Kaname Hirai

Human gingival fibroblasts are critical in sustaining inflammation in periodontal disease.

BACKGROUND AND OBJECTIVE A major factor in the pathogenesis of periodontal disease, which is one of the biofilm infectious diseases, is thought to be lipopolysaccharide (LPS), owing to its ability to cause inflammation and promote tissue destruction. Moreover, the elimination of pathogens and their component LPSs is essential for the successful treatment of periodontal disease. Lipopolysaccharide tolerance is a mechanism that prevents excessive and prolonged responses of monocytes and macrophages to LPS. Since persistence of inflammation is necessary for inflammatory cytokine production, cells other than monocytes and macrophages are thought to maintain the production of cytokines in the presence of LPS. In this study, we investigated whether human gingival fibroblasts (HGFs), the most abundant structural cell in periodontal tissue, might be able to maintain inflammatory cytokine production in the presence of LPS bynot displaying LPS tolerance. MATERIAL AND METHODS Human gingival fibroblasts were pretreated with LPS (from Porphyromonas gingivalis and Escherichia coli) and then treated with LPS, and the amounts of interleukin (IL)-6 and IL-8 in the cell culture supernatants were measured. The expression of negative regulators of LPS signalling (suppressor of cytokine signalling-1, interleukin-1 receptor-associated-kinase M and SH2 domain-containing inositol-5-phosphatase-1) was also examined in LPS-treated HGFs. RESULTS Human gingival fibroblasts did not display LPS tolerance but maintained production of IL-6 and IL-8 when pretreated with LPS, followed by secondary LPS treatment. Lipopolysaccharide-treated HGFs did not express negative regulators. CONCLUSION These results demonstrate that HGFs do not show LPS tolerance and suggest that this characteristic of HGFs sustains the inflammatory response in the presence of virulence factors.

Sixteen Homologs of the Mex-Type Multidrug Resistance Efflux Pump in Bacteroides fragilis

ABSTRACT Sixteen homologs of multidrug resistance efflux pump operons of the resistance-nodulation-cell division (RND) family were found in the Bacteroides fragilis genome sequence by homology searches. Disruption mutants were made to the mexB homologs of the four genes most similar to Pseudomonas aeruginosa mexB. Reverse transcription-PCR was conducted and indicated that the genes were transcribed in a polycistronic fashion and that the promoter was upstream of bmeA (the mexA homolog). One of these disruption mutants (in bmeB, the mexB homolog) was more susceptible than the parental strain to certain cephems, polypeptide antibiotics, fusidic acid, novobiocin, and puromycin. The gene for this homolog and the adjacent upstream gene, bmeA, were cloned in a hypersensitive Escherichia coli host. The resultant transformants carrying B. fragilis bmeAB were more resistant to certain agents; these agents also had lower MICs for the B. fragilis bmeB disruption mutants than for the parental strain. The putative efflux pump operon is composed of bmeA, bmeB, and bmeC (a putative outer membrane channel protein homologous with OprM). Addition of the efflux pump inhibitors, carbonyl cyanide m-chlorophenylhydrazone (a proton conductor that eliminates the energy source) and Phe-Arg β-naphthylamide (MC-207,110) (the first specific inhibitor described for RND pumps in P. aeruginosa), resulted in lowered MICs in the parental strain but not in the bmeB disruption mutant, indicating that the bmeB pump is affected by these inhibitors. This is the first description of RND type pumps in the genus Bacteroides.

The short-term effects of various oral care methods in dependent elderly: Comparison between toothbrushing, tongue cleaning with sponge brush and wiping on oral mucous membrane by chlorhexidine

OBJECTIVES To explore the short-term effects from toothbrushing, tongue cleaning with sponge brush and wiping on oral mucous membrane by chlorhexidine. BACKGROUND Numerous reports have been seen in recent years proving the effectiveness of mouth cleaning with a toothbrush for the prevention of respiratory infections among the dependent elderly. However, the short-term effects from each oral care method have not yet been clarified. Hence, an investigation was conducted by having each subject independently perform various oral care methods for five consecutive days. MATERIALS AND METHODS The subjects consisted of 12 assistance-dependent elderly who have difficulties with tooth brushing by themselves, have 10 or more residual teeth and are not yet using plate dentures. After the pre-intervention examination, each of the following oral care methods were performed on the same subject on an approximately three week basis: 1) Tooth brushing 2) Tongue cleaning with sponge brush 3) Wiping on oral mucous with sponge brush by chlorhexidine. Each method was performed independently, once a day for 5 consecutive days and the subjects were reexamined on the sixth day for comparative verification. RESULTS Consequently, toothbrushing decreased the plaque index and gingival index significantly and an improvement of oral malodour was also acknowledged (p < 0.01). Tongue cleaning with a sponge brush decreased the tongue coat score significantly (p < 0.05) and oral malodour was also improved (p < 0.01). Wiping on oral mucous with a sponge brush soaked in chlorhexidine significantly decreased opportunistic infections in the pharynx region (p < 0.05). CONCLUSIONS It was suggested that the use of not only a toothbrush but also chlorhexidine gluconate may be indicated for dependent elderly people in whom pathogens of opportunistic infection are detected.

Porphyromonas gingivalis mutant defective in a putative extracytoplasmic function sigma factor shows a mutator phenotype.

INTRODUCTION Porphyromonas gingivalis is implicated as a major pathogen in the development and progression of chronic periodontitis. P. gingivalis must possess the ability to tolerate stress signals outside the cytoplasmic membrane by transcriptional activation of genes encoding proteins involved in defense or repair processes. Some bacteria utilize a distinct subfamily of sigma factors to regulate extracytoplasmic function (hence termed the ECF subfamily). METHODS To elucidate their role in P. gingivalis, a chromosomal mutant carrying a disruption of an ECF sigma factor PG1318-encoding gene was constructed. Hemagglutination and proteolytic activities were measured in the PG1318-defective mutant. Reverse transcription-polymerase chain reaction (RT-PCR) analysis and southern blot analysis were used to assess transcription of kgp in the PG1318-defective mutant. Frequency of spontaneous mutation that conferred resistance to l-trifluoromethionine was measured in the PG1318-defective mutant. RESULTS The PG1318-defective mutant formed non-pigmented colonies on blood agar plates at a relatively high frequency. Arginine-specific and lysine-specific proteinase activities of the non-pigmented variants were remarkably decreased compared with those of the parent strain and the pigmented variants. RT-PCR analysis showed that kgp was not transcribed in some non-pigmented variants and southern blot analysis revealed that there was a deletion in their kgp region. Frequency of mutation conferring resistance to l-trifluoromethionine was significantly higher in the PG1318-defective mutant than in the wild-type. CONCLUSION These results suggest that PG1318 plays a role in the regulation of mutation frequency in the bacterium.

Protein kinase A enhances lipopolysaccharide-induced IL-6, IL-8, and PGE2 production by human gingival fibroblasts

ObjectivePeriodontal disease is accompanied by inflammation of the gingiva and destruction of periodontal tissues, leading to alveolar bone loss in severe clinical cases. Interleukin (IL)-6, IL-8, and the chemical mediator prostaglandin E2 (PGE2) are known to play important roles in inflammatory responses and tissue degradation.Recently, we reported that the protein kinase A (PKA) inhibitor H-89 suppresses lipopolysaccharide (LPS)-induced IL-8 production by human gingival fibroblasts (HGFs). In the present study, the relevance of the PKA activity and two PKA-activating drugs, aminophylline and adrenaline, to LPS-induced inflammatory cytokines (IL-6 and IL-8) and PGE2 by HGFs were examined.MethodsHGFs were treated with LPS from Porphyromonas gingivalis and H-89, the cAMP analog dibutyryl cyclic AMP (dbcAMP), aminophylline, or adrenaline. After 24 h, IL-6, IL-8, and PGE2 levels were evaluated by ELISA.ResultsH-89 did not affect LPS-induced IL-6 production, but suppressed IL-8 and PGE2 production. In contrast, dbcAMP significantly increased LPS-induced IL-6, IL-8, and PGE2 production. Up to 10 μg/ml of aminophylline did not affect LPS-induced IL-6, IL-8, or PGE2 production, but they were significantly increased at 100 μg/ml. Similarly, 0.01 μg/ml of adrenaline did not affect LPS-induced IL-6, IL-8, or PGE2 production, but they were significantly increased at concentrations of 0.1 and 1 μg/ml. In the absence of LPS, H-89, dbcAMP, aminophylline, and adrenaline had no relevance to IL-6, IL-8, or PGE2 production.ConclusionThese results suggest that the PKA pathway, and also PKA-activating drugs, enhance LPS-induced IL-6, IL-8, and PGE2 production by HGFs. However, aminophylline may not have an effect on the production of these molecules at concentrations used in clinical settings (8 to 20 μg/ml in serum). These results suggest that aminophylline does not affect inflammatory responses in periodontal disease.

Isolation and properties of a tripeptidyl peptidase from a periodontal pathogen Prevotella nigrescens

Prolyltripeptidyl amino peptidase activity was found in a crude extract of Prevotella nigrescens and this enzyme was purified by procedures including concentration with ammonium sulfate, ion exchange chromatography, gel filtration, and isoelectric focusing. This peptidase hydrolyzed Ala-Ala-Pro-p-nitroanilide as well as Ala-Phe-Pro-p-nitroanilide. Furthermore, several p-nitroanilide derivatives of dipeptides with a proline residue in the second position from the amino-terminal end (Xaa-Pro) were also cleaved detectably. The molecular mass of this tripeptidase was calculated as 56 kDa and its isoelectric point was 5.8. The enzyme was inactivated completely by heating at 60 degrees C for 5 min and inhibited significantly by specific serine enzyme inhibitors.

Cellular locations of proteinases and association with vesicles in porphyromonas gingivalis

We found that locations of arginine-specific gingipain (RGP) in the cellular fractions in the crude extract, envelope, vesicles, and culture supernatants were 48%, 16%, 17%, and 31%, respectively, and the corresponding values of lysine-specific gingipain (KGP) were 47%, 10%, 7%, and 36%, respectively. Although the molecular mass of RGP in the culture supernatant had been determined as 43 kDa, and that of KGP had been as 48 kDa, molecular masses of both proteinases solubilized from the vesicles were estimated to be over 1,500 kDa, since they eluted in the void volume of the column in the gel filtration on Sephacryl S-300. There was no reduction of molecular size by the following treatment with SDS, high-concentration NaCl, or urea. Interestingly, the occurrence of the macromolecular forms could not observed in other enzymes tested such as monopeptidyl, dipeptidyl, and tripeptidyl peptidases, as well as alkaline phosphatase. Therefore, occurrence of the macromolecular forms may be restricted to the proteinases. When the vesicle and culture supernatants containing free RGP and KGP were mixed and incubated, neither RGP nor KGP seemed to bind to vesicles. RGP bound to the vesicle was found to be more stable to heat treatment than the free form, suggesting that association of RGP with the vesicle caused heat stability of this enzyme.

Research articleSome binding properties of the envelope of Porphyromonas gingivalis to hemoglobin

Porphyromonas gingivalis was found to bind to hemoproteins (hemoglobin, myoglobin, catalase, cytochrome c) and the binding properties of the envelope of P. gingivalis to hemoglobin were investigated. Maximum amount of hemoglobin bound to 1 mg of the envelope was 58 μg. No significant binding was observed at 4°C and the binding was inhibited strongly by tosyl-l-lysine chloromethyl ketone, Leupeptin, EDTA and partially by meta-periodate. Heating of the envelope at 70°C for 15 min resulted in complete loss of the binding activity. The binding activity of the envelope was not influenced by the treatment with the endogenous proteases. The envelope saturated with hemoglobin could no longer bind to other hemoproteins tested, indicating that binding site for these hemoproteins are common.

Hemoglobin binding activity and hemoglobin-binding protein of Prevotella nigrescens

Prevotella nigrescens, lacking siderophores was found to bind to the hemoproteins. The binding was observed also in the envelope which was prepared by sonication of the cell. The binding occurred in the pH-dependent manner; the binding was observed below neutral pHs of the incubation mixtures but only slightly observed in the neutral and alkaline pHs. Furthermore, hemoglobin bound to the envelope was dissociated at high pHs buffers. Maximum amounts of hemoglobin bound to 1 mg envelope was 51.2 μg. Kd for the reaction at pH 5.0 was 2.1 × 10-10M (210 pM). From the dot blot assay, hemoglobin could bind to a protein solubilized from the envelope by a detergent, referred to as hemoglobin-binding protein (HbBP), then it was purified by the sequential procedures of ion exchange chromatography, affinity chromatography and isoelectric focusing. Molecular weight and isoelectric point of the HbBP were 46 kDa and 6.1, respectively.