (RHE) led us to rule out the possibility that the band could be due to adsorbed superoxide O(2)(-).

Molecular and enzymatic characterization of betaC-S lyase in Streptococcus constellatus.

BACKGROUND/AIMS: Streptococcus anginosus and Streptococcus constellatus are frequently isolated from dental abscesses and other suppurative lesions. We previously reported that betaC-S lyase from a strain of S. anginosus produced significantly more hydrogen sulfide than betaC-S lyases from other streptococci. The purpose of this study was to establish the molecular and enzymatic features of the betaC-S lyase in S. constellatus and to elucidate whether this unique capacity is common to many strains of S. constellatus and S. anginosus. METHODS: The capacity of crude extract to produce hydrogen sulfide was evaluated among 16 strains of S. constellatus, S. anginosus, and Streptococcus gordonii. The lcd gene encoding betaC-S lyase was cloned from the genomic DNA of each strain to compare the deduced amino acid sequences. The recombinant betaC-S lyases of three representative strains were purified and characterized. RESULTS: Incubation of crude extracts from all strains of S. constellatus and S. anginosus with l-cysteine resulted in the production of a large amount of hydrogen sulfide. The primary sequence of betaC-S lyase was very similar among strains of S. constellatus and S. anginosus. The kinetic properties of the betaC-S lyases purified from S. constellatus resembled those for betaC-S lyases purified from S. anginosus. In contrast, the betaC-S lyases of S. constellatus and S. gordonii differed in terms of their hydrogen sulfide production, with the former producing much more. CONCLUSION: A high level of hydrogen sulfide production, which appears to be a common feature in both S. constellatus and S. anginosus, may be associated with their abscess formation.

Long-term prognosis of parathyroid function after successful percutaneous ethanol injection therapy (PEIT) guided by color Doppler flow mapping in chronic dialysis patients.

Secondary hyperparathyroidism (2 HPT) is a representative disease of dialysis osteopathy, with the lesion that makes fibrous osteitis and the parathyroid hyperplasia by the hyper secretion of parathyroid hormone (PTH). This research examines the usefulness of selective percutaneous ethanol injection therapy (PEIT) of parathyroid glands in order to treat and control for 2 HPT. PEIT was performed in 46 patients resistant to calcitriol pulse therapy and all glands larger than 5 mm in diameter were destroyed by ethanol guided by power Doppler flow mapping. Serum intact-PTH (iPTH) levels fell from 633.3 +/- 359.9 to 226.3 +/- 204.7 pg/mL at three weeks and were maintained at 289.9 +/- 222.4 pg/mL at one year after PEIT. Total alkaline phosphatase activity fell from 384.9 +/- 160.1 to 234.0 +/- 110.5 IU/L at one year after PEIT. In 19 patients, i-PTH levels fell into relative hypoparathyroidism (iPTH < 160 pg/mL) at three weeks after PEIT: however, they recovered at one year after PEIT (191.1 +/- 29.6 pg/mL). In total, parathyroid function was maintained at optimal range (160 < iPTH < 360 pg/mL) in 80.4% of patients at one year after PEIT with appropriate medical therapy. As for the complications, recurrent nerve palsy was observed in only one patient, but was reversible. In conclusion, selective PEIT appears to be able to control appropriate parathyroid function and to be the method of choice to treat 2 HPT prior to parathyroidectomy.

Role of medullasin in nifedipine-induced gingival overgrowth in rats.

To clarify the possible pathophysiological role of medullasin, a neutrophil elastase-like proteinase, in nifedipine (NF)-induced gingival overgrowth, a rat model of gingival overgrowth was first established using a diet containing NF. The relation between histopathological changes and the distribution of the proteinase was then investigated. Thirty-two, specific pathogen-free 20 day-old, male, Fisher 344 rats were fed a diet containing NF and killed at 2, 8, 16 and 32 weeks. Control rats (n = 32) were fed the same diet but without the drug. The mandible of each rat was resected and sectioned at 4-microm thickness buccolingually between the first and second molars. Computer image analysis was used to evaluate the extent of overgrowth in the approximal gingiva of each sample. To examine medullasin activity, the mean percentage of medullasin-positive cells per total cells counted in the pocket epithelium and the connective tissue adjacent to the epithelium of approximal gingiva was determined immunohistochemically. The height of the mid-portion and the area in NF-treated group increased significantly with time (with the exception of area at 2 weeks) compared with the corresponding regions in the control group. A marked inflammatory-cell infiltration and elongated rete pegs, especially in the mid-portion of approximal gingiva, were seen in the NF-treated group. The mean percentages of medullasin-positive cells in the NF-treated group at 8, 16 and 32 weeks were significantly higher than those of the control. Although medullasin-positive cells were mainly neutrophils, in several samples of the NF-treated group they were recognized as macrophage-like. These findings suggest that medullasin may be involved in host defence and immunoregulation in a NF-induced rat model of gingival overgrowth.

Rice bifunctional alpha-amylase/subtilisin inhibitor: characterization, localization, and changes in developing and germinating seeds.

A bifunctional alpha-amylase/subtilisin inhibitor (RASI) was purified to electrophoretic homogeneity from rice (Oryza sativa L.) bran. Its molecular mass was 21 kDa by SDS-PAGE and its isoelectric point was 9.05. Purified RASI inhibited subtilisin Carlsberg strongly and inhibited alpha-amylase from germinating rice seeds weakly. It inhibited rice alpha-amylase more than barley alpha-amylase, and the inhibition of rice alpha-amylase was greater at higher pHs. RASI did not inhibit trypsin, chymotrypsin, cucumisin, or mammalian alpha-amylase. The RASI was in the outermost part of the rice grain and its subcellular site seemed to be aleurone particles in aleurone cells. SDS-PAGE and western blotting showed that RASI was synthesized in the late milky stage in developing seeds, and it remained fairly constant during the first 7 days of germination.

Immunohistochemical study of cathepsin G and medullasin in inflamed gingival tissues from periodontal patients.

Cathepsin G and medullasin are 2 major serine proteinases associated with the granular fraction of polymorphonuclear leukocytes (PMNs). To know their possible involvement in the pathophysiological gingival connective tissue turnover, we have determined the distribution and localization of these 2 enzymes in inflamed gingival tissues from periodontal patients by immunohistochemistry with discriminating antibodies specific for each enzyme. The gingival connective tissues were obtained from periodontitis patients with various inflammatory conditions and control healthy subjects without any clinical signs of periodontal inflammation. In all gingival specimens examined, cathepsin G and medullasin were found mainly in neutrophil-like cells and partly in macrophage-like cells. No positive staining for both enzymes was obtained in endothelial cells and fibroblasts in every part of the gingival tissues. Immunoreactivity for each enzyme in the gingival tissues from the periodontitis group was stronger and greater in the intensity and frequency than that from the control group and appeared to be increased with the severity of the disease In both groups, the number of immunoreactive cells for each enzyme was greater in the vicinity of pocket epithelium (zone I) than in the area of central connective tissue (zone II) or the area subjacent to the oral epithelium (zone III). While both enzymes in zones II and III were exclusively found in coarse granules, their stainings in zone I were not only coarse but also diffuse. These results strongly suggest that both enzymes may have some association with inflamed gingival tissue degradation.

Cathepsin B in gingival crevicular fluid of adult periodontitis patients: identification by immunological and enzymological methods.

Cathepsin B (EC 3.4.22.1), a typical lysosomal cysteine proteinase was identified immunologically with anti-human cathepsin B antibody in inflammatory exudate, gingival crevicular fluid (GCF) of adult periodontitis patients. The sensitive enzyme immunoassay (EIA) system initially developed, was rarely influenced by the presence of endogenous cysteine proteinase inhibitors, cystatin(s), indicating that it is possible to quantify the gross amount of cathepsin B including free enzyme forms and enzyme-inhibitor complex forms using this EIA system. The cathepsin B levels in GCF as determined by EIA and the activity measured with Z-Arg-Arg-MCA showed positive and significant correlation with various clinical parameters. Immunoblotting analysis revealed that the molecular form was a 29 kDa mature enzyme. More than 95% of Z-Arg-Arg-MCA hydrolytic activity in each GCF sample was inhibited by CA-074, specific inhibitor of cathepsin B. These results strongly suggested that the gross amount of cathepsin B in GCF as well as its activity level is closely associated with the severity of the disease and that cathepsins B play an important role in the pathogenesis of periodontitis.

Possible roles of medullasin in nifedipine-induced human gingival overgrowth.

In order to clarify a possible pathophysiological role of medullasin, a neutrophil elastase-like proteinase, in nifedipine (NF)-induced gingival overgrowth, the distributions of medullasin-positive cells immunostained in specimens from patients with NF-induced gingival overgrowth and chronic marginal gingivitis were compared in three different biopsy areas. Twenty gingival biopsies were obtained from five patients with gingival overgrowth and 20 biopsies from another five patients with chronic marginal gingivitis. In the marginal gingivitis group, the mean percentage of positive cells in the vicinity of pocket epithelium (zone I) was significantly higher than in the areas of connective tissue of the mid-portion (zone II) and adjacent to oral epithelium ( zone III) (p < 0.05). In the gingival overgrowth group, on the contrary, the positive cells significantly increased in zone II as compared with zones I and III (p < 0.05). Further, medullasin-positive cells of zones II and III in the overgrowth group had infiltrated more extensively than those in the gingivitis group (p < 0.001), indicating the participation of this enzyme in the mechanism of NF-induced gingival overgrowth. These observations suggest that medullasin may play a part in NF-induced overgrowth both in host defence and in immunoregulation, possibly cytotoxically.

Identification and possible function of cathepsin G in gingival crevicular fluid from chronic adult periodontitis patients and from experimental gingivitis subjects.

The levels of cathepsin G in gingival crevicular fluid (GCF) from chronic adult periodontitis patients and experimental gingivitis subjects were determined both by activity measurement using N-benzoyl-(DL)-phenylalanine-2-naphthyl ester as a substrate and by enzyme immunoassay using anti-human cathepsin G IgG. The activity level of cathepsin G in GCF of both periodontitis and experimental gingivitis has no significant correlation with all measured clinical parameters. Western immunoblotting using antibodies specific for cathepsin G or alpha 1-proteinase inhibitor revealed that the difficulty in demonstrating the association of its activity with the severity of these diseases was due largely to formation of the enzyme-inhibitor complexes. By contrast, statistically significant positive correlation was found between cathepsin G content in GCF of periodontitis, which was determined by enzyme immunoassay, and such clinical parameters as the GCF volume, the gingival index and probing depth. The increased cathepsin G content with increasing severity of periodontal inflammation was markedly diminished by the initial treatment. Although no significant activity was detectable in GCF of experimental gingivitis, a rapid increase of the immunoreactive cathepsin G was found in GCF at 3-5 d after refraining from oral hygiene measures, which rapidly decreased by 10 d. The progressively increased cathepsin G between 10th and 21st d rapidly decreased by cleaning of the teeth. The results indicate that cathepsin G is involved in the host's defensive mechanism against the invasion of etiologic microbes and/or the development of either periodontitis or gingivitis.

A cross-sectional study on osteocalcin levels in gingival crevicular fluid from periodontal patients.

The purpose of the present study was to determine the levels of osteocalcin, a bone specific matrix protein, in gingival crevicular fluid (GCF) from periodontal disease patients and to investigate the relationship between GCF osteocalcin levels and clinical parameters. Nineteen initial visit patients, 5 patients with gingivitis and 14 patients with adult periodontitis, participated in this study. The clinical parameters including probing depth, attachment level, gingival index, and tooth mobility were recorded following careful sampling of GCF with a filter paper strip harvested for 3 minutes. Osteocalcin adsorbed on a strip was extracted in a plastic tube containing 150 microliters of 10 mM sodium phosphate buffer (pH 6.5). GCF osteocalcin was determined by a newly-developed, high sensitive enzyme immunoassay which could recognize the N-terminal 20 residue peptide. In gingivitis patients, no significant amounts of osteocalcin were detected. In periodontitis patients, on the other hand, osteocalcin levels were detected, ranging between 0 and 540 pg/tube and positively correlated with these clinical parameters (P < 0.01). Moreover, in several sites in GI = 3 group, extremely higher levels of GCF osteocalcin were detected. These results strongly suggest that in addition to the presence of GCF osteocalcin the levels of osteocalcin may reflect the degree of the periodontal inflammation at the sampled sites.

Characterization of hemoglobin-hydrolyzing acidic proteinases in human and rat neutrophils.

The nature and levels of hemoglobin (Hb)-hydrolyzing acidic proteinases including cathepsin D and cathepsin E, which were most active at pH 3.5-4.0, were enzymatically and immunochemically compared between human and rat neutrophils. By subcellular fractionation and immunoprecipitation with discriminative antibodies specific for each enzyme, cathepsin D was shown to be present in the granular content fraction of both human and rat neutrophils and to account for about 35% of the total Hb-hydrolyzing activity. Cathepsin E was observed mainly in the cytoplasmic fraction of rat neutrophils from peripheral blood and peritoneal exudates and accounted for about 65% of the total activity, but it was not detected in human blood neutrophils. Immunoelectron microscopy on rat neutrophils revealed that cathepsin D was exclusively confined to lysosomes, whereas cathepsin E was localized mainly in the cytoplasmic matrix and often in the perinuclear spaces and the rough endoplasmic reticulum. The non-cathepsin D activity in human neutrophils, which represented about 65% of the total activity, appeared to be due to a serine proteinase, since it was inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride and was not inhibited by agents specific for aspartic-, cysteine-, or metallo proteinases. The enzyme(s) responsible for this activity was largely associated with the granular membranes, and a half of it could be described as an integral membrane protein on the basis of phase separation with Triton X-114 at 35 degrees C. The levels of these Hb-hydrolases in gingival crevicular fluid from human chronic inflammatory periodontitis patients were examined in order to clarify their participation in the periodontal tissue breakdown.(ABSTRACT TRUNCATED AT 250 WORDS)

Granulocyte medullasin levels in gingival crevicular fluid from chronic adult periodontitis patients and experimental gingivitis subjects.

The concentration of medullasin, an elastase-like serine proteinase, in gingival crevicular fluid (GCF) from chronic adult periodontitis patients and experimental gingivitis subjects was determined by the highly sensitive immunoassay method. In periodontitis patients, the medullasin content increased with increase of the GCF volume and then attained a maximum value at a relatively mildly inflamed stage. The value was maintained through more serious stages of disease activity. However, the medullasin content was independent of the probing depth. The medullasin content of the patients was markedly decreased after periodontal treatment, indicating that the enzyme participates in the development of the chronic periodontitis. Large amounts of medullasin were also detected in GCF from experimental gingivitis subjects, although it was not detected by the activity measurements. There was a rapid increase in the medullasin content during the 4-day period after abstention from oral hygiene measures, which corresponded to those of severely inflamed periodontitis patients. The peak value decreased up to the 7th-d followed by a gradual increase during the 21-d experimental period. The increased medullasin level rapidly decreased following resumption of oral hygiene measures. The results suggest that medullasin plays important roles both in the defence mechanism against the gingival inflammation and in the development of the acute inflammation.

Cathepsins B, H and L activities in gingival crevicular fluid from chronic adult periodontitis patients and experimental gingivitis subjects.

To clarify roles of lysosomal cysteine proteinases cathepsins B, H and L in pathological destructive process of periodontal tissues, levels of their enzymatic activities were determined in gingival crevicular fluid (GCF) from chronic adult periodontitis patients and from experimental gingivitis subjects. In periodontitis patients, higher levels of cathepsins B, H and L activities were found at sites with more serious signs of the disease activity. The total activity of each enzyme (per unit time) was positively correlated with the GCF volume. However, it had little or no correlation with the probing depth (PD). In contrast, the specific activity of each enzyme in GCF (activity units per mg protein), which reflects the selectivity of enzyme exudation, was negatively correlated with the GCF volume. These results suggest that the cysteine proteinases are selectively released into gingival crevices at a relatively mild stage of periodontitis. In experimental gingivitis subjects, no significant activity of each enzyme was detected in GCF, even when the quantity of GCF was comparable to that from periodontitis patients. These data suggest that no significant amounts of these enzymes are released at experimental gingivitis sites or that a homeostatic mechanism, including regulation by protease inhibitors, may control activities of these enzymes in GCF with acute inflammation.

[Epidemiological study of periodontal disease. Mass examination in university students with CPITN].

The purpose of this study was to investigate the prevalence and treatment needs of periodontal disease in the 1,150 university students (male: 726, female: 424) by using simplified Community Periodontal Index of Treatment Needs (CPITN-S). The results were as follows: 1. Approximately 85% of all the subjects have something wrong in periodontal tissue. If examined in detail; 1) Code 4: a group (1.6%) who had a deep pocket more than 6 mm. 2) Code 3: a group (20.1%) who had a shallow pocket 4 to 5 mm. 3) Code 2: a group (55.9%) who had supra- or subgingival calculus. 4) Code 1: a group (7.5%) who had bleeding on probing only. 2. As for treatment needs (TN), approximately 78% of the subjects need to receive professional prophylaxis and more complex treatment including periodontal surgery (TN 2-3). 3. Average time required to examine one subject was about one minute, so we could cut down the measuring time. From this point, we have understood that simplified CPITN was greatly effective to curtail the time for the primary screening of periodontal disease in the young age group.