Toxicological effect of ivermectin on the survival, reproduction, and feeding activity of four species of dung beetles (Coleoptera: Scarabaeidae and Geotrupidae) in Japan.

We investigated the effects of the antiparasitic drug ivermectin on the dung beetles Copris acutidens Motschulsky, Onthophagus bivertex Heyden, O. lenzii Harold and Phelotrupes auratus auratus Motschulsky in Japan. Ivermectin was detected in cattle dung from 1 to 3 or 7 days post-treatment, with a peak at 3 days post-treatment in two pour-on administrations (500 µg kg-1). In C. acutidens, adult survivals and numbers of brood balls were significantly reduced in dung collected at 3 and 7 days post-treatment, and adult emergence rates were significantly decreased in dung collected at 7 and 14 days post-treatment. Feeding activity of C. acutidens was inhibited in dung collected at 3 days post-treatment, but was not significantly different from that seen in control dung at 7 and 14 days post-treatment. In O. bivertex and O. lenzii, there were no effects of ivermectin on adult survival or feeding activities, but the numbers of brood balls of O. bivertex constructed in dung collected at 3 and 7 days post-treatment were significantly lower than observed with control dung. The adult emergence rates of O. bivertex and O. lenzii were significantly reduced in dung collected at 1 to 3 and 1 to 7 days post-treatment, respectively. In P. auratus, there were no effects of ivermectin on adult survival, oviposition, feeding activity, or larval survival (until the third instar) in dung at 3 days post-treatment. The environmental risks affecting the populations of dung beetles in Japan are discussed.

Effects of eprinomectin on the survival, reproduction and feeding activity of the dung beetles, Onthophagus lenzii Harold, and rare species, Copris ochus Motschulsky (Coleoptera: Scarabaeidae).

Effects of the antiparasitic drug eprinomectin were studied on the dung beetles, Onthophagus lenzii Harold and the rare species, Copris ochus Motschulsky by pour-on administrations (500 µg kg-1) in Japan. Eprinomectin was detected in cattle dung from 1 to 7 or 14 days after treatment, with a peak at 1 day after treatment in two experiments. In O. lenzii, adult survivals and numbers of brood balls constructed were significantly reduced in dung from eprinomectin-treated cattle at 1 and 3 days post-treatment in experiment 1, and adult emergence rates were extremely reduced in dung at 1, 3, and 7 days post-treatment. In C. ochus, adult survivals were significantly reduced in dung at 3 days post-treatment (experiment 1), and equivalent levels to the control were restored in dung at 7 and 14 days post-treatment (experiment 2). Numbers of brood balls of C. ochus were nil in dung at 3 days (experiment 1), and significantly reduced in dung at 7 days (experiment 2) post-treatment compared with control. Adult emergence rates of C. ochus were 100 and 71.6% in dung from control cattle in experiments 1 and 2, respectively. However, no oviposition was observed in dung at 3 days post-treatment, and all offspring died at egg or the first instar larval stage in dung from 7 and 14 days post-treatment. Feeding activities of O. lenzii and C. ochus were significantly inhibited in dung from treated cattle at 1-3 days and 3 days post-treatment, respectively, returning to levels of the control at 7 days post-treatment.

Histological evaluation of alveolar ridge augmentation using injectable calcium phosphate bone cement in dogs.

Alveolar ridge augmentation is an important procedure to restore tooth loss. Several types of graft materials have been used for augmenting the alveolar ridge. An injectable calcium phosphate cement (CPC) has been applied to periodontal bone defects and has shown favourable results. Thus, this CPC may work as an effective graft material for alveolar ridge augmentation. The aim of this study was to evaluate the effectiveness of the CPC for large-scaled (about 7 x 8 x 6 mm) ridge augmentation in dogs. Alveolar ridge defects were created bilaterally in the maxilla of six beagle dogs. The CPC was applied to one of the bilateral maxillary defects. The untreated defect on the contralateral side served as control. The animals were sacrificed at 6 months after surgery and decalcified histological specimens of the alveolar ridge were prepared histometrically and evaluated under a light microscope. Newly formed and reconstructed alveolar ridges covering the CPC were observed in all experimental sites. In the control sites, only slight newly bone formation was observed. Histomorphometrical analysis indicated that the CPC grafted group exhibited significantly (P = 0.0001) increased area and height in new bone formation compared with those of the control group. The results indicate that the CPC appears to be an effective material for alveolar ridge augmentation and may act as a space maintainer to conduct new bone formation.

Regulatory roles of beta-catenin and AP-1 on osteoprotegerin production in interleukin-1alpha-stimulated periodontal ligament cells.

BACKGROUND: Periodontitis is a chronic inflammatory disease characterized by the enhanced expression of inflammatory mediators leading to alveolar bone resorption. Osteoprotegerin (OPG) plays a suppressive role in cytokine-induced osteoclastogenesis. In osteoblasts, OPG expression is upregulated by beta-catenin but downregulated by the transcription factor activator protein-1 (AP-1; c-fos/c-jun). The purpose of this study was to examine the roles of beta-catenin and AP-1 in interleukin-1alpha (IL-1alpha) -induced OPG production in human gingival fibroblasts (hGFs) and periodontal ligament (PDL) cells. METHODS: Expression of c-fos and c-jun messenger RNA was measured by reverse transcription-polymerase chain reaction and OPG production was analysed by enzyme-linked immunosorbent assay. The nuclear AP-1 activity was quantified using an AP-1 microplate assay. The effect of the Wnt canonical pathway on OPG production was evaluated using small interfering (si) RNA for beta-catenin and the effect of AP-1 on OPG production was evaluated using the AP-1 inhibitor curcumin. RESULTS: Levels of c-fos messenger RNA and nuclear AP-1 activity were higher in PDL cells than in hGFs. When stimulated with IL-1alpha, PDL cells had significantly higher c-fos expression and lower OPG production compared with hGFs. The siRNA for beta-catenin suppressed the IL-1alpha-induced OPG production in both PDL cells and hGFs, whereas the AP-1 inhibitor curcumin augmented the IL-1alpha-induced OPG production in PDL cells, but not in hGFs. CONCLUSION: The present study suggests that beta-catenin enhances IL-1alpha-induced OPG production in both PDL cells and hGFs, whereas AP-1 suppresses IL-1alpha-induced OPG production in PDL cells. Higher expression of c-fos in PDL cells than in hGFs may implicate a role of PDL cells in alveolar bone resorption 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.

Red bacterial complex is associated with the severity of chronic periodontitis in a Thai population.

BACKGROUND: The distribution of periodontal pathogens differs in various geographic locations and racial/ethnic groups. This study investigated the microbiological features of chronic periodontitis (CP) patients in Thailand. METHODS: Subgingival plaque samples from 20 non-periodontitis subjects, 20 patients with mild CP, and 20 patients with moderate to severe CP were examined using polymerase chain reaction (PCR) to identify Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Aggregatibacter actinomycetemcomitans. RESULTS: In the moderate to severe CP patients, there was high prevalence of P. gingivalis (95%), T. forsythia (95%), T. denticola (80%), as well as the red complex (coexistence of all three species at the same lesion) (75%). A. actinomycetemcomitans was detected in only 35% of the patients in this study group. P. gingivalis was detected in as high as 45% of the non-periodontitis controls. CP and disease severity were significantly related to the presence of T. forsythia together with T. denticola and the red complex. The red complex was not found in any non-periodontitis site. CONCLUSION: Red complex bacteria were predominant periodontal pathogens of the moderate to severe form of CP in this Thai population. The presence of T. forsythia together with T. denticola, and the red complex species at the same site were significantly associated with the disease severity.

Cyclooxygenase-2-derived prostaglandin E2 is involved in vascular endothelial growth factor production in interleukin-1alpha-stimulated human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Prostaglandin E(2), which exerts its actions via EP receptors (EP1, EP2, EP3 and EP4), is a bioactive metabolite of arachidonic acid produced by cyclooxygenase-1 and/or cyclooxygenase-2. Interleukin-1alpha induces prostaglandin E(2) production via cyclooxygenase-2 in human periodontal ligament cells. Vascular endothelial growth factor is a key regulator of physiologic as well as pathologic angiogenesis and has been indicated to be involved in the pathology of periodontal diseases. In the present study, we investigated whether interleukin-1alpha induced vascular endothelial growth factor production in human periodontal ligament cells and whether cyclooxygenase-2-derived prostaglandin E(2) regulated interleukin-1alpha-induced vascular endothelial growth factor production. MATERIAL AND METHODS: Human periodontal ligament cells were obtained from extracted teeth of periodontally healthy subjects. After pre-incubation with a nonselective cyclooxygenase-1/2 inhibitor, indomethacin or a selective cyclooxygenase-2 inhibitor (NS-398), periodontal ligament cells were treated with or without interleukin-1alpha, prostaglandin E(2), various EP receptor agonists and dibutyryl cAMP (a cAMP analogue). The levels of vascular endothelial growth factor and prostaglandin E(2) in the culture supernatant were measured by enzyme-linked immunosorbent assay. The vascular endothelial growth factor mRNA expression was evaluated by semiquantitative reverse transcription-polymerase chain reaction. RESULTS: Interleukin-1alpha induced vascular endothelial growth factor production in a dose-dependent and time-dependent manner. The interleukin-1alpha-induced vascular endothelial growth factor mRNA and protein expression was inhibited to the same extent by indomethacin and NS-398. Indomethacin and NS-398 completely inhibited interleukin-1alpha-induced prostaglandin E(2) production. Exogenous prostaglandin E(2), butaprost (an EP2 receptor agonist) and dibutyryl cAMP abolished the inhibitory effect of indomethacin on interleukin-1alpha-induced vascular endothelial growth factor production. CONCLUSION: We suggest that interleukin-1alpha induced vascular endothelial growth factor production via cyclooxygenase-2-derived prostaglandin E(2) in human periodontal ligament cells. The interleukin-1alpha/prostaglandin E(2) pathway might regulate vascular endothelial growth factor production in periodontal lesions.

Multi-center intervention study on glycohemoglobin (HbA1c) and serum, high-sensitivity CRP (hs-CRP) after local anti-infectious periodontal treatment in type 2 diabetic patients with periodontal disease.

The purpose of this study was to examine whether periodontal treatment incorporating topical antibiotic therapy affects on levels of glycohemoglobin (HbA1c) and serum high-sensitivity C-reactive protein (hs-CRP) in type 2 diabetic patients with periodontal disease, and to explore the relationship between CRP and glycemic control. The whole intervention group (n=32), which underwent anti-infectious periodontal treatment, showed only transient reduction in HbA1c levels without any change in hs-CRP, while the control group (n=17) did not show any changes in HbA1c or hs-CRP. Multiple regression analysis of all subjects revealed that BMI and change in hs-CRP correlated significantly with the reduction of HbA1c at 6 months after the periodontal treatment. Based on the results of multiple regression analysis, the intervention group was subdivided into two groups: those in which hs-CRP levels decreased (CRP-D group), and those in which hs-CRP levels unchanged or increased (CRP-N group) (n=16, respectively), and re-analysis was conducted based upon these subgroups. In the CRP-D subgroup, HbA1c was significantly reduced at the end of the study, but it did not decrease in the CRP-N subgroup. The decrease of HbA1c in the CRP-D subgroup following periodontal treatment was significantly greater than that in the CRP-N subgroup. BMI of each group remained unchanged in this study at the end of the study. Thus, the results suggested that periodontal treatment with topical antibiotics improves HbA1c through reduction of CRP, which may relate to amelioration of insulin resistance, in type 2 diabetic patients with periodontal disease.

IL-6 induces osteoblastic differentiation of periodontal ligament cells.

Interleukin (IL)-6 has been considered as an osteolytic factor involved in periodontal disease. However, the function of IL-6 in osteoblastic differentiation of periodontal ligament cells is not clear. We examined the effects of IL-6 and its soluble receptor (sIL-6R) on osteoblastic differentiation of periodontal ligament cells. Osteoblastic differentiation was induced by ascorbic acid. Osteoblast markers, including alkaline phosphatase activity and Runx2 gene expression, were examined. The mechanism of action of IL-6 on osteoblastic differentiation was evaluated by insulin-like growth factor (IGF)-I production and specific inhibitors for the IL-6-signaling molecule. IL-6/sIL-6R enhanced alkaline phosphatase activity and Runx2. Alkaline phosphatase activity was reduced by anti-IGF-I antibody. Mitogen-activated protein kinase and Janus protein tyrosine kinase inhibitors diminished alkaline phosphatase induced by IL-6/sIL-6R. We conclude that IL-6/sIL-6R increases ascorbic-acid-induced alkaline phosphatase activity through IGF-I production, implying that IL-6 acts not only as an osteolytic factor, but also as a mediator of osteoblastic differentiation in periodontal ligament cells.

Effect of rhBMP-2 with PLGA/gelatin sponge type (PGS) carrier on alveolar ridge augmentation in dogs.

Recombinant human bone morphogenetic protein-2 has been shown to promote bone formation because of its osteoinductive property. The purpose of this study was to evaluate the efficacy of rhBMP-2 delivered on a poly (D, L-lactic-co-glycolic acid) copolymer/gelatin sponge (PGS) in vertical alveolar ridge augmentation on height-reduced edentulous mandible to verify the retention of rhBMP-2 withstanding the pressure of soft tissues. Coronal defects of the alveolar bone were created in six adult beagle dogs. After a healing period of 9 weeks, PGSs with or without rhBMP-2 (0 or 0.4 mg mL(-1)) were implanted on the defects(6 mm in height, 30 mm in length, 8 mm in width). Sixteen weeks after implantation, the bone mineral content (BMC) and the total bone area were measured by peripheral quantitative computed tomography. The BMC and the total bone area of the defect sites with rhBMP-2 group were significantly greater (133+/-33 mg mm(-1), 277+/-54 mm2, respectively) than those of the control group (80+/-19 mg mm(-1), 155+/-49 mm2, respectively) (P<0.01, P<0.0001, respectively; paired t-test). From the histological analyses, the height of newly formed bone in the experimental group was greater than that of the control group (4.3+/-0.9 mm, 0.22+/-0.28 mm, P<0.0001, n=6, paired t-test). These results indicate that PGS has characteristics of effective bone graft substitutes for implantation of rhBMP-2 on vertical alveolar ridge augmentation in huge defect of mandibles in dogs.

Microbial changes in patients with acute periodontal abscess after treatment detected by PadoTest.

AIMS: To investigate changes in bacterial counts in subgingival plaque from patients with acute periodontal abscess by IAI-PadoTest. MATERIALS AND METHODS: Ninety-one patients were randomly allocated to either test or control groups. In all the patients, pockets with acute periodontal abscess were irrigated with sterilized physiological saline, and in the test group, 2% minocycline hydrochloride ointment was applied once into the pocket in addition. Subgingival plaque samples were collected by paper point before treatment and 7 days after treatment. The total bacterial count was determined and Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia, were detected using IAI-PadoTest, a DNA/RNA probe method. RESULTS: The total bacterial count decreased in both groups, with a significant decrease in the test group. The counts and number of sites positive for P. gingivalis, T. forsythia and T. denticola significantly decreased in the test group after treatment, compared with those in the control group. Pocket depth decreased in the both groups, with a statistically significant decrease in the test group. CONCLUSION: Topical treatment with minocycline in pockets with acute periodontal abscess was effective in reducing the bacterial counts as shown by the microbiological investigation using PadoTest 4.5.

Cementum-periodontal ligament complex regeneration using the cell sheet technique.

BACKGROUND AND OBJECTIVE: In the present study we evaluated if a multilayered human periodontal ligament cell sheet could reconstruct the physiological architecture of a periodontal ligament-cementum complex. MATERIAL AND METHODS: Human periodontal ligament cells were isolated and then cultured in dishes coated with a temperature-responsive polymer to allow cell detachment as a cell sheet. In the control group, human periodontal ligament cells were cultured in Dulbecco's modified Eagle's minimal essential medium containing 10% fetal bovine serum and 1% antibiotics. In the experimental group, human periodontal ligament cells were cultured in Dulbecco's modified Eagle's minimal essential medium and osteodifferentiation medium containing dexamethasone, ascorbic acid and beta-glycerophosphate. After 3 wk, scanning electron microscopy was carried out, in addition to staining for alkaline phosphatase activity and for calcium (using the Von Kossa stain). Then human periodontal ligament cell sheets were multilayered and placed onto dentin blocks. The constructs were transplanted subcutaneously into the back of immunodeficient rats. At 1 and 6 wk after transplantation, the animals were killed. Demineralized tissue sections were stained using hematoxylin and eosin, and Azan, and then analyzed. RESULTS: After 3 wk of culture in osteodifferentiation medium, human periodontal ligament cells produced mineral-like nodules and also showed positive staining for alkaline phosphatase, calcium (Von Kossa) and mRNA expression of type I collagen. By contrast, in the control group only weak alkaline phosphatase staining was observed, the Von Kossa stain was negative and there was no mRNA expression of type I collagen. Six weeks after transplantation with human periodontal ligament cells cultured in osteodifferentiation medium, most of the dentin surfaces showed a newly immature cementum-like tissue formation and periodontal ligament with perpendicular orientation inserted into the newly deposited cementum-like tissue. CONCLUSION: This study suggests that the multilayered temperature-responsive culture system can be used as a novel strategy for periodontal regeneration. The human periodontal ligament cell sheet technique may be applicable for regeneration of the clinical periodontal ligament-cementum complex.

Periodontitis may increase the risk of peripheral arterial disease.

OBJECTIVES: The aim of this case control study was to evaluate whether periodontitis was associated with peripheral arterial disease (PAD). SUBJECTS AND METHODS: Twenty-five patients diagnosed with aorto-iliac and/or femoro-popliteal occlusive disease and thirty-two generally healthy control subjects were enrolled in this study. Polymerase chain reaction (PCR) was used to identify Porphyromonas gingivalis, Treponema denticola, Actinobacillus actinomycetemcomitans, Prevotella intermedia, Cytomegalovirus (CMV), Chlamydia pneumoniae, and Helicobacter pylori in tissue specimens taken from the anastomotic site of distal bypasses. Periodontal status was evaluated; serum IgG titres against the four listed bacteria were measured. RESULTS: Periodontopathic bacteria were detected in 13/25 (52%) atherosclerotic specimens. CMV or C. pneumoniae was detected in 1/25 (4%) specimens; H. pylori was not detected from any of these specimens. Fontaine grade III or IV patients showed higher detection frequency of P. gingivalis than Fontaine grade II patients (57.1% vs 22.2%, P=0.09). After adjusting for age, gender, diabetes and smoking, periodontitis increased 5-fold the risk of having PAD (OR 5.45). There were preliminary indications that periodontitis was associated with increased serum IL-6 and TNF-alpha concentrations. CONCLUSIONS: This study suggests that periodontitis may be associated with an increased risk of PAD. This association could result from the increased concentration of serum inflammatory cytokines in those with periodontitis.

Osteogenic effect of interleukin-11 and synergism with ascorbic acid in human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Human periodontal ligament cells are considered to be a key cell type in the regeneration of periodontal tissues because of their unique localization and stem cell-like properties. Interleukin-11 is a multifunctional cytokine known to participate actively in bone metabolism. The purpose of this study was to examine the effect of interleukin-11 on the osteoblastic differentiation of periodontal ligament cells. MATERIAL AND METHODS: Cultured periodontal ligament cells were stimulated with interleukin-11 and/or ascorbic acid, with or without inhibitors for type 1 collagen, janus kinase/signal transducers and activator of transcription, and mitogen-activated protein kinase (MAPK). Osteoblastic differentiation was investigated by examining the alkaline phosphatase activity and gene expression of Runx2, osteocalcin and bone sialoprotein using reverse transcription-polymerase chain reaction. Type 1 collagen and tissue inhibitor of metalloproteinase-1 production were measured using enzyme-linked immunosorbent assays. RESULTS: Interleukin-11 enhanced alkaline phosphatase activity and Runx2, osteocalcin and bone sialoprotein gene expression in the presence of ascorbic acid. Interleukin-11 induced type 1 collagen and tissue inhibitor of metalloproteinase-1 production in periodontal ligament cells. Type 1 collagen inhibitor completely inhibited the alkaline phosphatase activity enhanced by interleukin-11 and ascorbic acid. Furthermore, janus kinase/signal transducers and activator of transcription and MAPK signaling inhibitors reduced interleukin-11/ascorbic acid-induced alkaline phosphatase activity in periodontal ligament cells. CONCLUSION: Interleukin-11/ascorbic acid induced the osteoblastic differentiation of periodontal ligament cells through type 1 collagen production and janus kinase/signal transducers and activator of transcription, and MAPK signaling pathways were involved in this process. These findings suggest that interleukin-11 may function as an osteopromotive cytokine, stimulating the osteoblastic differentiation of periodontal ligament cells mainly through the synthesis of type 1 collagen and possibly by the induction of tissue inhibitor of metalloproteinase-1.

Internal prostaglandin synthesis augments osteoprotegerin production in human gingival fibroblasts stimulated by lipopolysaccharide.

Periodontitis is an inflammatory bone disease caused by Gram-negative anaerobic bacteria. Osteoclast differentiation is regulated by the balance between receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG). The purpose of this study was to examine the mechanism of OPG production in human gingival fibroblasts (HGF) stimulated by lipopolysaccharide (LPS) from periodontopathic bacteria. The expressions of Toll-like receptor 2 (TLR-2) and TLR-4 in HGF were examined using flow-cytometry. HGF were stimulated with whole cell extracts or LPS from Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis with or without polymyxin B, a LPS inhibitor. In addition, HGF were stimulated with LPS, prostaglandin E(2) (PGE(2)), various agonists of PGE receptors (EP1, EP2, EP3 and EP4 agonists) with or without indomethacin (IND), a prostaglandin synthesis inhibitor. OPG and PGE(2) production was measured using an enzyme-linked immunosorbent assay (ELISA). HGF expressed both TLR-2 and TLR-4. Both A. actinomycetemcomitans and P. gingivalis LPS augmented OPG expression in HGF. Whole cell extracts from A. actinomycetemcomitans and P. gingivalis augmented OPG production by HGF; the augmentation was suppressed by polymyxin B. IND suppressed OPG production in LPS-stimulated HGF. PGE(2) stimulated HGF to produce OPG. EP1 and EP2 agonists, but not EP3 and EP4 agonists, increased OPG production by HGF. These results suggest that LPS-induced OPG production by HGF is regulated via EP1 and/or EP2 receptors by endogenously generated PGE(2).

Identification of periodontopathic bacteria in gingival tissue of Japanese periodontitis patients.

INTRODUCTION: The identification of invading periodontopathic bacteria in tissues is important to determine their role in the pathogenesis of periodontal disease. The objective of this study was to identify periodontopathic bacteria in diseased gingival tissue of periodontitis patients. METHODS: Subgingival plaque and gingival tissue were collected from 32 generalized chronic periodontitis (CP), 16 generalized aggressive periodontitis (GAgP) and eight localized aggressive periodontitis (LAgP) patients. Detection frequencies and quantities of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Tannerella forsythensis were investigated by polymerase chain reaction. The prevalences of Streptococcus oralis and Streptococcus sobrinus were also examined and the distribution of A. actinomycetemcomitans serotypes was observed. RESULTS: P. gingivalis and T. forsythensis were detected in approximately 70% of tissue samples and 50% of plaque samples in the three periodontitis groups. Prevalence of A. actinomycetemcomitans in tissue samples was higher in the LAgP (63%) group than in either the CP (16%) or the GAgP (38%) group. A. actinomycetemcomitans serotype c was detected in 50% of LAgP patients. Detection frequencies of S. oralis and S. sobrinus were markedly low in both plaque and tissue samples from all three periodontitis groups. Amounts of P. gingivalis, A. actinomycetemcomitans and T. forsythensis in the tissue samples were not different among the three periodontitis groups. CONCLUSION: P. gingivalis, A. actinomycetemcomitans and T. forsythensis can localize in diseased gingival tissue and may be involved in periodontal tissue destruction. Serotype c is the predominant serotype of A. actinomycetemcomitans in Japanese LAgP patients.

Oral bacteria are a possible risk factor for valvular incompetence in primary varicose veins.

OBJECTIVES: To investigate a possible link between valvular incompetence in primary varicose veins and chronic infection of periodontal disease by assessing the presence of oral bacteria in the great saphenous vein from patients with varicose veins and control subjects. MATERIAL AND METHODS: Forty-four primary varicose vein patients were enrolled in the study. 12 control saphenous veins were obtained from patients undergoing peripheral arterial bypass without clinical evidence of venous reflux. In total, 56 saphenous vein specimen (44 varicose veins and 12 control veins) were examined for 7 periodontal bacteria using a polymerase chain reaction (PCR) method. RESULTS: Of the 44 primary varicose vein patients, 31 patients were women and mean age was 59 years (range, 39-79 years). PCR examination of the diseased vein specimens showed that 48% were positive for at least one of 7 periodontal bacterial DNA. No bacteria were detected in the control specimens. CONCLUSION: Bacterial colonisation or infection of varicose veins is a frequent event although we were not able to establish whether this is a cause or consequence of the development of varices but this could be considered a risk factor for the development of varices.

Processing of ameloblastin by MMP-20.

Ameloblastin (AMBN) cleavage products are the most abundant non-amelogenin proteins in the enamel matrix of developing teeth. AMBN N-terminal cleavage products accumulate in the sheath space between enamel rods, while AMBN C-terminal products localize within rods. We tested the hypothesis that MMP-20 is the protease that cleaves AMBN. Glycosylated recombinant porcine AMBN (rpAMBN) was expressed in human kidney 293F cells, and recombinant porcine enamelysin (rpMMP-20) was expressed in bacteria. The purified proteins were incubated together at an enzyme:substrate ratio of 1:100. N-terminal sequencing of AMBN digestion products determined that rpMMP-20 cleaved rpAMBN after Pro(2), Gln(130), Gln(139), Arg(170), and Ala(222). This shows that MMP-20 generates the 23-kDa AMBN starting at Tyr(223), as well as the 17-kDa (Val(1)-Arg(170)) and 15-kDa (Val(1)-Gln(130)) AMBN cleavage products that concentrate in the sheath space during the secretory stage. We conclude that MMP-20 processes ameloblastin in vitro and in vivo.

A time-dependent effect of PDGF-BB on adhesion and growth of cultured fibroblasts to root surfaces.

OBJECTIVE: Platelet-derived growth factor (PDGF-BB) is suggested to be a potent stimulator and a strong mitogenic agent for human periodontal ligament cells (PDL). This study aimed at assessing the effectiveness of PDGF-BB application on periodontally diseased root surfaces through attachment and growth of fibroblast cells. MATERIALS AND METHODS: Fifteen periodontally involved and five healthy teeth were selected, prepared from proximal surfaces and distributed into four groups (10 specimens per group): I: healthy; II: untreated diseased; III: scaling and root planning (SRP); and IV: SRP and PDGF-BB. Each group had three subdivisions (three specimens per group) which were incubated at three different time periods. The remaining specimen for each group was used to examine surface topography. Fibroblasts were pooled on root specimens and incubated. Results were evaluated by using scanning electron microscopy. Repeated cell counting was done within a representative standard area. RESULTS: The best results regarding PDL cell shape and density were obtained at day 3 in all experimental groups, except the diseased group. Although SRP samples showed slightly higher results in numbers of attached fibroblasts than diseased samples, they demonstrated a similar negative effect denoting incompatible root surfaces for fibroblast attachment. SRP plus PDGF-BB and healthy samples showed a comparable positive effect, suggesting a good root surface biocompatibility. Inter-group differences showed no significant differences on day 1, but statistically significant differences were found on both day 3 and day 7 incubation periods favoring groups I and IV over groups II and III. CONCLUSIONS: Platelet-derived growth factor showed a positive effect on adhesion and growth of cultured fibroblasts to periodontally diseased surfaces. Thus, PDGF-BB may have a promising role in clinical periodontics.