Actinobacillus actinomycetemcomitans induces apoptosis in human monocytic THP-1 cells.

It has previously been reported that the murine macrophage cell line J774.1 and the human oral epithelial cell line KB undergo apoptosis as a result of Actinobacillus actinomycetemcomitans infection. Recent studies have demonstrated that apoptosis regulation is modulated by multiple phosphorylation of several different protein kinases, including the major subtypes of the mitogen-activated protein kinase (MAPK) family. The MAPK family promotes cell survival and/or proliferation in response to growth factor stimulation, or apoptosis in response to various stress stimuli. The primary objective of the present investigation was to clarify whether human immune cells undergo apoptosis following A. actinomycetemcomitans infection and, if so, to establish the involvement of the MAPK family. Human monocytic THP-1 cells were infected with A. actinomycetemcomitans in microtubes. Lactate dehydrogenase release into the culture supernatant and DNA fragmentation in the cells were monitored. DNA fragmentation was also identified by agarose gel electrophoresis. Cell death following A. actinomycetemcomitans infection occurred by apoptosis, shown by an increase in the proportion of fragmented DNA and the typical ladder pattern of DNA fragmentation indicative of apoptosis. Furthermore, p38 MAPK activity and tumour necrosis factor alpha (TNF-alpha) levels increased following A. actinomycetemcomitans infection. In contrast, cell death and TNF-alpha levels in infected cells decreased upon addition of a p38 inhibitor or an anti-TNF-alpha antibody. However, exogenous TNF-alpha could not induce apoptosis in uninfected THP-1 cells. Interestingly, p38 MAPK activity diminished in the presence of anti-TNF-alpha antibody. These findings indicated that A. actinomycetemcomitans infection induces apoptosis in THP-1 cells and that p38 MAPK activity is directly involved in apoptosis. TNF-alpha may play an indirect role in apoptosis via enhanced p38 MAPK activity. A. actinomycetemcomitans-induced apoptosis of human immune cells may be important in terms of initiation and progression of periodontal diseases.

Periodontal conditions in an elderly Japanese population influenced by smoking status and serum immunoglobulin G2 levels.

BACKGROUND: A Japanese population residing in the same area may be suitable for evaluation of the association among IgG subclass levels, smoking status, and periodontal disease due to similar racial and environmental factors, as these factors can interact to influence serum IgG subclass levels. The present cross-sectional investigation attempted to examine the influence of serum IgG subclass levels and smoking status on periodontal condition in a population of elderly Japanese subjects. METHODS: Elderly individuals (N = 451, age, 71 years) residing in Niigata City, Japan participated in the present study. Clinical evaluations, which consisted of probing depth (PD), clinical attachment level (CAL), bleeding on probing (BOP), and calculus, and serological determinations including serum IgG subclass levels and anti-Porphyromonas gingivalis fimbriae IgG subclass titers were conducted. All participants were asked to complete questionnaires regarding smoking status and were then divided into one of three groups: never smoker, former smoker, or current smoker. RESULTS: Distributions of the number of teeth did not differ significantly across the groups. The proportion of sites with CAL > or =4 mm (%CAL4) in current smokers was significantly higher in comparison with never smokers. Multiple linear regression analysis revealed that %CAL4 was influenced by number of teeth, serum IgG2 levels, gender, and smoking status (R(2) = 0.253, P < 0.001). The effect of IgG2 was greater than that of smoking status. Distribution of IgG2 was not significantly different across the three groups. CONCLUSION: These data indicate that serum IgG2 levels influences periodontal conditions in an elderly Japanese population independent of smoking status.

Human epithelial cell death caused by Actinobacillus actinomycetemcomitans infection.

The gingival sulcus is the shallow crevice around the tooth, and its epithelium is a gateway for initial bacterial infection in periodontal disease. Recent studies have shown that Actinobacillus actinomycetemcomitans invades an epithelial cell line, KB cells, in vitro. The aim of the present study was to clarify the changes in KB cells after A. actinomycetemcomitans infection. The cytotoxic effects of A. actinomycetemcomitans on KB cells were determined at 72, 96 and 120 h after infection by an MTT assay. Nuclear morphological changes were observed by staining with Hoechst 33258. Cytoplasmic histone-associated DNA fragmentation in the infected KB cells was determined by ELISA. A. actinomycetemcomitans was cytotoxic on KB cells, and condensation and degradation of the nuclei were observed. DNA fragmentation was increased after the infection. In addition, A. actinomycetemcomitans showed similar cytotoxic effects on human gingival epithelial cells. The present study demonstrated that A. actinomycetemcomitans induces apoptotic cell death of oral epithelial cells in an in-vitro culture system. This induced apoptosis might be involved in the initiation and progression of periodontitis.

Physical and biological evaluations of sintered hydroxyapatite/silicone composite with covalent bonding for a percutaneous implant material.

A composite (HA/silicone) of hydroxyapatite (HA) microparticles with an average diameter of 2.0 micro m covalently linked to a silicone substrate has been developed, and its physical and biological properties as a percutaneous soft-tissue-compatible material have been evaluated. In tensile property measurement, samples of HA/silicone and the original silicone were similar in tensile strength, ca. 7.8 MPa, and elongation at break, ca. 570%. It was found that chemical surface modification with HA particles presented no mechanical disadvantage. In an adhesive-tape peeling test, scanning electron microscopic (SEM) observation showed that HA particles coupled directly to the substrate were not removed. HA particles may bond strongly with the substrate. In human periodontal ligament fibroblast attachment and proliferation experiments, the number of cells attached to HA/silicone was 14 times greater than that attached to the original silicone after 24 h of incubation. The value on HA/silicone was ca. 80% versus that on a tissue-culture plastic used as a positive control. After 72 h of incubation, the number of cells grown on HA/silicone increased to the level of the positive control. In observation of fluorescence microscopy stained by Hoechst 33342, cells appeared to tightly adhere to HA particles coupled to the silicone sheet due to intact nuclear morphology. Observation of cells by fluorescence dye with rhodamin phalloidin showed an extensive F-actin cytoskeleton on HA/silicone. In a 4-week animal implant test, force required to pull out the HA/silicone sheet was 15 times that of the original silicone. HA-particle coating on silicone with covalent linkage gave the inert surface bioactivity. The HA composite thus effectively prevents germ infection percutaneously.

Periodontal tissue regeneration using fibroblast growth factor-2: randomized controlled phase II clinical trial.

BACKGROUND: The options for medical use of signaling molecules as stimulators of tissue regeneration are currently limited. Preclinical evidence suggests that fibroblast growth factor (FGF)-2 can promote periodontal regeneration. This study aimed to clarify the activity of FGF-2 in stimulating regeneration of periodontal tissue lost by periodontitis and to evaluate the safety of such stimulation. METHODOLOGY/PRINCIPAL FINDINGS: We used recombinant human FGF-2 with 3% hydroxypropylcellulose (HPC) as vehicle and conducted a randomized double-blinded controlled trial involving 13 facilities. Subjects comprised 74 patients displaying a 2- or 3-walled vertical bone defect as measured > or = 3 mm apical to the bone crest. Patients were randomly assigned to 4 groups: Group P, given HPC with no FGF-2; Group L, given HPC containing 0.03% FGF-2; Group M, given HPC containing 0.1% FGF-2; and Group H, given HPC containing 0.3% FGF-2. Each patient underwent flap operation during which we administered 200 microL of the appropriate investigational drug to the bone defect. Before and for 36 weeks following administration, patients underwent periodontal tissue inspections and standardized radiography of the region under investigation. As a result, a significant difference (p = 0.021) in rate of increase in alveolar bone height was identified between Group P (23.92%) and Group H (58.62%) at 36 weeks. The linear increase in alveolar bone height at 36 weeks in Group P and H was 0.95 mm and 1.85 mm, respectively (p = 0.132). No serious adverse events attributable to the investigational drug were identified. CONCLUSIONS: Although no statistically significant differences were noted for gains in clinical attachment level and alveolar bone gain for FGF-2 groups versus Group P, the significant difference in rate of increase in alveolar bone height (p = 0.021) between Groups P and H at 36 weeks suggests that some efficacy could be expected from FGF-2 in stimulating regeneration of periodontal tissue in patients with periodontitis. TRIAL REGISTRATION: ClinicalTrials.gov NCT00514657.

Interaction of Actinobacillus actinomycetemcomitans outer membrane vesicles with HL60 cells does not require leukotoxin.

Outer membrane derived vesicles (MVs) secreted by Actinobacillus actinomycetemcomitans JP2 contain a membranolytic leukotoxin and are toxic to human HL60 cells. To determine how MVs interact with human target cells, HL60 cells were incubated with vesicles, reacted with anti-vesicle antibodies and a FITC-labelled reporter, and visualized by confocal scanning laser microscopy. Target cells rapidly became reactive with anti-vesicle antibodies upon exposure to vesicles. Confocal microscopy showed that labelling occurred primarily in the cytoplasmic membrane and that very little internal fluorescence was observed. The cytoplasmic membrane of HL60 cells was also strongly labelled after exposure to MVs that contained the fluorescent phospholipid, SP-DiOC18. In contrast, incubation of cells with free SP-DiOC18 resulted primarily in the labelling of internal structures of HL60 cells. These results suggest that A. actinomycetemcomitans MVs associate with, or are incorporated into the cytoplasmic membrane of HL60 cells. The leukotoxin is a membranolytic cytotoxin and cells exposed to MVs were lysed by vesicle-associated toxin in a time and dose-dependent manner. However, cells became reactive with anti-vesicle antibodies when MVs were added in the presence of inhibitors of leukotoxin-mediated lysis or when sublytic doses of MVs were analysed. In addition, MVs produced by an isogenic leukotoxin-deficient strain of A. actinomycetemcomitans JP2 were non-toxic but rapidly interacted with HL60 cells. These results suggest that A. actinomycetemcomitans MVs can deliver leukotoxin to HL60 cells but that the association of vesicles with the cytoplasmic membrane occurs independently of the leukotoxin polypeptide.

Outer membrane-like vesicles secreted by Actinobacillus actinomycetemcomitans are enriched in leukotoxin.

Actinobacillus actinomycetemcomitans is associated with early onset periodontal diseases and secretes membranous vesicles that appear to contain several virulence-associated proteins. However, the composition of these vesicles and the process leading to their secretion are not well defined. Electron micrographs of thin sectioned bacterial cells and purified vesicle preparations showed that vesicles are spherical lipid bilayers, 50-100 nm in diameter, that appear to form by budding from the outer membrane of the bacterium. Thin layer chromatography identified the predominant lipid components of vesicles as lipopolysaccharide, phosphatidylethanolamine and cardiolipin, similar to the main lipid constituents of the outer membrane. However, vesicles also contained minor lipids that were not detected in outer membrane samples. The major protein constituents of vesicles co-migrated with proteins in outer membrane extracts of A. actinomycetemcomitans, but the outer membrane preparations possessed polypeptides that were not detected in vesicles. Three vesicle proteins were identified; the heat-modifiable OmpA homologue of A. actinomycetemcomitans, a 28 kDa lipoprotein related to the major outer membrane lipoprotein of Mannheimia haemolytica and leukotoxin. Incubation of leukotoxin-sensitive human HL60 cells with vesicles from A. actinomycetemcomitans strains JP2 and 652 resulted in cell lysis, indicating that vesicle-associated leukotoxin is biologically active. Vesicles from the highly leukotoxic strain JP2 were five- to 10-fold more toxic than vesicles from the minimally leukotoxic 652 strain. Furthermore, the specific leukotoxic activity of JP2 vesicles was approximately four- to five-fold higher than isolated outer membrane preparations from JP2, suggesting that vesicles are enriched in leukotoxin. Together, these results suggest that the formation of A. actinomycetemcomitans vesicles occurs by a process that results in the enrichment of leukotoxin.

Nitric oxide-mediated protection of A. actinomycetemcomitans-infected murine macrophages against apoptosis.

We investigated apoptotic cell death in murine macrophage cell line J774.1 following Actinobacillus actinomycetemcomitans infection. Infected macrophages generally kill bacteria within phagosomes with nitric oxide (NO). Our previous study demonstrated that DNA fragmentation in infected cells increased significantly on addition of S-Methylisothiourea (SMT), a selective inhibitor of inducible NO synthetase (iNOS). The purpose of the present study was to determine the mechanism via which NO affects apoptosis of infected macrophages. J774.1 cells were infected with A. actinomycetemcomitans Y4 at a bacterium/cell ratio of 500:1. The infected cells were then cultured in the presence or absence of SMT (400 microM). Culture supernatant was removed 21 h after the infection to measure LDH activity. Additionally, cellular proteins were extracted from the infected cells and measured for histone-associated DNA fragmentation and caspase-1, -3, -5, -6, -8, -9 activities. LDH activity and DNA fragmentation were significantly elevated by the infection; moreover, levels increased further on addition of SMT. Caspase activity of infected cells, particularly caspase-3, was significantly higher than that of uninfected cells. Furthermore, caspase activity increased on addition of SMT. These findings indicate that NO protects infected J774.1 cells, at least in part, against apoptotic cell death via a decrease in caspase activity.

DNA microarray analysis of human gingival fibroblasts from healthy and inflammatory gingival tissues.

In the inflammatory gingival tissues of patients with periodontitis, cytokines such as interleukin (IL)-1 alpha, IL-1 beta, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha have been detected. Gingival fibroblasts are the major constituents of gingival tissue. We recently demonstrated that lipopolysaccharide (LPS) from periodontopathic bacteria induces inflammatory reactions in various tissues via CD14 and/or Toll-like receptors (TLRs) in gingival tissues [Biochem. Biophys. Res. Commun. 273 (2000) 1161]. To confirm this, we examined the expression of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF-alpha, CD14, TLR2, and TLR4 in human gingival fibroblasts (HGFs) obtained from patients with healthy or inflammatory gingiva using DNA microarray analysis. We also studied the expression levels of these proteins by flow cytometric analysis (FACS). The expression levels of all eight genes in the HGFs of the Inflammatory group were significantly higher than those in the Healthy group on DNA microarray analysis. FACS revealed that the expression levels of all eight proteins on the HGFs of the Inflammatory group were higher than those on the Healthy group. Our data indicated that these eight proteins in HGFs are involved in inflammatory conditions in the gingiva, including periodontal disease. Our results suggested that these eight proteins, in turn, act directly or indirectly on the immune response by activating host cells involved in inflammatory processes.

Effect of anti-CD14 antibody on experimental periodontitis induced by Porphyromonas gingivalis lipopolysaccharide.

The lipopolysaccharide (LPS) released by Porphyromonas gingivalis, a Gram-negative bacterium found in the periodontal pockets of patients with periodontitis, induces bone resorbing activity in vivo. We previously showed that a receptor for LPS on human gingival fibroblasts and gingival epithelial cells is CD14. In this study, we established a mouse model of experimental periodontitis by applying a P. gingivalis LPS solution to the buccal region of mice. P. gingivalis LPS-induced bone resorption and interleukin-6 production in the gingival tissues were significantly inhibited by pretreatment with anti-CD14 antibody for 5 weeks prior to LPS treatment. This result suggests that anti-CD14 antibody may be usable as a prototype for the development of drugs for the treatment of periodontal disease.