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Isao Ishikawa

Publications and source records attributed to Isao Ishikawa.

At least 19 recordsLinked to original sources
PubMed2005

Identification of genes preferentially expressed in periodontal ligament: specific expression of a novel secreted protein, FDC-SP.

Gene expression in human periodontal ligament (PDL) was examined by suppression subtractive hybridization to identify genes that are preferentially expressed in tissue compared to cultured PDL fibroblasts. The most enriched genes in a subtracted cDNA library are primarily genes for extracellular matrix components, types I and III collagen, lumican, periostin, and asporin, among others, whose expression conveys unique mechanical properties to the PDL. Also within this group is the gene for follicular dendritic cell secreted protein (FDC-SP), a small protein like statherin in saliva, not previously found in PDL. FDC-SP's presence in PDL was confirmed by in situ hybridization in mouse which also showed that it was definitely present in the parotid gland, but, surprisingly, not in the other salivary glands: submandibular and sublingual. Since only normal tissue was examined, these findings suggest that FDC-SP plays an important but previously unsuspected role within oral connective tissue.

Cells, Cultured↗
PubMed2005

Characterization of the mutant (A115V) tissue-nonspecific alkaline phosphatase gene from adult-type hypophosphatasia.

Hypophosphatasia (HOPS) is a clinically heterogeneous heritable disorder characterized by defective skeletal mineralization, deficiency of tissue-nonspecific alkaline phosphatase (TNSALP) activity, and premature loss of deciduous teeth. To date, various mutations in the TNSALP gene have been identified. Especially, A115V located in exon 5 has been detected in a Japanese patient with severe periodontitis and adult-type HOPS. In this study, we have characterized the protein translated from the mutant A115V gene. Wild-type and A115V mutant-type TNSALP cDNA expression vector pcDNA3 have been constructed and transfected to COS-1 cells by lipofectin technique. After 48-h transfection, the cells were subjected to assay ALP activity. In order to identify possible dominant effect of the mutation, we performed co-transfections of wild-type and mutated cDNA, and evaluated the residual activities of each mutation. Detection of TNSALP synthesized by COS-1 cells transfected with the wild- or the mutated-type was also performed by using an immunofluorescent method. ALP activity of cell transfected with the mutant cDNA (A115V) plasmid after 48-h transfection exhibited 0.399+/-0.021 U/mg. As the enzymatic activity of the wild type was taken as 100%, the value of the mutant was estimated as 16.9%. When co-transfected this mutant showed no inhibition of the wild-type enzyme. TNSALP in COS-1 cells with transfected with the mutant exhibited strong fluorescence at the surface of cells as wild-type. This study indicated that the mutant (A115V) TNSALP gene produced the defective ALP enzyme and it could be recessively transmitted and be a disease-causing mutation of the adult-type hypophosphatasia.

Alanine↗
PubMed2005

S100A4 inhibition by RNAi up-regulates osteoblast related genes in periodontal ligament cells.

Periodontal ligament (PDL) is a thin fibrous connective tissue located between alveolar bone and cementum that remains unmineralized physiologically. It is thus thought that PDL cells possess mechanisms to inhibit mineralization. It has been demonstrated that S100A4, a member of the S100 calcium-binding protein family, is synthesized and secreted by PDL cells, and that it may act as an inhibitor of mineralization. However, the mechanisms of action of S100A4 in mineralization have not been thoroughly clarified. In the present study we investigated the effects of S100A4 inhibition by a short interfering RNA (siRNA) on the expression of osteoblast related genes by human PDL cells. Inhibition of S100A4 by siRNA resulted in increased expression of osteoblastic markers such as osteopontin and osteocalcin, and the osteoblast-specific transcription factors, Runx2/Cbfa1 and Osterix. These results indicate that S100A4 suppresses the expression of osteoblastic genes in PDL cells and may thus inhibit mineralization in the PDL.

Cell Differentiation↗
PubMed2005

Oral bacteria in the occluded arteries of patients with Buerger disease.

OBJECTIVE: Recent studies have suggested that infectious organisms play a role in vascular diseases. In this study, to explore a possible link between oral infection and Buerger disease, we investigated whether oral (periodontal) bacteria were present in occluded arteries removed from patients with characteristic Buerger disease. METHODS: Fourteen male patients with a smoking history who had developed characteristics of Buerger disease before the age of 50 years were included in this study. Occluded arteries, including superficial femoral (n = 4), popliteal (n = 2), anterior tibial (n = 4), and posterior tibial (n = 4) arteries, were removed and studied. A periodontist performed a periodontal examination on each patient and collected dental plaque and saliva samples from them at the same time. The polymerase chain reaction method was applied to detect whether seven species of periodontal bacteria--Porphyromonas gingivalis, Tannerella forsythensis, Treponema denticola, Campylobacter rectus, Actinobacillus actinomycetemcomitans, Prevotella intermedia , and Prevotella nigrescens--were present in the occluded arteries and oral samples. In addition, arterial specimens from seven control patients were examined by polymerase chain reaction analysis. RESULTS: DNA of oral bacteria was detected in 13 of 14 arterial samples and all oral samples of patients with Buerger disease. Treponema denticola was found in 12 arterial and all oral samples. Campylobacter rectus, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis, and Prevotella nigrescens were found in 14% to 43% of the arterial samples and 71% to 100% of the oral samples. A pathologic examination revealed that arterial specimens showed the characteristics of an intermediate-chronic-stage or chronic-stage lesion of Buerger disease. All 14 patients with Buerger disease had moderate to severe periodontitis. None of the control arterial samples was positive for periodontal bacteria. CONCLUSIONS: This is the first study to identify oral microorganisms in the lesions of Buerger disease. Our findings suggest a possible etiologic link between Buerger disease and chronic infections such as oral bacterial infections.

Adult↗
PubMed2005

Prevotella multiformis sp. nov., isolated from human subgingival plaque.

Four bacterial strains isolated from the human oral cavity, PPPA19, PPPA21(T), PPPA28 and PPPA30, were characterized by determining phenotypic and biochemical features, cellular fatty acid profiles and the phylogenetic position based on 16S rRNA gene sequence analysis. 16S rRNA gene sequence analysis showed that each of the isolates was a member of the genus Prevotella. These strains were related to Prevotella denticola with about 95 % similarity. The strains were obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-negative rods. However, the cells of these strains were often cocci (coccobacilli), depending on the cultivation time. Colonies of different sizes were detected on Eggerth Gagnon agar plates for these strains. The cells forming large colonies were cocci, whereas those forming small colonies were cocci and rods. However, 16S rRNA gene sequence comparison of colonies of different sizes revealed that only a single organism was present. Although these strains had phenotypic characteristics that were similar to those of P. denticola JCM 8528, they could be differentiated from P. denticola JCM 8528 by aesculin hydrolysis and d-cellobiose fermentation in API 20A tests. DNA-DNA hybridization experiments revealed the genomic distinction of these four strains with respect to P. denticola JCM 8528. On the basis of these data, a novel Prevotella species, Prevotella multiformis sp. nov., is proposed, with PPPA21(T) (=JCM 12541(T)=DSM 16608(T)) as the type strain.

Bacteroidaceae Infections↗
PubMed2005

Prevotella multisaccharivorax sp. nov., isolated from human subgingival plaque.

Six bacterial strains isolated from the human oral cavity, PPPA16, PPPA20(T), PPPA24, PPPA31, EPPA6 and EPPA7, were characterized by determining phenotypic and biochemical features, cellular fatty acid profiles, menaquinone profiles and phylogenetic position based on 16S rRNA gene sequence analysis. 16S rRNA gene sequence analysis showed that the isolates represented the same species of the genus Prevotella. The strains were related to Prevotella dentalis with about 89% similarity. In addition, the isolates were related to Prevotella sp. oral clone IDR-CEC-0032, which is a representative of the numerically dominant cluster VI in carious dentine lesions [Nadkarni et al. (2004). J Clin Microbiol 42, 5238-5244], with about 99% similarity. The strains were obligately anaerobic, non-pigmenting, non-spore-forming, non-motile, Gram-negative rods. The isolates could be differentiated from other Prevotella species by D-mannitol, D-melezitose, D-sorbitol and D-trehalose fermentation in API 20A tests. The cellular fatty acid composition of strains PPPA16, PPPA20(T), PPPA24, PPPA31, EPPA6 and EPPA7 was significantly different from that of other Prevotella species. Compared with other Prevotella species, only these six strains contained dimethyl acetals. The major menaquinones of the clinical isolates were MK-12 and MK-13, whereas the major menaquinones of other Prevotella species were MK-10 and MK-11. On the basis of these data, a novel Prevotella species, Prevotella multisaccharivorax sp. nov., is proposed, with PPPA20(T) (=JCM 12954(T)=DSM 17128(T)) as the type strain.

Bacterial Typing Techniques↗
PubMed2005

Twist is required for establishment of the mouse coronal suture.

Cranial sutures are the growth centres of the skull, enabling expansion of the skull to accommodate rapid growth of the brain. Haploinsufficiency of the human TWIST gene function causes the craniosynostosis syndrome, Saethre-Chotzen syndrome (SCS), in which premature fusion of the coronal suture is a characteristic feature. Previous studies have indicated that Twist is expressed in the coronal suture during development, and therefore that it may play an important role in development and maintenance of the suture. The Twist-null mouse is lethal before the onset of osteogenesis, and the heterozygote exhibits coronal suture synostosis postnatally. In this study we investigated the function of Twist in the development of the mouse coronal suture, by inhibiting Twist synthesis using morpholino antisense oligonucleotides in calvarial organ culture. Decreased Twist production resulted in a narrow sutural space and fusion of bone domains within 48 h after the addition of the morpholino oligonucleotides. Proliferation activity in the sutural cells was decreased, and the expression of osteogenic marker genes such as Runx2 and Fgfr2 was up-regulated in the developing bone domain within 4 h. These results suggest that during establishment of the suture area, Twist is required for the regulation of sutural cell proliferation and osteoblast differentiation.

Acrocephalosyndactylia↗
PubMed2005

Effects of single-visit full-mouth ultrasonic debridement versus quadrant-wise ultrasonic debridement.

BACKGROUND: The aim of this randomized controlled clinical trial was to determine the effects of single-visit full-mouth ultrasonic debridement versus quadrant-wise therapy. MATERIAL AND METHODS: Thirty-six subjects with chronic periodontitis, were randomly allocated to three groups--quadrant-wise ultrasonic debridement, single-visit full-mouth ultrasonic debridement with povidone iodine and single-visit full-mouth ultrasonic debridement with water. Whole-mouth plaque, bleeding on probing (BOP), pocket depth and attachment level were recorded before treatment and 1, 3 and 6 months post-treatment. Plaque and saliva samples were collected for microbiological analysis. RESULTS: After treatment, all groups showed significant improvement in clinical parameters. Full-mouth treatments resulted in similar improvements in full-mouth mean plaque percentage, probing pocket depth and probing attachment level as conventional therapy. When data were analysed based on pocket depth and tooth type, there was no difference between groups in probing depth reduction or attachment gains. The full-mouth groups demonstrated greater reduction in BOP% and number of pockets > or =5 mm and the total treatment time was significantly shorter. The detection frequencies of periodontal pathogens in plaque and saliva showed slight changes with no difference between groups. CONCLUSION: Single-visit full-mouth mechanical debridement may have limited additional benefits over quadrant-wise therapy in the treatment of periodontitis, but can be completed in a shorter time.

Adult↗
PubMed2005

Application of periodontal ligament cell sheet for periodontal regeneration: a pilot study in beagle dogs.

OBJECTIVE: The ultimate goal of periodontal treatment is to regenerate the damaged periodontal support. Although periodontal ligament (PDL) cells are essential for periodontal regeneration, few studies have reported the transplantation of periodontal ligament cells to periodontal defects. We developed a new method to apply periodontal ligament cells as a sheet to the defect. The aim of this study was to investigate the periodontal healing after application of the periodontal ligament cell sheet in beagle dogs. METHODS: Autologous periodontal ligament cells were obtained from extracted premolars of each beagle dog. Periodontal ligament cell sheets were fabricated using a temperature-responsive cell culture dish. Dehiscence defects were surgically created on the buccal surface of the mesial roots of bilateral mandibular first molars of each dog. In the experimental group (five defects), periodontal ligament cell sheet with reinforced hyaluronic acid carrier was applied to the defect. Only the hyaluronic acid carrier was applied to the contralateral side as a control (five defects). Eight weeks after surgery, the animals were sacrificed and decalcified specimens were prepared. Healing of the periodontal defects was evaluated histologically and histometrically. RESULTS: No clinical signs of inflammation or recession of gingiva were observed in both experimental and control groups. In the experimental group, periodontal tissue healing with bone, periodontal ligament and cementum formation was observed in three out of five defects. In the control group, such periodontal tissue formation was not observed except in one defect. Histometric analysis revealed that the formation of new cementum in the experimental group was significantly higher than that in the control group. CONCLUSION: The periodontal ligament cell sheet has a potential to regenerate periodontal tissue and may become a novel regenerative therapy.

Adjuvants, Immunologic↗
PubMed2005

Effect of low-level Er:YAG laser irradiation on cultured human gingival fibroblasts.

BACKGROUND: Low-level laser irradiation has been reported to enhance wound healing. Activation of gingival fibroblasts (GF) has a potential for early wound healing in periodontal treatment. The present study aimed to investigate the direct effect of low-level Er:YAG laser irradiation on gingival fibroblasts proliferation in order to clarify the laser effect on healing. METHODS: Cultured human gingival fibroblasts (hGF) were exposed to low-power, pulsed Er:YAG laser irradiation with different energy densities ranging from 1.68 to 5.0 J/cm(2). The cultures were analyzed by means of trypan blue staining and counted under a light microscope. The effect of Er:YAG laser on hGF was also evaluated using a transmission electron microscope (TEM). RESULTS: Cultures irradiated with Er:YAG laser presented faster cell growth when compared with untreated controls. This difference was statistically significant. Transmission electron microscopy revealed rough endoplasmic reticulum, prominent Golgi complexes, and mitochondria after laser irradiation. CONCLUSIONS: Our results showed that the low-level Er:YAG laser irradiation stimulates the proliferation of cultured gingival fibroblasts. The optimal stimulative energy density was found to be 3.37 J/cm(2). This result suggests that Er:YAG laser irradiation may be of therapeutic benefit for wound healing.

Analysis of Variance↗
PubMed2005

A novel mutation of the cathepsin C gene in a thai family with Papillon-Lefevre syndrome.

BACKGROUND: Papillon-Lefevre syndrome (PLS) is a rare autosomal recessive disorder characterized by palmar- plantar hyperkeratosis and rapid periodontal destruction of both primary and permanent dentitions. It has been shown that the disease is caused by cathepsin C gene (CTSC) mutation leading to the deficiency of cathepsin C enzymatic activity. This study demonstrates the clinical manifestations and CTSC mutational and enzymatic activity analyses in a 5-year-old Thai male PLS patient and his parents. METHODS: Peripheral blood samples were obtained for genomic DNA isolation. All exons of the CTSC gene were amplified by polymerase chain reaction (PCR) using specific primers. Mutations were identified by DNA sequencing. Verification of the mutation was performed by digestion of PCR products by restriction endonucleases. The cathepsin C enzymatic activity was determined using the synthetic substrate glycyl- L-arginine-7-amino-4-methylcoumarin. RESULTS: The patient demonstrated classical characteristics of PLS, including hyperkeratotic skin lesions. By the age of 5, all of his primary teeth were extracted due to severe periodontal infection. The parents had no physical abnormalities. The periodontal examination revealed localized mild periodontal destruction. Sequence analysis showed a nucleotide change at position 90 from C >A (c.90C >A) which resulted in a change from cysteine residue to a premature stop codon at the amino acid position 30 in the exon 1. The HpyCH4V digestion revealed that the patient was homozygous, whereas both the father and mother were heterozygous carriers of this mutation. The cathepsin C activity was reduced in the patient's mother, and the activity in the patient was almost completely lost. CONCLUSIONS: This is the first study to demonstrate a CTSC gene mutation in a Thai family with PLS. The identified mutation is novel and potentially leads to the drastic reduction of the cathepsin C enzymatic activity. This suggests that the mutation is pathogenetic, causing the PLS. Mutational analysis in more members of the family is warranted to identify whether the mutation is inherited from a common ancestor.

Adenine↗
PubMed2005

Syndecan-1 expression during the formation of junctional epithelium.

BACKGROUND: Syndecans are cell surface heparan sulfate proteoglycans (PG) which can bind to and modulate the action of growth factors and extracellular matrix components (ECM). Syndecan- 1 has been shown to play important roles during early tooth development and wound healing and repair. Among diverse cells and tissues that comprise the periodontium, the junctional epithelium (JE) constitutes a region of significant anatomic and clinical importance, but the nature of inductive signals and molecules involved in its formation is still unclear. Therefore, this work examines if syndecan-1 is associated with formation of JE, and the distribution of other syndecan family members in the epithelium. METHODS: In situ hybridization and immunohistochemical techniques were performed using oral tissues from 4-day-old to 10-week-old mice to investigate the expression of syndecan- 1, -2, -3 and -4 mRNAs and their corresponding proteins. RESULTS: Based on in situ hybridization experiments, all syndecan mRNAs were detected in sulcular epithelium (SE), gingival epithelium (GE), and JE with varying intensity and distribution. Syndecan-1 immunostaining was localized on the cell surface while that of syndecan-2 did not show clear membrane localization. Our experiments in the developing tooth demonstrated that syndecan-1 protein followed characteristic patterns of expression during JE formation and that immunoreactivity for syndecan-1 protein decreased with age when JE cells underwent terminal differentiation. CONCLUSION: Results of syndecan-1 mRNA and protein expression patterns suggested that this proteoglycan might be an important molecule during the formation of JE.

Ameloblasts↗
PubMed2005

Cyclooxygenase-2-dependent prostaglandin (PG) E2 downregulates matrix metalloproteinase-3 production via EP2/EP4 subtypes of PGE2 receptors in human periodontal ligament cells stimulated with interleukin-1alpha.

BACKGROUND: Prostaglandin E2 (PGE2), which exerts its actions via EP receptors (EP1, EP2, EP3, and EP4), is a bioactive metabolite produced by cyclooxygenase (COX)-1 and/or COX-2 from arachidonic acid. In the present study, we investigated whether COX-2-derived PGE2 regulated matrix metalloproteinase (MMP)-3 production in human periodontal ligament (PDL) cells stimulated with interleukin (IL)-1alpha and which EP receptors were involved in PGE2 regulation of IL-1alpha-induced MMP-3 production. METHODS: Human PDL cells obtained from periodontally healthy subjects were stimulated with vehicle or IL-1alpha in the presence or absence of indomethacin (a COX-1/COX-2 inhibitor), NS-398 (a specific COX- 2 inhibitor), PGE2, EP receptor agonists, dibutyryl cAMP, and forskolin. PGE2 levels were assayed by enzyme-linked immunosorbent assay (ELISA). MMP-3 levels and caseinolytic activities were evaluated by ELISA and casein zymography, respectively. RESULTS: IL-1alpha enhanced both MMP-3 and PGE2 production. Indomethacin and NS-398 enhanced IL-1alpha-induced MMP-3 production in PDL cells, to the same extent, although both the agents completely inhibited IL-1alpha-induced PGE2 production. Exogenous PGE2 reduced IL-1alpha-induced MMP-3 production in a dose-dependent manner. Butaprost, a selective EP2 agonist, and ONO-AE1-329, a selective EP4 agonist, significantly inhibited IL-1alpha-induced MMP-3 production, although butaprost was less potent than ONO-AE-1-329. Dibutyryl cAMP, a cAMP analog, and forskolin, an adenylate cyclase activator, significantly inhibited IL-1alpha-stimulated MMP-3 production in PDL cells. CONCLUSIONS: These data suggest that COX-2-dependent PGE2 downregulates IL-1alpha-elicited MMP-3 production by cAMP-dependent pathways via EP2/EP4 receptors in human PDL cells. cAMP-elevating agents such as EP2/EP4 receptor activators may regulate the destruction of extracellular matrix components in periodontal tissue.

Analysis of Variance↗
PubMed2005

Treatment of human class II furcation defects using connective tissue grafts, bioabsorbable membrane, and resorbable hydroxylapatite: a comparative study.

OBJECTIVE: To evaluate clinical outcomes and assess digital subtraction radiographic changes after using bioabsorbable membrane (polyglycolic acid/polylactic acid--PGA/PLA) or a connective tissue graft (CTG) as a barrier, with or without resorbable hydroxylapatite (HA Resorb) in the treatment of mandibular class II furcations. METHODS: Fifty furcations in twenty patients with chronic periodontitis were divided into five treatment groups: (I) PGA/PLA; (II) PGA/PLA and resorbable hydroxylapatite; (III) CTG; IV) CTG and resorbable hydroxylapatite; and (V) flap debridement alone (control). Vertical and horizontal probing depths, vertical probing attachment level, gingival recession and standardized periapical radiographs were obtained at baseline and at 3, 6 and 12 months post-operatively. At 12 months, the treated teeth were exposed by re-entry to verify clinical results. RESULTS: All experimental groups (I, II, III and IV) showed statistically significant improvement in the clinical parameters and bone density as compared to the control group. However, no statistically significant differences were observed among any of the experimental groups. Percentages of complete furcation closure and sites still defined as class II furcations were, respectively, as follows: 40% and 20-30% for groups II and IV; 20% and 40% for groups I and III; 0% and 80% for control. Groups II and IV showed significant radiographic changes in bone gain at both 6 and 12 months, while groups I and III showed significant changes only at 12 months. CONCLUSIONS: (1) Guided tissue regeneration (GTR) therapy with or without resorbable hydroxylapatite showed significantly favorable results clinically and radiographically compared to flap debridement alone. (2) GTR plus resorbable hydroxylapatite (groups II and IV) showed higher percentages of complete furcation closure and more bone gain than GTR alone (groups I and III). (3) CTG may be used as an alternative to PGA/PLA bioabsorbable membrane with comparable potential. (4) Digital subtraction radiography may give higher accuracy in assessing results of periodontal therapy.

Absorbable Implants↗
PubMed2004

Expression of membrane-bound and soluble receptor activator of NF-kappaB ligand (RANKL) in human T cells.

The receptor activator of NF-kappaB ligand (RANKL) and its receptor RANK are critical regulators for immune responses as well as bone remodeling. RANKL is a type II transmembrane protein that has two forms-a membrane-anchored protein and a secreted protein. In this report, we demonstrate for the first time the kinetical expression of two forms of RANKL in human T cells using two monoclonal antibodies (mAbs) against human RANKL, which we newly derived. Freshly isolated T cells rarely expressed mRANKL, while the activation of T cells induced a substantial but minimal level of mRANKL as well as the accumulation of considerable amounts of sRANKL. The addition of the metalloprotease inhibitor KB-R8301 efficiently suppressed the release of sRANKL from activated T cells or RANKL-transfectants, and reciprocally enhanced the mRANKL expression. The membrane form of RANKL was also expressed on the infiltrating T cells in the rheumatoid synovial fluid and in the gingival tissues of patients with periodontitis. Our results demonstrate that the expression of mRANKL on T cells is strictly limited, and the majority of RANKL protein produced by T cells may be active in the soluble form after shedding. The mAbs that were derived in this study may be useful for investigating the regulation and function of RANKL in immune responses and bone remodeling.

Animals↗
PubMed2004

Costimulation via glucocorticoid-induced TNF receptor in both conventional and CD25+ regulatory CD4+ T cells.

The glucocorticoid-induced TNF receptor (GITR), which is a member of the TNF receptor family, is expressed preferentially at high levels on CD25+CD4+ regulatory T cells and plays a key role in the peripheral tolerance that is mediated by these cells. GITR is also expressed on conventional CD4+ and CD8+ T cells, and its expression is enhanced rapidly after activation. In this report we show that the GITR provides a potent costimulatory signal to both CD25+ and CD25- CD4+ T cells. GITR-mediated stimulation induced by anti-GITR mAb DTA-1 or GITR ligand transfectants efficiently augmented the proliferation of both CD25-CD4+ and CD25+CD4+ T cells under the limited dose of anti-CD3 stimulation. The augmentation of T cell activation was further confirmed by the enhanced cell cycle progression; early induction of the activation Ags, CD69 and CD25; cytokine production, such as IL-2, IFN-gamma, IL-4, and IL-10; anti-CD3-induced redirected cytotoxicity; and intracellular signaling, assessed by translocation of NF-kappaB components. GITR costimulation showed a potent ability to produce high amounts of IL-10, which resulted in counter-regulation of the enhanced proliferative responses. Our results highlight evidence that GITR acts as a potent and unique costimulator for an early CD4+ T cell activation.

Animals↗