Characterization of the Cell Surface Substances of Fusobacterium nucleatum that Induce a Chemiluminescence Response by Human Neutrophils Masato Mikami, Chihomi Kato and Kazuko Saito Department of Microbiology, The Nippon Dental University, School of Dentistry at Niigata Accepted for publication 5 July 2003 We previously reported that the surface fraction derived from Fusobacterium nucleatum is responsible for induction of the chemiluminescence (CL) response generated by reactive oxygen intermediates (ROIs) produced by human neutrophils. ROIs are thought to not only kill bacteria but to cause host-cell injury. The purpose of this study was to identify the species of ROIs released by neutrophils stimulated with the bacterial fraction called CL-inducing surface materials (CSM), and to identify the CL-inducing factor in CSM and the molecules involved in signal transduction in neutrophils. The results of inhibition assays using superoxide dismutase and sodium azide demonstrated that superoxide anion produced in neutrophils stimulated with CSM was mainly converted to hypochlorite by the myeloperoxidase-H₂O₂-Cl- system. A mannose-sensitive or mannan-sensitive lectin-like interaction between CSM and neutrophils was confirmed by sugar inhibition of the CL-stimulating activity of CSM. Studies with specific inhibitors suggested that phosphatidylinositol 3-kinase (PI3K) and phospholipase C (PLC) were involved in the signal transduction in neutrophils in response to CSM stimulation but that pertussis-toxin-sensitive heterotrimeric G protein was not. CSM contains proteins, lipopolysaccharide (LPS), and other polysaccharides, and the 11 kD protein in CSM was found to be essential to the CL-stimulating activity. LPS had an effect that increased the activity of the protein, but neither CD14 nor Toll-like receptor 4 participated in the LPS signaling. A binding assay by the blotting method showed that the 11 kD protein of CSM bound to the 12 kD and 18 kD cell membrane protein of neutrophils. Thus, the results suggested that 11 kD protein and LPS in the surface of F.nucleatum stimulate neutrophils cooperatively and activate PI3K and PLC in neutrophils to produce ROIs. J Jpn Soc Periodontol, 45 : 215∼228, 2003.

View full abstract

The gingival bleeding index (BI) is frequently used as a sign of gingival disease. However, the normal range and its clinical applications have never been clearly descried. The purpose of the present study was to determine the normal range and to consider the possible clinical applications of the BI. A PS probe was used to keep the measurement pressure constant during determinations of the BI. To define the normal range of BI values, 50 healthy volunteers were instructed to mamtain thorough plaque control for one month before measurement of the BI. The BI value was 17.4±8.3% at baseline, and 6.6±3.1% after one month of plaque control, and the values wad comerted to a normal distibution to a normal distribution. BI values were therefore statistically estimated to range from 0% to about 12% in healthy individuals under adequate plaque control. Determination of the state of plaque control was considered for possible clinical applica-tions of BI. Gingivitis was experimentally induced in 30 healthy volunteers, and the Corelation between the plaque index and BI was tested. The results indicated that continuous plaque control for at least 3 to 7 days was necessary to re-establish the improvement in BI.
The plaque index was hypothesized to indicate the cument status of plaque control, whereas the BI reflects and can be used to determine whether plaque is being continuously controlled. To test our hypothesis, the BI was serially determined in 26 patients who required long-term maintenance.
While the BI values varied at the first visit and after treatment and maintenance, they converged to with in the normal range as plaque control progressed. It was concluded that the normal range of BI values can be used as a variable not only to detect gingival inflammation but to monitor whether adequate plaque control is being maintained. J Jpn Soc Periodontol, 45 : 229-240, 2003.

View full abstract

Occlusal trauma causes alveolar bone resorption around the root in healthy periodontal tissue, and occlusal trauma is considered to be a factor that accelerates alveolar bone resorption caused by bacterial infection in periodontitis. In an experimental model of occlusal trauma in beagle dogs, since the occurrence of thrombosis in the microcirculation of the periodontal ligament was demonstrated histopathologically, it was hypothesized that occlusal trauma causes hypoxia in the periodontal ligament. To test this hypothesis, the present study examined the effect of hypoxia on the production of bone-resorbing factors by cultured human periodontal ligament cells. Cultured periodontal ligament cells were divided into 2 groups, a group cultured under hypoxic conditions in 1% O₂ (hypoxia group), and a group cultured under 20% O₂ (normoxia group). The concentrations of bone-resorbing factors, vascular endothelial growth factor (VEGF), interleukin (IL) -6, IL-1β tumor necrosis factor-α (TNF-α), and prostaglandin E₂ (PGE₂) in the cell culture super-natants were determined by enzyme-linked immunosorbent assay. Expression of the mRNAs for the bone-resorbing factors was detected by performing the reverse transcription-polymerase chain reaction. Higher concentrations of VEGF and IL-6 were found in the culture media of the hypoxia group than in the normoxia group (p < 0.01), and the expression of the mRNAs for these proteins in the hypoxia group was also higher than in the normoxia group. Expression of IL-1β was observed in the hypoxia group alone. TNF-α and PGE₂ were undetectable in the culture medium of both the hypoxia group and the normoxia group, whereas COX-2 mRNA was detected in the hypoxia group. However, PGE₂ production became detectable 3 hours after the addition of recombinant human VEGF, even under normoxic conditions. Similarly, PGE₂ became detectable in the culture medium 9 hours after the oxygen level had been increased from hypoxic to normoxic. These results suggest that hypoxia induced by occlusal trauma may stimulate the periodontal ligament to produce bone resorbing factors and result in resorption of alveolar bone. J Jpn Soc periodontol, 45: 241-251, 2003.

View full abstract

Periodontal diseases are caused by changes in the balance among oral microorganisms, the host's immune system and environmental factors. Specific microorganisms are implicated in the destruction of periodontal tissue in patients with periodontitis. Candida species (Candida spp.) have been detected in various sites, including the oral cavity, oral mucosa, intestine, and pharynx of periodontitis patients, and the frequency of detection of Candida spp. in periodontal pockets has been reported to be 2-2. 5%. However, no correlation between Candida spp. in periodontal pockets and their pathology causing periodontal diseases has ever been clearly demonstrated.
The aim of the present study to determine whether Candida spp. are present in the periodontal pockets of patients with chronic periodontitis by means of PCR coupled with microbial culture and to clarify the relationship between Candida spp. and periodontal diseases.
We investigated the ginigival sulcus of 100 patients with adult periodontitis (chronic periodontitis) for the presence of Candida spp. The patients were divided into two groups: in 40 patients the tooth surface was polished with Prophy-point to remove supragingival plaque before sample collection (tooth-surface-polished group), while in the remaining 60 subjects the tooth surface was cleaned with sterilized cotton swabs (non-polished group).
Candida spp. were detected in 68% of the oral mucosal samples, and in 4% of the periodontal pocket samples. Polishing the tooth surface before specimen collection significantly lowered the frequency of detection of Candida spp. Whenever Candida spp. were detected in a periodontal pocket sample, a second sample was obtained from the same patient, however, Candida spp. were never detected in the second specimen.
The results suggest that Candida spp. are not present in the periodontal pocket of patients with adult periodontitis. J Jpn Soc Periodontol 45: 252-259, 2003.

View full abstract

PRP (Platelet Rich Plasma) has been reported to be effective for accelerating wound healing when used in conjunction with skin grafts in medical practice and bone grafts in dental practice. We report that PRP was effective for increasing the width of the attached gingiva in peri-implant tissue during free autogenous gingival graft surgery. The patient was a 47-year-old woman. The recipient site was 7654 in the buccal mucosa and the donor site was 654 in the palatal mucosa. After the graft tissue was removed from the donor site, PRP paste was inserted into the wound and a bendable wound protection splint was placed over the donor bed. PRP was also injected into the graft tissue using a syringe and into the recipient bed. Thereafter, the graft was sutured to the recipient site. After the suturing was complete, bleeding was not observed at either the donor or the recipient site. Hemostatic pressure was not necessary. A periodontal pack was then placed over the wound. Four days after the surgery, granulomatous tissue had developed to the same height as the surrounding tissue at the donor site. Periodontal packs are generally removed on postoperative day 14, by which time blood circulation has been established between the graft and the recipient bed. In the present case, the periodontal pack was removed five days after the surgery, and the graft site was found to be healthy. Moreover, the patient did not experience any pain or unpleasant adverse effects at either the donor site or the recipient site. In conclusion, the present case demonstrates that PRP is useful for accelerating wound healing by enhancing blood circulation when applied to free autogenous gingival grafts in peri-implant tissue. J Jpn Soc Periodontol, 45: 2603∼266, 2003.

View full abstract

Periodontitis is considered to be a polygenic disease. Susceptibility to aggressive periodontitis (AgP), formerly known as early-onset periodontitis, might be associated with genetic polymorphisms. Tumor necrosis factor receptor (TNFR) 1 and 2 are cell surface receptors for TNF-α. Recent studies have suggested that TNFRs may modulate TNF-α-mediated inflammatory responses in periodontal disease. The aim of the present study was to evaluate whether TNFR1 and TNFR2 gene polymorphisms are associated with AgP in Japanese patients.
Forty-five generalized AgP (G-AgP) patients and 100 age-matched healthy Japanese subjects were selected according to established clinical criteria. Single nucleotide polymorphisms at positions -383 (A/C) and +36 (A/G) in the TNFR1 gene, at positions +587 (T/G) and +694 (G/A) in the TNFR2 gene, and the variable number of tandem repeats (VNTR) promoter polymorphism in the TNFR 2 gene were detected using polymerase chain reactions, restriction fragment length polymorphisms, single-strand conformation polymorphisms, and direct sequencing methods. Statistically significant differences between the AgP and healthy groups were determined using chi-square and Fisher exact tests, equivalent results were assessed using the equivalence-delta test and further adjusted using the Mantel-Haenszel method.
The frequency of the -383C allele was significantly lower in the G-AgP patients group than in the healthy group (p=0.04). On the other hand, equivalent allele frequencies between the two groups were found at three positions (+587 (T/G), +694 (G/A) and VNTR) in the TNFR2 gene. After adjusting for a confounding factor, the +587 (T/G) and VNTR allele frequencies of the two groups were found to be significantly equivalent (p<0.01).
These findings suggest that the TNFR1 (-383A/C) gene polymorphism might be associated with G-AgP, whereas the TNFR2 +587 (T/G) and VNTR gene polymorphisms are not likely to result in a susceptibility to G-AgP. J Jpn Soc Periodontol, 45: 267-278, 2003.

View full abstract

Early-onset periodontitis (aggressive periodontitis) is characterized by rapid attachment loss and alveo-lar bone loss, despite a healthy systemic condition. The severity of this disease is closely associated with both highly virulent pathogens and highly susceptible subjects. Although the tissue destruction takes place very rapidly, the progression of attachment loss and bone loss may be self-arresting in some patients. This case report describes the 18-year clinical history of an early-onset periodontitis patient who did not receive continuous treatment but who experienced the self-arrest of alveolar bone loss after puberty. The patient first visited the Orthodontic Department of Tokushima University Dental Hospital complaining of maloc-culusion when she was 11 years old. Clinical findings at that time showed moderate gingivitis with no alveolar bone loss. She refused orthodontic therapy because of the need to undergo tooth extraction prior to the therapy. She returned to our hospital when she was 17 years old, complaining of a pain in her lower left first molar. This time, severe generalized periodontitis was found, and a radiographic examination revealed marked alveolar bone loss in several teeth, including the lower left first molar. While local irrigation and medication relieved the pain, she visited our hospital once again at the age of 21 years, complaining of the same symptoms in the same molar. The lower left first molar was extracted at this time. When the patient was 29 years old, she returned to our hospital once again because of pulpitis of her upper molar. Although gingival inflammation and deep periodontal pockets were still present, the degree of alveolar bone loss had not changed since the examinations that were performed when she was 17 and 21 years old. These data indicate that periodontal destruction progressed while she was a teen-ager and then arrested, despite the absence of cause-related therapy. These results suggest that the progression of attach-ment and bone loss may be self-arresting in some patients. J Jpn Soc Periodontol, 45 : 279∼288, 2003.

View full abstract

Nifedipine (NF), a calcium channel blocker commonly used for treatming essential hypertension, has been reported in many studies to cause gingival hyperplasia. To clarify the mechanism of NF-induced gingival overgrowth in animals, we examined angiotensin II levels in gingiva and the expression of transforming growth factor (TGF) -β protein in relation to the tissue renin-angiotensin (RA) system in spontaneously hypertensive rats (SHRs) and Wistar Kyoto rats (WKYs) with normal blood pressure. We also studied the inhibitory effect of captopril, an angiotensin-converting enzyme (ACE) inhibitor, on NF-induced gingival overgrowth in both hypertensive and normotensive animals.
1) Ang II levels in gingiva were significantly higher in the SHR group than in the WKY group.
2) Although hyperplasia of the gingival endothelium and connective tissue was enhanced in both groups receiving NF (SHR NF and WKY NF) compared to that in animals not receiving it (SHR and WKY controls), gingival hyperplasia tended to be more marked in the SHR NF group than in the WKY NF group.
3) TGF-β protein expression was higher in the SHR control group than in the WKY control group. The expression of this protein was significantly higher in both groups receiving NF than in control groups (SHR and WKY).
4) No tendency toward gingival overgrowth was observed in animals receiving NF plus CP or CP alone in either the SHR or WKY group. In both groups, TGF-β protein expression was inhibited more significantly in animals receiving NF plus CP than in those receiving NF alone.
These findings suggest that the tissue RA system may result from overexpression of the TGF-β protein due to an increase in tissue Ang II, and thus also in the pathogenesis of gingival overgrowth in SHRs receiving NF. J Jpn Soc Periodontol 45 : 289-299, 2003.

View full abstract