Kack-Kyun Kim

Upregulation of Intercellular Adhesion Molecule 1 and Proinflammatory Cytokines by the Major Surface Proteins of Treponema maltophilum and Treponema lecithinolyticum, the Phylogenetic Group IV Oral Spirochetes Associated with Periodontitis and Endodontic Infections

ABSTRACT Treponema maltophilum and Treponema lecithinolyticum belong to the group IV oral spirochetes and are associated with endodontic infections, as well as periodontitis. Recently, the genes encoding the major surface proteins (Msps) of these bacteria (MspA and MspTL, respectively) were cloned and sequenced. The amino acid sequences of these proteins showed significant similarity. In this study we analyzed the functional role of these homologous proteins in human monocytic THP-1 cells and primary cultured periodontal ligament (PDL) cells using recombinant proteins. The complete genes encoding MspA and MspTL without the signal sequence were cloned into Escherichia coli by using the expression vector pQE-30. Fusion proteins tagged with N-terminal hexahistidine (recombinant MspA [rMspA] and rMspTL) were obtained, and any possible contamination of the recombinant proteins with E. coli endotoxin was removed by using polymyxin B-agarose. Flow cytometry showed that rMspA and rMspTL upregulated the expression of intercellular adhesion molecule 1 (ICAM-1) in both THP-1 and PDL cells. Expression of proinflammatory cytokines, such as interleukin-6 (IL-6) and IL-8, was also induced significantly in both cell types by the Msps, as determined by reverse transcription-PCR and an enzyme-linked immunosorbent assay, whereas IL-1β synthesis could be detected only in the THP-1 cells. The upregulation of ICAM-1, IL-6, and IL-8 was completely inhibited by pretreating the cells with an NF-κB activation inhibitor, l-1-tosylamido-2-phenylethyl chloromethyl ketone. This suggests involvement of NF-κB activation. The increased ICAM-1 and IL-8 expression in the THP-1 cells obtained with rMsps was not inhibited in the presence of the IL-1 receptor antagonist (IL-1ra), a natural inhibitor of IL-1. Our results show that the Msps of the group IV oral spirochetes may play an important role in amplifying the local immune response by continuous inflammatory cell recruitment and retention at an infection site by stimulation of expression of ICAM-1 and proinflammatory cytokines.

Cloning and sequence analysis of the gene encoding the crystalline surface layer protein of Rickettsia typhi

Abstract The nucleotide sequence of the gene (slpT) encoding the crystalline surface layer protein (SLP) of Rickettsia typhi was determined. The slpT gene consists of 4935 bp coding for a 1645-amino-acid (aa) protein containing a predicted signal peptide at the N terminus. The size of the predicted SLP exceeds the observed size (135kDa) on SDS-PAGE. The N-terminal aa sequence of the 32-kDa protein of R. typhi reported by Hackstadt et al. [Infect. Immun. 60 (1992) 159–165] was found in the C-terminal portion of the deduced aa sequence, suggesting that the product of slpT is processed into the mature SLP and the 32-kDa protein.

Adhesion of oral streptococci to experimental bracket pellicles from glandular saliva.

The aim of this study was to evaluate the functions of bracket pellicles as the binding receptors for Streptococcus mutans and Streptococcus gordonii. Four different types of orthodontic brackets were used: stainless steel, monocrystalline sapphire, polycrystalline alumina, and plastic. The bracket pellicles were formed by incubating orthodontic brackets with fresh submandibular-sublingual saliva or parotid saliva for 2 hours. The pellicles were extracted, and their components were confirmed by gel electrophoresis, immunodetection, and amino acid composition analysis. The roles of the bracket pellicles in the adhesion of oral streptococci were evaluated by incubating tritium-labeled streptococci with pellicle-transfer blots. The results showed that the salivary components adhered selectively according to type of bracket and glandular saliva. The selective adsorption was also proven by the amino acid composition profiles. Among the several salivary proteins, MG2, alpha-amylase, and the acidic proline-rich proteins provided the binding sites for S gordonii. However, none of these proteins in the bracket pellicles contributed to the adhesion of S mutans. These findings suggest that numerous salivary proteins can adhere selectively to the orthodontic brackets, and some of them contribute to the binding of S gordonii.

Synergistic Inhibitory Effect of Cationic Peptides and Antimicrobial Agents on the Growth of Oral Streptococci

Although chlorhexidine is one of the most efficacious antimicrobial agents used for the prevention of dental caries, side effects limit its application. The effects of gaegurin 6 (GGN6), an animal-derived cationic peptide, and its derivatives PTP6 and PTP12 on the growth of oral streptococci were investigated to assess the potential of these agents for use in the prevention of dental caries. The minimal inhibitory concentrations of the peptides for inhibition of the growth of oral streptococci (Streptococcus mutans, S. sobrinus, S. sanguis and S. gordonii) ranged from 1.2 to 8.2 µM. The peptides also exhibited marked synergistic antibacterial effects with chlorhexidine or xylitol. The most effective combinations (fractional inhibitory concentration index of 0.5) were xylitol with GGN6 against S. gordonii 10558 and chlorhexidine with either GGN6 or PTP6 against S. sobrinus OMZ-175. These results indicate that cationic peptides alone or in combination with chlorhexidine or xylitol might prove effective for the inhibition of the growth of cariogenic oral streptococci in situ.

In Vitro Analysis of the Efficacy of Ultrasonic Scalers and a Toothbrush for Removing Bacteria from Resorbable Blast Material Titanium Disks

BACKGROUND A resorbable blast material (RBM) surface is reported to have a higher bone-to-implant contact percentage than machined surfaces, but modified surfaces with rougher textures have been shown to favor colonization by bacteria and development of peri-implantitis. Therefore, this in vitro study compares the effects of different instruments on surface roughness and removal of bacteria from RBM titanium implant disks. METHODS RBM titanium disks were treated with various ultrasonic scaler tips and a toothbrush, and change in surface roughness was measured by confocal microscopy. The disks were incubated with bacteria, and instruments made of carbon or plastic, two metal ultrasonic scaler tips, or a toothbrush were used to remove the attached bacteria. The amount of remaining bacteria was evaluated using a crystal violet assay. RESULTS The change in surface structure following different treatment modalities was analyzed by confocal microscopy. A statistically significant decrease in the arithmetic mean value of RBM surfaces (R(a)) was observed after treatment with an ultrasonic scaler with a metal tip. The use of a metal tip (rather than a carbon or plastic tip) and brushing with dentifrice was more efficient in removing bacteria from the contaminated titanium surface according to the crystal violet assay. CONCLUSION Within the limits of this study, the use of a metal tip may be effective in removing bacteria from contaminated surfaces.

Use of Insertion Sequence Element IS1126 in a Genotyping and Transmission Study of Porphyromonas gingivalis

ABSTRACT Porphyromonas gingivalis is strongly associated with periodontal diseases and is regarded as one of the risk factors for periodontitis. Insertion sequence element IS1126-based PCR was used to investigate the genetic heterogeneity of P. gingivalis from periodontitis patients and to examine the frequency of the parent-child and spouse-spouse transmission. Two sets of IS1126-specific primers were used for the PCR. The inward primer set (PI1 and PI2), which amplifies the IS1126 fragment of approximately 690 bp, was used to identify P. gingivalis. The outward primer set (PI1RC and PI2RC), which is reverse complementary to PI1 and PI2, respectively, and amplifies the gene fragments between the adjacent IS1126 elements was used to characterize the genotypes of the P. gingivalis strains. PCR of P. gingivalis with PI1RC and PI2RC resulted in the production of two to seven amplicons, which showed a unique electrophoretic pattern in each strain (4 laboratory strains and 37 clinical isolates cultured from 12 patients with aggressive periodontitis). The usefulness of the method for transmission study was confirmed by detecting identical genotypes between the isolates and the plaque samples from which the isolates were cultured and between the plaque samples from different tooth sites in the same patient. Thirty probands with periodontal diseases and their thirty immediate family members were included in the transmission study. In 11 of 14 parent-child pairs (78.6%), P. gingivalis revealed an identical or similar band pattern, whereas 5 of 16 spouse pairs (31.25%) had this similarity. These results show that IS1126-based PCR for genotyping P. gingivalis has a highly discriminating potential with reproducible data and is a simple and reliable method for a transmission study.

A new method for rapid screening of bacterial species- or subspecies-specific DNA probes

A simple assay for the rapid screening of bacterial species- or subspecies-specific DNA probes for the random cloning method is presented, involving the use of genomic DNAs as probes and recombinant plasmid DNAs containing genomic DNA digested with HindIII as targets. The optimal amount of target DNAs and the concentration of digoxigenin-labeled genomic DNA probes were 20 ng and 100 ng ml(-1) (or 10 ng and 200 ng ml(-1)), respectively. The method was applied to the development of Fusobacterium nucleatum subspecies-specific probes. Our results showed that four out of 96 probes were F. nucleatum subspecies-specific, which was confirmed by Southern blot analysis. Our results indicate that the new method can be used for the rapid screening of species- or subspecies-specific probes.

Treatment with various ultrasonic scaler tips affects efficiency of brushing of SLA titanium discs.

PurposeThe dental implant surface will be colonized by bacteria once it is exposed to the oral cavity. It is necessary to keep the titanium surface clean to prevent peri-implant diseases. Mechanical instrumentation is widely used, but this may cause damage to the implant surfaces. There is limited information whether surface change resulting from instrumentation influences the adherence of bacteria to the implant surface or influences the ease of removal of bacteria from the titanium surface by daily brushing. Therefore, this in vitro study was performed (1) to evaluate removal of Porphyromonas gingivalis from sand-blasted and acid-etched (SLA) titanium discs after the discs were instrumented by various ultrasonic scaler tips or brushed with a toothbrush with dentifrice using crystal violet assay and scanning electron microscopy (SEM), and (2) to assess the change of surface roughness after the treated discs were brushed with a toothbrush with dentifrice. Materials and MethodsSLA discs were treated with various ultrasonic scaler tips and a toothbrush. The titanium discs were incubated with P. gingivalis for 2 days after treatment (ultrasonic scales tips and brush) and then the disc surfaces were brushed for total of 40 seconds (20 seconds, two cycles) with a toothbrush with dentifrice. Differences in adhering bacteria were evaluated using crystal violet assay and SEM. Surface roughness of the treated discs after brushing with dentifrice was measured using confocal microscopy. ResultsThe change of surface structure was observed after different treatment modalities. Removal of bacteria was increased with the longer time of brushing, and the ultrasonic metal tip group displayed a significantly lower number of bacteria after brushing when compared to other groups. ConclusionsWithin the limits of this study, it may be suggested that when SLA surface is exposed to the oral cavity, it should firstly be treated with metal tips to smoothen the rough surface and thereby reduce attachment of bacteria and facilitate the removal of bacteria by daily oral hygiene procedures.

Protection against Ovariectomy-Induced Bone Loss by Tranilast

Background Tranilast (N-(3′,4′-dimethoxycinnamonyl) anthranilic acid) has been shown to be therapeutically effective, exerting anti-inflammatory and anti-oxidative effects via acting on macrophage. We hypothesized that Tranilast may protect against oxidative stress-induced bone loss via action in osteoclasts (OCs) that shares precursors with macrophage. Methodology and Principal Findings To elucidate the role of Tranilast, ovariectomy (OVX)-induced bone loss in vivo and OC differentiation in vitro were evaluated by µCT and tartrate-resistant acid phosphatase staining, respectively. Oral administration of Tranilast protected against OVX-induced bone loss with decreased serum level of reactive oxygen species (ROS) in mice. Tranilast inhibited OC formation in vitro. Decreased osteoclastogenesis by Tranilast was due to a defect of receptor activator of nuclear factor-κB ligand (RANKL) signaling, at least partly via decreased activation of nuclear factor-κB and reduced induction and nuclear translocation of nuclear factor of activated T cells, cytoplasmic 1 (or NFAT2). Tranilast also decreased RANKL-induced a long lasting ROS level as well as TGF-β to inhibit osteoclastogenesis. Reduced ROS caused by Tranilast was due to the induction of ROS scavenging enzymes (peroxiredoxin 1, heme oxygenase-1, and glutathione peroxidase 1) as well as impaired ROS generation. Conclusions/Significance Our data suggests the therapeutic potential of Tranilast for amelioration of bone loss and oxidative stress due to loss of ovarian function.

Instrumentation With Ultrasonic Scalers Facilitates Cleaning of the Sandblasted and Acid-Etched Titanium Implants

Mechanical instrumentation is widely used to debride dental implants, but this may alter the surface properties of titanium, which in turn may influence bacterial adhesion and make it more difficult to remove the biofilm. This in vitro study was performed (1) to assess the amount of biofilm formation on a sand-blasted and acid-etched titanium fixture treated with ultrasonic scalers with metal, plastic, and carbon tips and (2) to evaluate how this treatment of titanium surfaces affects implant cleaning by brushing with dentifrice. The titanium fixtures were treated with various ultrasonic scaler tips, and surface roughness parameters were measured by confocal microscopy. Biofilm was formed on the treated fixtures by using pooled saliva from 10 subjects, and the quantity of the adherent bacteria was compared with crystal violet assay. The fixture surfaces with biofilm were brushed for total of 30 seconds with a toothbrush with dentifrice. The bacteria remaining on the brushed fixture surfaces were quantified by scanning electron microscopy. Surface changes were evident, and the changes of the surfaces were more discernible when metal tips were used. A statistically significant decrease in roughness value (arithmetic mean height of the surface) was seen in the 2 metal-tip groups and the single plastic-tip group. After brushing with dentifrice, the treated surfaces in all the treatment groups showed significantly fewer bacteria compared with the untreated surfaces in the control group, and the parts of the surfaces left untreated in the test groups. Within the limits of this study, treatment of titanium fixture surfaces with ultrasonic metal, plastic, or carbon tips significantly enhanced the bacterial removal efficacy of brushing. Thorough instrumentation that smooths the whole exposed surface may facilitate maintenance of the implants.