The role of 5-lipoxygenase in Aggregatibacter actinomycetemcomitans-induced alveolar bone loss.

AIM: Leukotrienes (LTs) are pro-inflammatory lipid mediators formed by the enzyme 5-lipoxygenase (5-LO). The involvement of 5-LO metabolites in periodontal disease (PD) is not well defined. This study aimed to assess the role of 5-LO in experimental PD induced by Aggregatibacter actinomycetemcomitans (Aa). MATERIAL AND METHODS: In vivo experiments were carried out using SV129 wild-type (WT) and 5-LO-deficient (5lo-/- ) mice inoculated with Aa. Osteoclasts were stimulated in vitro with AaLPS in the presence or not of selective inhibitors of the 5-LO pathway, or LTB4 or platelet-activating factor (PAF), as PAF has already been shown to increase osteoclast activity. RESULTS: In 5lo-/- mice, there were no loss of alveolar bone and less TRAP-positive osteoclasts in periodontal tissues, after Aa inoculation, despite local production of TNF-α and IL-6. The differentiation and activity of osteoclasts stimulated with AaLPS were diminished in the presence of BLT1 antagonist or 5-LO inhibitor, but not in the presence of cysteinyl leukotriene receptor antagonist. The osteoclast differentiation induced by PAF was impaired by the BLT1 antagonism. CONCLUSION: In conclusion, LTB4 but not CysLTs is important for Aa-induced alveolar bone loss. Overall, LTB4 affects osteoclast differentiation and activity and is a key intermediate of PAF-induced osteoclastogenesis.

Periodontal disease and gene-expression levels of metalloendopeptidases in human buccal mucosal epithelium.

BACKGROUND AND OBJECTIVE: Endopeptidases, such as neutral endopeptidase (NEP), endothelin-converting enzyme-1 (ECE-1) and a disintegrin and metalloprotease 17 (ADAM17), are believed to have various important roles in oral mucosal and epidermal tissue for the regulation of defensive biological responses in the oral cavity, and their expression and activity are influenced by various factors, including oral diseases. However, knowledge concerning these endopeptidases in the oral cavity has been minimal until now. This study focused on three metalloendopeptidases - NEP, ECE-1 and ADAM17 - in the oral buccal mucosal epithelium of patients with periodontal diseases and investigated the relationship between their gene-expression levels and periodontal disease. MATERIAL AND METHODS: The levels of expression of NEP, ECE-1 and ADAM17 mRNAs in tissue samples collected from the oral buccal mucosal epithelium of 61 patients were investigated by relative quantification using real-time RT-PCR analysis. information on oral and systemic health was obtained from the clinical record of each patient. RESULTS: Among the three groups, classified based on the diagnosis of periodontal diseases (healthy/gingivitis, early periodontitis and moderate/advanced periodontitis), the relative expression level of NEP mRNA was significantly increased in the early periodontitis group and in the moderate/advanced periodontitis group compared with that in the healthy/gingivitis group. Moreover, the relative expression levels of ECE1 and ADAM17 mRNAs were significantly increased in the moderate/advanced periodontitis group compared with those in the healthy/gingivitis group. The correlation coefficients between the mean relative expression levels of NEP and ECE1 mRNAs, NEP and ADAM17 mRNAs, and ECE1 and ADAM17 mRNAs were r = 0.758, r = 0.707 and r = 0.934, respectively (p < 0.001). Furthermore, among the oral-related factors, there was a significant correlation between the number of sites with probing pocket depths of more than 4 mm and of more than 6 mm and the relative expression levels of NEP, ECE1 and ADAM17 mRNAs. In stepwise logistic regression models, high relative expression levels of ECE1 and ADAM17 mRNAs were significantly associated with moderate/advanced periodontitis. CONCLUSION: The present study suggests that the severity of periodontal disease may be associated with the expression of metalloendopeptidase genes, including NEP, ECE1 and ADAM17, in the buccal mucosal epithelium.

Temporal expression of metalloproteinase-8 and -13 and their relationships with extracellular matrix metalloproteinase inducer in the development of ligature-induced periodontitis in rats.

BACKGROUND AND OBJECTIVE: Matrix metalloproteinases (MMPs) play important roles in extracellular matrix degradation and may be regulated by extracellular matrix metalloproteinase inducer (EMMPRIN). The aim of this study was to investigate the temporal expression and localization of MMP-8 and MMP-13 during the development of ligature-induced periodontitis in rats, and to analyze the correlations of EMMPRIN with MMP-8 and MMP-13 in periodontitis. MATERIAL AND METHODS: Periodontitis was simulated in rats by ligaturing the cervix of the lower first molars, as described in our previous method. The rats were killed 0, 3, 5, 7, 11, 15 and 21 d after ligation. Micro-computed tomography examinations were performed to detect alveolar bone loss. Semiquantitative western blotting was used to assess the temporal changes in the levels of MMP-8, MMP-13 and EMMPRIN proteins in gingival tissue. Immunohistochemistry was applied to detect the expression and locations of MMP-8 and MMP-13 in gingival tissue and alveolar bone. RESULTS: Alveolar bone loss showed an exponential increase from days 3 to 11, followed by a slower rate of loss at subsequent study time points. MMP-8 showed a rapid increase of expression from baseline to a peak on day 3, a gradual decrease from days 5 to 7 and then stabilized thereafter. MMP-8 was predominantly located in neutrophil-like cells. Statistically, the expression of MMP-8 was not correlated with the expression of EMMPRIN. The expression of MMP-13 and of EMMRPIN increased from days 3 to 7, and showed a moderate decrease thereafter. The immunoreactivity of MMP-13 was mainly detected in monocytes/macrophages, on the alveolar bone surface, in osteoclasts and in gingival epithelial cells. Statistically, MMP-13 had a strong, positive correlation with EMMPRIN (r = 0.855, p < 0.01). CONCLUSION: The levels of expression of MMP-8 and MMP-13 are temporally varied at different periods during the development of experimental periodontitis. The level of expression of EMMPRIN is closely associated with the expression of MMP-13, but not with the expression of MMP-8. In addition, MMP-13 might be involved in alveolar bone destruction, as well as in physiological bone remodeling.

Inhibition of GSK3 abolishes bacterial-induced periodontal bone loss in mice.

The tissue destruction that characterizes periodontitis is driven by the host response to bacterial pathogens. Inhibition of glycogen synthase kinase 3ß (GSK3ß) in innate cells leads to suppression of Toll-like receptor (TLR)-initiated proinflammatory cytokines under nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 transcriptional control and promotion of cyclic adenosine monophosphate response element-binding (CREB)-dependent gene activation. Therefore, we hypothesized that the cell permeable GSK3-specific inhibitor, SB216763, would protect against alveolar bone loss induced by the key periodontal pathogen, Porphyromonas gingivalis (P. gingivalis), in a murine model. B6129SF2/J mice either were infected orally with P. gingivalis ATCC 33277; or treated with SB216763 and infected with P. gingivalis; sham infected; or exposed to vehicle only (dimethyl sulfoxide [DMSO]); or to GSK3 inhibitor only (SB216763). Alveolar bone loss and local (neutrophil infiltration and interleukin [IL]-17) and systemic (tumor necrosis factor [TNF], IL-6, Il-1ß and IL-12/IL-23 p40) inflammatory indices also were monitored. SB216763 unequivocally abrogated mean P. gingivalis-induced bone resorption, measured at 14 predetermined points on the molars of defleshed maxillae as the distance from the cementoenamel junction to the alveolar bone crest (p < 0.05). The systemic cytokine response, the local neutrophil infiltration and the IL-17 expression were suppressed (p < 0.001). These data confirm the relevance of prior in vitro phenomena and establish GSK3 as a novel, efficacious therapeutic preventing periodontal disease progression in a susceptible host. These findings also may have relevance to other chronic inflammatory diseases and the systemic sequelae associated with periodontal infections.

S-nitrosoglutathione decreases inflammation and bone resorption in experimental periodontitis in rats.

BACKGROUND: S-nitrosoglutathione (GSNO) is a nitric oxide donor that may exert antioxidant, anti-inflammatory, and microbicidal actions and is thus a potential drug for the topical treatment of periodontitis. In this study, the effect of intragingival injections of GSNO-containing polyvinylpyrrolidone (PVP) formulations is evaluated in a rat model of periodontitis. METHODS: Periodontal disease was induced by placing a sterilized nylon (000) thread ligature around the cervix of the second left upper molar of the animals, which received intragingival injections of PVP; saline; or PVP/GSNO solutions which corresponded to GSNO doses of 25, 100, and 500 nmol; 1 hour before periodontitis induction, and thereafter, daily for 11 days. RESULTS: PVP/GSNO formulations at doses of 25 and/or 100, but not 500 nmol caused significant inhibition of alveolar bone loss, increase of bone alkaline phosphatase, decrease of myeloperoxidase activity, as well as significant reduction of inflammatory and oxidative stress markers when compared to saline and PVP groups. These effects were also associated with a decrease of matrix metalloproteinases 1 and 8, inducible nitric oxide synthase, and nuclear factor-κB immunostaining in the periodontium. CONCLUSION: Local intragingival injections of GSNO reduces inflammation and bone loss in experimental periodontal disease.

Presence of aspartate aminotransferase in peri-implant crevicular fluid with and without mucositis.

The aim of this study was to assess the presence of aspartate aminotransferase (AST) in peri-implant crevicular fluid, with or without clinical signs of mucositis, to determine its predictive diagnostic value, sensitivity, and specificity. The AST levels were determined (at a threshold of 1200 µIU/mL) for 60 clinically successful implants in 25 patients with or without peri-implant mucositis. Samples were taken prior (AST1) to peri-implant probing with a manual constant-pressure probe (0.2 N) and 15 minutes after probing (AST2). Clinical assessments included radiographic determination of preexisting bone loss, probing, and the evaluation of mucositis, plaque, and bleeding upon probing. Analysis was performed at both the level of the implant and the patient as a unit. We detected a significant difference between AST1 and AST2 at both levels. A significant difference was observed at AST1 between implants that bled upon probing and those that did not. However, when we considered the patient as a unit, there were no significant differences. The plaque index was not significant at either level. AST1 had high specificity and positive predictive diagnostic value (80%) for bleeding upon probing. Probing induces a greater release of AST from inflamed tissues compared with healthy tissues in situ but not at the systemic level. At the implant level, the implant position could be responsible for this difference. Aspartate aminotransferase was a reliable predictor of patients with mucositis.

Analysis of cathepsin-K activity at tooth and dental implant sites and the potential of this enzyme in reflecting alveolar bone loss.

BACKGROUND: Cathepsin-K is an enzyme involved in bone metabolism which may make this feature important for both natural teeth and dental implants. The aims of the present study are to comparatively analyze the gingival crevicular fluid (GCF)/peri-implant sulcus fluid (PISF) cathepsin-K levels of natural teeth and dental implants, and to assess the potential relationship between this biochemical parameter and alveolar bone loss around natural teeth and dental implants. METHODS: Probing depth, bleeding on probing, gingival index, and plaque index clinical parameters were assessed, and GCF/PISF samples were obtained from natural teeth/dental implants presenting with either clinical health, gingivitis/peri-implant mucositis, or chronic periodontitis/peri-implantitis. Cathepsin-K activity levels of 42 GCF samples and 54 PISF samples were determined, and marginal bone loss (MBL) measures were calculated from digitalized standardized intraoral periapical radiographs obtained from natural teeth and dental implants by using cemento-enamel junction and the actual distance between two consecutive threads of the dental implant as reference points for natural teeth and dental implants, respectively. RESULTS: Comparing the natural teeth group with dental implant group with regard to MBL measure, cathepsin-K activity, and GCF/PISF volume revealed no significant differences. In both natural teeth and dental implant groups, despite higher MBL measures, cathepsin-K activity, and GCF/PISF volumes with the presence of inflammation, it was the presence of alveolar bone loss that lead to significantly higher values for these parameters. CONCLUSION: We suggest cathepsin-K as a biochemical parameter for monitoring periodontal/peri-implant alveolar bone loss.

Sequential IL-23 and IL-17 and increased Mmp8 and Mmp14 expression characterize the progression of an experimental model of periodontal disease in type 1 diabetes.

Molecular mechanisms responsible for periodontal disease (PD) and its worsening in type 1 Diabetes Mellitus (DM1) remain unknown. Cytokine profile and expression levels of collagenases, Mmp14, and tissue inhibitors were determined, as were the numbers of neutrophils and macrophages in combined streptozotocin-induced DM1 and ligature-induced PD models. Increased IL-23 (80-fold) and Mmp8 expression (25-fold) was found in DM1. Ligature resulted in an IL-1ß/IL-6 profile, increased expression of Mmp8, Mmp13, and Mmp14 (but not Mmp1), and transient expression of Timp1 and Reck in non-diabetics. PD in DM1 involved IL-1ß (but not IL-6) and IL-23/IL-17, reduced IL-6 and IL-10, sustained Mmp8 and Mmp14, increased Mmp13 and reduced Reck expression in association with 20-fold higher counts of neutrophils and macrophages. IL-23 and Mmp8 expression are hallmarks of DM1. In association with the IL-1/IL-6 (Th1) response in PD, one found a secondary IL-17 (Th17) pathway in non-diabetic rats. Low IL-6/TNF-α suggest that the Th1 response was compromised in DM1, while IL-17 indicates a prevalence of the Th17 pathway, resulting in high neutrophil recruitment. Mmp8, Mmp13, and Mmp14 expression seems important in the tissue destruction during PD in DM1. PD-associated IL-1/IL-6 (Th1), IL-10, and Reck expression are associated with the acute-to-chronic inflammation transition, which is lost in DM1. In conclusion, IL-23/IL-17 are associated with the PD progression in DM1.

Analysis of cathepsin-K levels in biologic fluids from healthy or diseased natural teeth and dental implants.

PURPOSE: Cathepsin-K is an enzyme involved in bone metabolism. This feature may make it important both for natural teeth and dental implants. The aims of the present study were to comparatively analyze cathepsin-K levels in gingival crevicular fluid (GCF) and peri-implant sulcus fluid (PISF) and to determine whether GCF and PISF cathepsin-K profiles reflect the clinical periodontal/peri-implant status. MATERIALS AND METHODS: Clinical parameters (probing depth, Gingival Index, Plaque Index, and bleeding on probing) were recorded, and GCF/PISF samples were obtained from natural teeth (group T) and dental implants (group I), which were divided into groups based on health (clinically healthy, gingivitis/peri-implant mucositis, and periodontitis/peri-implantitis). Cathepsin-K activity was determined with a commercially available cathepsin-K activity assay kit (BioVision). RESULTS: Sixty natural teeth and 68 dental implants were examined. Teeth with periodontitis (group T-3) showed significantly higher total cathepsin-K activity (10.39 units) than teeth with gingivitis (group T-2, 1.71 units) and healthy teeth (group T-1, 1.90 units). The difference in cathepsin-K activity between groups T-2 and T-1 was not significant. Implants with peri-implantitis (group I-3) had higher total enzyme activity (10.26 units) than healthy implants (group I-1) (3.44 units). Although the difference between clinical parameters was not significant, group I-3 had higher cathepsin-K levels than group I-2 (4.74 units). When natural teeth (T-1, T-2, T-3) were compared to implants (I-1, I-2, I-3), no significant differences were observed for cathepsin-K levels. CONCLUSION: More cathepsin-K activity was clearly observed with inflammatory periodontal and peri-implant destruction. The highest cathepsin-K levels detected in GCF and PISF samples, obtained from sites with periodontitis and peri-implantitis, suggests the potential involvement of cathespin-K in increased bone metabolism around natural teeth and dental implants.

The role of iNOS and PHOX in periapical bone resorption.

Nitric oxide (NO) and reactive oxygen species (ROS) are key molecules in resistance to pathogens. Little is known about their role in pathogenesis of periapical lesions. To address this issue, we induced periapical lesions in mice lacking nitric oxide synthase (iNOS(-/-)) or phagocyte oxidase (PHOX(-/-)). iNOS(-/-) mice expressed higher levels of IL-1ß, TNF-α, RANK, RANKL, and MCP-1 than C57BL/6 and PHOX(-/-). Apical thickening of the periodontal ligament was also greater in iNOS(-/-) compared with other groups. Interestingly, ROS production did not interfere in periapical lesion progression, but seemed to be essential for the appearance of multinucleated TRAP-positive cells. Thus, periapical lesion progression in iNOS(-/-) was associated with an imbalance of pro-inflammatory cytokines (IL-1ß and TNF-α), bone-resorptive modulators (RANK and RANKL), and MCP-1. We conclude that NO, but not ROS, controls progression of bone resorption in a murine experimental model of apical periodontitis.

Silencing mitogen-activated protein kinase-activated protein kinase-2 arrests inflammatory bone loss.

p38 mitogen-activated protein kinases (MAPKs) are critical for innate immune signaling and subsequent cytokine expression in periodontal inflammation and bone destruction. In fact, previous studies show that systemic p38 MAPK inhibitors block periodontal disease progression. However, development of p38 MAPK inhibitors with favorable toxicological profiles is difficult. Here, we report our findings regarding the contribution of the downstream p38 MAPK substrate, mitogen-activated protein kinase-activated protein kinase 2 (MK2 or MAPKAPK-2), in immune response modulation in an experimental model of pathogen-derived lipopolysaccharide (LPS)-induced periodontal bone loss. To determine whether small interfering RNA (siRNA) technology has intraoral applications, we initially validated MK2 siRNA specificity. Then, gingival tissue surrounding maxillary molars of rats was injected with MK2 siRNA or scrambled siRNA at the palatal regions of bone loss. Intraoral tissues treated with MK2 siRNA had significantly less MK2 mRNA expression compared with scrambled siRNA-treated tissues. MK2 siRNA delivery arrested LPS-induced inflammatory bone loss, decreased inflammatory infiltrate, and decreased osteoclastogenesis. This proof-of-concept study suggests a novel target using an intraoral RNA interference strategy to control periodontal inflammation.

Effect of venlafaxine on bone loss associated with ligature-induced periodontitis in Wistar rats.

BACKGROUND: The present study investigated the effects of venlafaxine, an antidepressant drug with immunoregulatory properties on the inflammatory response and bone loss associated with experimental periodontal disease (EPD). MATERIALS AND METHODS: Wistar rats were subjected to a ligature placement around the second upper left molar. The treated groups received orally venlafaxine (10 or 50 mg/kg) one hour before the experimental periodontal disease induction and daily for 10 days. Vehicle-treated experimental periodontal disease and a sham-operated (SO) controls were included. Bone loss was analyzed morphometrically and histopathological analysis was based on cell influx, alveolar bone, and cementum integrity. Lipid peroxidation quantification and immunohistochemistry to TNF-alpha and iNOS were performed. RESULTS: Experimental periodontal disease rats showed an intense bone loss compared to SO ones (SO = 1.61 +/- 1.36; EPD = 4.47 +/- 1.98 mm, p < 0.001) and evidenced increased cellular infiltration and immunoreactivity for TNF-alpha and iNOS. Venlafaxine treatment while at low dose (10 mg/kg) afforded no significant protection against bone loss (3.25 +/- 1.26 mm), a high dose (50 mg/kg) caused significantly enhanced bone loss (6.81 +/- 3.31 mm, p < 0.05). Venlafaxine effectively decreased the lipid peroxidation but showed no significant change in TNF-alpha or iNOS immunoreactivity. CONCLUSION: The increased bone loss associated with high dose venlafaxine may possibly be a result of synaptic inhibition of serotonin uptake.

MAP kinase phosphatase-1 protects against inflammatory bone loss.

The mitogen-activated protein (MAP) kinase phosphatase (MKP) family plays an important function in regulating the pro-inflammatory cytokines by deactivating MAP kinases. MKP-1 is essential for the dephosphorylation of p38 MAP kinase that regulates expression of IL-6, TNF-alpha, and IL-1 beta. We hypothesized that MKP-1 regulates inflammatory bone loss in experimental periodontitis. Wild-type and Mkp-1(-/-) mice received A. actinomycetemcomitans LPS injection in the palatal region or PBS control 3 times/wk for 30 days. Mice were killed, and maxillae were assessed by microcomputed tomography, histological analysis, and TRAP staining for measurement of bone loss, extent of inflammation, and degree of osteoclastogenesis. Results indicated that, in LPS-injected Mkp-1(-/-) mice, significantly greater bone loss occurred with more inflammatory infiltrate and a significant increase in osteoclastogenesis compared with Mkp-1(-/-) control sites or either wild-type group. Analysis of these data indicates that MKP-1 plays a key role in the regulation of inflammatory bone loss.

Lipopolysaccharide-induced epithelial monoamine oxidase mediates alveolar bone loss in a rat chronic wound model.

Reactive oxygen species (ROS) production is an antimicrobial response to pathogenic challenge that may, in the case of persistent infection, have deleterious effects on the tissue of origin. A rat periodontal disease model was used to study ROS-induced chronic epithelial inflammation and bone loss. Lipopolysaccharide (LPS) was applied for 8 weeks into the gingival sulcus, and histological analysis confirmed the onset of chronic disease. Junctional epithelium was collected from healthy and diseased animals using laser-capture microdissection, and expression microarray analysis was performed. Of 19,730 genes changed in disease, 42 were up-regulated >/=4-fold. Three of the top 10 LPS-induced genes, monoamine oxidase B (MAO/B) and flavin-containing monooxygenase 1 and 2, are implicated in ROS signaling. LPS-associated induction of the ROS mediator H(2)O(2), as well as MAO/B and tumor necrosis factor (TNF)-alpha levels were validated in the rat histological sections and a porcine junctional epithelial cell culture model. Topical MAO inhibitors significantly counteracted LPS-associated elevation of H(2)O(2) production and TNF-alpha expression in vivo and in vitro, inhibited disease-associated apical migration and proliferation of junctional epithelium and inhibited induced systemic H(2)O(2) levels and alveolar bone loss in vivo. These results suggest that LPS induces chronic wounds via elevated MAO/B-mediated increases in H(2)O(2) and TNF-alpha activity by epithelial cells and is further associated with more distant effects on systemic oxidative stress and alveolar bone loss.

Vascular endothelial growth factor and e-nitric oxide synthase-mediated regenerative response occurring upon autologous and heterologous bone grafts.

Bone regeneration procedures allow oral rehabilitation with dental implants also in edentulous ridges with severe bone atrophy. The integration of grafted materials with the host tissue can initiate regenerative, inflammatory and apoptotic response. Since molecular mechanisms exist at the basis of such response, the aim of this work is to investigate, by immunohistochemical analyses, the expression of proteins involved in the graft integration process, in parallel to clinical and histological modifications, occurring on sites treated with extraoral autologous bone graft deriving from the parietal region of the calvaria (eAB), intraoral autologous bone graft deriving from mandibular ramus (iAB) and heterologous bone graft from swine (hB) in human patients. In our study, the immunohistochemical expression of BSP, VEGF, eNOS in eAB samples was significantly higher (p < 0.05) compared to values recorded in iAB and hB samples. The inflammatory response, investigated by iNOS expression, was found lower in all autologous samples (eAB and iAB) compared to hB, at statistically significant values. Moreover, the expression of the pro-apoptotic molecule, Bax, resulted significantly lower (p < 0.05) in eAB than in iAB and hB samples. These values, together with the low number of apoptotic cells detected in autologous samples, suggest a good regenerative response when extraoral autologous bone graft is used in comparison to the response from the other grafts, and also suggest the use of calvaria graft as a predictable therapeutic procedure for repairing severe bone defects in oral and maxillofacial surgery, not only by clinical and biomechanical criteria, but also from a biomolecular aspect.

iNOS-derived nitric oxide modulates infection-stimulated bone loss.

Nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) plays an important role in host defense, as well as in inflammation-induced tissue lesions. Here we evaluated the role of NO in bone loss in bacterial infection-induced apical periodontitis by using iNOS-deficient mice (iNOS(-/-)). The iNOS(-/-) mice developed greater inflammatory cell recruitment and osteolytic lesions than WT mice. Moreover, tartrate-resistant acid-phosphatase-positive (TRAP(+)) osteoclasts were significantly more numerous in iNOS(-/-) mice. Furthermore, the increased bone resorption in iNOS(-/-) mice also correlated with the increased expression of receptor activator NF-kappaB (RANK), stromal-cell-derived factor-1 alpha (SDF-1 alpha/CXCL12), and reduced expression of osteoprotegerin (OPG). These results show that NO deficiency was associated with an imbalance of bone-resorption-modulating factors, leading to severe infection-stimulated bone loss.

The expression of macrophage and neutrophil elastases in rat periradicular lesions.

Macrophage elastase and neutrophil elastase are involved in tissue destruction in periradicular lesions. The purpose of this study was to examine these elastases immunohistochemically during development of periradicular lesions induced in rat mandibular first molar by pulpal exposure for 7, 14, 21, 28, and 42 days. Histologically, periapical inflammation developed from 7 to 21 days and then subsided after 28 days. The area of these lesions gradually increased from 7 to 28 days and subsequently decreased at 42 days. Macrophage elastase was first detected at 7 days and predominantly shown from 14 to 28 days, whereas neutrophil elastase gradually increased from 14 to 28 days. Macrophage elastase was significantly greater than neutrophil elastase from 7 to 21 days. These results suggest that macrophage elastase was enhanced from an early stage during the development of these lesions and that neutrophil elastase was related to the expansion of periapical tissue destruction including bone resorption.

Subantimicrobial-dose doxycycline modulates gingival crevicular fluid biomarkers of periodontitis in postmenopausal osteopenic women.

BACKGROUND: We recently demonstrated that a 2-year subantimicrobial-dose doxycycline (SDD) regimen (double-masked, placebo-controlled clinical trial) in postmenopausal (PM) women exhibiting mild systemic bone loss (osteopenia) and local bone loss (periodontitis) reduced the progression of periodontal attachment loss (intent-to-treat analysis) and the severity of gingival inflammation and alveolar bone loss (subgroups) without producing antibiotic side effects. We now describe SDD effects on biomarkers of collagen degradation and bone resorption in the gingival crevicular fluid (GCF) of the same vulnerable subjects. METHODS: GCF was collected from SDD- and placebo-treated PM subjects (n=64 each) at the baseline and 1- and 2-year appointments; the volume was determined; and the samples were analyzed for collagenase activity (using a synthetic peptide as substrate), relative levels of three genetically distinct collagenases (Western blot), a type-1 collagen breakdown product/bone resorption marker (a carboxyterminal telopeptide cross-link fragment of type I collagen [ICTP]; radioimmunoassay), and interleukin-1beta (enzyme-linked immunosorbent assay). Statistical analyses were performed using generalized estimating equations; primary analyses were intent-to-treat. RESULTS: Collagenase activity was significantly reduced by SDD treatment relative to placebo based on intent-to-treat (P=0.01). ICTP showed a similar pattern of change during SDD treatment, and GCF collagenase activity and ICTP were positively correlated at all time periods (P<0.001). Matrix metalloproteinase (MMP)-8 accounted for approximately 80% of total collagenase in GCF, with much less MMP-1 and -13, and SDD reduced the odds of elevated MMP-8 by 60% compared to placebo (P=0.006). CONCLUSION: These observations support the therapeutic potential of long-term SDD therapy to reduce periodontal collagen breakdown and alveolar bone resorption in PM women; effects on serum biomarkers of systemic bone loss in these subjects are being analyzed.

Tumor necrosis factor-alpha-converting enzyme (TACE) levels in periodontal diseases.

Tumor necrosis factor-alpha-converting enzyme (TACE) is a metalloprotease which can shed several cytokines from the cell membrane, including receptor activator of NF-kappaB ligand (RANKL). This study aimed to investigate the hypothesis that TACE would be elevated in the gingival crevicular fluid (GCF) of persons with periodontitis. Total TACE amounts in GCF were higher in persons with chronic and aggressive periodontitis than in those with gingivitis or in healthy persons. TACE concentrations in GCF were higher in persons with chronic and aggressive periodontitis than in those with gingivitis, although not significantly higher than in healthy persons. Persons with chronic periodontitis receiving immunosuppressive treatment exhibited over 10-fold lower TACE levels than the other periodontitis groups. TACE was positively correlated with probing pocket depth, clinical attachment levels, and RANKL concentrations in GCF. In conclusion, the increased GCF TACE levels in persons with periodontitis and their positive correlation with RANKL may indicate an association of this enzyme with alveolar bone loss, and may warrant special attention in future therapeutic approaches.

Hyper-reactive PMNs in FcgammaRIIa 131 H/H genotype periodontitis patients.

BACKGROUND: Receptors for the Fc part of IgG (FcgammaRIIa) on polymorphonuclear leukocytes (PMN) mediate phagocytosis and cell activation. Previous results show that one of the genetic variants of the FcgammaRIIa, the 131 H/H, is associated with more periodontal breakdown than the R/R. This may be due to hyper-reactivity of the H/H-PMNs upon interaction with bacteria. AIM: To study whether the FcgammaRIIa genotype modifies the PMN reactivity in periodontitis patients. MATERIAL AND METHODS: A cohort of 98 periodontitis patients was genotyped. From these, 10 H/H and 10 R/R consented to participate. PMNs were incubated with immune serum-opsonized Actinobacillus actinomycetemcomitans (A.a.). Phagocytosis, degranulation (CD63 and CD66b expression), respiratory burst and elastase release were assessed. RESULTS: Patients of the H/H genotype showed more bone loss than those with the H/R or R/R genotype (p=0.038). H/H-PMNs phagocytosed more opsonized A.a. than did R/R-PMNs (p=0.019). The H/H-PMNs also expressed more CD63 and CD66b than did the R/R-PMNs (p=0.004 and 0.002, respectively) and released more elastase (p=0.001). CONCLUSIONS: The genotyping results confirm previous reports that more periodontal destruction occurs in the H/H genotype than in the H/R or R/R genotype. The functional studies indicate a hyper-reactivity of the H/H-PMN in response to bacteria, which may be one of several pathways leading to more periodontal breakdown.