Sensitivity of genera Porphyromonas and Prevotella to the bactericidal action of C-terminal domain of human CAP18 and its analogues.

This paper reports the effect of the synthesized 27-amino acid sequence in the C-terminal domain of human CAP18 (hCAP18), a human cationic antibacterial protein or cathelicidin, on certain strains belonging to the genera Porophyromonas and Prevotella. The domain binds lipopolysaccharides (LPS) from Porophyromonas gingivalis and Porophyromonas circumdentaria as well as enterobacterial LPS. Two analogues of hCAP18, designated LL/CAP18 and FF/CAP18, were also tested to determine whether additional activity was obtained. The analogue peptides replaced with hydrophobic and cationic amino acid residues showed more potent bactericidal and LPS-binding activities than the original one.

Heterogeneous expression of Toll-like receptor 4 and downregulation of Toll-like receptor 4 expression on human gingival fibroblasts by Porphyromonas gingivalis lipopolysaccharide.

Porphyromonas gingivalis (P. gingivalis) is implicated in the initiation and progression of periodontitis. Human gingival fibroblasts (HGFs) are the major constituent of gingival connective tissue. P. gingivalis or its components such as lipopolysaccharide (LPS) upregulate the production of various inflammatory cytokines including interleukin (IL)-1 and IL-6 in HGFs. Recently, we demonstrated that the binding of P. gingivalis LPS to Toll-like receptor 4 (TLR4) on HGFs activates various second messenger systems (Biochem. Biophys. Res. Commun. 273, 1161-1167, 2000). In the present study, we examined the level of TLR4 expression on HGFs by flow cytometric analysis (FACS), and studied the levels of IL-1 and IL-6 in the culture medium upon LPS stimulation of HGFs by enzyme-linked immunosorbent assay (ELISA). Upon stimulation by P. gingivalis LPS for 24 h, HGFs that expressed a high level of TLR4 secreted significantly higher levels of IL-1 and IL-6 than HGFs that expressed a low level of TLR4. On the other hand, after stimulation with P. gingivalis LPS for 24 h, the level of TLR4 on the surface of HGFs decreased. These results suggest that the level of TLR4 expression on HGFs reflects the extent of inflammation in the gingival tissue, and that P. gingivalis LPS downregulates TLR4 expression on HGFs. These findings may be used to control inflammatory and immune responses in periodontal disease.

Porphyromonas gingivalis gingipain-R enhances interleukin-8 but decreases gamma interferon-inducible protein 10 production by human gingival fibroblasts in response to T-cell contact.

Proteases produced by Porphyromonas gingivalis, an oral pathogen, are considered important virulence factors and may affect the responses of cells equipped with proteinase-activated receptors. The aim of this study was to investigate the effect of the arginine-specific cysteine protease gingipain-R produced by P. gingivalis on chemokine production by human gingival fibroblasts (HGF) and the effect of gingipain-R treatment on the subsequent contact-dependent activation of HGF by T cells. HGF incubated in the presence of purified 47-kDa gingipain-R showed increased levels of interleukin-8 (IL-8) mRNA. Cyclooxygenase-2 (COX-2) mRNA was also induced. Further exposure of HGF to activated T cells resulted in the dose- and time-dependent enhancement of IL-8 transcription and release. T-cell membrane-bound tumor necrosis factor (TNF) was the ligand inducing IL-8 production by HGF, since TNF neutralization abrogated HGF responses to T-cell contact. The enhanced IL-8 release was due, at least in part, to prostaglandin-E(2) production, which was mostly blocked by indomethacin. Gingipain-R proteolytic activity was required since heat inactivation, specific synthetic protease inhibitors, and the natural substrate competitor histatin 5 abrogated its effects. The enhanced production of IL-8 in response to T-cell contact was specific since monocyte chemotactic protein-1 (MCP-1) production was unaffected while interferon-gamma-inducible protein-10 (IP-10) was inhibited. The sum of these activities may result in the recruitment of differential cell types to sites of inflammation since IL-8 preferentially recruits neutrophils and IP-10 attracts activated T cells and may be relevant to the pathogenesis of periodontitis.

Involvement of caspases in apoptotic cell death of murine macrophages infected with Actinobacillus actinomycetemcomitans.

Infection of murine macrophages in vitro with periodontopathic bacterium Actinobacillus actinomycetemcomitans induces apoptotic cell death. In this study, we investigated the involvement of caspases in apoptotic cell death of A. actinomycetemcomitans-infected macrophages. Two peptide inhibitors of caspases, benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone (Z-VAD-FMK) and benzyloxycarbonyl-Asp-Glu-Val-Asp (OMe)-fluoromethyl ketone (Z-DEVD-FMK), inhibited apoptotic cell death of murine macrophage cell line J774.1 infected with A. actinomycetemcomitans. During the process of apoptosis, interleukin-1beta (IL-1beta) was detected in the culture supernatants of J774.1 cells. IL-1beta secretion was blocked by the caspase-1 inhibitor, Z-VAD-FMK, indicating that caspase-1 is involved in not only the induction of apoptosis but also the IL-1beta secretion from A. actinomycetemcomitans-infected J774.1 cells. Immunoblot analysis revealed that the infection of A. actinomycetemcomitans to J774.1 cells induced the cleavage of retinoblastoma protein (Rb), suggesting that caspase-3 was activated by A. actinomycetemcomitans infection. The cytosol from A. actinomycetemcomitans-infected J774.1 cells induced Rb proteolysis in vitro, which was inhibited by the caspase-3 inhibitor, Z-DEVD-FMK. Furthermore, caspase-3-like activity was markedly increased in J774.1 cells infected with A.actinomycetemcomitans between 12 h and 24 h, which was subsequently inhibited by the addition of caspase-3 inhibitor, Z-DEVD-FMK. These findings indicate that caspase-3 induces apoptosis in J774.1 cells infected with A. actinomycetemcomitans. Taken together, these results suggest that caspase-1 and caspase-3 are involved in the induction of apoptosis in A. actinomycetemcomitans-infected macrophages.

In situ detection of gelatinolytic activity in developing craniofacial tissues.

Frozen tissue sections of developing and adult rat heads were incubated on a film coated with a gelatin-containing colloidal silver emulsion in order to detect gelatinolytic activity present in the different tissues. The method, termed film in situ zymography, is based on the ability of the thiol group of the propeptide released from the degraded gelatin to induce a structural change in the colloidal silver and thereby a visible change in color. The frozen tissue sections mounted on the coated film were incubated at 37 degrees C overnight. Gelatinolytic activity was detected as a color change from yellow to red. The activity of gelatinase was completely blocked by phenanthroline, which inhibits matrix metalloproteinases. Gelatinolytic activity was widely present in the oral epithelium, tooth buds, tongue, Meckel's cartilage, salivary glands, and other tissues. The intensity of the gelatinolytic activity varied among the different tissue types. The present study demonstrated gelatinolytic activity in both developing and adult craniofacial tissues. These results suggest that gelatinolytic activity plays an important role in normal turn-over in several tissues. Whereas some of the activity also in the developing rats may be related to this turn-over process, some of it is probably directly associated with developmental changes.

Inhibitory effect of lipopolysaccharide on apoptotic cell death in macrophages infected with Actinobacillus actinomycetemcomitans.

We have reported that the macrophage-like cell line J774.1, when infected with the periodontopathic bacterium Actinobacillus actinomycetemcomitans, undergoes apoptosis. In this study, we examined whether stimulation of J774.1 cells with lipopolysaccharide (LPS) before the infection affects the subsequent apoptosis. Cytotoxicity on the LPS-stimulated cells was about half of the unstimulated control cells. DNA fragmentation in the LPS-stimulated cells was also significantly lower than in the control cells, whereas it was increased to a level similar to that of the control cells by addition of a nitric oxide (NO) inhibitor. In addition, significantly smaller numbers of live A. actinomycetemcomitans were recovered from the LPS-stimulated macrophages at 8 h after the infection as compared with the control cells. These findings suggest that the inhibitory effect of LPS on apoptosis results from an enhanced NO-mediated bactericidal activity.

Purification and characterization of a trypsin-like protease from the culture supernatant of Actinobacillus actinomycetemcomitans Y4.

We purified and characterized a protease from Actinobacillus actinomycetemcomitans. The protease was isolated from the culture supernatant by sonication in phosphate-buffered 3-[(3 cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). The protease was purified by acetone precipitation, followed by column chromatography with Arginine Sepharose 4B, DEAE Sepharose CL-6B, Sephacryl S-200HR and HiTrap Q. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protease showed a clear band at approximately 50 kDa. The protease showed trypsin-like activity with hydrolytic activity for the synthetic substrates N alpha-benzoyl-DL-arginine p-nitroanilide (BApNA) and N alpha benzoyl-DL-lysine p-nitroanilide (BLpNA). The activity of the protease was stable at pH 7.0 to approximately 8.0. The activity of the protease was inhibited by leupeptin, phenylmethylsulfonyl fluoride (PMSF), and EDTA, but was not affected by dithiothreitol (DTT), cysteine, 2-mercaptoethanol, pepstatin or soybean trypsin inhibitor. These data suggest that this protease is a serine protease or metallo protease. This enzyme extensively degraded collagen type I and fibronectin.

Induction of apoptosis in human gingival fibroblasts by a Porphyromonas gingivalis protease preparation.

Proteases produced by Porphyromonas gingivalis are believed to contribute to the pathogenesis of periodontal diseases. Here the cytotoxic effects of a purified preparation of a P. gingivalis protease with trypsin-like specificity were tested on human gingival fibroblasts in vitro. The active protease induced apoptotic cell death in the fibroblasts, as indicated by DNA fragmentation and the expression of 7A6 antigen. Thus, the production of proteases by periodontopathic bacteria could be an important factor in the induction of apoptosis of host cells in the aetiology of periodontal diseases.

Scanning electron microscopic study of the effects of Er:YAG laser on root cementum.

Use of Er:YAG laser has been proposed for the removal of microbial deposits and calculus present on teeth affected by periodontal disease. However, the influence of Er:YAG laser irradiation on root surfaces has not yet been fully investigated. The aim of the present study was to evaluate the effects of Er:YAG laser irradiation on root cementum by scanning electron microscopy (SEM). Specimens were obtained from extracted human periodontally-diseased teeth using a water-cooled high-speed bur. An Er:YAG laser beam was then applied at various powers ranging from 25 to 100 mJ/ pulse/sec. The laser irradiation was performed under water irrigation, with the tip held perpendicular to the root surface in the contact mode. Following laser exposure, specimens were fixed, dehydrated, and dried at critical-point in liquid CO2. After mounting on SEM plates and sputter-coating with gold, the cementum surface was examined by SEM. Observations of the root surface showed a relatively flat surface in control specimens. In Er:YAG exposed specimens, the laser beam created a circular, notched-edge, crater-like defect on the root. The bottom of the lesion showed an irregular and sharp-pointed surface. Subsequently, the specimens were fractured with a sharp scalpel perpendicularly to the surface. SEM observations of these specimens showed a 15 microm layer of damaged tissue within the laser-irradiated cementum. The tissue presented an amorphous appearance and the Sharpey's and matrix fiber bundles were not clearly distinguishable. These observations indicate that cementum tissue could be damaged by Er:YAG laser irradiation.

Involvement of CD14 on human gingival fibroblasts in Porphyromonas gingivalis lipopolysaccharide-mediated interleukin-6 secretion.

The lipopolysaccharides (LPS) of Porphyromonas gingivalis are implicated in the initiation and development of periodontal diseases. However, the mechanisms underlying P. gingivalis LPS-mediated periodontal destruction are still unknown. Here, it was found that P. gingivalis LPS activates human gingival fibroblasts (HGF) to release interleukin 6 (IL-6) via CD14. Flow-cytometric analysis showed that HGFs bind to fluorescein-isothiocyanate (FITC)-labelled LPS, and express CD14 on their surfaces. The binding of FITC LPS was competitively suppressed by unlabelled synthetic lipid A as well as by LPS. LPS-induced IL-6 production was inhibited by anti-CD14 monoclonal antibody in a dose-dependent manner. The binding of FITC LPS to HGF was abrogated by anti-CD14 monoclonal antibody. Engagement of LPS initiated the protein tyrosine phosphorylation of several intracellular proteins including extracellular signal-regulated kinase (ERK) 1 and 2, and these events were suppressed by the anti-CD14 monoclonal. These results suggest that CD14 is a cell surface binding site for LPS and is involved in the LPS-mediated activation of HGF.

Possible involvement of protein kinase C in apoptotic cell death of macrophages infected with Actinobacillus actinomycetemcomitans.

We have previously reported the evidence for apoptosis in the mouse macrophage cell line J774.1 by the periodontopathic bacterium Actinobacillus actinomycetemcomitans. In this study, we examined the role of protein kinases in the induction of apoptosis in A. actinomycetemcomitans-infected J774.1 cells by the MTT assay, fluorescence microscopy and flow cytometric analysis. After J774.1 cells were precultured with protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), J774.1 cells infected with A. actinomycetemcomitans showed the increased percentage of apoptotic cells. On the contrary, protein kinase A (PKA) activators, such as forskolin and dibutyryl cAMP, do not mimic the effect of PMA. PKC inhibitors, such as staurosporine, calphostin C, chelerythrine chloride, and H7 were found to suppress apoptotic cell death in J774.1 cells infected with A. actinomycetemcomitans. However, HA1004, known as PKA inhibitor, had no effect on apoptosis in infected macrophages. The results presented here suggest that the signals through PKC may play crucial roles in the modulation of apoptosis in macrophages infected with A. actinomycetemcomitans.

Role of CD14 molecules in internalization of Actinobacillus actinomycetemcomitans by macrophages and subsequent induction of apoptosis.

We report the evidence for apoptosis in J774.1 cells by the periodontopathic bacterium Actinobacillus actinomycetemcomitans, suggesting that the ability of A. actinomycetemcomitans to promote apoptosis might be important in the initiation and development of periodontitis. In this study, we examined the role of macrophage CD14, anchored by a glycerophosphatidylinositol tail, in the induction of apoptosis by A. actinomycetemcomitans infection by using the parent J774.1 cells and CD14-defective mutant (LR-9) cells. A small number of A. actinomycetemcomitans Y4 cells inside the LR-9 cells compared with the number in J774.1 cells was detected by confocal scanning microscopy. We found that LR-9 cells showed a weak cytotoxic effect after being infected with A. actinomycetemcomitans Y4. Apoptotic cell death of LR-9 cells infected with A. actinomycetemcomitans Y4, compared with that of the parent J774.1 cells was almost undetectable, as shown by the proportion of fragmented DNA in agarose gel electrophoresis and by the terminal deoxynucleotidyl transferase-mediated dUTP end-labeling method. Flow cytometric cell cycle analysis of J774.1 cells infected with A. actinomycetemcomitans Y4 revealed the increased percentage of apoptotic cells with hypodiploid DNA. However, LR-9 cells infected with A. actinomycetemcomitans Y4 showed no increase in population of apoptotic nuclei compared with the noninfected cells. These findings suggest that the CD14 molecules may contribute to the phagocytosis of A. actinomycetemcomitans by J774.1 cells and regulate, at least in part, apoptotic cell death of macrophages infected with A. actinomycetemcomitans.

Evidence for apoptosis of murine macrophages by Actinobacillus actinomycetemcomitans infection.

The gram-negative bacterium Actinobacillus actinomycetemcomitans is considered an important etiological agent in periodontal diseases. In this study, we show that A. actinomycetemcomitans strains are cytotoxic for the murine macrophage cell line J774.1. On the other hand, Porphyromonas gingivalis strains, other gram-negative oral species implicated in adult periodontitis, showed weak cytotoxic effects. For this to occur, A. actinomycetemcomitans had to gain entry into the macrophages, since cytotoxicity was prevented by cytochalasin D. We demonstrate that cell death induced by A. actinomycetemcomitans Y4 occurs through apoptosis, as shown by changes in nuclear morphology, an increase in the proportion of fragmented DNA, and the typical ladder pattern of DNA fragmentation indicative of apoptosis. We further sought to determine whether the cytotoxicity induced by A. actinomycetemcomitans Y4 could be modulated by the protein kinase inhibitors H7 and HA1004. Apoptotic cell death induced by A. actinomycetemcomitans Y4 was suppressed by H7 but was relatively unaffected by HA1004. These findings suggest that the signals of protein kinases may regulate apoptosis induced by A. actinomycetemcomitans Y4. The ability of A. actinomycetemcomitans to promote the apoptosis of macrophages may be important for the initiation of infection and the development of periodontal diseases.

[Silent period and initial occlusal sliding time in patients with premature contacts].

This study was designed to examine the relationship between frequency of silent period and initial occlusal sliding time. The subjects consisted of three volunteers with normal occlusal contacts and three patients with premature contacts. Electromyograms of the bilateral masseter muscles, initial occlusal contacts and jaw movements during the habitual tapping (20-25) were synchronously recorded and replayed using Takamatsu's technique. Results were as follows: 1. The frequency of silent period in the bilateral masseter muscles was 95-100% in subjects with normal occlusal contacts and 34-53% in patients with premature contacts. 2. The silent period latency in three normal subjects was 8.9 +/- 1.3 msec in the left masseter and 13.9 +/- 2.2 msec in the right masseter. The silent period latency in three patients with premature contacts was 12.4 +/- 2.6 msec in the left masseter and 13.9 +/- 8.2 msec in the right masseter. 3. The duration of silent period in three normal subjects was 12.4 +/- 2.6 msec in the left masseter and 11.1 +/- 3.7 msec in the right masseter. The duration of silent period in three patients with premature contacts was 9.1 +/- 1.7 msec in the left masseter and 11.1 +/- 5.6 msec in the right masseter. 4. Initial occlusal sliding time in three subjects with normal occlusal contacts was less than 30 msec, and 98% of all slidings lasted for less than 15 msec. Initial occlusal sliding times in patients with premature contacts were distributed between 6 and 80 msec (40%: 6 to 15 msec, 60%: greater than 15 to 80 msec).(ABSTRACT TRUNCATED AT 250 WORDS)

[Clinical study of bruxism. Comparison of muscle activity during sleep between patients conscious of bruxism and those not conscious of the condition].

Bruxism has been considered to be one of the most important factors in accelerating the progression of established periodontal lesions. However the objective diagnostic method has not yet been established. At present, diagnosing bruxism might mainly be dependent on interview. The purpose of this study was to compare and analyze the differences in frequency and duration of bruxism between a group of patients conscious of the problem and a group not conscious of bruxism. After interviewing, the subjects were divided into two groups; 1) group A consisted of 8 subjects who were conscious of bruxism and 2) group B of 8 subjects who were not conscious of bruxism. The frequencies and durations of muscle activity during sleep at night were compared between groups A and B using EMG with a telemetric method. In the one-night observation, muscle activity supposed to be bruxism was observed in both groups. Significant differences in frequencies of muscle activity were not found between the two groups. A similar result was obtained in the durations of muscle activity. In a seven-day observation, muscle activity was seen in all three volunteers, although marked differences were not found among them. A long duration of muscle activity was found under conditions of physical or mental stress. The results of this study showed the difficulty of diagnosing bruxism by interview and the necessity of an objective method.

[Connective tissue attachment to root surfaces in periodontal disease. Initial attachment of human gingival fibroblasts].

We studied the effect of periodontal treatment (scaling, SC; root planning, RP; scaling followed by citric acid, SC + CA; and root planing followed by citric acid, RP + CA) of periodontally diseased root surfaces on the initial attachment of human gingival fibroblasts in vitro. Root slices were prepared from surgically extracted human normal and periodontally involved teeth. Each treated root slice was placed in a well of a 24-well plate containing a PBS-antibiotic solution (penicillin, 200 units/ml; streptomycin, 200 micrograms/ml) for 1 hr. at 4 degrees C. Then, to each well was added 1 x 10(4) cells in 1 ml of alpha-MEM, and the plates were incubated for 24 hr. After the root slices were fixed and stained, the morphological changes and the numbers of attached cells were determined under a dissecting microscope. The fibroblasts on the controls (untreated normal roots) and the RP-roots appeared spindle-shaped with a few cell processes. The cells on the SC-roots were rounded or slightly elongated. The cells on the roots treated with citric acid (SC + CA and RP + CA) had were flat and seemed well attached to the surface. The number of cells attached on RP- and RP + CA-roots was increased to the control level, but those on SC- and SC + CA-roots were showed about 60% of the control. No significant differences in the numbers of cells were found between RP and RP + CA treatment or between SC and SC + CA treatment.(ABSTRACT TRUNCATED AT 250 WORDS)