Regenerative effect of azithromycin on periodontitis with different levels of gingival inflammation: three case reports.

BACKGROUND: Azithromycin is an antibiotic belonging to the macrolides. Previous case reports showed that azithromycin has a regenerative effect on periodontal tissue in addition to improving periodontal gingival inflammation. Recently, we experienced three periodontitis cases, all of which showed severe bone loss. However, their gingival inflammatory signs differed greatly. The present case reports evaluated the regenerative effects of azithromycin on periodontitis sites with different clinical signs of gingival inflammation. METHODS: In Case 1, generalized chronic periodontitis with severe gingival inflammation was treated with azithromycin before periodontal treatment. In contrast, Case 2 presented with few clinical signs of gingival inflammation, but was treated with azithromycin prescribed within a day of scaling and root planing. In Case 3, teeth with moderate gingival inflammation were treated with azithromycin after a series of scaling and root planing. RESULTS: Remarkable alveolar bone growth, regardless of baseline gingival inflammation, was noted in all three cases. CONCLUSIONS: The use of adjunctive azithromycin in scaling and root planing may be effective for periodontal tissue regeneration. This property may be independent of the degree of baseline gingival inflammation.

Porphyromonas gingivalis gingipain is involved in the detachment and aggregation of Aggregatibacter actinomycetemcomitans biofilm.

Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are major periodontal pathogens that cause several types of periodontal disease. Our previous study suggested that P. gingivalis gingipains secreted in the subgingival environment are related to the detachment of A.actinomycetemcomitans biofilms. However, it remains unclear whether arginine-specific cysteine proteinase (Rgp) and lysine-specific proteinase (Kgp) play different roles in the detachment of A. actinomycetemcomitans biofilm. The aim of this study was to investigate possible disruptive roles of Kgp and Rgp in the aggregation and attachment of A. actinomycetemcomitans. While P. gingivalis ATCC33277 culture supernatant has an ability to decrease autoaggregation and coaggregation of A. actinomycetemcomitans cells, neither the boiled culture supernatant of ATCC33277 nor the culture supernatant of KDP136 showed this ability. The addition of KYT-1 and KYT-36, specific inhibitors of Rgp and Kgp, respectively, showed no influence on the ability of P. gingivalis culture supernatant. The result of gelatin zymography suggested that other proteases processed by gingipains mediated the decrease of A. actinomycetemcomitans aggregations. We also examined the biofilm-destructive effect of gingipains by assessing the detachment of A. actinomycetemcomitans from polystyrene surfaces. Scanning electron microscope analysis indicated that A. actinomycetemcomitans cells were detached by P. gingivalis Kgp. The quantity of A. actinomycetemcomitans in biofilm was decreased in co-culture with P. gingivalis. However, this was not found after the addition of KYT-36. These findings suggest that Kgp is a critical component for the detachment and decrease of A. actinomycetemcomitans biofilms.

The pga gene cluster in Aggregatibacter actinomycetemcomitans is necessary for the development of natural competence in Ca(2+) -promoted biofilms.

Natural competence is the ability of bacteria to incorporate extracellular DNA into their genomes. This competence is affected by a number of factors, including Ca(2+) utilization and biofilm formation. As bacteria can form thick biofilms in the presence of extracellular Ca(2+) , the additive effects of Ca(2+) -promoted biofilm formation on natural competence should be examined. We evaluated natural competence in Aggregatibacter actinomycetemcomitans, an important periodontal pathogen, in the context of Ca(2+) -promoted biofilms, and examined whether the pga gene cluster, required for bacterial cell aggregation, is necessary for competence development. The A. actinomycetemcomitans cells grown in the presence of 1 mm CaCl2 exhibited enhanced cell aggregation and increased levels of cell-associated Ca(2+) . Biofilm-derived cells grown in the presence of Ca(2+) exhibited the highest levels of natural transformation frequency and enhanced expression of the competence regulator gene, tfoX. Natural competence was enhanced by the additive effects of Ca(2+) -promoted biofilms, in which high levels of pga gene expression were also detected. Mutation of the pga gene cluster disrupted biofilm formation and competence development, suggesting that these genes play a critical role in the ability of A. actinomycetemcomitans to adapt to its natural environment. The Ca(2+) -promoted biofilms may enhance the ability of bacteria to acquire extracellular DNA.

Porphyromonas gingivalis displays a competitive advantage over Aggregatibacter actinomycetemcomitans in co-cultured biofilm.

BACKGROUND AND OBJECTIVE: Biofilm formation occurs through the events of cooperative growth and competitive survival among multiple species. Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are important periodontal pathogens. The aim of this study was to demonstrate competitive or cooperative interactions between these two species in co-cultured biofilm. MATERIAL AND METHODS: P. gingivalis strains and gingipain mutants were cultured with or without A. actinomycetemcomitans. Biofilms formed on glass surfaces were analyzed by crystal violet staining and colony counting. Preformed A. actinomycetemcomitans biofilms were treated with P. gingivalis culture supernatants. Growth and proteolytic activities of gingipains were also determined. RESULTS: Monocultured P. gingivalis strains exhibited a range of biofilm-formation abilities and proteolytic activities. The ATCC33277 strain, noted for its high biofilm-formation ability and proteolytic activity, was found to be dominant in biofilm co-cultured with A. actinomycetemcomitans. In a time-resolved assay, A. actinomycetemcomitans was primarily the dominant colonizer on a glass surface and subsequently detached in the presence of increasing numbers of ATCC33277. Detachment of preformed A. actinomycetemcomitans biofilm was observed by incubation with culture supernatants from highly proteolytic strains. CONCLUSION: These results suggest that P. gingivalis possesses a competitive advantage over A. actinomycetemcomitans. As the required biofilm-formation abilities and proteolytic activities vary among P. gingivalis strains, the diversity of the competitive advantage is likely to affect disease recurrence during periodontal maintenance.

A novel gene required for natural competence in Aggregatibacter actinomycetemcomitans.

BACKGROUND AND OBJECTIVE: Natural competence is the ability of bacteria to take up extracellular DNA and incorporate it into their genomes. Some strains of Aggregatibacter actinomycetemcomitans, a critical periodontal pathogen, are naturally competent for transformation. However, information on natural competence genes is limited for this species. The aim of this study was to confirm the involvement of a novel gene identified near the fimbriae gene cluster in natural competence. MATERIAL AND METHODS: The functions of putative open reading frames (ORFs), designated AA00863-AA00865, in the Oralgen project database for A. actinomycetemcomitans strain HK1651, have not been determined. Using naturally transformable A. actinomycetemcomitans strains D7S-1 and ATCC29523, we created deletion mutants of homologous genes of these ORFs. Natural competence in the study strains was determined using an agar-based transformation frequency assay. RESULTS: Mutation of the AA00865 homolog, which we named urpA in A. actinomycetemcomitans strain D7S-1, resulted in the loss of natural competence, whereas mutations of the AA00864 and AA00863 homologs, located downstream of urpA gene, did not. Similar results were also observed in the mutants of A. actinomycetemcomitans ATCC29523. Complementation of the deleted sequence in the urpA mutant restored natural competence. CONCLUSION: The urpA gene is a novel gene required for natural competence in A. actinomycetemcomitans and does not exhibit significant homology with any natural competence genes previously identified in other bacterial species.

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.

Expression levels of adiponectin receptors and periodontitis.

BACKGROUND AND OBJECTIVE: We recently showed that adiponectin, an adipocyte-derived cytokine, may function as a negative regulator of the Toll-like receptor signaling pathway and of osteoclast formation in periodontal disease. In this study, we investigated whether the expression levels of adiponectin receptors (AdipoR1 and AdipoR2) are related to the presence of periodontitis. MATERIAL AND METHODS: We initially examined, using RT-PCR, the expression of the AdipoR1 and AdipoR2 genes at the mRNA level in several oral tissues of C57BL mice. Next, we investigated (using real-time PCR assays) whether inflammatory cytokines, such as tumor necrosis factor-alpha, could affect the expression levels of these genes in human gingival fibroblasts. Lastly, we compared the expression levels of these receptor proteins in gingival tissues between two healthy subjects and five patients with severe periodontal disease using western blotting analysis. RESULTS: The AdipoR1 and AdipoR2 receptors were ubiquitously expressed in the oral tissues of mice. We observed that treatment with tumor necrosis factor-alpha could significantly reduce the expression levels of both AdipoR1 and AdipoR2 genes in human gingival fibroblasts. Moreover, we found that the expression of both receptors was lower in periodontal tissues from patients with severe periodontitis than in patients with healthy periodontal tissues. CONCLUSION: These observations suggest that adiponectin may not function efficiently in sites of periodontal disease because of a decrease in the number of its receptors, and this probable dysfunction may play a role in worsening periodontitis in patients.

The role of macrophages in the periodontal regeneration using Emdogain gel.

BACKGROUND AND OBJECTIVE: Emdogain gel is clinically used as a periodontal regenerative material. However, the mechanism of the regeneration has not been completely elucidated. Although many studies have focused on the regenerative effect of Emdogain on connective tissue attachment and alveolar bone, the role of macrophages and the expression of growth factors remains unclear in the regeneration stimulated by Emdogain gel in vivo. The aim of this study was to investigate the effect of Emdogain gel on the expression of cytokines and growth factors by macrophages in vivo using a newly devised rat experimental periodontitis model. MATERIAL AND METHODS: Rat experimental periodontitis was induced by elevating a full-thickness gingival flap and ligating silk threads around the first molars of the mandible. At 14 d after inducing experimental periodontitis, Emdogain gel or propylene glycol alginate was applied to the furcation area. The rats were killed 7 and 14 d after treatment with propylene glycol alginate or Emdogain gel. The expression of cytokines and growth factors, and the regeneration of periodontal tissue, were examined by histochemical and immunohistochemical methods. RESULTS: Fourteen days after the induction of periodontitis, the resorption of alveolar bone at furcation was observed and cytokines such as interleukin-1beta, transforming growth factor-beta1, receptor activator of nuclear factor-kappaB ligand, receptor activator of nuclear factor-kappaB and osteoprotegerin were found. In the Emdogain-treatment group, the formation of new acellular cementum and, more remarkably, recovery of the bone, were observed. The new bone formation ratio in the Emdogain treatment group was significantly higher than that of the propylene glycol alginate treatment group. Although the expression of cytokines such as interleukin-1beta, transforming growth factor-beta1, receptor activator of nuclear factor-kappaB ligand and receptor activator of nuclear factor-kappaB was very low, bone morphogenetic protein-2- and bone morphogenetic protein-4-expressing macrophages were observed close to the root, and bone morphogenetic protein-4-expressing macrophages were mainly observed close to the bone surface at the furcation in the Emdogain-treatment group. CONCLUSION: These results suggest that wound-healing macrophages may express bone morphogenetic protein and play an important role in the regeneration of periodontal tissue at the furcation following the application of Emdogain gel.

Relationship between the presence of periodontopathic bacteria and the expression of chemokine receptor mRNA in inflamed gingival tissues.

BACKGROUND AND OBJECTIVE: Periodontal disease is a chronic disease characterized by the interaction between periodontopathic bacteria and the host immune response. The aim of this study was to investigate the correlation between periodontopathic bacteria and host immune cell infiltrates. MATERIAL AND METHODS: Twenty-two patients with chronic periodontitis were included in this study. Gingival tissues were taken at the periodontal surgery after completion of initial therapy. Three types of periodontopathic bacteria were detected by polymerase chain reaction, and the prevalence of mRNA expression of chemokine receptors was examined by reverse-transcription-polymerase chain reaction in the gingival tissues. The infiltration of T and B cells was determined by an immunohistochemical method. RESULTS: In the patients, both Porphyromonas gingivalis and Tanerella forsythia were detected, and the mRNA expression of chemokine receptors CXCR1&2, CXCR4, CCR1, CCR2, CCR3 and CCR4 were more prevalent. The mean number of infiltrated B cells was significantly larger than that of T cells in the sites harboring both P. gingivalis and T. forsythia. Similarly, in the sites where P. gingivalis was detected but T. forsythia was not, the mean number of B cells was significantly larger than that of T cells. In the sites with mRNA expression of CCR2 and CCR3, the mean number of B cells was significantly larger. CONCLUSION: These results suggest that a high proportion of T helper 2-associated chemokine receptor-positive T cells may be associated with the predominance of B cells and may play an important role in the formation of chronic periodontitis in sites where both P. gingivalis and T. forsythia are detected.

Deficient cell proliferation in palatal shelf mesenchyme of CL/Fr mouse embryos.

How secondary palate formation is affected in the cleft lip genotype remains poorly understood. The purpose of this study was to analyze regional patterns of cell proliferation in CL/Fr mouse embryos with or without cleft lip. Pairs of palatal shelves were dissected at E13.5 from CL/Fr normal embryos (CL/Fr-N), CL/Fr embryos with bilateral cleft lip (CL/Fr-BCL), and a control strain of C57BL embryos (C57BL). The explants were examined histologically after 48 hrs of organ culture. Cell kinetics for proliferation in the palatal shelves was examined at E13.5 by the bromodeoxyuridine method in vivo. The CL/Fr-BCL palates fused as well as the CL/Fr-N palates in vitro. There were inter-group differences in the absolute number of BrdU-positive cells and the ratio of positive/(positive+negative) cells in the palate's mesenchyme (C57BL > CL/Fr-N > CL/Fr-BCL) and epithelium (C57BL > CL/Fr-N = CL/Fr-BCL). These findings indicate that a cleft palate follows reduced cell proliferation of secondary palatal mesenchyme in CL/Fr mice.

Periodontal management of an adolescent with Down's syndrome--a case report.

A case of periodontitis in a young adolescent Japanese girl with Down's syndrome is presented in this report. The patient received a monthly preventive course of dental care consisting of mechanical plaque control and oral hygiene instruction. After 2.5 years she recovered from progression of periodontal disease both clinically and microbiologically. The importance of clinical care for periodontitis in Down's syndrome patients is discussed.

Time required to achieve a stable cuff pressure by repeated aspiration of the cuff during anaesthesia with nitrous oxide.

BACKGROUND AND OBJECTIVE: When the endotracheal tube cuff is repeatedly aspirated to avoid excessive cuff pressure during nitrous oxide anaesthesia, a stable cuff pressure is eventually achieved. We assessed the time required to achieve a stable cuff pressure after repeated cuff deflation. METHODS: During 67% nitrous oxide and oxygen anaesthesia, air-filled cuffs of a standard tracheal tube (Mallinckrodt Hi-Contour) were repeatedly deflated every 30 min for the first 3 or 4 h to inhibit excessive pressure (Groups Def-3 or Def-4, respectively, n = 10 for each); the cuff pressure was monitored for an additional 3 h. In some patients, the study was terminated at 1, 2, 3 and 4 h (n = 6 for each). RESULTS: Cuff pressure in Group Def-3, but not in Group Def-4, > 22 mmHg after stopping cuff aspiration. Intracuff nitrous oxide concentrations increased during repeated cuff deflation and increased further in Group Def-3 during an additional 3 h (from 39.8 +/- 4.7% to 44.3 +/- 3.8%; P < 0.05), whereas intracuff nitrous oxide concentrations at 4 h were not different from those in Group Def-4 at the end of the study (43.7 +/- 4.5% versus 42.3 +/- 4.8%; P = 0.579). CONCLUSIONS: When the air-filled cuff of the standard endotracheal tube is repeatedly deflated every 30 min for 4 h, but not for only 3 h, during nitrous oxide anaesthesia, a stable cuff pressure can be achieved without further deflation of the cuff. Our data also suggest that achieving an equilibrating nitrous oxide concentration in the cuff provides a subsequent stable cuff pressure.

Systemic administration of interferon-gamma-expressing plasmid reduces late allergic bronchitis in a mouse model of asthma.

Asthma might be caused by a helper T(Th)2 immune response. We hypothesized that the systemic administration of the Th1 cytokines may reduce the Th2 type late asthmatic response (LAR). We examined the effect of the intraperitoneal injection of interferon(IFN)-gamma-expressing plasmid, a Th1 cytokine, or interleukin(IL)-4-expressing plasmid, a Th2 cytokine, at the time of sensitization on a mouse model of asthma induced by ovalbumin in BALB/c mice. We demonstrated that the IFN-gamma-expressing plasmid reduced the LAR, whereas the IL-4-expressing plasmid enhanced the LAR as compared with the saline or plasmid-only treated group. The present study suggests that the systemic administration of IFN-gamma-expressing plasmid may have a modulating ability of Th1/Th2 balance to down-regulate Th2 response by a mutual inhibitory mechanism between Th1 and Th2 cells, leading to the reduction of the LAR.

Bronchial lesions of the late asthmatic response in BALB/c and C57BL/6 mice.

In this study, histopathological bronchial-bronchiolar lesions of the late asthmatic responses induced by ovalbumin in BALB/c and C57BL/6 mice were compared. Prominent goblet cell hyperplasia and metaplasia with mucous secretion, and desquamation of epithelial cells with severe infiltration of eosinophils and lymphocytes, were observed in the BALB/c mice; in the C57BL/6 mice, however, these changes were less severe. The reduced histopathological changes in the C57BL/6 mice were associated with a decreased infiltrate of eosinophils, decreased serum immunoglobulin-E (IgE) concentrations and increased serum interferon-gamma concentrations. The results suggest that the reduced bronchial lesions in C57BL/6 mice were due, at least in part, to suppression of the T-helper (Th)2 immune response that underlies the decreased infiltration of lymphocytes and eosinophils into the bronchial mucosa.

Mixed infection of Porphyromonas gingivalis and Bacteroides forsythus in a murine abscess model: involvement of gingipains in a synergistic effect.

Several microorganisms including Porphyromonas gingivalis and Bacteroides forsythus have been implicated to be etiologically important agents of periodontal disease. In this study, we determined the ability of combinations of periodontopathogenic microorganisms to cause tissue destruction in a murine abscess model. Although all bacterial combinations used in this study produced larger abscesses than did monoinfection of each bacterium, the combination of P. gingivalis and B.forsythus showed a synergistic effect on abscess formation. Since these two bacteria have been frequently found together in lesions of periodontitis, these results suggest the significance of their co-infection in the progression of periodontitis. P. gingivalis produces extracellular and cell-associated cysteine proteinases (gingipains) which appear to be involved in its virulence. The rgpA rgpB double and kgp mutants induced significantly smaller abscesses than the wild type. Moreover, the rgpA rgpB kgp triple (gingipain-null) mutant hardly showed lesion formation at all with the experimental conditions used in this study, indicating that these genes encoding gingipains are important for virulence of P. gingivalis. Mixed infection of these P. gingivalis mutants with B. forsythus showed an additive effect on abscess formation, indicating that the gingipains of P. gingivalis may play an important role in the pathological synergism between P. gingivalis and B. forsythus.

Effects of a combination of an antibacterial agent (ofloxacin) and a collagenase inhibitor (FN-439) on the healing of rat periapical lesions.

To investigate the effects of a combination of an antibacterial agent (ofloxacin) and a collagenase inhibitor (FN-439) in the root canal treatment of apical periodontitis, we studied the healing process of experimentally induced periapical lesions in rats by using immunohistochemical methods. With a topical application of a combination of ofloxacin and FN-439 following experimentally induced periapical lesions, both neutrophils and macrophages became significantly decreased in number, while active cementogenesis and extensive bone formation were seen in the periapical region. However, the use of ofloxacin alone also demonstrated a beneficial effect on periapical inflammation and healing. Therefore, it is suggested that ofloxacin is powerful against bacterial infection whether FN-439 is added. The only observed effect of a combination of ofloxacin and FN-439 is that it may more effectively inhibit osteoclastic bone resorption and activate the remodeling of the apical periodontal tissue if this combined medicament is used in a stage of active bone destruction characterized by high production of tissue collagenase.

Colorimetric microtiter plate based assay for detection and quantification of amplified Actinobacillus actinomycetemcomitans DNA.

We developed a colorimetric microtiter plate-based assay for the detection and quantification of polymerase chain reaction-amplified DNA fragment specific for Actinobacillus actinomycetemcomitans. We amplified the 396-bp leukotoxin-specific DNA fragment by using two oligonucleotide primers, one carrying a biotin group at the 5' end and another one with a digoxigenin at the 5' end. Following amplification, the biotinylated polymerase chain reaction products were applied to a microtiter well precoated with avidin. The colorimetric detection and quantification were achieved by an enzyme-linked immunosorbent assay using alkaline phosphatase-conjugated anti-digoxigenin antibody. The detection limit of the colorimetric assay was found to be as little as 500 fg of purified A. actinomycetemcomitans DNA and as few as 50 A. actinomycetemcomitans. Therefore, this colorimetric assay was able to estimate the amount of A. actinomycetemcomitans in subgingival plaque samples. We concluded that the colorimetric assay of the PCR product is a very useful method not only to detect the presence of A. actinomycetemcomitans but also to quantify the amount of A. actinomycetemcomitans in large numbers of subgingival plaque samples.

Detection of interleukin-1 beta mRNA in rat periapical lesions.

Cells expressing interleukin-1 beta (IL-1 beta) mRNA were demonstrated by in situ hybridization in rat periapical lesions. A great number of osteoclasts with significant tartrate-resistant acid phosphatase activity were observed on the bone surfaces, and numerous IL-1 beta mRNA-expressing cells were widely distributed in the periodontal ligaments. IL-1 beta mRNA-expressing cells were mainly observed around the blood vessels in the vicinity of the bone resorption sites and occasionally found near the osteoblasts. Immunohistochemistry and enzyme histochemistry assays showed that IL-1 beta mRNA-expressing cells were not bone cells, but that they had the characteristic features of macrophages. These results suggested that macrophages may contribute to the production of IL-1 beta and play an important role in activation of osteoclastic bone resorption.

A histochemical study of the behavior of macrophages during experimental apical periodontitis in rats.

The behavior of macrophages from experimentally induced periapical lesions of rats was studied in paraffin sections using nonspecific esterase and a monoclonal antibody, ED1. Macrophages were seen near the regularly arranged osteoblasts in controls and the detached osteoblasts at the initiation phase of bone resorption. In addition, numerous macrophages were widely distributed throughout the periodontium at the activation phase of bone resorption. On the other hand, macrophages were rarely seen near the bone formation surfaces, but large numbers of macrophages were localized in microabscess at the activation phase of bone formation. It is suggested that macrophages may play an important role in activation of osteoclastic bone resorption and inhibition of complete bone repair in bone remodeling during experimental apical periodontitis.

Analysis of the molecular mimicry between HLA-B27 and a bacterial OmpA protein using synthetic peptides.

In spite of a lack of sequence 'homology' between HLA-B27 and the bacterial OmpA outer membrane proteins, they both react with the Ye-2 monoclonal anti-HLA-B27 antibody. The Ye-2 antibody also reacted positively in ELISA with a synthetic peptide derived from the segment spanning residues 63-84 of B*2705. The critical peptide residues were determined by testing first with overlapping peptides, followed by a replacement set made according to the determined epitope. The results were compared with those with overlapping eight mers made to span a carboxyl fragment of the Escherichia coli OmpA protein. They indicate the reason why Ye-2 reacts with both sets of peptides is because it has a preference for polymers of arginine.