Extracellular ATP is a key modulator of alveolar bone loss in periodontitis

Periodontal diseases are initiated by pathogenic bacterial biofilm activity that induces a host inflammatory cells immune response, degradation of dento gingival fibrous tissue and its detachment from root cementum. It is well accepted, that osteoclastic alveolar bone loss is governed exclusively through secretion of proinflammatory cytokines. Nevertheless, our findings suggest that once degradation of collagen fibers by MMPs occurs, a drop of cellular strains cause immediate release of ATP from marginal gingival fibroblasts, cell deformation and influx of Ca + 2. Increased extracellular ATP (eATP) by interacting with P2 × 7 purinoreceptors, present on fibroblasts and osteoblasts, induces generation of receptor activator of nuclear factor kB ligand (RANKL) that further activates osteoclastic alveolar bone resorption and bone loss. In addition, increased eATP levels may amplify inflammation by promoting leukocyte recruitment and NALP3-inflammasome activation via P2 × 7. Then, the inflammatory cells secrete cytokines, interleukin IL-1, TNF and RANKL that further trigger alveolar bone resorption. Moreover, eATP can be secreted from periodontal bacteria that may further contribute to inflammation and bone loss in periodontitis. It seems therefore, that eATP is a key modulator that initiates the pathway of alveolar bone resorption and bone loss in patients with periodontal disease. In conclusion, we propose that strain release in gingival fibroblasts aligned on collagen fibers, due to activity of MMP, activates release of ATP that triggers the pathway of alveolar bone resorption in periodontitis. We predict that by controlling the eATP interaction with its cellular purinoreceptors will reduce significantly bone loss in periodontitis.     

Human gingival fibroblasts release high‐mobility group box‐1 protein through active and passive pathways

Introduction:  The nuclear protein high‐mobility group box‐1 (HMGB1) acts as a late mediator of inflammation when secreted in the extracellular milieu. In this study, we examined the effect of lipopolysaccharides from periodontal pathogens and apoptotic and necrotic cell death on HMGB1 production in human gingival fibroblasts (HGF). Methods:  HGF from healthy periodontal tissue were cultured and stimulated with lipopolysaccharides (LPS) from Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Escherichia coli. We also initiated apoptotic and necrotic cell deaths in HGF. The HMGB1 released in the supernatants from stimulated or dying cells was measured. Immunocytochemical staining against HMGB1 was performed in LPS‐stimulated HGF. Results:  A significantly higher amount of HMGB1 was detected from necrotic and apoptotic HGF. LPS from A. actinomycetemcomitans, P. gingivalis, and E. coli significantly induced the production of HMGB1 in a time‐dependent manner. After 6 h of LPS stimulation, HMGB1 was present in the cytoplasm of cells whereas its location was mainly nuclear after 24 h. Conclusions:  LPS from two major periodontal pathogens, A. actinomycetemcomitans and P. gingivalis, induced HMGB1 secretion from HGF. Apoptotic and necrotic cell deaths resulted in the enhancement of HMGB1. Our results suggest that HGF can be a source of HMGB1 by both active secretion and passive release, and that HMGB1 from HGF may contribute to periodontal tissue destruction.     

Biology and pathogenesis of cytomegalovirus in periodontal disease

Human periodontitis is associated with a wide range of bacteria and viruses and with complex innate and adaptive immune responses. Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, Treponema denticola, cytomegalovirus and other herpesviruses are major suspected pathogens of periodontitis, and a combined herpesvirus–bacterial periodontal infection can potentially explain major clinical features of the disease. Cytomegalovirus infects periodontal macrophages and T‐cells and elicits a release of interleukin‐1β and tumor necrosis factor‐α. These proinflammatory cytokines play an important role in the host defense against the virus, but they also have the potential to induce alveolar bone resorption and loss of periodontal ligament. Gingival fibroblasts infected with cytomegalovirus also exhibit diminished collagen production and release of an increased level of matrix metalloproteinases. This article reviews innate and adaptive immunity to cytomegalovirus and suggests that immune responses towards cytomegalovirus can play roles in controlling, as well as in exacerbating, destructive periodontal disease.     

Induction of matrix metalloproteinases and a collagen-degrading phenotype in fibroblasts and epithelial cells by secreted Porphyromonas gingivalis proteinase

Periodontitis is characterized by advancement of a narrow band of epithelium (1–10 cells wide) through the collagenous periodontal ligament in response to bacterial accumulation and infection. A modulating role by epithelial cells in the progression of periodontitis was hypothesized due to the close proximity of the advancing epithelium to both the etiological bacteria and to the collagen fibers of the ligament. We demonstrate that rat mucosal epithelial cells and human fibroblasts are similarly stimulated to degrade a collagen type I cellular substrate by thiol-dependent activity released by the major periodontal pathogen Porphyromonas gingivalis. A purified, extracellular bacterial thiolproteinase from P. gingivalis ATCC 33277 stimulated mucosal epithelial cells to upregulate expression of collagenase and stromelysin, and to degrade a collagen type I fibril matrix. Stimulation of the epithelial cells with this purified proteinase was associated with morphological changes in the cells and with accumulation of secreted latent procollagenase throughout the culture medium. Release of active collagenase was minimal and collagen degradation by the epithelial cells was discreet and localized subcellularly suggesting the possibility that activation of the secreted procollagenase was cell-associated. We conclude that a collagen-degrading phenotype can be stimulated in relatively quiescent mucosal epithelial cells and fibroblasts by the presence of bacterial proteinase. These experiments suggest roles for the P. gingivalis thiol-proteinase and the epithelial cell in the pathogenesis of periodontal disease and demonstrate the potential for dysregulation of extracellular matrix remodeling events during healing of other bacterially infected wounds.     

Interaction of oral bacteria with gingival epithelial cell multilayers

Primary gingival epithelial cells were cultured in multilayers as a model for the study of interactions with oral bacteria associated with health and periodontal disease. Multilayers maintained at an air–liquid interface in low‐calcium medium displayed differentiation and cytokeratin properties characteristic of junctional epithelium. Multilayers were infected with fluorescently labeled Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum or Streptococcus gordonii, and bacterial association was determined by confocal microscopy and quantitative image analysis. Porphyromonas gingivalis invaded intracellularly and spread from cell to cell; A. actinomycetemcomitans and F. nucleatum remained extracellular and showed intercellular movement through the multilayer; whereas S. gordonii remained extracellular and predominantly associated with the superficial cell layer. None of the bacterial species disrupted barrier function as measured by transepithelial electrical resistance. P. gingivalis did not elicit secretion of proinflammatory cytokines. However, A. actinomycetemcomitans and S. gordonii induced interleukin‐1β (IL‐1β), tumor necrosis factor‐α (TNF‐α), IL‐6 and IL‐8 secretion; and F. nucleatum stimulated production of IL‐1β and TNF‐α. Aggregatibacter actinomycetemcomitans, F. nucleatum and S. gordonii, but not P. gingivalis, increased levels of apoptosis after 24 h infection. The results indicate that the organisms with pathogenic potential were able to traverse the epithelium, whereas the commensal bacteria did not. In addition, distinct host responses characterized the interaction between the junctional epithelium and oral bacteria.     

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 Ca2+‐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 Ca²⁺ utilization and biofilm formation. As bacteria can form thick biofilms in the presence of extracellular Ca²⁺, the additive effects of Ca²⁺‐promoted biofilm formation on natural competence should be examined. We evaluated natural competence in Aggregatibacter actinomycetemcomitans, an important periodontal pathogen, in the context of Ca²⁺‐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 Ca²⁺. Biofilm‐derived cells grown in the presence of Ca²⁺ 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 Ca²⁺‐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 Ca²⁺‐promoted biofilms may enhance the ability of bacteria to acquire extracellular DNA.     

Activation of toll‐like receptors 2 and 4 by gram‐negative periodontal bacteria

Background/aims: Periodontitis is a chronic infectious disease associated with a gram‐negative subgingival microflora. Bacterial components stimulate, among other receptors, Toll‐like receptor (TLR) 2 and/or TLR4. Accumulating evidence indicates that both qualitatively and quantitatively distinct immune responses result from the triggering of TLR2 as compared to TLR4 triggering. The aim was to study the interaction of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Tannerella forsythensis, Prevotella intermedia, Prevotella nigrescens, Fusobacterium nucleatum and Veillonella parvula with TLR2 and TLR4. We investigated all known serotypes (K⁻, K1–K6) of P. gingivalis and A. actinomycetemcomitans serotype a–e strains for their potency to stimulate cytokine production. Methods: Human embryonic kidney (HEK) cells, stably transfected with CD14, CD14‐TLR2, or CD14‐TLR4 and whole blood were stimulated with bacterial sonicates. Cytokine production (interleukin‐6, ‐8, ‐10 and ‐12) was measured in the supernatant by enzyme‐linked immunosorbent assay. Results: All test bacteria stimulated HEK‐CD14‐TLR2, but only A. actinomycetemcomitans and V. parvula stimulated HEK‐CD14‐TLR4. No differences were found in the activation of HEK‐CD14‐TLR2/4, or cytokine production in whole blood between serotypes of P. gingivalis and A. actinomycetemcomitans. Conclusion: Gram‐negative periodontal bacteria predominantly stimulated TLR2, which may be of importance for the Th1/Th2 cell orientation of the immune response in periodontitis.     

Extracellular deoxyribonuclease production by periodontal bacteria

Palmer LJ, Chapple ILC, Wright HJ, Roberts A, Cooper PR. Extracellular deoxyribonuclease production by periodontal bacteria. J Periodont Res 2012; 47: 439–445. © 2011 John Wiley & Sons A/S Background and Objective: Whilst certain bacteria have long been known to secrete extracellular deoxyribonuclease (DNase), the purpose in microbial physiology was unclear. Recently, however, this enzyme has been demonstrated to confer enhanced virulence, enabling bacteria to evade the host’s immune defence of extruded DNA/chromatin filaments, termed neutrophil extracellular traps (NETs). As NETs have recently been identified in infected periodontal tissue, the aim of this study was to screen periodontal bacteria for extracellular DNase activity. Material and Methods: To determine whether DNase activity was membrane bound or secreted, 34 periodontal bacteria were cultured in broth and on agar plates. Pelleted bacteria and supernatants from broth cultures were analysed for their ability to degrade DNA, with relative activity levels determined using an agarose gel electrophoresis assay. Following culture on DNA‐supplemented agar, expression was determined by the presence of a zone of hydrolysis and DNase activity related to colony size. Results: Twenty‐seven bacteria, including red and orange complex members Porphyromonas gingivalis, Tannerella forsythia, Fusobacterium nucleatum, Parvimonas micra, Prevotella intermedia, Streptococcus constellatus, Campylobacter rectus and Prevotella nigrescens, were observed to express extracellular DNase activity. Differences in DNase activity were noted, however, when bacteria were assayed in different culture states. Analysis of the activity of secreted DNase from bacterial broth cultures confirmed their ability to degrade NETs. Conclusion: The present study demonstrates, for the first time, that DNase activity is a relatively common property of bacteria associated with advanced periodontal disease. Further work is required to determine the importance of this bacterial DNase activity in the pathogenesis of periodontitis.     

Mast Cells Act as Phagocytes Against the Periodontopathogen Aggregatibacter Actinomycetemcomitans

Background: Evidence to date shows that mast cells play a critical role in immune defenses against infectious agents, but there have been no reports about involvement of these cells in eliminating periodontopathogens. In this study, the phagocytic ability of mast cells against Aggregatibacter actinomycetemcomitans compared with macrophages is evaluated. Methods: In vitro phagocytic assays were conducted using murine mast cells and macrophages, incubated with A. actinomycetemcomitans, either opsonized or not, with different bacterial load ratios. After 1 hour, cells were stained with acridine orange and assessed by confocal laser‐scanning electron microscopy. Results: Phagocytic ability of murine mast cells against A. actinomycetemcomitans was confirmed. In addition, the percentage of mast cells with internalized bacteria was higher in the absence of opsonization than in the presence of opsonization. Both cell types showed significant phagocytic activity against A. actinomycetemcomitans. However, the percentage of mast cells with non‐opsonized bacteria was higher than that of macrophages with opsonized bacteria in one of the ratios (1:10). Conclusions: This is the first report about the participation of murine mast cells as phagocytes against A. actinomycetemcomitans, mainly in the absence of opsonization with human serum. Our results may indicate that mast cells act as professional phagocytes in the pathogenesis of biofilm‐associated periodontal disease.     

Suppression of the periodontopathic microflora in localized juvenile periodontitis by systemic tetracycline

Abstract. Since recent studies have implicated Actinobacillus actinomycetemcomitans in the etiology of localized juvenile periodontitis, this investigation determined the effectiveness of subgingival debridement, topical Betadine Solution®, and systemic tetiacycline in suppressing subgingival A. actinomycetemcomitans and other microorganisms. A total of 20 deep periodontal pockets and 10 normal periodontal sites of 6 localized juvenile periodontitis patients was included in the study. Each patient was treated in 3 stages over a period of 22 weeks, and the result of treatment was monitored for an additional 38 weeks. The first stage of treatment included plaque control, as well as thorough scaling and root planing, composed of at least 6 h of debridement. No concomitant periodontal surgery was performed. In the second stage, Betadine saturated cotton gauze was inserted into the periodontal pockets for 10 min. Stage 3 involved systemic tetracycline therapy (1 g/day) for J4 days. The subgingival microflora was determined at frequent intervals by selective culturing of A. actinomycetemcomitans and Capnocytophaga and by direct microscopic examination. The clinical effect was assessed by measuring changes in probing periodontal attachment level, probing periodontal pocket depth, radiographic alveolar bone mass, and other relevant clinical parameters. Scaling and root planing reduced the total subgingival bacterial counts and the proportions of certain Gram-negative bacteria, but no periodontal pocket became free of A actinomycetemcomitans. Betadine application had little or no effect on the subgingival microflora. In contrast, tetracycline administered via the systemic route suppressed. A actinomycetemcomitans, Capnocytophaga, and spirochetes to low or undetectable levels in all test periodontal pockets. A, actinomycetemcomitans reappeared in 9 of the deep periodontal pockets after the administration of tetracycline. Most of these 9 pockets became free of detectable A. actinomycetemcomitans during the second week of tetracycline administration, whereas pockets which yielded no A. actinomycetemcomitans after tetracycline therapy became free of the organisms during the first week of tetracycline treatment. This data suggests that systemic tetracycline therapy of localized juvenile periodontitis should, as a practical rate, be continued for 3 weeks. Periodontal destruction continued in 4 deep pockets which all showed high posttetracycline A, actinomycetemcomitans counts. All 6 pockets which demonstrated a marked gain in periodontal attachment yielded no cultivable A. actinomycetemcomitans. No association was found between periodontal disease status and subgingival Capnocytophaga, spirochetes or motile rods. The present study indicates that A. actinomycetemcomitans is an important etiologic agent in localized juvenile periodontitis. Also, this study demonstrates that the effectiveness of therapy can be monitored by subgingival A. actinomycetemcomitans counts, and that periodontal A, actinomycetemcomitans infections cannot be resolved by root surface debridement alone but can be cured by systemic tetracycline therapy.     

Antimicrobial Activity of Platelet‐Rich Plasma and Other Plasma Preparations Against Periodontal Pathogens

Background: In addition to releasing a pool of growth factors during activation, platelets have many features that indicate their role in the anti‐infective host defense. The antimicrobial activities of platelet‐rich plasma (PRP) and related plasma preparations against periodontal disease–associated bacteria were evaluated. Methods: Four distinct plasma fractions were extracted in the formulation used commonly in dentistry and were tested for their antibacterial properties against three periodontal bacteria: Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum. The minimum inhibitory concentration of each plasma preparation was determined, and in vitro time‐kill assays were used to detect their abilities to inhibit bacterial growth. Bacterial adhesion interference and the susceptibility of bacterial adherence by these plasma preparations were also conducted. Results: All plasma preparations can inhibit bacterial growth, with PRP showing the superior activity. Bacterial growth inhibition by PRP occurred in the first 24 hours after application in the time‐kill assay. PRP interfered with P. gingivalis and A. actinomycetemcomitans attachment and enhanced exfoliation of attached P. gingivalis but had no influences on F. nucleatum bacterial adherence. Conclusions: PRP expressed antibacterial properties, which may be attributed to platelets possessing additional antimicrobial molecules. The application of PRP on periodontal surgical sites is advisable because of its regenerative potential and its antibacterial effects.     

Nucleases from Prevotella intermedia can degrade neutrophil extracellular traps

Periodontitis is an inflammatory disease caused by periodontal bacteria in subgingival plaque. These bacteria are able to colonize the periodontal region by evading the host immune response. Neutrophils, the host's first line of defense against infection, use various strategies to kill invading pathogens, including neutrophil extracellular traps (NETs). These are extracellular net‐like fibers comprising DNA and antimicrobial components such as histones, LL‐37, defensins, myeloperoxidase, and neutrophil elastase from neutrophils that disarm and kill bacteria extracellularly. Bacterial nuclease degrades the NETs to escape NET killing. It has now been shown that extracellular nucleases enable bacteria to evade this host antimicrobial mechanism, leading to increased pathogenicity. Here, we compared the DNA degradation activity of major Gram‐negative periodontopathogenic bacteria, Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans. We found that Pr. intermedia showed the highest DNA degradation activity. A genome search of Pr. intermedia revealed the presence of two genes, nucA and nucD, putatively encoding secreted nucleases, although their enzymatic and biological activities are unknown. We cloned nucA‐ and nucD‐encoding nucleases from Pr. intermedia ATCC 25611 and characterized their gene products. Recombinant NucA and NucD digested DNA and RNA, which required both Mg²⁺ and Ca²⁺ for optimal activity. In addition, NucA and NucD were able to degrade the DNA matrix comprising NETs.     

Herpesvirus-bacteria synergistic interaction in periodontitis

The etiopathogenesis of severe periodontitis includes herpesvirus-bacteria coinfection. This article evaluates the pathogenicity of herpesviruses (cytomegalovirus and Epstein-Barr virus) and periodontopathic bacteria (Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis) and coinfection of these infectious agents in the initiation and progression of periodontitis. Cytomegalovirus and A. actinomycetemcomitans/P. gingivalis exercise synergistic pathogenicity in the development of localized (“aggressive”) juvenile periodontitis. Cytomegalovirus and Epstein-Barr virus are associated with P. gingivalis in adult types of periodontitis. Periodontal herpesviruses that enter the general circulation may also contribute to disease development in various organ systems. A 2-way interaction is likely to occur between periodontal herpesviruses and periodontopathic bacteria, with herpesviruses promoting bacterial upgrowth, and bacterial factors reactivating latent herpesviruses. Bacterial-induced gingivitis may facilitate herpesvirus colonization of the periodontium, and herpesvirus infections may impede the antibacterial host defense and alter periodontal cells to predispose for bacterial adherence and invasion. Herpesvirus-bacteria synergistic interactions, are likely to comprise an important pathogenic determinant of aggressive periodontitis. However, mechanistic investigations into the molecular and cellular interaction between periodontal herpesviruses and bacteria are still scarce. Herpesvirus-bacteria coinfection studies may yield significant new discoveries of pathogenic determinants, and drug and vaccine targets to minimize or prevent periodontitis and periodontitis-related systemic diseases.     

Predation of oral pathogens by Bdellovibrio bacteriovorus 109J

Periodontal diseases are multifactorial infections elicited by a complex of primarily gram‐negative bacteria that interact with host tissues and lead to the destruction of the periodontal structures. Bdellovibrio bacteriovorus is a gram‐negative bacterium that preys upon other gram‐negative bacteria. It was previously shown that B. bacteriovorus has an ability to attack and remove surface‐attached bacteria or biofilms. In this study, we examined the host specificity of B. bacteriovorus strain 109J and its ability to prey on oral pathogens associated with periodontitis, including; Aggregatibacter actinomycetemcomitans, Eikenella corrodens, Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis and Tannerella forsythia. We further demonstrated that B. bacteriovorus 109J has an ability to remove biofilms of Ei. corrodens as well as biofilms composed of A. actinomycetemcomitans. Bdellovibrio bacteriovorus was able to remove A. actinomycetemcomitans biofilms developed on hydroxyapatite surfaces and in the presence of saliva, as well as to detach metabolically inactive biofilms. Experiments aimed at enhancing the biofilm removal aptitude of B. bacteriovorus with the aid of extracellular‐polymeric‐substance‐degrading enzymes demonstrated that proteinase‐K inhibits predation. However, treating A. actinomycetemcomitans biofilms with DspB, a poly‐N‐acetylglucosamine (PGA) ‐hydrolysing enzyme, increased biofilm removal. Increased biofilm removal was also recorded when A. actinomycetemcomitans PGA‐defective mutants were used as host cells, suggesting that PGA degradation could enhance the removal of A. actinomycetemcomitans biofilm by B. bacteriovorus.     

Clarithromycin accumulation by phagocytes and its effect on killing of Aggregatibacter actinomycetemcomitans

Background: Clarithromycin inhibits several periodontal pathogens and is concentrated inside gingival fibroblasts and epithelial cells by an active transporter. We hypothesized that polymorphonuclear leukocytes (PMNs) and less mature myeloid cells possess a similar transporter for clarithromycin. It is feasible that clarithromycin accumulation inside PMNs could enhance their ability to kill Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans). Methods: To test the first hypothesis, purified PMNs and cultured HL‐60 cells were incubated with [³H]‐clarithromycin. Clarithromycin transport was assayed by measuring changes in cell‐associated radioactivity over time. The second hypothesis was examined with PMNs loaded by incubation with clarithromycin (5 μg/ml). Opsonized bacteria were incubated at 37°C with control and clarithromycin‐loaded PMNs. Results: Mature human PMNs, HL‐60 cells differentiated into granulocytes, and undifferentiated HL‐60 cells all took up clarithromycin in a saturable manner. The kinetics of uptake by all yielded linear Lineweaver‐Burk plots. HL‐60 granulocytes transported clarithromycin with a K_m of ≈250 μg/ml and a V_max of 473 ng/min/10⁶ cells, which were not significantly different from the values obtained with PMNs. At steady state, clarithromycin levels inside HL‐60 granulocytes and PMNs were 28‐ to 71‐fold higher than extracellular levels. Clarithromycin‐loaded PMNs killed significantly more A. actinomycetemcomitans and achieved shorter half‐times for killing than control PMNs when assayed at a bacteria‐to‐PMN ratio of 100:1 (P <0.04). At a ratio of 30:1, these differences were not consistently significant. Conclusions: PMNs and less mature myeloid cells possess a transporter that takes up and concentrates clarithromycin. This system could help PMNs cope with an overwhelming infection by A. actinomycetemcomitans.     

Chronic Periodontal Disease,Periodontal Pathogen Colonization,and Increased Risk of Precancerous Gastric Lesions

Background: This study assesses the association between periodontal pathogen colonization and the potential risk of developing precancerous lesions of gastric cancer (PLGC) in a clinical setting. Methods: Included were 35 newly diagnosed patients with PLGC and 70 age‐matched individuals without PLGC. A full‐mouth intraoral examination was performed to assess periodontal conditions. Stimulated whole saliva and pooled plaque samples were collected to evaluate colonization by Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Aggregatibacter actinomycetemcomitans and to characterize oral microbial diversity in saliva and dental plaque. Results: Compared with the control group, patients with PLGC experienced higher prevalence of bleeding on probing (31.5% versus 22.4%; P <0.05), higher levels of T. denticola (P <0.01) and A. actinomycetemcomitans (P <0.01), and less bacterial diversity in their saliva (P <0.01). The final multivariate logistic regression model consisting of all key sociodemographic characteristics, oral health behavioral factors, and periodontal assessments revealed that elevated colonization with periodontal pathogens, specifically T. forsythia, T. denticola, and A. actinomycetemcomitans, decreased bacterial diversity in dental plaque, and not flossing teeth regularly was a significant predictor of increased risk of PLGC (P = 0.022). Conclusion: Findings of the present study provide new evidence suggesting that periodontal pathogen burdens and bacterial diversity in the oral cavity are important factors contributing to a potentially increased risk of developing precancerous gastric lesions.     

Microbiological features of Papillon-Lefèvre syndrome periodontitis

Abstract. Papillon-Lefèvre syndrome patients exhibit hyperkeratosis palmoplantaris and severe periodontitis. The syndrome is an autosomal recessive trait, but the mechanism of periodontal destruction is not known. This report presents the clinical and microbiological features of an 11-year old girl with Papillon-Lefěvre syndrome. Clinical examination included conventional periodontal measurements and radiographic analysis. In samples from 3 deep periodontal lesions, the occurrence of major suspected periodontopathic bacteria was determined by selective and non-selective culture and polymerase chain reaction (PCR) identification, and the presence of cytomegalovirus and Epstein-Barr type 1 virus by a nested-PCR detection method. 10 of 22 available teeth demonstrated severe periodontal breakdown. Major cultivable bacteria included Actinobacillus actinomycetemcomitans (3.4% of total isolates), Prevotella nigrescens (16.4%), Fusobacterium nucleatum (14.3%) and Peptostreptococcus micros (10.6%). A. actinomycetemcomitans, P. nigrescens, Porphyromonas gingivalis and Eikenella corrodens were identified by PCR analysis. The patient's non-affected parents and older brother revealed several periodontal pathogens but not A. actinomycetemcomitans. The viral examination demonstrated cytomegalovirus and Epstein-Barr type 1 virus in the subgingival sample of the Papillon-Lefèvre syndrome patient. The father and brother yielded subgingival cytomegalovirus but not Epstein-Barr type 1 virus. We hypothesize that human herpesviruses in concert with A. actinomycetemcomitans play important rǒles in the development of Papillon-Lefèvre syndrome periodontitis.     

Cross‐reactive adaptive immune response to oral commensal bacteria results in an induction of receptor activator of nuclear factor‐κB ligand (RANKL)‐dependent periodontal bone resorption in a mouse model

Introduction: The present study examined whether induction of an adaptive immune response to orally colonizing non‐pathogenic Pasteurella pneumotropica by immunization with the phylogenetically closely related bacterium, Actinobacillus actinomycetemcomitans, can result in periodontal bone loss in mice. Methods: BALB/c mice harboring P. pneumotropica (P. pneumotropica⁺ mice) in the oral cavity or control P. pneumotropica‐free mice were immunized with fixed A. actinomycetemcomitans. The animals were sacrificed on day 30, and the following measurements were carried out: (i) serum immunoglobulin G and gingival T‐cell responses to A. actinomycetemcomitans and P. pneumotropica; (ii) periodontal bone loss; and (iii) identification of receptor activator of nuclear factor‐κB ligand (RANKL) ‐positive T cells in gingival tissue. Results: Immunization with A. actinomycetemcomitans induced a significantly elevated serum immunoglobulin G response to the 29‐kDa A. actinomycetemcomitans outer membrane protein (Omp29), which showed strong cross‐reactivity with P. pneumotropica OmpA compared to results in the control non‐immunized mice. The A. actinomycetemcomitans‐immunized P. pneumotropica⁺ mice developed remarkable periodontal bone loss in a RANKL‐dependent manner, as determined by the abrogation of bone loss by treatment with osteoprotegerin‐Fc. The T cells isolated from the gingival tissue of A. actinomycetemcomitans‐immunized P. pneumotropica⁺ mice showed an in vitro proliferative response to both A. actinomycetemcomitans and P. pneumotropica antigen presentation, as well as production of soluble(s)RANKL in the culture supernatant. Double‐color confocal microscopy demonstrated that the frequency of RANKL⁺ T cells in the gingival tissue of A. actinomycetemcomitans‐immunized P. pneumotropica⁺ mice was remarkably elevated compared to control mice. Conclusion: The induction of an adaptive immune response to orally colonizing non‐pathogenic P. pneumotropica results in RANKL‐dependent periodontal bone loss in mice.