Randomized Placebo-Controlled and Controlled Non-Inferiority Phase III Trials Comparing Trafermin, a Recombinant Human Fibroblast Growth Factor 2, and Enamel Matrix Derivative in Periodontal Regeneration in Intrabony Defects.

We investigated the efficacy, safety, and clinical significance of trafermin, a recombinant human fibroblast growth factor (rhFGF)-2, for periodontal regeneration in intrabony defects in Phase III trials. Study A, a multicenter, randomized, double-blind, placebo-controlled study, was conducted at 24 centers. Patients with periodontitis with 4-mm and 3-mm or deeper probing pocket depth and intrabony defects, respectively, were included. A total of 328 patients were randomly assigned (2:1) to receive 0.3% rhFGF-2 or placebo, and 323 patients received the assigned investigational drug during flap surgery. One of the co-primary endpoints, the percentage of bone fill at 36 weeks after drug administration, was significantly greater in the rhFGF-2 group at 37.131% (95% confidence interval [CI], 32.7502 to 41.5123; n = 208) than it was in the placebo group at 21.579% (95% CI, 16.3571 to 26.8011; n = 100; p < 0.001). The other endpoint, the clinical attachment level regained at 36 weeks, was not significantly different between groups. Study B, a multicenter, randomized, blinded (patients and evaluators of radiographs), and active-controlled study was conducted at 15 centers to clarify the clinical significance of rhFGF-2. Patients with 6-mm and 4-mm or deeper probing pocket depth and intrabony defects, respectively, were included. A total of 274 patients were randomly assigned (5:5:2) to receive rhFGF-2, enamel matrix derivative (EMD), or flap surgery alone. A total of 267 patients received the assigned treatment during flap surgery. The primary endpoint, the linear alveolar bone growth at 36 weeks, was 1.927 mm (95% CI, 1.6615 to 2.1920; n = 108) in the rhFGF-2 group and 1.359 mm (95% CI, 1.0683 to 1.6495; n = 109) in the EMD group, showing non-inferiority (a prespecified margin of 0.3 mm) and superiority of rhFGF-2 to EMD. Safety problems were not identified in either study. Therefore, trafermin is an effective and safe treatment for periodontal regeneration in intrabony defect, and its efficacy was superior in rhFGF-2 compared to EMD treatments.

Sphingosine-1-phosphate promotes the nuclear translocation of ß-catenin and thereby induces osteoprotegerin gene expression in osteoblast-like cell lines.

Sphingosine-1-phosphate (S1P) is a well-known signaling sphingolipid and bioactive lipid mediator. Recently, it was reported that S1P inhibits osteoclast differentiation and bone resorption. On the other hand, S1P effects on osteoblasts and bone formation are little known. In this study, we investigated the effects of S1P on osteoblasts, using two osteoblast-like cell lines, SaOS-2 and MC3T3-E1. S1P activated phosphatidylinositol 3-kinase (PI3K)/Akt signaling, leading to the inhibition of glycogen synthase kinase-3ß and the nuclear translocation of ß-catenin, followed by the increase of the transcriptional activity by ß-catenin/T-cell factor complex formation in both SaOS-2 cells and MC3T3-E1 cells. The inhibitors of PI3K and Akt suppressed S1P-induced nuclear localization of ß-catenin. We further investigated the effects of PI3K/Akt signaling on the Wnt/ß-catenin signaling pathway, since ß-catenin takes a central role in this signaling pathway. Both inhibitors for PI3K and Akt suppressed the nuclear localization of ß-catenin and T-cell factor transcriptional activity induced by Wnt-3a. S1P increased the amount of osteoprotegerin at both mRNA and protein levels, and increased the activity of alkaline phosphatase, leading to the mineralization. These findings suggest that S1P activates the PI3K/Akt signaling pathway leading to the promotion of nuclear translocation of ß-catenin in osteoblast-like cells, resulting in the upregulation of osteoptotegerin and osteoblast differentiation markers including alkaline phosphatase, probably relating to the inhibition of osteoclast formation and the mineralization, respectively.

Taste responses in mice lacking taste receptor subunit T1R1.

The T1R1 receptor subunit acts as an umami taste receptor in combination with its partner, T1R3. In addition, metabotropic glutamate receptors (brain and taste variants of mGluR1 and mGluR4) are thought to function as umami taste receptors. To elucidate the function of T1R1 and the contribution of mGluRs to umami taste detection in vivo, we used newly developed knock-out (T1R1(-/-)) mice, which lack the entire coding region of the Tas1r1 gene and express mCherry in T1R1-expressing cells. Gustatory nerve recordings demonstrated that T1R1(-/-) mice exhibited a serious deficit in inosine monophosphate-elicited synergy but substantial residual responses to glutamate alone in both chorda tympani and glossopharyngeal nerves. Interestingly, chorda tympani nerve responses to sweeteners were smaller in T1R1(-/-) mice. Taste cell recordings demonstrated that many mCherry-expressing taste cells in T1R1(+/-) mice responded to sweet and umami compounds, whereas those in T1R1(-/-) mice responded to sweet stimuli. The proportion of sweet-responsive cells was smaller in T1R1(-/-) than in T1R1(+/-) mice. Single-cell RT-PCR demonstrated that some single mCherry-expressing cells expressed all three T1R subunits. Chorda tympani and glossopharyngeal nerve responses to glutamate were significantly inhibited by addition of mGluR antagonists in both T1R1(-/-) and T1R1(+/-) mice. Conditioned taste aversion tests demonstrated that both T1R1(-/-) and T1R1(+/-) mice were equally capable of discriminating glutamate from other basic taste stimuli. Avoidance conditioned to glutamate was significantly reduced by addition of mGluR antagonists. These results suggest that T1R1-expressing cells mainly contribute to umami taste synergism and partly to sweet sensitivity and that mGluRs are involved in the detection of umami compounds.

Serotype-dependent expression patterns of stabilized lipopolysaccharide aggregates in Aggregatibacter actinomycetemcomitans strains.

Above a critical concentration, amphiphilic lipopolysaccharide (LPS) molecules in an aqueous environment form aggregate structures, probably because of interactions involving hydrophobic bonds. Ionic bonds involving divalent cations stabilize these aggregate structures, making them resistant to breakdown by detergents. The aim of this study was to examine expression patterns of stabilized LPS aggregates in Aggregatibacter actinomycetemcomitans, a microorganism that causes periodontitis. A. actinomycetemcomitans strains of various serotypes and truncated LPS mutants were prepared for this study. Following treatment with a two-phase separation system using the detergent Triton X-114, crude LPS extracts of the study strains were separated into detergent-phase LPS (DP-LPS) and aqueous-phase LPS (AP-LPS). Repeated treatment of the aqueous phase with the two-phase separation system produced only a slight decrease in AP-LPS, suggesting that AP-LPS was resistant to the detergent and thus distinguishable from DP-LPS. The presence of divalent cations increased the yield of AP-LPS. AP-LPS expression patterns were serotype-dependent; serotypes b and f showing early expression, and serotypes a and c late expression. In addition, highly truncated LPS from a waaD (rfaD) mutant were unable to generate AP-LPS, suggesting involvement of the LPS structure in the generation of AP-LPS. The two-phase separation was able to distinguish two types of LPS with different physical states at the supramolecular structure level. Hence, AP-LPS likely represents stabilized LPS aggregates, whereas DP-LPS might be derived from non-stabilized aggregates. Furthermore, time-dependent expression of stabilized LPS aggregates was found to be serotype-dependent in A. actinomycetemcomitans.

The usefulness of three-dimensional imaging in the diagnosis and treatment of clinically ambiguous gingival swelling.

We evaluated and treated a 54-year-old woman with gingival swelling. Conventional intraoral and panoramic radiography did not provide sufficient information for either determining the cause of gingival swelling or planning treatment of clinical symptoms. The 3D Accuitomo XYZ Slice View Tomograph (3DX) is a compact dental computed tomography device that allowed for accurate identification and optimal treatment of the causes of gingival swelling. At four years after treatment, 3DX radiographs showed no abnormalities in treated teeth or healing of surrounding structures. We conclude that high-resolution 3D images obtained with 3DX promise to be very effective for diagnosing oral diseases and determining effective treatment.

Periodontal tissue regeneration using fibroblast growth factor-2: randomized controlled phase II clinical trial.

BACKGROUND: The options for medical use of signaling molecules as stimulators of tissue regeneration are currently limited. Preclinical evidence suggests that fibroblast growth factor (FGF)-2 can promote periodontal regeneration. This study aimed to clarify the activity of FGF-2 in stimulating regeneration of periodontal tissue lost by periodontitis and to evaluate the safety of such stimulation. METHODOLOGY/PRINCIPAL FINDINGS: We used recombinant human FGF-2 with 3% hydroxypropylcellulose (HPC) as vehicle and conducted a randomized double-blinded controlled trial involving 13 facilities. Subjects comprised 74 patients displaying a 2- or 3-walled vertical bone defect as measured > or = 3 mm apical to the bone crest. Patients were randomly assigned to 4 groups: Group P, given HPC with no FGF-2; Group L, given HPC containing 0.03% FGF-2; Group M, given HPC containing 0.1% FGF-2; and Group H, given HPC containing 0.3% FGF-2. Each patient underwent flap operation during which we administered 200 microL of the appropriate investigational drug to the bone defect. Before and for 36 weeks following administration, patients underwent periodontal tissue inspections and standardized radiography of the region under investigation. As a result, a significant difference (p = 0.021) in rate of increase in alveolar bone height was identified between Group P (23.92%) and Group H (58.62%) at 36 weeks. The linear increase in alveolar bone height at 36 weeks in Group P and H was 0.95 mm and 1.85 mm, respectively (p = 0.132). No serious adverse events attributable to the investigational drug were identified. CONCLUSIONS: Although no statistically significant differences were noted for gains in clinical attachment level and alveolar bone gain for FGF-2 groups versus Group P, the significant difference in rate of increase in alveolar bone height (p = 0.021) between Groups P and H at 36 weeks suggests that some efficacy could be expected from FGF-2 in stimulating regeneration of periodontal tissue in patients with periodontitis. TRIAL REGISTRATION: ClinicalTrials.gov NCT00514657.

Wound healing process of injured pulp tissues with emdogain gel.

This study aimed to investigate the wound healing process of injured pulp tissues with Emdogain gel (EMD). Pulpotomy was performed for the first molars of the mandibles in rats. EMD or Vitapex (VIT)-containing calcium hydroxide was applied to the exposed pulp tissues. The treated teeth were extracted after 7, 14, and 28 days and prepared for histologic examination. In the VIT-treated group, the number of interleukin-1 beta (IL-1 beta)-expressing macrophages initially increased, followed by that of transforming growth factor-beta1 (TGF-beta1)-expressing macrophages. The number of cells expressing bone morphogenetic proteins (BMPs) gradually increased with reparative dentin formation. Meanwhile, in the EMD-treated group, cells expressing IL-1 beta or TGF-beta1 were few. However, the number of BMP-expressing cells, partly macrophages, increased in the early phase, and large amounts of reparative dentin were observed. This study demonstrated that different healing processes existed for EMD and VIT. BMP-expressing macrophages might play important roles in reparative dentin formation.

Risk of Porphyromonas gingivalis recolonization during the early period of periodontal maintenance in initially severe periodontitis sites.

BACKGROUND: Porphyromonas gingivalis is considered a critical pathogen of periodontal diseases including recurrent periodontitis. The profound effects of active periodontal treatment (APT) on P. gingivalis elimination were previously demonstrated and revealed that the subsequent P. gingivalis-free or -suppressed status seems to be maintained during early periodontal maintenance (PMT). The aim of the present study was to show the occurrence of microbial recolonization during this early PMT period. METHODS: In total, 128 sites from 11 generalized chronic periodontitis patients and one generalized aggressive periodontitis patient underwent clinical and microbiologic examination at baseline (Exam-I), after APT (Exam-II), and in PMT (Exam-III). Exam-III was carried out an average of 4.5 +/- 3.5 months after Exam-II. Detection and quantification of putative pathogens were performed using a polymerase chain reaction-based method. RESULTS: The PMT used was effective in maintaining the clinical conditions improved by APT. However, in microbiological examinations, Exam-III showed higher detection frequency and levels of P. gingivalis than Exam-II. This suggests that a P. gingivalis recolonization started in the early PMT period. P. gingivalis-increased sites then showed significantly more severe signs of periodontitis in Exam-I than P. gingivalis-stable sites (bleeding on probing frequency: 76.7% versus 56.5%; suppuration frequency: 41.9% versus 12.9%). On the other hand, in Exam-II, no significant differences of clinical parameters were noted between P. gingivalis-increased and -stable sites. CONCLUSION: Severe periodontitis sites before APT seemed to place them at risk of P. gingivalis recolonization in the early PMT period, and this microbial restoration could be a cause of recurrent periodontitis.

Differentiation-inducing factor-1 alters canonical Wnt signaling and suppresses alkaline phosphatase expression in osteoblast-like cell lines.

UNLABELLED: Because DIF-1 has been shown to affect Wnt/beta-catenin signaling pathway, the effects of DIF-1 on osteoblast-like cell lines, SaOS-2 and MC3T3-E1, were examined. We found that DIF-1 inhibited this pathway, resulting in the suppression of ALP promoter activity through the TCF/LEF binding site. INTRODUCTION: Differentiation-inducing factor-1 (DIF-1), a morphogen of Dictyostelium, inhibits cell proliferation and induces cell differentiation in several mammalian cells. Previous studies showed that DIF-1 activated glycogen synthase kinase-3beta, suggesting that this chemical could affect the Wnt/beta-catenin signaling pathway. This pathway has been shown to be involved in bone biology. MATERIALS AND METHODS: We studied the effects of DIF-1 on SaOS-2 and MC3T3-E1, osteosarcoma cell lines widely used as a model system for ostoblastic cells and murine osteoblast-like cell line, respectively. Reporter gene assays were also carried out to examine the effect of DIF-1 on the Wnt/beta-catenin signaling pathway. RESULTS: DIF-1 inhibited SaOS-2 proliferation and reduced alkaline phosphatase (ALP) activity in a concentration- and a time-dependent manner. The expression of ALP was markedly suppressed by DIF-1-treatment in protein and mRNA levels. DIF-1 also suppressed the expression of other osteoblast differentiation markers, including core binding factor alpha1, type I collagen, and osteocalcin, in protein and mRNA levels and inhibited osteoblast-mediated mineralization. Subsequently, we examined the effect of DIF-1 on the Wnt/beta-catenin signaling pathway. We found that DIF-1 suppressed the expression of beta-catenin protein and the activity of the reporter gene containing T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) consensus binding sites. We examined the effect of DIF-1 on a reporter gene driven by the human ALP promoter and found that DIF-1 significantly reduced the ALP reporter gene activity through the TCF/LEF binding site (-1023/-1017 bp). Furthermore, the effect of DIF-1 on MC3T3-E1, a murine osteoblast-like cell line, was examined, and it was found that DIF-1 suppressed ALP mRNA expression by the reduction of the ALP reporter gene activity through the TCF/LEF binding site. CONCLUSIONS: Our data suggest that DIF-1 inhibits Wnt/beta-catenin signaling, resulting in the suppression of ALP promoter activity. To our knowledge, this is the first report to analyze the role of the TCF/LEF binding site (-1023/-1017 bp) of the ALP gene promoter in osteoblast-like cell lines.

Resolution of furcation bone loss after non-surgical root canal treatment: application of a peptidase-detection kit for treatment of type I endoperiodontal lesion.

Here, we report the management of a type I endoperiodontal lesion with furcation bone loss. A 59-year-old female attended our hospital with the chief complaint of mobility of tooth 46 and recurrent gingival swelling around the tooth. She previously received dental treatment from two dentists, but her condition did not improve. The tooth manifested the symptoms of typical periodontitis, such as gingival swelling, tooth mobility, pus discharge from the periodontal pocket and furcation bone loss. The tooth had no caries and the pulp reacted to an electric pulp test. Careful examination of the gingiva revealed traces of dental fistula. X-ray examination via a gutta percha inserted into the fistula revealed that furcation bone loss was associated with the periapical lesion. We diagnosed a type I endoperiodontal lesion, and applied Periocheck, a detection kit for peptidase-producing bacteria, to check for decreases in bacteria in the furcation and root canals. Soon after non-surgical root canal treatment, the condition of tooth 46 improved without periodontal treatment. After confirming a negative score with Periocheck, the root canal was filled. After 3 months, the furcation bone loss was on the way to recovery. These results indicate that proper diagnosis and confirmation of a decrease in root canal bacteria are important for treating endoperiodontal lesions.

Involvement of Porphyromonas gingivalis fimA genotype in treatment outcome following non-surgical periodontal therapy.

BACKGROUND: Porphyromonas gingivalis is considered a critical pathogen in periodontal diseases. It is classified into six genotypes based on diversity of the fimA gene encoding fimbrillin. The present study evaluated the involvement of the fimA genotype in treatment outcome following non-surgical periodontal therapy. METHODS: Chronic periodontitis patients were enrolled in this study; all received clinical and microbiological examinations at baseline. The detection of subgingival species and identification of P. gingivalis fimA genotypes were performed using polymerase chain reaction based methods. In total, 160 P. gingivalis positive sites with bleeding on probing (BOP) and a probing depth of > or =4 mm were accepted. They were followed up after scaling and root planing. RESULTS: Longitudinal investigation indicated that fimA type I positive sites at baseline were followed by a significantly higher frequency of persistent BOP after treatment than type I negative sites (51.6% versus 27.9%), while types Ib and II were not. Type I positive sites also showed more persistence of Tannerella forsythensis and P. gingivalis after treatment than type I negative sites. In post-treatment investigation, type I positive sites showed higher frequencies of BOP and T. forsythensis detection than type I negative sites (77.8% versus 43.5% and 100% versus 76.1%, respectively). CONCLUSIONS: BOP in initially type I positive sites showed little improvement with treatment, and the combined persistence of fimA type I and T. forsythensis seemed to be involved in this poor treatment outcome. The present study demonstrated the potential of P. gingivalis fimA type I as a predictor of persistent BOP after treatment.

Local anesthetics inhibit priming of neutrophils by lipopolysaccharide for enhanced release of superoxide: suppression of cytochrome b558 expression by disparate mechanisms.

Local anesthetics have anti-inflammatory effects in vivo and inhibit neutrophil functions in vitro, but how these agents act on neutrophils remains unclear. Phagocytosis and bactericidal activity of neutrophils are enhanced by exposure to bacterial components such as lipopolysaccharide (LPS); this process is termed priming, which for enhanced release of superoxide (O2-) causes mobilization of intracellular granules that contain cytochrome b558, a component of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. We studied whether local anesthetics affected LPS priming for enhanced release of O2- in response to triggering by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), and we investigated which element in the LPS signaling pathway might be the target of local anesthetics. Neutrophils were incubated with 10 ng/ml LPS and 1% plasma+/-local anesthetics, washed, and triggered with fMLP. Local anesthetics all inhibited LPS priming, and 50% inhibition was at 0.1 mM tetracaine, 0.5 mM bupivacaine, 3.0 mM lidocaine, or 4.0 mM procaine. Local anesthetics inhibited LPS-induced mobilization of specific granules and secretory vesicles. Local anesthetics inhibited LPS-induced up-regulation of cytochrome b558 but not LPS-induced translocation of p47phox. Inhibition of priming by local anesthetics was reversed by washing and incubating for 5 min. Tetracaine alone, but not the other local anesthetics, inhibited LPS activation of p38 mitogen-activated protein kinase (MAPK) and MAPK kinase 3 (kinases in the LPS signaling pathway). The p38 MAPK inhibitors SB203580 and PD169316 also blocked LPS priming. Thus, tetracaine and the other local anesthetics inhibit by disparate mechanisms, but all the local anesthetics impaired up-regulation of cytochrome b558 and all impaired priming of NADPH oxidase by LPS.

Stimulation of growth of Porphyromonas gingivalis by cell extracts from Tannerella forsythia.

OBJECTIVE: In order to examine if Tannerella forsythia stimulates the growth of Porphyromonas gingivalis, an in vitro study was performed. BACKGROUND: P. gingivalis and T. forsythia are often isolated simultaneously from active periodontitis sites, indicating that these bacteria somewhat interact in the periodontal environment. We reported previously that mixed infection of P. gingivalis and T. forsythia synergistically induced lesion formation in a murine abscess model, and gingipains of P. gingivalis played an important role in this synergism. One of the possible mechanisms of this synergism is growth promotion by coinfection of the two bacteria. METHODS: Cell extracts of T. forsythia were added to the nutrition-decreased medium and the promotion of growth of P. gingivalis was examined. RESULTS: Sonicated extract of T. forsythia stimulated growth of P. gingivalis in nutrition-decreased medium in a dose-dependent manner. Proteins appeared to be the nature of growth-promoting factor, and the cell extract of T. forsythia had no stimulating effect on the growth of P. gingivalis strain devoid of gingipain activities. CONCLUSION: A product or a component of T. forsythia seemed to stimulate growth of P. gingivalis under nutrition-limited conditions. Gingipains are considered to play an important role in digestion or uptake of this growth-promoting factor. The interaction between T. forsythia and P. gingivalis in growth may be in part related with the synergistic virulence in a murine model.

The prevalence and pathogenic differences of Porphyromonas gingivalis fimA genotypes in patients with aggressive periodontitis.

BACKGROUND: The fimA gene, which encodes fimbrillin (FimA), is found in Porphyromonas gingivalis and has been classified into six genotypes based on nucleotide sequence. P. gingivalis that possesses the type II fimA gene is prevalent in adult periodontitis. OBJECTIVES: The aim of this study was to investigate the prevalence of P. gingivalis fimA genotypes in Japanese aggressive periodontitis patients, and to examine their virulence. METHODS: Subgingival plaque samples were obtained from 223 sites in 18 aggressive periodontitis patients and 95 sites in 22 periodontally healthy young adults. Actinobacillus actinomycetemcomitans, P. gingivalis and Tannerella forsythensis detection, determination of the fimA genotype in P. gingivalis, and the quantification of P. gingivalis were analyzed by polymerase chain reaction (PCR) methods. The proteolytic activities of the P. gingivalis fimA type I and fimA type II were also examined. RESULTS: In aggressive periodontitis patients, the most prevalent fimA genotype was the type II (46.7%), followed by the type Ib and type I, whereas in healthy subjects, the type I fimA was the only genotype detected. The number of P. gingivalis pathogens was the greatest in the type I fimA positive sites, and the frequency of coexisting A. actinomycetemcomitans and T. forsythensis was highest in the type II fimA positive sites in the aggressive periodontitis patients. Both the arginine-specific cysteine proteinase (Arg-gingipain) and lysine-specific cysteine proteinase (Lys-gingipain) activity of the P. gingivalis fimA type I strain were significantly higher than those of the fimA type II strains. CONCLUSIONS: These results suggest that differences in virulence exist among different fimA genotypes. Coadherence with other pathogens in P. gingivalis fimA type II-associated aggressive periodontitis and quantitative increases in P. gingivalis in fimA type I-associated aggressive periodontitis are related to this virulence.

Role of PRIP-1, a novel Ins(1,4,5)P3 binding protein, in Ins(1,4,5)P3-mediated Ca2+ signaling.

PRIP-1 was isolated as a novel inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] binding protein with a domain organization similar to phospholipase C-delta1 (PLC-delta1) but lacking the enzymatic activity. Further studies revealed that the pleckstrin homology (PH) domain of PRIP-1 is the region responsible for binding Ins(1,4,5)P3. In this study we aimed to clarify the role of PRIP-1 at the physiological concentration in Ins(1,4,5)P3-mediated Ca2+ signaling, as we had previously used COS-1 cells overexpressing PRIP-1 (Takeuchi et al., 2000, Biochem J 349:357-368). For this purpose we employed PRIP-1 knock out (PRIP-1-/-) mice generated previously (Kanematsu et al., 2002, EMBO J 21:1004-1011). The increase in free Ca2+ concentration in response to purinergic receptor stimulation was lower in primary cultured cortical neurons prepared from PRIP-1-/- mice than in those from wild type mice. The relative amounts of [3H]Ins(1,4,5)P3 measured in neurons labeled with [3H]inositol was also lower in cells from PRIP-1-/- mice. In contrast, PLC activities in brain cortex samples from PRIP-1-/- mice were not different from those in the wild type mice, indicating that the hydrolysis of Ins(1,4,5)P3 is enhanced in cells from PRIP-1-/- mice. In vitro analyses revealed that type1 inositol polyphosphate 5-phosphatase physically interacted with a PH domain of PRIP-1 (PRIP-1PH) and its enzyme activity was inhibited by PRIP-1PH. However, physical interaction with these two proteins did not appear to be the reason for the inhibition of enzyme activity, indicating that binding of Ins(1,4,5)P3 to the PH domain prevented its hydrolyzation. Together, these results indicate that PRIP-1 plays an important role in regulating the Ins(1,4,5)P3-mediated Ca2+ signaling by modulating type1 inositol polyphosphate 5-phosphatase activity through binding to Ins(1,4,5)P3.

Presence of CXCR3-positive cells and IFN-gamma-producing cells in human periapical granulomas.

In an attempt to understand further the balance between the types of helper T (Th) cells in human apical periodontitis, we examined the difference in the expression of the chemokine receptor and cytokine in samples obtained from human subjects by means of immunohistochemical methods. Chemokine receptor CXCR3-positive cells and IFN-gamma-producing cells were found to be present in human periapical granulomas, whereas chemokine receptor CCR3-positive cells and IL-4-producing cells could not be detected. By contrast, no factor expression was observed in a clinically healthy periodontal ligament serving as a negative control. Our findings suggest that Th1 cells may play an important role in the pathological process of local inflammation such as apical periodontitis.

Follistatin restricts bone morphogenetic protein (BMP)-2 action on the differentiation of osteoblasts in fetal rat mandibular cells.

UNLABELLED: We tested whether FS secretion might modulate BMP-2 actions by measuring FS levels and counting bone numbers of rat mandibular cells. In the presence of Dex, BMP-2 stimulated FS secretion at the early phase and augmented bone nodule by neutralizing with FS antibody. We concluded that BMP-2 facilitates FS secretion, and the FS restricts BMP-2 action on osteoblastogenesis. INTRODUCTION: Bone morphogenetic proteins (BMPs) promote the differentiation of osteoprogenitor cells into osteoblasts. Activin A is involved in the regulation of bone formation. Follistatin (FS) antagonizes the bioactivities of BMP and activin A extracellularly. MATERIALS AND METHODS: In this study, we tested whether the induction of FS secretion might modulate the effects of BMP-2 on osteoblast development, using the bone nodule-forming cultures of fetal rat mandibular cells. RESULTS AND CONCLUSIONS: In the presence of dexamethasone (Dex), BMP-2 stimulated the secretion of FS at the early phase (days 3-9) of the culture. Dex alone had no effect, and BMP-2 alone was less effective than the combination of the two. BMP-4 and -6 had little effect on FS secretion. Activin A inhibited the early upregulation of FS secretion when added with BMP-2 and Dex. In the presence of Dex, BMP-2 increased bone nodule numbers when added to early cultures. The addition of anti-FS antibody to cultures with BMP-2 and Dex augmented bone nodule formation. These results show that BMP-2 facilitates the secretion of FS in the presence of Dex, and the increased FS secretion restricts the action of BMP-2 on osteoblast differentiation.

Characterization of the 5'-untranslated region of YB-1 mRNA and autoregulation of translation by YB-1 protein.

The eukaryotic Y-box binding protein YB-1 is involved in various biological processes, including DNA repair, cell proliferation and the regulation of transcription and translation. YB-1 protein is abundant and expressed ubiquitously in human cells, functioning in cell proliferation and transformation. Its concentration is thought to be highly regulated at both the levels of transcription and translation. Therefore, we investigated whether or not the 5'-UTR of YB-1 mRNA affects the translation of YB-1 protein, thus influencing expression levels. Luciferase mRNA ligated to the YB-1 mRNA 5'-UTR was used as a reporter construct. Ligation of the full-length YB-1 5'-UTR (331 bases) enhanced translation as assessed by in vitro and in vivo translation assays. Deletion constructs of the YB-1 5'-UTR also resulted in a higher efficiency of translation, especially in the region mapped to +197 to +331 from the major transcription start site. RNA gel shift assays revealed that the affinity of YB-1 for various 5'-UTR probe sequences was higher for the full-length 5'-UTR than for deleted 5'-UTR sequences. An in vitro translation assay was used to demonstrate that recombinant YB-1 protein inhibited translation of the full-length 5'-UTR of YB-1 mRNA. Thus, our findings provide evidence for the autoregulation of YB-1 mRNA translation via the 5'-UTR.

Antibody responses to Porphyromonas gingivalis infection in a murine abscess model--involvement of gingipains and responses to re-infection.

BACKGROUND: Porphyromonas gingivalis is one of the most important periodontopathogens. It produces cysteine proteinases named gingipains. We previously examined the effect of gingipains on abscess formation in a murine model. The rgpA rgpB double and kgp mutants induced smaller abscesses than the wild type. Moreover, the rgpA rgpB kgp triple (gingipain-null) mutant hardly showed lesion formation at all under the experimental conditions used, indicating that genes encoding gingipains are important for P. gingivalis virulence. OBJECTIVES: Here, we further report the humoral immune responses induced by P. gingivalis strains. METHODS: After the lesions were apparently cured, sera were collected from the mice and immunoglobulin G (IgG) responses against the whole cell antigens of wild-type P. gingivalis were measured. RESULTS: Wild-type strain was found to induce a strong antibody reaction. On the other hand, the rgpA rgpB kgp triple and kgp mutants induced significantly lower antibody responses compared to the wild type. Western blotting analysis confirmed the differences in antibody production. Next, these mice were re-infected with wild-type strain. Mice that were first infected with wild-type strain showed significantly smaller lesion formation than control mice that were first infected with medium only. On the other hand, mice that were first infected with mutant strains devoid of gingipain activities did not show resistance to re-infection and immunoglobulins directed against gingipains may be protective. CONCLUSIONS: These results suggest that gingipains play an important role in abscess formation in mice, and humoral immune responses seem to be partly responsible for the resistance to re-infection by P. gingivalis.

Dental root resorption and repair: histology and histometry during physiological drift of rat molars.

OBJECTIVE: The process of dental root resorption and subsequent cementum regeneration has not been sufficiently elucidated. This study aimed to examine the process of the root resorption and cementum regeneration during physiological tooth drift using a rat model, and to evaluate this experimental model. METHODS: Distal roots in mandibular first molars and the surrounding periodontal tissues were investigated with light and electron microscopy. The light microscopic approach included histochemical and histometric analyses utilizing the tartrate-resistant acid phosphatase (TRAP) reaction. RESULTS: Root resorption was observed in the distal side of the roots and was most active in 5- to 6-week-old rats, and gradually decreased hereafter. An increase in the number of TRAP-positive mononuclear cells, which seemed to be odontoclast precursor cells, preceded the increase in the number of odontoclasts. Root resorption was transient, and was followed by the new formation of acellular extrinsic fiber cementum accompanied with only a slight inflammation, and therefore classified as external surface resorption. Preparation for new cementum started adjacent to the resorption areas when root resorption was most active. CONCLUSIONS: The root resorption during drift in rats is transient and followed by acellular extrinsic fiber cementum regeneration. Cellular kinetics suggested that odontoclast precursor cells are supplied as mononuclear cells from vascular spaces.