Bacteriology of Most Frequent Oral Anaerobic Infections

The anaerobic infections most frequently found in the oral cavity are gingivoperiodontal diseases and pulpal and periapical infections. Gingivitis and adult periodontitis are the most frequent forms. In adult periodontitis the subgingival microbiota are complex and there is a prevalence of Porphyromonas gingivalis, Prevotella intermedia,Prevotella nigrescens , Actinobacillus actinomycetemcomitans, Bacteroides forsythus,Peptostreptococcus micros , Campylobacter rectus, and species of Fusobacterium, Eikenella andTreponema .The microflora associated with peri-implant infections are similar to the microflora found in periodontal diseases, particularly in partially edentulous patients. Implant placement is therefore not recommended in patients presenting with uncontrolled periodontal disease.Likewise, there is a similarity between the genera identified in periodontal pockets and infected root canals, and in periapical infections. However, some species are more prevalent than others in both infections. The following were predominantly observed inside the root canals: Prevotella intermedia, Prevotella nigrescens,Peptostreptococcus anaerobius , Peptostreptococcus micros, Eubacterium lentum, Eubacterium alactolyticum and Porphyromonas endodontalis, with strong associations among some species.Pericoronaritis is another infection associated with anaerobic Gram-negative bacilli and treponeme. There are a great number of methods for microbiological diagnosis, and treatment of some oral infections depends on close interaction between the microbiologist and the dentist.     

Porphyromonas gingivalis and Treponema denticola Synergistic Polymicrobial Biofilm Development

Chronic periodontitis has a polymicrobial biofilm aetiology and interactions between key bacterial species are strongly implicated as contributing to disease progression. Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia have all been implicated as playing roles in disease progression. P. gingivalis cell-surface-located protease/adhesins, the gingipains, have been suggested to be involved in its interactions with several other bacterial species. The aims of this study were to determine polymicrobial biofilm formation by P. gingivalis, T. denticola and T. forsythia, as well as the role of P. gingivalis gingipains in biofilm formation by using a gingipain null triple mutant. To determine homotypic and polymicrobial biofilm formation a flow cell system was employed and the biofilms imaged and quantified by fluorescent in situ hybridization using DNA species-specific probes and confocal scanning laser microscopy imaging. Of the three species, only P. gingivalis and T. denticola formed mature, homotypic biofilms, and a strong synergy was observed between P. gingivalis and T. denticola in polymicrobial biofilm formation. This synergy was demonstrated by significant increases in biovolume, average biofilm thickness and maximum biofilm thickness of both species. In addition there was a morphological change of T. denticola in polymicrobial biofilms when compared with homotypic biofilms, suggesting reduced motility in homotypic biofilms. P. gingivalis gingipains were shown to play an essential role in synergistic polymicrobial biofilm formation with T. denticola.     

Identification of Dipeptidyl-Peptidase (DPP)5 and DPP7 in Porphyromonas endodontalis,Distinct from Those in Porphyromonas gingivalis

Dipeptidyl peptidases (DPPs) that liberate dipeptides from the N-terminal end of oligopeptides are crucial for the growth of Porphyromonas species, anaerobic asaccharolytic gram negative rods that utilize amino acids as energy sources. Porphyromonas endodontalis is a causative agent of periapical lesions with acute symptoms and Asp/Glu-specific DPP11 has been solely characterized in this organism. In this study, we identified and characterized two P. endodontalis DPPs, DPP5 and DPP7. Cell-associated DPP activity toward Lys-Ala-4-methylcoumaryl-7-amide (MCA) was prominent in P. endodontalis ATCC 35406 as compared with the Porphyromonas gingivalis strains ATCC 33277, 16-1, HW24D1, ATCC 49417, W83, W50, and HNA99. The level of hydrolysis of Leu-Asp-MCA by DPP11, Gly-Pro-MCA by DPP4, and Met-Leu-MCA was also higher than in the P. gingivalis strains. MER236725 and MER278904 are P. endodontalis proteins belong to the S9- and S46-family peptidases, respectively. Recombinant MER236725 exhibited enzymatic properties including substrate specificity, and salt- and pH-dependence similar to P. gingivalis DPP5 belonging to the S9 family. However, the k _cat/K _m figure (194 µM⁻¹·sec⁻¹) for the most potent substrate (Lys-Ala-MCA) was 18.4-fold higher as compared to the P. gingivalis entity (10.5 µM⁻¹·sec⁻¹). In addition, P. endodontalis DPP5 mRNA and protein contents were increased several fold as compared with those in P. gingivalis. Recombinant MER278904 preferentially hydrolyzed Met-Leu-MCA and exhibited a substrate specificity similar to P. gingivalis DPP7 belonging to the S46 family. In accord with the deduced molecular mass of 818 amino acids, a 105-kDa band was immunologically detected, indicating that P. endodontalis DPP7 is an exceptionally large molecule in the DPP7/DPP11/S46 peptidase family. The enhancement of four DPP activities was conclusively demonstrated in P. endodontalis, and remarkable Lys-Ala-MCA-hydrolysis was achieved by qualitative and quantitative potentiation of the DPP5 molecule.     

Porphyromonas gingivalis and Treponema denticola Exhibit Metabolic Symbioses

Porphyromonas gingivalis and Treponema denticola are strongly associated with chronic periodontitis. These bacteria have been co-localized in subgingival plaque and demonstrated to exhibit symbiosis in growth in vitro and synergistic virulence upon co-infection in animal models of disease. Here we show that during continuous co-culture a P. gingivalis:T. denticola cell ratio of 6∶1 was maintained with a respective increase of 54% and 30% in cell numbers when compared with mono-culture. Co-culture caused significant changes in global gene expression in both species with altered expression of 184 T. denticola and 134 P. gingivalis genes. P. gingivalis genes encoding a predicted thiamine biosynthesis pathway were up-regulated whilst genes involved in fatty acid biosynthesis were down-regulated. T. denticola genes encoding virulence factors including dentilisin and glycine catabolic pathways were significantly up-regulated during co-culture. Metabolic labeling using ¹³C-glycine showed that T. denticola rapidly metabolized this amino acid resulting in the production of acetate and lactate. P. gingivalis may be an important source of free glycine for T. denticola as mono-cultures of P. gingivalis and T. denticola were found to produce and consume free glycine, respectively; free glycine production by P. gingivalis was stimulated by T. denticola conditioned medium and glycine supplementation of T. denticola medium increased final cell density 1.7-fold. Collectively these data show P. gingivalis and T. denticola respond metabolically to the presence of each other with T. denticola displaying responses that help explain enhanced virulence of co-infections.     

Polymicrobial Infection with Major Periodontal Pathogens Induced Periodontal Disease and Aortic Atherosclerosis in Hyperlipidemic ApoEnull Mice

Periodontal disease (PD) and atherosclerosis are both polymicrobial and multifactorial and although observational studies supported the association, the causative relationship between these two diseases is not yet established. Polymicrobial infection-induced periodontal disease is postulated to accelerate atherosclerotic plaque growth by enhancing atherosclerotic risk factors of orally infected Apolipoprotein E deficient (ApoE^null) mice. At 16 weeks of infection, samples of blood, mandible, maxilla, aorta, heart, spleen, and liver were collected, analyzed for bacterial genomic DNA, immune response, inflammation, alveolar bone loss, serum inflammatory marker, atherosclerosis risk factors, and aortic atherosclerosis. PCR analysis of polymicrobial-infected (Porphyromonas gingivalis [P. gingivalis], Treponema denticola [T. denticola], and Tannerella forsythia [T. forsythia]) mice resulted in detection of bacterial genomic DNA in oral plaque samples indicating colonization of the oral cavity by all three species. Fluorescent in situ hybridization detected P. gingivalis and T. denticola within gingival tissues of infected mice and morphometric analysis showed an increase in palatal alveolar bone loss (p<0.0001) and intrabony defects suggesting development of periodontal disease in this model. Polymicrobial-infected mice also showed an increase in aortic plaque area (p<0.05) with macrophage accumulation, enhanced serum amyloid A, and increased serum cholesterol and triglycerides. A systemic infection was indicated by the detection of bacterial genomic DNA in the aorta and liver of infected mice and elevated levels of bacterial specific IgG antibodies (p<0.0001). This study was a unique effort to understand the effects of a polymicrobial infection with P. gingivalis, T. denticola and T. forsythia on periodontal disease and associated atherosclerosis in ApoE^null mice.     

Association between Polycystic Ovary Syndrome,Oral Microbiota and Systemic Antibody Responses

Polycystic ovary syndrome (PCOS) is a hormonal disorder of women that not only is the leading cause of infertility but also shows a reciprocal link with oral health. This study aimed to investigate the hypothesis that the levels of putative periodontal pathogens in saliva and their antibody response in serum are elevated in PCOS, compared to systemic health. A total of 125 women were included in four groups; 45 women with PCOS and healthy periodontium, 35 women with PCOS and gingivitis, 25 systemically and periodontally healthy women, 20 systemically healthy women with gingivitis. Salivary levels of seven putative periodontal pathogens were analyzed by quantitative real-time polymerase chain reaction and serum antibody levels were analyzed by ELISA. In women with PCOS, salivary Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus oralis and Tannerella forsythia levels were higher than matched systemically healthy women, particularly in the case of gingivitis. Aggregatibacter actinomycetemcomitans and Treponema denticola levels were similar among study groups. The presence of PCOS also enhanced P. gingivalis, Prevotella intermedia and S. oralis serum antibody levels, when gingivitis was also present. Gingival inflammation correlated positively with levels of the studied taxa in saliva, particularly in PCOS. The presence of P. gingivalis and F. nucleatum in saliva also exhibited a strong positive correlation with the corresponding serum antibody levels. In conclusion, as an underlying systemic endocrine condition, PCOS may quantitatively affect the composition of oral microbiota and the raised systemic response to selective members of this microbial community, exerting a confounding role in resultant gingival inflammation and periodontal health. The most consistent effect appeared to be exerted on P. gingivalis.     

Possible translocation of periodontal pathogens into the lymph nodes draining the oral cavity

Numerous publications have reported the presence of periodontopathogenic bacteria in peripheral and central vascular lesions. However, it is unclear how this bacterial translocation occurs. The objective of this study was to investigate whether periodontopathic bacteria are translocated to lymph nodes proximal to the oral cavity. Obtaining lymph node samples is not ethically feasible unless they are excised as part of the surgical management of patients with cancer. This study analyzed formalin-fixed and paraffin-embedded lymph nodes, histologically negative for cancer cell invasion, that were excised from 66 patients with histories of head and neck cancer. Real-time PCR was performed to amplify the 16S ribosomal DNA fragments from Porphyromonas gingivalis, Treponema denticola, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, and Prevotella intermedia. The relationship between bacterial detection and cancer severity, gender, and the use of anti-cancer therapy was examined by Fisher??s exact test. P. gingivalis, T. forsythia, and P. intermedia were present in 17%, 8%, and 8% of the samples of submandibular and submental lymph nodes, respectively. There were no significant relationships between bacterial detection and the cancer disease status, patient gender or use of anticancer therapy. According to these data, it appears that the translocation of periodontopathic bacteria may occur via lymphatic drainage, irrespective of the cancer disease status, gender or anticancer therapy.     

Impact of Early Colonizers on In Vitro Subgingival Biofilm Formation

The aim of this study was to investigate the impact of early colonizing species on the structure and the composition of the bacterial community developing in a subgingival 10-species biofilm model system. The model included Streptococcus oralis, Streptococcus anginosus, Actinomycesoris, Fusobacterium nucleatum subsp. nucleatum, Veillonella dispar, Campylobacter rectus, Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. Based on literature, we considered Streptococcus oralis, Streptococcus anginosus, and Actinomyces oris as early colonizers and examined their role in the biofilms by either a delayed addition to the consortium, or by not inoculating at all the biofilms with these species. We quantitatively evaluated the resulting biofilms by real-time quantitative PCR and further compared the structures using confocal laser scanning microscopy following fluorescence in situ hybridisation. The absence of the early colonizers did not hinder biofilm formation. The biofilms reached the same total counts and developed to normal thickness. However, quantitative shifts in the abundances of individual species were observed. In the absence of streptococci, the overall biofilm structure appeared looser and more dispersed. Moreover, besides a significant increase of P. intermedia and a decrease of P. gingivalis , P. intermedia appeared to form filamented long chains that resembled streptococci. A. oris, although growing to significantly higher abundance in absence of streptococci, did not have a visible impact on the biofilms. Hence, in the absence of the early colonizers, there is a pronounced effect on P. intermedia and P. gingivalis that may cause distinct shifts in the structure of the biofilm. Streptococci possibly facilitate the establishment of P. gingivalis into subgingival biofilms, while in their absence P. intermedia became more dominant and forms elongated chains.     

PCR analysis of Actinobacillus actinomycetemcomitans,Porphyromonas gingivalis,Treponema denticola andFusobacterium nucleatum in middle ear effusion

Anaerobes contribute to the severity and chronicity of infections that occur in and around the oral cavity. One of the factors involved in the pathogenesis of otitis media with effusion (OME) is the retrograde movement of bacteria from the oropharynx into the middle ear cavity. OME is one of the most common causes of hearing loss in children. We have used a PCR-based method to identify Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola andFusobacterium nucleatum in 65 middle ear effusion (MEE) samples obtained from paediatric patients seen for myringotomy and tube placement. DNA was extracted from MEE samples and PCR was initially done with DNA extracts by using the universal primers within the 16S rRNA gene sequence common to all bacterial species. The positive samples were further assessed with four species-specific primers. With the universal primers, 27 of 65 samples (41.5%) showed positive reaction indicating the presence of bacterial DNA. F. nucleatum was present in 10 out of 27 PCR-positive samples (37%) while one sample was positive for both T. denticola and F. nucleatum (3.7%). A. actinomycetemcomitans and P. gingivalis were not detected in any of the samples. The results of this study suggest that oral bacterial species may also play a role in the aetiopathogenesis of paediatric MEE.     

Mixture of periodontopathogenic bacteria influences interaction with KB cells

IntroductionThe purpose of this study was to investigate the adhesion and invasion of periodontopathogenic bacteria in varied mixed infections and the release of interleukins from an epithelial cell line (KB cells).MethodsKB cells were co-cultured with Porphyromonas gingivalis ATCC 33277 and M5-1-2, Tannerella forsythia ATCC 43037, Treponema denticola ATCC 35405 and Fusobacterium nucleatum ATCC 25586 in single and mixed infections. The numbers of adherent and internalized bacteria were determined up to 18 h after bacterial exposure. Additionally, the mRNA expression and concentrations of released interleukin (IL)-6 and IL-8 were measured.ResultsAll periodontopathogenic bacteria adhered and internalized in different numbers to KB cells, but individually without any evidence of co-aggregation also to F. nucleatum. High levels of epithelial mRNA of IL-6 and IL-8 were detectable after all bacterial challenges. After the mixed infection of P. gingivalis ATCC 33277 and F. nucleatum ATCC 25586 the highest levels of released interleukins were found. No IL-6 and IL-8 were detectable after the mixed infection of P. gingivalis M5-1-2 and F. nucleatum ATCC 25586 and the fourfold infection of P. gingivalis ATCC 33277, T. denticola ATCC 35405, T. forsythia ATCC 43037 and F. nucleatum ATCC 25586.ConclusionAnaerobic periodontopathogenic bacteria promote the release of IL-6 and IL-8 by epithelial cells. Despite a continuous epithelial expression of IL-8 mRNA by all bacterial infections these effects are temporary because of the time-dependent degradation of cytokines by bacterial proteases. Mixed infections have a stronger virulence potential than single bacteria. Further research is necessary to evaluate the role of mixed infections and biofilms in the pathogenesis of periodontitis.     

Quantification of five putative periodontal pathogens in female patients with and without chronic periodontitis by real-time polymerase chain reaction

Chronic periodontitis is a highly prevalent endogenous polymicrobial disease. To better understand the etiology of the disease a quantitative approach is mandatory and real-time PCR is the molecular technique currently preferred to achieve this purpose. Taking into account that such a kind of study is still scarce, we aimed to evaluate the association between periodontal microbiota and chronic periodontitis. A total of 60 low-income age-matched female adults, 30 with chronic periodontitis and 30 without periodontal disease, were enrolled. DNA obtained from subgingival specimens was used for quantification of Aggregatibacter actinomycetemcomitans, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia by real-time PCR. A. actinomycetemcomitans, E. corrodens, and F. nucleatum were detected in all subjects, P. gingivalis was observed in 70.0% and 46.6% and P. intermedia in 90.0% and 80.0% of chronic periodontitis patients and periodontally healthy subjects, respectively. P. gingivalis mean count was significantly higher in patients with chronic periodontitis than in periodontally healthy individuals. Accurate detection and quantification of five putative periodontal pathogens was feasible using a simple and fast real-time PCR protocol. Although P. gingivalis and P. intermedia have been found more commonly in chronic periodontitis patients, no statistical difference was observed between periodontally diseased and healthy groups. Quantitative data indicated association between P. gingivalis and chronic periodontitis. However, because of its uneven distribution, it should not be solely taken as a marker of periodontal status.     

Porphyromonas gingivalis with either Tannerella forsythia or Treponema denticola induces synergistic IL-6 production by murine macrophage-like J774.1 cells

BackgroundChronic periodontitis is caused by mixed bacterial infection. Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola are frequently detected in deep periodontal pockets. We demonstrate that these bacteria induce proinflammatory cytokine production by the mouse macrophage-like cell line J774.1.Materials and methodsJ774.1 cells were incubated with and without bacteria for 24 h in 96-well flat-bottomed plates. The culture supernatants were analyzed by enzyme-linked immunosorbent assay for secreted mouse interleukin (IL)-6, monocyte chemoattractant protein-1, IL-23, IL-1β and tumor necrosis factor-α. The cytokine concentrations were determined using a standard curve prepared for each assay.ResultsMixed infection with P. gingivalis and either T. forsythia or T. denticola at 10⁵ CFU/ml acted synergistically to increase IL-6 production, but not monocyte chemoattractant protein-1, IL-23, IL-1β or tumor necrosis factor-α production. Gingipain inhibitors KYT-1 and KYT-36 inhibited IL-6 production by J774.1 cells incubated with 10⁵ CFU/ml of mixed bacteria.ConclusionThese results suggest that P. gingivalis with either T. forsythia or T. denticola directly induces synergistic IL-6 protein production and that gingipains play a role in this synergistic effect.     

Identification of a Methioninase Inhibitor,Myrsinoic Acid B,from Myrsine seguinii Lév., and Its Inhibitory Activities

A methioninase inhibitor from Myrsine seguinii was purified and identified as myrsinoic acid B. Its inhibitory activities as to crude methioninase from periodontal bacteria such as Fusobacterium nucleatum, Porphyromonas gingivalis, and Treponema denticola were determined. The IC₅₀ values were 10.5, 82.4, and 30.3 μM respectively.     

Identification and Localization of Extraradicular Biofilm-Forming Bacteria Associated with Refractory Endodontic Pathogens

Bacterial biofilms have been found to develop on root surfaces outside the apical foramen and be associated with refractory periapical periodontitis. However, it is unknown which bacterial species form extraradicular biofilms. The present study aimed to investigate the identity and localization of bacteria in human extraradicular biofilms. Twenty extraradicular biofilms, used to identify bacteria using a PCR-based 16S rRNA gene assay, and seven root-tips, used to observe immunohistochemical localization of three selected bacterial species, were taken from 27 patients with refractory periapical periodontitis. Bacterial DNA was detected from 14 of the 20 samples, and 113 bacterial species were isolated. Fusobacterium nucleatum (14 of 14), Porphyromonas gingivalis (12 of 14), and Tannellera forsythensis (8 of 14) were frequently detected. Unidentified and uncultured bacterial DNA was also detected in 11 of the 14 samples in which DNA was detected. In the biofilms, P. gingivalis was immunohistochemically detected in all parts of the extraradicular biofilms. Positive reactions to anti-F. nucleatum and anti-T. forsythensis sera were found at specific portions of the biofilm. These findings suggested that P. gingivalis, T. forsythensis, and F. nucleatum were associated with extraradicular biofilm formation and refractory periapical periodontitis.     

The identification of genes specific to Prevotella intermedia and Prevotella nigrescens using genomic subtractive hybridization

Prevotella intermedia and Prevotella nigrescens, which are often isolated from periodontal sites, were once considered two different genotypes of P. intermedia. Although the genomic sequence of P. intermedia was determined recently, little is known about the genetic differences between P. intermedia and P. nigrescens. The subtractive hybridization technique is a powerful method for generating a set of DNA fragments differing between two closely related bacterial strains or species. We used subtractive hybridization to identify the DNA regions specific to P. intermedia ATCC 25611 and P. nigrescens ATCC 25261. Using this method, four P. intermedia ATCC 25611-specific and three P. nigrescens ATCC 25261-specific regions were determined. From the species-specific regions, insertion sequence (IS) elements were isolated for P. intermedia. IS elements play an important role in the pathogenicity of bacteria. For the P. intermedia-specific regions, the genes adenine-specific DNA-methyltransferase and 8-amino-7-oxononanoate synthase were isolated. The P. nigrescens-specific region contained a Flavobacterium psychrophilum SprA homologue, a cell-surface protein involved in gliding motility, Prevotella melaninogenica ATCC 25845 glutathione peroxide, and Porphyromonas gingivalis ATCC 33277 leucyl-tRNA synthetase. The results demonstrate that the subtractive hybridization technique was useful for distinguishing between the two closely related species. Furthermore, this technique will contribute to our understanding of the virulence of these species.     

Comparative genome analysis and identification of competitive and cooperative interactions in a polymicrobial disease

Polymicrobial diseases are caused by combinations of multiple bacteria, which can lead to not only mild but also life-threatening illnesses. Periodontitis represents a polymicrobial disease; Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia, called ‘the red complex'', have been recognized as the causative agents of periodontitis. Although molecular interactions among the three species could be responsible for progression of periodontitis, the relevant genetic mechanisms are unknown. In this study, we uncovered novel interactions in comparative genome analysis among the red complex species. Clustered regularly interspaced short palindromic repeats (CRISPRs) of T. forsythia might attack the restriction modification system of P. gingivalis, and possibly work as a defense system against DNA invasion from P. gingivalis. On the other hand, gene deficiencies were mutually compensated in metabolic pathways when the genes of all the three species were taken into account, suggesting that there are cooperative relationships among the three species. This notion was supported by the observation that each of the three species had its own virulence factors, which might facilitate persistence and manifestations of virulence of the three species. Here, we propose new mechanisms of bacterial symbiosis in periodontitis; these mechanisms consist of competitive and cooperative interactions. Our results might shed light on the pathogenesis of periodontitis and of other polymicrobial diseases.     

A morphological and immunolabeling study of freeze-substituted Bacteroides forsythus

We used a rapid freezing and freeze-substitution technique without glutaraldehyde and OsO₄ fixation for the electron microscopic immunocytochemical demonstration of the surface structure of Bacteroides forsythus, an anaerobic Gram-negative periodontopathogen. Cells were applied to pieces of filter paper and freeze-substituted by plunge-freezing in liquid propane, substituted in methanol containing 0.5% uranyl acetate, and infiltrated with LR White resin. The membrane ultrastructure of B. forsythus was preserved well, and the labeling density of the freeze-submitted cells was compared to a conventional processing method. Our results show the usefulness of the freeze-substitution method for immunohistochemical studies of B. forsythus.     

Enhancement of Toxicity of Periodontal Anaerobes by Cotinine

Smoking is a risk factor in periodontal disease where nicotine acts synergistically with toxins of periodontopathogens. This study tested the hypothesis that another product of smoking, cotinine, also acts synergistically with bacterial toxins in a toxin bioassay. Prevotella intermedia (3 out of 4 isolates), Prevotella nigrescens (3 out of 4 isolates) and a single strain of Porphyromonas gingivalis all showed synergy between extracellular toxins and cotinine (χ²test, P< 0.05). Similar results were obtained using cell-free lysates in place of extracellular toxins. It is concluded that toxicity of certain periodontal anaerobes can be enhanced by levels of cotinine associated with smoking less than one cigarette.     

Electrophoresis of periodontal pathogens in poly(ethyleneoxide) solutions with uncoated capillary

Periodontitis is a prevalent inflammatory disease caused by different species of anaerobic bacteria such as Porphyromonas gingivalis (P.g), Treponema denticola (T.d), and Tannerella forsythia (T.f). We compared the separation result of DNA ladders in hydroxyethyl cellulose, poly(ethyleneoxide) (PEO), and polyethylene glycol and analyzed the effect of polymer concentration, electric field, and temperature of the background electrolyte on the separation performance. Results demonstrated that there was a linear relationship (R = 0.942) for 100 to 700 bp of DNA and its migration time. Finally, the polymerase chain reaction products of P.g, T.d, and T.f were successfully identified within 8.5 min in 0.5% PEO with uncoated capillary.     

miR-146a regulates inflammatory cytokine production in Porphyromonas gingivalis lipopolysaccharide-stimulated B cells by targeting IRAK1 but not TRAF6

It has been suggested that microRNAs (miRs) are involved in the immune regulation of periodontitis. However, it is unclear whether and how miRs regulate the function of B cells in the context of periodontitis. This study is to explore the role of miR-146a on the inflammatory cytokine production of B cells challenged by Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS). Primary B cells were harvested from mouse spleen. Quantitative real-time polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of inflammatory cytokines in B cells in the presence or absence of P. gingivalis LPS and/or miR-146a. Bioinformatics, luciferase reporter assay and overexpression assay were used to explore the binding target of miR-146a. Our results showed that miR-146a level in B cells was elevated by P. gingivalis LPS stimulation, and the mRNA expressions of interleukin (IL)-1β, 6 and 10, and IL-1 receptor associated kinase-1 (IRAK1), but not TNF receptor associated factor 6 (TRAF6), were also upregulated. The expression levels of IL-1β, 6, 10 and IRAK1 were reduced in the presence of miR-146a mimic, but were elevated by the addition of miR-146a inhibitor. MiR-146a could bind with IRAK1 3′ untranslated region (UTR) but not TRAF6 3′-UTR. Overexpression of IRAK1 reversed the inhibitory effects of miR-146a on IL-1β, 6 and 10. In summary, miR-146a inhibits inflammatory cytokine production in B cells through directly targeting IRAK1, suggesting a regulatory role of miR-146a in B cell-mediated periodontal inflammation.