Search

Treponema denticola, an anaerobic spirochete found mainly in the oral cavity, is associated with periodontal disease and has a variety of virulence factors. Although in vitro studies have shown that T. denticola is able to penetrate epithelial cell monolayers, its effect on the epithelial barrier junction is not known. Human gingival epithelial cells are closely associated with adjacent membranes, forming barriers in the presence of tight junction proteins, including zonula occludens-1 (ZO-1), claudin-1, and occludin. Tight junction proteins are also expressed by Madin-Darby canine kidney (MDCK) cells in culture. In this study, the MDCK cell profile was investigated following infection with T. denticola (ATCC 35405) wild-type, as well as with its dentilisin-deficient mutant, K1. Basolateral exposure of MDCK cell monolayers to T. denticola at a multiplicity of infection (MOI) of 104 resulted in a decrease in transepithelial electrical resistance (TER). Transepithelial electrical resistance in MDCK cell monolayers also decreased following apical exposure to T. denticola (MOI=104), although this took longer with basolateral exposure. The effect on the TER was time-dependent and required the presence of live bacteria. Meanwhile, MDCK cell viability showed a decrease with either basolateral or apical exposure. Immunofluorescence analysis demonstrated decreases in the amounts of immunoreactive ZO-1 and claudin-1 in association with disruption of cell-cell junctions in MDCK cells exposed apically or basolaterally to T. denticola. Western blot analysis demonstrated degradation of ZO-1 and claudin-1 in culture lysates derived from T. denticola-exposed MDCK cells, suggesting a bacteria-induced protease capable of cleaving these tight junction proteins.

The objective of this study was to investigate the relationship between various clinical factors and bacterial contamination of bone chips (BC) collected during dental implant surgery and to elucidate how bacterial contamination might be minimized.Implants were installed in 55 partially edentulous patients (36 men and 19 women), among whom the relationship between various clinical factors and bacterial contamination of BC collected by bone trap was investigated in 37. The effect of rinsing with a saline on BC was determined in 18 patients. Number of contaminating microorganisms was expressed as colony-forming units (CFUs).CFUs in the maxilla were lower than those in the mandible (P0.01). CFUs at the incisors or canines were lower than those at the premolars or molars (P0.01). Logistic regression analysis revealed a relationship between average bacterial count and duration of surgery (odds ratio, 1.046; 95% CI, 1.012-1.081). Rinsing of BC reduced bacterial contamination.Duration of surgery is a major clinical factor affecting contamination risk in BC, and rinsing of BC with a sterile saline solution reduces bacterial number.

Porphyromonas gingivalis, a pathogen involved in the development of chronic periodontitis, has a number of major virulence factors, among which are its surface cysteine protease gingipains. The purpose of this study was to investigate the feasibility of inducing protective antibodies against P. gingivalis by means of immunization with recombinant Lactococcus lactis expressing the 44-kDa gingipain adhesion/hemagglutinin domain (Hgp44). Part of the Hgp44 sequence encoding the first 314 amino acid residues, residues 188-251, and residues 354-393 was amplified and inserted into shuttle plasmid pSGANC332, with the resulting chimeric plasmids designated as pISTY210, pCOL, and pSHGRP44A, respectively. After confirming the clone sequences, expression of recombinant proteins was investigated by immunoblot. The results revealed that while pISTY210 and pCOL both expressed the Hgp44 antigen on the surface of L. lactis, the level of expression was quite low. To enhance expression of the protein on the surface of the cells, cysteine residues were changed to serine residues by site-directed mutagenesis. Replacement of 3 out of 5 cysteine residues (pISTY213) significantly increased expression of the recombinant protein on the surface of the bacteria. Interestingly, replacement of the 4th cysteine residue (pISTY215) reduced antigenicity of the recombinant protein. These results indicate that expression of Hgp44 on the surface of L. lactis cells requires the replacement of several key cysteine residues, and that L. lactis expressing this antigen could be a promising candidate for immunization against P. gingivalis-induced periodontitis.

Periapical periodontitis usually results from microbial infection, with these microorganisms occasionally migrating to the root canal, which can lead to further, potentially life-threatening, complications. Here, the susceptibility of 27 bacterial strains to various antimicrobial agents was evaluated. These strains comprised 13 species; 16 of the strains were clinical isolates from periapical lesions. Each strain was inoculated onto blood agar plates containing one of the antimicrobial agents. The plates were incubated anaerobically at 37°C for 96 hr and the minimal inhibitory concentrations (MICs) determined. Ten strains required an MIC of 32 μg/ml or greater for amoxicillin, 6 for cefmetazole, and 5 for cefcapene among β-lactam antibiotics; 8 strains required an MIC of 32 μg/ml or greater for clindamycin, 4 for azithromycin, and 11 for clarithromycin among macrolide antibiotics; 3 strains required an MIC of 32 μg/ml or greater for ciprofloxacin and 2 for ofloxacin among fluoroquinolones. The effect of cefcapene on 5 strains was evaluated after biofilm formation to investigate the relationship between biofilm formation and susceptibility. All strains showed a decrease in susceptibility after biofilm formation. The results revealed that several antimicrobial agents commonly used in a clinical setting, including amoxicillin, cefmetazole, and clindamycin, are potentially effective in the treatment of orofacial odontogenic infections. The development of resistant strains, however, means that this can no longer be guaranteed. In addition, azithromycin, ciprofloxacin, and ofloxacin were more effective than the 3 β-lactam antibiotics tested. These results suggest that sensitivity testing is needed if odontogenic infections are to be treated safely and effectively.

The formation of biofilm by anaerobic, Gram-negative bacteria in the subgingival crevice plays an important role in the development of chronic periodontitis. The aim of this study was to characterize the role of coaggregation between Fusobacterium nucleatum and Prevotella species in biofilm formation. Coaggregation between F. nucleatum and Prevotella species was determined by visual assay. Effect of co-culture of the species on biofilm formation was assessed by crystal violet staining. Effect of soluble factor on biofilm formation was also examined using culture supernatant and two-compartment co-culture separated by a porous membrane. Production of autoinducer-2 (AI-2) by the organisms was evaluated using Vibrio harveyi BB170. Cells of all F. nucleatum strains coaggregated with Prevotella intermedia or Prevotella nigrescens with a score of 1-4. Addition of ethylenediamine tetraacetic acid or l-lysine inhibited coaggregation. Coaggregation disappeared after heating of P. intermedia or P. nigrescens cells, or Proteinase K treatment of P. nigrescens cells. Co-culture of F. nucleatum ATCC 25586 with P. intermedia or P. nigrescens strains increased biofilm formation compared with single culture (p < 0.01); co-culture with culture supernatant of these strains, however, did not enhance biofilm formation by F. nucleatum. Production of AI-2 in Prevotella species was not related to enhancement of biofilm formation by F. nucleatum. These findings indicate that physical contact by coaggregation of F. nucleatum strains with P. intermedia or P. nigrescens plays a key role in the formation of biofilm by these strains.

Objective Periodontitis, an infectious disease caused by periodontopathic bacteria, including Porphyromonas gingivalis, is reported to be accelerated by stress, under which noradrenaline levels are increased in the bloodstream. The purpose of this study was to evaluate the effects of noradrenaline on P. gingivalis. Design P. gingivalis was incubated in the presence of 25 μM, 50 μM, or 100 μM adrenaline or noradrenaline at 37 °C for 12, 24 or 36 h and growth was evaluated by OD660. Auto-inducer-2 (AI-2) was measured by luminescence of Vibrio harveyi BB 170. Expression of P. gingivalis genes was evaluated using a microarray and RT-PCR. Rgp activity of arg-gingipainA and B (Rgp) was measured with a synthetic substrate. Results Growth of P. gingivalis FDC381 was inhibited by noradrenaline at 24 and 36 h. Growth inhibition by noradrenaline increased dose-dependently. Inhibition of growth partially recovered with addition of propranolol. AI-2 production from P. gingivalis showed a marked decrease with addition of noradrenaline compared with peak production levels in the control group. Microarray analysis revealed an increase in expression in 18 genes and a decrease in expression in 2 genes. Amongst these genes, expression of the protease arg-gingipainB (RgpB) gene, a major virulence factor of P. gingivalis, was further analysed. Expression of rgpB showed a significant increase with addition of noradrenaline, which was partially reduced by addition of propranolol. Cell-associated Rgp activity also increased with addition of noradrenaline. Conclusions These results suggest that stressors influence the expression of the virulence factors of P. gingivalis via noradrenaline.

Porphyromonas gingivalis and Treponema denticola are major pathogens of periodontal disease. Coaggregation between microorganisms plays a key role in the colonization of the gingival crevice and the organization of periodontopathic biofilms. We investigated the involvement of surface ligands of P. gingivalis in coaggregation. Two triple mutants of P. gingivalis lacking Arg-gingipain A (RgpA), Lys-gingipain (Kgp) and Hemagglutinin A (HagA) or RgpA, Arg-gingipain B (RgpB) and Kgp showed significantly decreased coaggregation with T. denticola, whereas coaggregation with a major fimbriae (FimA)-deficient mutant was the same as that with the P. gingivalis wild-type parent strain. rgpA, kgp and hagA code for proteins that contain 44 kDa Hgp44 adhesin domains. The coaggregation activity of an rgpA kgp mutant was significantly higher than that of the rgpA kgp hagA mutant. Furthermore, anti-Hgp44 immunoglobulin G reduced coaggregation between P. gingivalis wild type and T. denticola. Treponema denticola sonicates adhered to recombinant Rgp domains. Coaggregation following co-culture of the rgpA kgp hagA mutant expressing the RgpB protease with the rgpA rgpB kgp mutant expressing the unprocessed HagA protein was enhanced compared with that of each triple mutant with T. denticola. These results indicate that the processed P. gingivalis surface Hgp44 domains are key adhesion factors for coaggregation with T. denticola.

Objective The radical anion of oxygen (O − ) is extremely oxidative and shows high reactivity. In this study, the antibacterial activity of water super-oxidised water containing high concentration of O − (O − -water) was tested against cultured planktonic cells of cariogenic bacteria, periodontopathic bacteria and Candida albicans . Methods O − -water was prepared using the AOE-750 (Oxy Japan Corporation, Japan) and its antibacterial activity against pure culture of Streptococcus sobrinus , Porphyromonas gingivalis , Prevotella intermedia , Aggregatibacter actinomycetemcomitans , Fusobacterium nucleatum and C. albicans evaluated. Each oral microorganism (10 4 to 10 8 CFU/ml) was exposed to three concentrations of O − -water at room temperature or 37 °C for 15 s to 24 h. Results Exposure to O − -water resulted in a bactericidal effect against all cariogenic and periodontopathic bacteria tested. No significant fungicidal effect was observed on C. albicans , however. Conclusion The results demonstrate that O − -water exerts an antibacterial effect on cariogenic and periodontopathic bacteria.

Yamazaki-Kubota T, Miyamoto M, Sano Y, Kusumoto M, Yonezu T, Sugita K, Okuda K, Yakushiji M, Ishihara K. Analysis of matrix metalloproteinase (MMP-8 and MMP-2) activity in gingival crevicular fluid from children with Down’s syndrome. J Periodont Res 2010; doi: 10.1111/j.1600-0765.2009.01214.x. © 2009 John Wiley & Sons A/S Background and Objective: High levels of colonization by periodontopathic bacteria and a high prevalence of chronic inflammatory periodontal disease have been reported in children with Down’s syndrome. Matrix metalloproteinases (MMPs) are mediators of extracellular matrix degradation and remodelling, and are deeply involved in the course of periodontal disease. To clarify the relationship between Down’s syndrome and periodontitis, we investigated levels of MMP-2 and MMP-8 in gingival crevicular fluid (GCF) and detection of periodontopathic bacteria from subgingival plaque. Material and Methods: Samples of GCF and plaque were isolated from central incisors. Levels of MMPs were evaluated by enzyme-linked immunosorbent assay, and periodontopathic bacteria were detected by polymerase chain reaction. Results: Levels of MMP-2 and MMP-8 in Down’s syndrome patients were higher than those in healthy control subjects. In the Down’s syndrome group, increases in these MMPs were observed in GCF from patients with an oral hygiene index score of

Aggregatibactor actinomycetemcomitans colonizes human periodontal lesions and is implicated in both aggressive periodontitis and chronic periodontitis. Clinical isolated colonies of A. actinomycetemcomitans were rough type. The rough type has a remarkable ability to adhere tenaciously to solid surfaces and colonize firmly. Rough type colonies change into smooth type colonies during the course of repeated inoculation and biofilm-forming activity ceases. Adherence by A. actinomycetemcomitans is mediated by the tight-adherence (tad) gene locus, which includes flp, rcpA and rcpB. In this study, we investigated the relationship between its biofilm-forming ability and expression of the flp, rcpA and rcpB genes associated with fimbriae protein production. First, we changed rough type strain organized biofilm on glass into smooth type and confirmed it by observation of biofilm on glass surfaces. Then, we carried out Real-Time PCR and found that expression of the rcpA and rcpB genes was clearly reduced in smooth type colonies. This suggests that expression of rcpA and rcpB plays a key role in biofilm formation by A. actinomycetemcomitans strains and the establishment of persistent infections in periodontal lesions.

Purpose: Prevention of peri-implantitis is essential for the success of implant rehabilitation. Infection by periodontopathic bacteria is a major cause of peri-implantitis. The aim of the present study was to identify the source of peri-implant colonization by periodontopathic bacteria. Materials and Methods: Twenty-one patients with implants were enrolled in the study. Subgingival plaque samples from the adjacent, occluding, and contralateral natural teeth were collected prior to second-stage surgery. Samples from implant sulci were then obtained 2 weeks later. Detection of periodontopathic bacteria was performed by the polymerase chain reaction. Results: The detection rates for Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium nucleatum in all subgingival samples from natural teeth were similar to that in the peri-implant sulci. Multiple logistic regression analysis revealed an association between the detection of A. actinomycetemcomitans, Prevotella intermedia, Porphyromonas gingivalis, Treponema denticola, and F. nucleatum in the gingival crevices of adjacent teeth and that of the peri-implant sulcus, but no association for Tannerella forsythia. Conclusions: The present findings suggest that colonization by A. actinomycetemcomitans, Prevotella intermedia, Porphyromonas gingivalis, Treponema denticola, and F. nucleatum at the implant sulcus was affected by these microorganisms in the gingival crevice of adjacent teeth rather than those on occluding and contralateral teeth.

Introduction: Microorganisms are able to survive and induce persistent infection in periapical tissues. The aim of this study was to investigate the composition of the microflora of persistent apical periodontitis lesions. Methods: Twenty apical lesion samples were obtained from 20 patients with chronic apical periodontitis by root end surgery and processed using aerobic or anaerobic culture techniques. All isolated strains were identified by 16S ribosomal DNA sequence analysis. Results: Seventy-four strains were isolated, belonging to 31 bacterial species obtained from the 20 apical lesions that were isolated. The majority of the strains were facultative anaerobes (51.6%). Propionibacterium acnes, Staphylococcus epidermidis, Pseudomonas aeruginosa and Fusobacterium nucleatum were isolated from 16.2, 9.5, 6.8 and 5.4% of the samples, respectively. Fifteen samples harboured more than one species. The predominant association was P. acnes, S. epidermidis and F. nucleatum. Conclusion: The microbiota of persistent apical periodontitis lesions is composed by diverse types of microorganisms with biofilm-forming capacity, including P. acnes, S. epidermidis and F. nucleatum.

The purpose of this study was to determine the time-window of infection for Streptococcus mutans between mother and child in Japan and which factors affected colonization by this microorganism. After obtaining informed consent, saliva was collected from the mothers and their children. These samples were serially diluted and inoculated onto Mitis-Salivarius agar and Mitis-Salivarius bacitracin plates, and then cultured under anaerobic conditions at 37°C for 5 days. Isolated mutans streptococci were identified by conventional biochemical characteristics and species-specific polymerase chain reaction based on the dextranase gene. No mutans streptococci groups were isolated from 0–5-month-old children with no erupted teeth. No statistically significant difference was found in detection rates in S. mutans among mother's milk, bottled milk or mixture. Cross-sectional study revealed that detection rates of S. mutans in the saliva samples increased with number of erupted teeth. Pulse-field gel-electrophoresis patterns obtained from isolated S. mutans using SmaI from the mothers and their children indicated that colonization by S. mutans in some Japanese children resulted from vertical transmission. In strains in which vertical transmission was observed, the mutacin IV and Smb A and B genes were detected from 8 out of 12 isolates. These results suggest that the window of infection for S. mutans is at around 6–23 months, and that colonization by this microorganism is mainly affected by number of erupted teeth.

Silent aspiration of oral microorganisms is a major cause of aspiration pneumonia. To establish oral hygiene criteria for the prevention of aspiration pneumonia in edentulous elderly persons, we investigated the relationship between presence of tongue-coating and number of oral bacteria in saliva and episodes of pneumonia. A total of 71 edentulous Japanese people aged 65 years or older living in nursing homes were enrolled in the study. A tongue plaque index (TPI) was used to evaluate quantity of tongue-coating, with TPI0 signifying no tongue-coating and TPI1 signifying presence of tongue-coating. Edentate elderly with TPI1 demonstrated significantly higher salivary bacterial counts than those with TPI0 (p0.05). The number of elderly patients developing aspiration pneumonia was larger (p0.005) in patients with TPI-based poor scores (average TPI0.5) than in those with TPI-based good scores. The relative risk of developing pneumonia in the good tongue hygiene group compared with in the poor tongue hygiene group was 0.12, 95% confidence interval (CI): 0.02-0.9. The results demonstrate that tongue-coating is associated with number of viable salivary bacterial cells and development of aspiration pneumonia, suggesting that tongue-coating is a risk indicator of aspiration pneumonia in edentate subjects.

Background and Objective: The purpose of this study was to determine the time of infection by anaerobic gram-negative rods associated with periodontal disease, and to clarify their transmission from mother to child. Material and Methods: Seventy-eight Japanese children (including 10 siblings), aged from 3 to 9 years, and 68 mothers, were enrolled in this study. Colonization by 11 periodontal bacterial species was determined using polymerase chain reaction amplification of samples of subgingival plaque obtained from the children and their mothers. Results: The detection rates of Porphyromonas gingivalis, Tannerella forsythensis and Treponema denticola increased in children after the age of 6 years. We found a high consistency in colonization by P. gingivalis, T. denticola, Prevotella intermedia and Prevotella nigrescens in 9 of the 10 siblings. The average number of bacterial species in plaque samples harboring Fusobacterium nucleatum and/or Fusobacterium periodonticum was significantly greater than in those without, in both children and mothers. Kappa statistical analysis revealed that the detection of Capnocytophaga gingivalis, Capnocytophaga ochracea, Campylobacter rectus and T. denticola in children was consistent with that in the mother. Conclusion: Periodontal bacterial colonization in Japanese children increased with age and was associated with F. nucleatum and/or periodonticum, and the bacterial flora in children was similar to that in their mothers.

Periodontal disease, for which smoking is a known risk factor, is infectious, and is associated with oral biofilm. Cytokines mediate and regulate immune and inflammatory responses. Lipopolysaccharide produced by periodontopathic bacteria plays a role in the progression of periodontitis. The effect of nicotine on cytokine production in mice was evaluated in this study. Nicotine (10 or 200 microg mouse(-1)) was administered intraperitoneally to 4-week-old female BALB/c mice, once a day, for 49 days. Control mice received injections of phosphate-buffered saline. Blood was collected from all mice and serum IL-6, IL-10, tumor necrosis factor (TNF)-alpha and IFN-gamma levels were measured by an enzyme-linked immunosorbent assay on the 42nd day. IL-6, IL-10 and IFN-gamma levels in the nicotine-treated mice were higher than those in the control mice. However, no differences were found in TNF-alpha levels between nicotine-treated and control mice. Lipopolysaccharide (20 microg mouse(-1)) purified from Aggregatibacter actinomycetemcomitans (formerly Actinobacillus actinomycetemcomitans) Y4 was administered intraperitoneally on the 49th day. A rapid increase in TNF-alpha was observed in the control mice at 2 h after administration of lipopolysaccharide. In contrast, no increase was noted in the nicotine-treated groups. Significantly higher levels of IFN-gamma were seen in the 200 microg nicotine-treated mice at 2 h after administration of lipopolysaccharide (P

The purpose of this study was to investigate the effect of cranberry polyphenol fraction on mutans streptococci. Hydrophobicity is an important factor in the adherence of bacteria to the tooth surface. We found that cranberry polyphenol fraction significantly decreased the hydrophobicity of Streptococcus sobrinus 6715, Streptococcus mutans MT8148R and JC2 in a dose-dependent manner (p0.05). Biofilm formation by S. sobrinus 6715 and S. mutans MT8148R was inhibited by 100 microg/ml cranberry polyphenol fraction (p0.01). When dosage was increased to 500 microg/ml, biofilm formation by S. mutans JC2 was significantly inhibited (p0.05). Addition of 500 microg/ml cranberry polyphenol fraction to medium inhibited growth of S. mutans MT8148R compared with the control (p0.05).

Background/aims: Bacterial infection is a major cause of periapical periodontitis. Eradication of these microorganisms from apical lesions is essential to the success of endodontic treatment. The aim of this study was to clarify the molecular interaction between Fusobacterium nucleatum, Porphyromonas gingivalis and other microorganisms associated with periapical periodontitis. Methods: Microorganisms isolated from periapical lesions were inoculated into type-I collagen-coated polystyrene microtiter plates and maintained at 37°C under anaerobic conditions for 2 days, after which, the quantity of organized biofilm on the plates was evaluated by crystal violet staining. Growth enhancement via soluble factor was evaluated by separated coculture using a 0.4-μm membrane filter. Results: F. nucleatum exhibited strong adherence to type-I collagen-coated polystyrene microplates. Biofilm formation by F. nucleatum was significantly enhanced by P. gingivalis. It was complemented by compartmentalized coculture with P. gingivalis. Enhancement of biofilm formation by P. gingivalis was only slightly reduced by inactivation of its autoinducer-2-producing gene luxS. Conclusion: The results suggest that P. gingivalis enhances biofilm formation by F. nucleatum by releasing diffusible signaling molecules other than autoinducer-2.

Background and Objective: The purpose of this study was to investigate the effects of cranberry polyphenol fraction on biofilm formation and activities of Arg-gingipain and Lys-gingipain in Porphyromonas gingivalis. Material and Methods: The polyphenol fraction was prepared by using a glass column packed with Amberlite XAD 7HP and 70% aqueous ethanol as an elution solvent. Results: Synergistic biofilm formation by P. gingivalis and Fusobacterium nucleatum was significantly inhibited by the polyphenol fraction at a concentration of 250 µg/mL compared with untreated controls (p

Treponema denticola, a major pathogen of periodontitis, has also been detected in the lesions of atherosclerosis. The aim of this study was to investigate induction of chemokine production in human umbilical vein endothelial cells (HUVECs) by T. denticola and determine whether those chemokines were degraded by a protease, dentilisin. T. denticola ATCC35405 or dentilisin-deficient mutant K1 were added to HUVECs and levels of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in the culture supernatants were determined by enzyme-linked immunosorbent assay. T. denticola ATCC35405 induced production of IL-8 in a time-dependent manner, with both production of IL-8 and expression of IL-8 mRNA showing higher levels than with exposure to dentilisin-deficient mutant K1. Although exposure to ATCC35405 induced expression of MCP-1 mRNA in the HUVECs, MCP-1 levels were remained similar to that in unstimulated cells. IL-8 and MCP-1 showed partial hydrolysis with exposure to T. denticola ATCC35405, but not with T. denticola K1. These results suggest that T. denticola can evade host defense mechanisms by modulating production of IL-8 and MCP-1, and that this play a role in the development of chronic infections such as periodontitis. The association of T. denticola infection to atherosclerosis was also discussed based on the present study.

Tannerella forsythia is a Gram-negative oral anaerobe implicated in the development of periodontitis, a chronic inflammatory disease induced by bacterial infections which leads to tooth loss if untreated. Since biofilms formed by periodontal bacteria are considered important in disease progression and pose difficulties in treatment, we sought to investigate the underlying mechanisms of T. forsythia biofilm formation. This was carried out by screening random insertion mutants of T. forsythia for alterations in biofilm development. This approach lead to the identification of an operon involved in exopolysaccharide (EPS) synthesis. An isogenic mutant of one of the genes, wecC, contained within the operon was constructed. The isogenic wecC mutant showed increased ability to form biofilms as compared to the parent strain. The wecC mutant also formed aggregated microcolonies and showed increased cell-surface associated hydrophobicity as compared to the parent strain. Moreover, biochemical characterization of the wecC mutant indicated that glycosylation of surface glycoproteins was reduced. Therefore, our results suggest that the wecC operon is associated with glycosylation of surface-glycoprotein expression and likely plays an inhibitory role in T. forsythia biofilm formation.

Influenza is a major cause of respiratory infection and has a high mortality rate in the elderly. Neuraminidase (NA) on the surface of the influenza virus and bacterial trypsin-like proteases (TLP) play key roles in influenza virus infections. We investigated the effects of oral care on influenza, evaluating in particular the activities of NA and TLP in saliva, as they may contribute to an increased risk of infection with influenza. One hundred ninety elderly patients who visited day care service facilities once a week were randomly assigned to either a professional oral care group or to an own oral care group as the control group. Nine individuals in the control group and one person in the professional oral care group were diagnosed with influenza during the follow-up period. The relative risk of developing influenza while under professional oral care compared to that in the control group was 0.1 (95% CI 0.01-0.81, p=0.008). Significant decreases in numbers of salivary anaerobic bacterial CFUs, and NA and TLP levels were observed in the professional oral care group compared to that in the control group (p

The aim of this investigation was to analyze the effects of early life exposure to periodontopathic bacterial lipopolysaccharides on immunoresponse. Newborn BALB/c mice were subcutaneously injected with 20 ng lipopolysaccharide of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans or Escherichia coli daily for 2 days, starting within 24 h after birth. The treated mice were given intraperitoneal injections of bovine serum albumin at 180 and 187 days of age. Seventeen hours after each injection, the mice were bled and sera were separated. Their sera were tested in an enzyme-linked immunosorbent assay system. The mean interleukin-4, interleukin-5, interleukin-6 and immunoglobulin E levels in the sera of mice treated neonatally with P. gingivalis lipopolysaccharide were significantly higher than those of the controls. However, in all cases, no significant difference was noted between mice treated neonatally with A. actinomycetemcomitans- or E. coli lipopolysaccharide and control mice. These data suggest that neonatal exposure to P. gingivalis lipopolysaccharide induces changes in immunological responses when the mice reach maturity.

We studied the effect of antibodies against Porphyromonas gingivalis gingipain domains, preparing them against three recombinant fragments of RgpA (catalytic domain, r-Rgp CAT; hemagglutinin domains, r-Rgp 44 and r-Rgps 15-27) and one fragment of Kgp (catalytic domain, r-Kgp CAT). Enhancement of opsonization and killing by human polymorphonuclear leukocytes were measured in the noninvasive FDC 381 and invasive W50 strains of P. gingivalis. Anti-r-Rgp 44 was the most effective in both strains of P. gingivalis. The present findings lead us to recommend RgpA 44 as a candidate immunogen for vaccines against P. gingivalis.

Objectives: The aim of this study was to analyse the prevalence of oral bacteria on the dorsum of the tongue. In addition, the relationship between the number of teeth and the microflora present on the coating of the tongue in a population of 85-year-old people was assessed. Subjects and methods: Two hundred and five individuals (89 males, 116 females) from the same geographical area who were 85 years of age were examined. Five periodontopathic bacteria (Porphyromonas gingivalis, Tannerella forsythia, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, and Treponema denticola) and one cariogenic bacterium (Streptococcus mutans) were analysed using a polymerase chain reaction assay of tongue samples from the population. Results: Periodontal bacteria-positive individuals have more teeth than that of periodontal bacteria-negative people. Between the periodontal bacteria-positive and -negative individuals, there were significant differences in the mean number of teeth for P. gingivalis (p

Treponema denticola is a dominant microorganism in human periodontal lesions. One of the major virulence factors of this microorganism is its chymotrypsin-like surface protease, dentilisin. The purpose of this study was to evaluate the effect of dentilisin on human polymorphonuclear leukocytes (PMNs). We used chemiluminescence to assess production of O(-)(2) by PMNs against T. denticola ATCC 35405 and dentilisin-deficient mutant K1. T. denticola ATCC 35405 induced production of O(-)(2), whereas dentilisin-deficient K1 did not. We found that chymostatin, a protease inhibitor, strongly reduced the ability of T. denticola ATCC 35405 to induce production of, O(-)(2), whereas K1 was relatively unaffected. We also used Immunoblot and ELISA to evaluate the activation of complement by this microorganism in relation to PMNs. T. denticola ATCC 35405 hydrolyzed the alpha-chain of C3, producing iC3b. Furthermore, strain ATCC 35405 induced a larger release of MMP-9 from PMNs than strain K1. Dentilisin activated PMNs via complement pathways and may play a role in establishing periodontal lesions.

Increasing evidence has linked the anaerobic bacteria forming periodontopathic biofilms with aspiration pneumonia in elderly persons. In experiments designed to eliminate the potent respiratory pathogens forming biofilms in the oral cavity, we have shown that the mechanical and chemical oral cleansing using povidone-iodine effectively reduced the detection rates and numbers of methicillin-sensitive Staphylococcus species, Streptococcus pneumoniae, and Haemophilus influenzae in patients scheduled to undergo oral surgery requiring endotracheal intubation. We confirmed the pathogenicity of periodontopathic anaerobic bacteria for aspiration pneumonia in an experimental mouse model. Based upon the finding of the coexistence of Porphyromonas gingivalis with Treponema denticola in chronic periodontitis lesions, we innoculated a mixed culture of P. gingivalis and T. denticola into the mouse trachea; the resulting infection induced inflammatory cytokine production and caused pneumonia. In another series of investigations, professional oral health care (POHC), mainly cleansing administered by dental hygienists once a week for 24 months to elderly persons requiring daily care, resulted in the reduction of the number of total anaerobes, Candida albicans, and Staphylococcus species and in the number of cases of fatal aspiration pneumonia. We also found that the POHC treatment of elderly persons for 6 months in the winter season reduced the salivary levels of protease, trypsin-like activity, and neuraminidase and also decreased the frequency of influenza cases.

To develop a diagnostic trial enabling the selective examination for a target cystatin in human body fluids, we attempted to prepare monoclonal antibodies against human cystatin SA1 (originally cystatin SA) and its variant form (cystatin SA2). BALB/c mice were immunized with recombinant (r-) cystatins SA1 and SA2. Two monoclonal antibodies designated Cys3F11 and Cys2E5 were selected. By ELISA analyses, the Cys2E5 was shown to react with r-cystatin SA2 but also somewhat with r-cystatin SA1 (22% cross-reactivity) and with plasma cystatin C (18% cross-reactivity), indicating a high specificity for cystatin SA2. The Cys3F11 reacted not only with r-cystatin SA1 but also with r-cystatin SA2 (89% cross-reactivity) and plasma cystatin C (47% cross-reactivity). This finding was further emphasized by immunoblotting of human submandibular–sublingual saliva samples. ELISA additivity test suggests that the two monoclonal antibodies bind to distinct epitopes. In conclusion, we have succeeded in producing two antibodies that discriminate the structural differences between salivary cystatins S and SN, which share more than 90% identity in amino acid sequence with cystatin SA.

Single HIV-1 subtype DNA vaccine is unlikely to provide reactive protection across a wide range of HIV strains since the HIV virus changes the antigenic sites, particularly, in env gene. To overcome these issues, we constructed a multivalent poly-epitope DNA vaccine. A polygenic DNA vaccine encoding 20 antigenic epitopes from the HIV-1 Env, Gag, and Pol proteins of several clades was constructed using humanized and optimized codons and it was named here hDNA vaccine. In mice, this hDNA vaccine stimulated the following strong (1) antigen-specific serum antibody (Ab) responses, (2) delayed-type hypersensitivity, (3) the activation of IFN-gamma secretion cells targeting gp120 and synthetic antigenic peptides, in addition (4) a significant level of several peptide specific cytotoxic T lymphocytes (CTL) responses. Challenged with modified vaccinia viruses vPE16 and vP1206 expressing HIV-1 env and gag.pol genes, respectively, demonstrated the viral titers in the ovary of the mice vaccinated with hDNA significantly less compared to the unvaccinated mice. Thus, the use of polygene DNA vaccine appears to induce a high level of HIV-specific immune responses and is very effective against challenge with recombinant HIV-vaccinia viruses.

A protease of Treponema denticola, dentilisin, is thought to be part of a complex with 43- and 38-kDa proteins. A sequence encoding a 43-kDa protein was located in the 3' region of the prcA gene upstream of the dentilisin gene (prtP). The 43-kDa protein was apparently generated from digestion of PrcA. To clarify the function of the protein, we constructed a mutant of the 43-kDa protein following homologous recombination. The mutant lacked detectable dentilisin activity. Immunoblot analysis demonstrated that the dentilisin protein was degraded in the mutant. The results of real-time polymerase chain reaction suggested that prtP mRNA expression in the mutant was somewhat decreased compared with the wild-type strain. These data suggest that the 43-kDa protein is involved in the stabilization of the dentilisin protein.

Cariogenic bacteria and periodontopathic bacteria are present in dental plaque as biofilms. In this study, we investigated the antibacterial effects of essential oils on the following oral bacteria: Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Streptococcus mutans, and Streptococcus sobrinus. We tested manuka oil, tea tree oil, eucalyptus oil, lavandula oil, and romarinus oil and determined their minimum inhibitory concentration and minimum bactericidal concentration. The essential oils inhibited the growth of the bacteria tested, manuka oil being the most effective. Minimum bactericidal concentration values showed that lavandula oil acts bacteriostatically, and the remaining oils, bactericidally. Periodontopathic bacterial strains tested were killed completely by exposure for 30 s to 0.2% manuka oil, tea tree oil or eucalyptus oil. Tea tree oil and manuka oil showed significant adhesion-inhibiting activity against P. gingivalis. All the essential oils tested inhibited the adhesion of S. mutans. This study showed that, among the essential oils tested, manuka oil and tea tree oil in particular had strong antibacterial activity against periodontopathic and cariogenic bacteria. From the viewpoint of safety, we also examined the effects of these essential oils on cultured human umbilical vein endothelial cells and found that, at a concentration of 0.2%, they had little effect on cultured cells.

Recent studies have shown that invasive and non-invasive strains of Porphyromonas gingivalis can both be isolated from patients with periodontitis. We examined the interaction between an invasive 16-1 P. gingivalis strain and phagocytes obtained from human peripheral blood and guinea pig peritoneal cavity. Phagocytes from human peripheral blood, mainly polymorphonuclear leukocytes (PMNs) isolated by centrifugation in Ficoll Hypaque, and macrophages collected from the peritoneal cavity of guinea pigs, were exposed to P. gingivalis cells. After this exposure, greater numbers of the non-invasive P. gingivalis ATCC 33277 were observed in human PMNs and guinea pig macrophages compared with the invasive P. gingivalis 16-1. Electron microscopic observations showed that invasive 16-1 within phagosomes in human PMNs and guinea pig macrophages retained their surface fibrous structures as well as their outer membranes. Electron microscopic examination showed that destruction and damage to the cell membranes and inner structures were clear in human PMNs and guinea pig macrophages after exposure to invasive 16-1 for 6 and 24 hours; this was a clear difference from exposure to the non-invasive ATCC 33277. Release of lactate dehydrogenase (LDH) activities into the culture supernatant of PMNs after exposure to the invasive 16-1 for 4 and 6 hours was significantly greater than that after exposure to the non-invasive ATCC 33277 (p0.05). On the other hand, the LDH activity after exposure for 21 hours to the invasive 16-1 was significantly lower than that of untreated cells and cells after exposure to the non-invasive ATCC 33277 strain (p0.05). The PMN viabilities after exposure to cells of the invasive 16-1 for 3, 4, and 6 hours as evaluated by trypan blue staining were similar to those after exposure to cells of the non-invasive ATCC 33277, but that after exposure to the invasive 16-1 strain for 21 hours was significantly lower than that after exposure to cells of the non-invasive ATCC 33277 strain.

Colonization by periodontopathic bacteria is a risk factor for peri-implantitis. The purpose of this study was to investigate the colonization by black-pigmented anaerobic bacteria that occurs between the time before fixture installation and 6 months after inserting superstructures in implant treatment in partial edentulous cases. Dental plaque was serially collected from around the natural teeth and implants in 12 patients in whom a dental implant was indicated, and Porphyromonas gingivalis and Prevotella intermedia were detected using polymerase chain reaction (PCR). One month after connecting the abutment, the detection rate of P. gingivalis per site from around the implants was 63.7% and that of P. intermedia was 50.8%. Six months after superstructure setting, the detection rate per site of P. gingivalis from around the implants was 56.8% and that of P. intermedia was 41.1%. When chromosomal DNA segmentation patterns in the isolated P. gingivalis and P. intermedia were compared using pulsed field gel electrophoresis (PFGE), the patterns in the natural teeth were in accordance with those in the implants in 3 of 4 cases (75.0%) in P. gingivalis and all cases in P. intermedia. This finding suggested that bacterial colonization around implants occurred early after the implant region was exposed to the intraoral cavity and that the bacteria were transmitted from the area around the natural teeth.

Arginine- and lysine-specific cysteine proteinases (arg-gingipain: Rgp, lys-gingipain: Kgp) are major virulence factors of Porphyromonas gingivalis. Recent reports have suggested that antibodies against gingipains can play a protective role against infection by P. gingivalis. The purpose of this study was to evaluate the IgG responses of patients with periodontitis to functional domains of gingipains.A group of 29 periodontitis patients and 10 periodontally healthy subjects (control group) were recruited into this study. We prepared three recombinant fragments of rgp A (catalytic domain; r-Rgp CAT) and two hemagglutinin domains (r-Rgp 44, and r-Rgps 15-27) corresponding to amino acid residues 228 to 719, 720 to 1136, and 1137 to 1704, respectively. One fragment of the Kgp catalytic domain (r-Kgp CAT) corresponding to amino acid residues 229 to 737 and expressed in Escherichia coli was also used. IgG antibody levels to these recombinant proteins in sera from the subjects were determined by an enzyme-linked immunosorbent assay (ELISA).We found that IgG levels against r-Rgp 44 and r-Rgps 15-27 in sera obtained from the patients were significantly higher than those in the healthy group (P0.01). In contrast, no significant differences in IgG levels against r-Rgp CAT and r-Kgp CAT were found between the control and patient groups. The IgG responses to P. gingivalis sonic extracts, r-Rgp 44 and r-Rgps 15-27, were related to probing depth in sera from patients, but those to r-Rgp CAT and r-Kgp CAT were not.The present findings suggest that the low responsiveness of IgG antibody against the catalytic domains of gingipain, r-Rgp CAT, and r-Kgp CAT is a key factor in infection by P. gingivalis.

Porphyromonas gingivalis is one of the major microbial pathogens associated with chronic periodontitis. To eradicate such pathogens by periodontal therapy, it is essential to clarify the source of infection. Recent findings suggest that the genotype of the fimbriae is one of the important factors in infection by P. gingivalis. The objectives of the present study were to investigate the transmission of P. gingivalis between spouses and to determine the relationship between P. gingivalis fimA type and colonization.A total of 14 couples were selected to investigate the transmission of P. gingivalis and its association with the fimA types. To examine the distribution of fimA type in the general population, 32 subgingival plaque samples from 47 patients with periodontitis were also tested. The transmission of P. gingivalis strains was determined by using pulsed field gel electrophoresis (PFGE). P. gingivalis strains isolated from the couples and subgingival dental plaque samples were studied for fimA classification.The PFGE patterns of P. gingivalis strains from matched husbands and wives were identical for six of the 14 couples. In five of these six couples (83.3%), P. gingivalis strains harboring the type II fimA gene were present. The proportion of type II fimA in the strains isolated from couples with probable intrafamilial transmission was significantly higher than that in patients with periodontitis or in the group of samples isolated from one member of a couple.This study suggests that fimA type II, even though widely distributed in patients with periodontitis, may be an important factor in the transmission of P. gingivalis between spouses.

Tuberculosis is a re-emerging infectious disease, and infection by Mycobacterium tuberculosis has been increasing in immunocompromised hosts, including elderly persons. M. tuberculosis-infected persons may receive dental treatment. To evaluate the risk of M. tuberculosis infection in dental clinics, we examined the detection rates of M. tuberculosis in sample of mixed saliva, dental plaque, extracted teeth, caries lesions, and denture plaque by nested polymerase chain reaction (PCR). The detection rates by PCR in samples from mixed saliva, dental plaque, caries lesions and denture plaque obtained from tuberculosis patients were 98.0%, 92.0%, 89.0%, and 100%, respectively. The detection rates by the culture method were 17.3%, 2.0%, 0%, and 0%, respectively. M. tuberculosis also was detected from the nontuberculous mycobacteria-infected group. Strains of Actinomyces naeslundii, Porphyromonas gingivalis, and Fusobacterium nucleatum inhibited the growth of clinical strains of M. tuberculosis, but strains of Streptococcus mutans, Streptococcus sanguinis, and Actinobacillus actinomycetemcomitans did not. The present study concludes that the PCR method is essential for detecting M. tuberculosis in oral samples.

Campylobacter rectus, a gram-negative, microaerophilic, and motile bacterium, has been proposed to play a pathogenic role in human periodontitis. Surface components, such as the flagellum, surface layer (S-layer), and cytotoxin, have been reported as possible virulence factors of the microorganism. In the present study, monoclonal antibodies against surface components of this bacterium were produced to detect and investigate the pathogenic potential of C. rectus in periodontitis. Two monoclonal antibodies, designated CRT-1 and CRT-2, recognized a peculiar 150 kDa S-layer protein by immunoblot analysis. The CRT-2 antibody reacted to all C. rectus strains tested, except for the S-layer negative strain of the species [C. rectus ATCC 33238 S-layer (-) strain]. The CRT-3 antibody reacted to a 60-kDa protein in C. rectus and also cross-reacted with Campylobacter showae ATCC 51164 and CCUG 11641 strains. Using the dot-blot method, we were able to detect C. rectus using the CRT-2 antibody when as few as 103 organisms were present in a subgingival dental plaque sample. Detection of C. rectus in plaque samples correlated significantly with clinical findings such as probing depth (P < 0.001), bleeding on probing (P < 0.001), and gingival index (P < 0.001). These findings indicate that infection by C. rectus may be an important indicator of periodontal disease status.

Porphyromonas gingivalis and Treponema denticola have been found together in lesions of human periodontitis. We examined the ability of a mixed infection by both bacteria to synergistically form abscesses and disturb immune responses in mice. Absorbance of an invasive P. gingivalis 16-1 strain grown in tryptic soy broth and T. denticola ATCC 33520 strain grown in TYGVS medium were adjusted. BALB/c mice were injected with 200 microliters of the cell suspension at a site on the lateral dorsal area. The sizes of the subsequent subcutaneous abscesses were measured with a caliper gauge, and the area was expressed in square mm. Mixed infections by P. gingivalis and T.denticola produced larger abscesses than those formed after mono-infections by either P. gingivalis or T.denticola. The abscesses caused by mixed infection reached their maxima on the 6th day and maintained that size for the subsequent 5 days. The delayed type hypersensitivities against extracted antigens of P.gingivalis in mixed infection mice were significantly lower than those in the mono-infected mice. However, the IgG response to sonicated antigen of P.gingivalis did not differ between the two groups. The sizes of the abscesses caused by mixed infections in mice immunized with whole cells of P.gingivalis 16-1 were compared to those caused in sham-immunized mice. The average size of the abscess caused by mixed infection in immunized mice did not differ from that in sham-immunized mice, but many of the abscesses in immunized mice ruptured on the 4th or 5th day, followed by recovery in two weeks. These results suggest that mixed infection with P.gingivalis and T.denticola attenuates protective immune responses.

Pustulosis palmaris et plantaris (PPP) is a chronic relapsing skin disease characterized by sterile intraepidermal pustules and, usually, by scaly erythematous skin on the palm, soles, or both. To determine the primary pathogenic factors involved in the onset of PPP, we analyzed the implications of heat shock proteins (HSPs), which are highly conserved immunogenic proteins, chronic microbial infections including marginal and periapical periodontitis, and delayed type hypersensitivity (DTH) against dental metals examined by patch tests. We found that the titers of IgG against HSPs of Escherichia coli GroEL and Actinobacillus actinomycetemcomitans DnaJ in the sera from PPP patients were higher than those in the control group (GroEL; p0.05). Two patients with PPP had more than 1,000 ng/ml serum IgE level. We found that the average serum IgG antibody level against human cytomegalovirus (HCMV) in the PPP patient group was higher than that of the healthy group (p0.05). DTH against dental metal was found in eight of 22 PPP patients. Six of eight patients reacted against more than two metals. Replacement of dental metal with resin in these patients resulted in the disappearance or remission of PPP. These findings suggest that the immune responses to HSPs produced by oral bacteria, HCMV infection, dental metal allergy, and their combinations are etiological factors for PPP. We concluded that various kinds of examinations, including oral cavity conditions and chronic infections, are essential to determine the major etiologic factors of PPP.

Increasing evidence has linked colonization by Helicobacter pylori with the development of gastritis and peptic ulcer disease. H. pylori resides primarily in the gastric mucosa without invading the gastric epithelium, causing persistent mild gastric inflammation. There are many reports examining the relationship between colonization by microorganisms in the stomach and oral cavity. We found that some oral bacteria are able to trap H. pylori cells, but oral bacteria inhibit H. pylori growth in vitro. In cases where H. pylori was detected in oral cavity samples, including oral cancer surface samples, we suggested that this species had colonized the stomach and were present in the oral cavity only as a transient organism. We demonstrated that periodontopathic Campylobacter rectus strains posses proteinaceous antigens, including heat shock proteins that share antigenicity with antigens of H. pylori strains. These cross-reactive antigens between H. pylori and C. rectus may be related to the induction of immunopathological responses in periodontal tissues and the stomach. We concluded that H. pylori could not survive in the human oral cavity; however, there would be an interrelationship between periodontal disease due to C rectus and stomach diseases due to H. pylori.

It has been suggested that infection by some mycoplasma species can act as possible cofactors in the acceleration of immunodeficiency in HIV-infected patients. The present study was designed to examine infections by oral mycoplasma species in HIV-seropositive (HIV(� )) patients. Mycoplasma salivarium and Mycoplasma orale were isolated from 59.5% and 16.7% of 42 HIV(� ) patients, respectively. Non-M. salivarium and non-M. orale species were isolated from 40.5% of saliva samples from the HIV(� ) group and 20.8% of those from 24 HIV-seronegative (HIV(� )) subjects, respectively. Although the production of superantigen by human peripheral lymphocytes in the isolated mycoplasma species from HIV(� ) and HIV (� ) subjects was evaluated, none of the examined mycoplasma strains, including ATCC strains of M.salivarium, M.orale, Mycoplasma buccae and Mycoplasma penetrans, were found to produce superantigen. Production of heat shock proteins (HSPs) by isolated mycoplasma strains was examined by immunoblotting using monoclonal antibodies against Helicobacter pylori HSP60. It was found that all the strains of M.salivarium, M.orale, and unidentified mycoplasma species isolated from HIV(� ) and HIV(� ) groups produced heat shock proteins. HSP production by oral mycoplasma may play a role in the immunomodulation of HIV(� ) patients.

Although the potential of DNA vaccination is now beginning to be greatly appreciated, no detailed study of its localization in tissue or its expression kinetics has been reported. In this study, we investigated these issues using HIV-1 DNA plasmids administered either intranasally or intramuscularly. Fluorescence in situ hybridization (FISH) revealed that the human immunodeficiency virus (HIV) plasmids administered intranasally localized in the alveoli, lung, liver, spleen, regional lymph nodes, kidney, fetus, and esophagus. These HIV plasmids were detected 2 to 4 weeks after administration. We detected messenger RNA production of HIV env gene in the lung, liver and spleen, and human immunodeficiency virus type 1 (HIV-1)-specific proteins were detectable in the lung. These observations may provide important information for understanding the mechanisms of strong immune activation induced by DNA vaccination via the intranasal route. This technology of DNA administration suggests possible practical applications for vaccination and probably for gene therapy.

DNA vaccination is characterized by its preferential induction of the cytotoxic T cell lymphocyte (CTL) response and is expected to be a useful means of protection against viral infection. We examined the protective effect of an expression plasmid (pME18S-M) containing M1 and M2 genes of influenza A/PR/8/34. We detected the CTL activity by introducing these plasmids into BALB/c mice by either the intramuscular or the intranasal route. The influenza-specific antibody response was also induced, although its neutralizing effect against influenza virus was not observed. From 70 to 80% protection was observed in the mice immunized with the pME18S-M plasmid followed by lethal infection with influenza viruses of the A/WSN/33 and A/PR/8/34 strains, whereas all mice without the plasmid vaccination failed to survive. This protective activity was significantly weakened when the CD8(+) cells of these immunized mice were eliminated by several injections of anti-CD8 antibody. The protective activity was also weakened when anti-CD4 antibody was injected in the early phase of DNA vaccination. These data suggest that the pME18S-M plasmid is useful as a DNA vaccine for overcoming highly mutational influenza viruses.

A new species, Porphyromonas gulae sp. nov., is proposed to include strains isolated from the gingival sulcus of various animal hosts which are distinct from related strains of Porphyromonas gingivalis of human origin. This bacterium exhibits the following characteristics: black-pigmented colonies; asaccharolytic, obligate anaerobic growth; and Gram-negative, non-motile and non-spore-forming, rod-shaped cells. Colonies do not fluoresce under UV light. Vitamin K1 and haemin are required for growth. Cells haemagglutinate sheep erythrocytes. Major fatty acid end products are butyric acid, isovaleric acid, succinic acid and phenylacetic acid. Strains are catalase-positive and indole is produced. Alkaline phosphatase, trypsin-like and N-acetyl-beta-glucosaminidase activities are strong. A beta-galactosidase and a glutamylglutamic acid arylamidase are also present. The G+C content of the chromosomal DNA is 51 mol%. DNA-DNA homology data and 16S rRNA gene sequence analysis provide strong evidence that strains from the animal biotype of P. gingivalis represent a Porphyromonas species that is distinct from P. gingivalis. The type strain of P. gulae is Loup 1T (= ATCC 51700T = NCTC 13180T).

Periodontopathic bacteria induce inflammation of periodontal tissues. The cytokines and nitric oxide released in periodontal lesions have been reported to play a protective role in bacterial infection and to relate to the process of inflammation. To clarify the relationship between colonization of periodontopathic bacteria and cytokines, we evaluated profiles of inflammatory cytokines, chemokine, anti-inflammatory cytokines, and inducible nitric oxide synthase (iNOS) and colonization by Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans, which are major pathogens of periodontitis.mRNA expression of cytokines and iNOS in inflamed and healthy gingival tissue was determined using reverse transcription-polymerase chain reaction (RT-PCR), and the relationship between their profiles and the detection of specific bacteria was analyzed.The relative expression of interleukin (IL)-6 and iNOS mRNAs in periodontal lesions was significantly higher than those in healthy individuals. IL-6 mRNA expression was also significantly higher at bleeding on probing (BOP)-positive sites than at BOP-negative sites. The expressions of IL-1alpha and IL-8 increased, but IL-10 expression decreased at sites where A. actinomycetemcomitans was detected. We found no correlation between the expression of cytokine and iNOS mRNA and infection by P. gingivalis.The expression of IL-6 may reflect inflammation in gingival tissue, and iNOS may be involved in the inflammatory process in periodontitis. The presence of A. actinomycetemcomitans or P. gingivalis might relate to the different cytokine profiles of IL-1alpha, IL-8, and IL-10.

Periodontopathic Campylobacter rectus strains possess 41- and 68-kDa proteinaceous antigens which share antigenicity with antigens of Helicobacter pylori strains. H. pylori strains have a 54-kDa antigen which reacts with C. rectus strains. We found that the salivary IgA levels against H. pylori were correlated with those against C. rectus. These cross-reactive antigens of C. rectus may affect the serological diagnosis of H. pylori infections, especially when saliva is used. It is possible that these cross-reacting antigens may relate to the induction of immunopathological responses against both microorganisms.

Porphyromonas gingivalis is a gram-negative rod associated with the progression of human periodontal disease. Inflammatory cytokines are believed to be the major pathological mediators in periodontal diseases. We therefore investigated the productions of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-alpha) in human gingival fibroblasts treated with lipopolysaccharide, polysaccharide and outer-membrane proteins from P. gingivalis ATCC 53977. Outer-membrane protein from P. gingivalis enhanced the production of IL-6 and IL-8 from the cells of periodontium in vitro as well as lipopolysaccharide did. The IL-8 production activity of polysaccharide from P. gingivalis was higher than that of other cell-surface components. The levels of IL-6 and IL-8 released from the P. gingivalis lipopolysaccharide-treated human gingival fibroblasts were lower than those of the same cells treated with lipopolysaccharides from Actinobacillus actinomycetemcomitans or Escherichia coli. Rabbit antisera against either outer-membrane protein or lipopolysaccharide inhibited the IL-6 and IL-8 production derived from human gingival fibroblasts stimulated sonicated supernatants from P. gingivalis. The present study suggests that, in addition to lipopolysaccharide, outer-membrane protein and polysaccharide of P. gingivalis are also pathological mediators in periodontal diseases.