Effect of nitro-conjugated linoleic acid on the inflammatory response of murine macrophages activated with lipopolysaccharide derived from Prevotella intermedia.

Nitro-conjugated linoleic acid (NO2-CLA) has been observed to manifest salutary signaling responses, including anti-inflammatory and antioxidant properties. Here, the authors have explored the influence and underlying mechanisms of NO2-CLA on the proinflammatory reaction of murine macrophages that were challenged with lipopolysaccharide (LPS) derived from Prevotella intermedia, a putative periodontopathic bacterium. Treatment of LPS-activated RAW264.7 cells with NO2-CLA notably dampened the secretion of iNOS-derived NO, IL-1ß and IL-6 as well as their gene expressions and significantly enhanced the markers for M2 macrophage polarization. NO2-CLA promoted the HO-1 expression in cells challenged with LPS, and tin protoporphyrin IX, an HO-1 inhibitor, significantly reversed the NO2-CLA-mediated attenuation of NO secretion, but not IL-1ß or IL-6. We found that cells treated with NO2-CLA significantly increased mRNA expression of PPAR-γ compared to control cells, and NO2-CLA significantly reverted the decrease in PPAR-γ mRNA caused by LPS. Nonetheless, antagonists to PPAR-γ were unable to reverse the NO2-CLA-mediated suppression of inflammatory mediators. In addition, NO2-CLA did not alter the p38 and JNK activation elicited by LPS. Both NF-κB reporter activity and IκB-α degradation caused by LPS were notably diminished by NO2-CLA. NO2-CLA was observed to interrupt the nuclear translocation and DNA binding of p50 subunits caused by LPS with no obvious alterations in p65 subunits. Further, NO2-CLA attenuated the phosphorylation of STAT1/3 elicited in response to LPS. We propose that NO2-CLA could be considered as a possible strategy for the therapy of periodontal disease, although additional researches are certainly required to confirm this.

Carbon monoxide-releasing molecule-401, a water-soluble manganese-based metal carbonyl, suppresses Prevotella intermedia lipopolysaccharide-induced production of nitric oxide in murine macrophages.

CONTEXT: Many reports in the literature have suggested the therapeutic value of carbon monoxide-releasing molecules (CORMs) against various diseases. However, to date, little is known about their possible influence on periodontal disease. OBJECTIVE: This study was performed to investigate the influence of CORM-401 on the generation of nitric oxide (NO) in murine macrophage cells activated with lipopolysaccharide (LPS) derived from Prevotella intermedia, a pathogen associated with periodontal disease. MATERIALS AND METHODS: LPS was isolated by the hot phenol-water method. Culture supernatants were analyzed for NO. Real-time PCR and immunoblotting were conducted to quantify mRNA and protein expression, respectively. NF-κB-dependent SEAP levels were estimated by reporter assay. DNA-binding of NF-κB was also analyzed. RESULTS: CORM-401 caused an apparent suppression of NO production through inhibition of iNOS at both the mRNA and protein levels in RAW264.7 cells stimulated with P. intermedia LPS. CORM-401 upregulated the expression of both the HO-1 gene and its protein in LPS-activated cells, and treatment with the HO-1 inhibitor significantly reversed the attenuating influence of CORM-401 against LPS-induced generation of NO. CORM-401 caused an apparent attenuation of NF-κB-dependent SEAP release induced by LPS. IκB-α degradation and nuclear translocation of NF-κB p50 subunit induced by LPS were significantly reduced by CORM-401. Additionally, CORM-401 significantly attenuated DNA-binding of p65 and p50 induced by LPS. CORM-401 attenuated NO generation induced by P. intermedia LPS independently of PPAR-γ, JNK, p38 and STAT1/3. CONCLUSION: The modulation of host inflammatory response by CORM-401 might be of help in the therapy of periodontal disease.

Tricarbonyldichlororuthenium(II) dimer, the lipid-soluble carbon monoxide-releasing molecule, attenuates Prevotella intermedia lipopolysaccharide-induced production of nitric oxide and interleukin-1ß in murine macrophages.

Carbon monoxide (CO) is increasingly being appreciated as an important mediator that has pleiotropic biological properties and appears to have a possible therapeutic application for a variety of disorders. Nevertheless, whether this gaseous molecule may be utilized as a therapeutic intervention for periodontal disease is unclear. Here, we examined the potential beneficial effect of CO-releasing molecule-2 (CORM-2), a tricarbonyldichlororuthenium(II) dimer, against the elaboration of proinflammatory mediators by murine macrophages challenged with lipopolysaccharide (LPS) isolated from Prevotella intermedia, a pathogenic bacterium implicated in inflammatory periodontal disease. We found that NO and IL-1ß production, iNOS protein expression and mRNA expressions of iNOS and IL-1ß were significantly down-regulated when LPS-challenged RAW264.7 cells were exposed to CORM-2. In addition, HO-1 expression was upregulated by CORM-2 in cells activated with P. intermedia LPS, and the inhibitory influence of CORM-2 upon NO production was attenuated by tin protoporphyrin IX, an inhibitor of HO activity. PPAR-γ did not function in the attenuation of NO and IL-1ß by CORM-2. JNK and p38 phosphorylation caused by LPS was not altered by CORM-2. CORM-2 reduced NF-κB reporter activity and IκB-α degradation elicited by P. intermedia LPS. Additionally, CORM-2 inhibited LPS-induced phosphorylation of STAT1/3. In conclusion, CORM-2 suppresses NO and IL-1ß production caused by P. intermedia LPS. CORM-2 exerts its effect by a mechanism involving anti-inflammatory HO-1 induction and attenuation of NF-κB and STAT1/3 activation, independently of PPAR-γ as well as JNK and p38. CORM-2 may hold promise as host response modulation agent for periodontal disease, though further research is indicated to verify the therapeutic effect.

Phenotypic identification of periodontal Prevotella intermedia/nigrescens group isolates validated by MALDI-TOF mass spectrometry.

The accuracy of a phenotypic scheme to recognize periodontal Prevotella intermedia/nigrescens group clinical isolates on primary isolation culture plates was assessed with matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). A total of 84 fresh subgingival isolates from 23 chronic periodontitis patients were presumptively recognized on anaerobically-incubated enriched Brucella blood agar primary isolation plates as P. intermedia/nigrescens based on their dark-pigmented colony morphology, brick-red autofluorescence under long-wave ultraviolet light, and a negative fluorescence test for lactose production. The presumptive P. intermedia/nigrescens clinical isolates were subjected to MALDI-TOF MS analysis using Bruker MALDI Biotyper analytic software containing mass spectra for P. intermedia and Prevotella nigrescens in its reference library of bacterial protein profiles. Using a ≥1.7 log score agreement threshold, 60 (71.4%) of the presumptive P. intermedia/nigrescens clinical isolates were confirmed as either P. intermedia (25 isolates) or P. nigrescens (35 isolates). All isolates with a <1.7 log score were also identified as P. intermedia or P. nigrescens from the top choice designated on the MALDI Biotyper most likely species identification list. These MALDI-TOF MS findings document the ability of the phenotypic scheme to correctly recognize most periodontal P. intermedia/nigrescens group clinical isolates on primary isolation culture plates.

Josamycin suppresses Prevotella intermedia lipopolysaccharide-induced production of nitric oxide and interleukin-1ß in murine macrophages.

AIMS: Josamycin has immunomodulatory properties independent of its antibacterial actions. This study was designed to explore the influences and associated mechanisms of josamycin upon the generation of proinflammatory mediators in murine macrophages activated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogenic bacterium associated with periodontal disease. MAIN METHODS: LPS was purified by employing phenol-water extraction protocol. Culture supernatants were analyzed for nitric oxide (NO) and interleukin (IL)-1ß. Real-time PCR and immunoblotting were conducted to quantify mRNA and protein expression, respectively. The expression levels of IL-1ß were analyzed by confocal laser scanning microscopy. NF-κB-dependent SEAP levels were estimated by reporter assay. KEY FINDINGS: Josamycin significantly attenuated NO production elicited by P. intermedia LPS as well as induction of iNOS protein and mRNA in RAW264.7 cells. While the release of IL-1ß from cells stimulated by LPS was suppressed in the presence of josamycin, josamycin failed to reduce the degree of IL-1ß mRNA expression. Josamycin did not reduce the stability of IL-1ß mRNA induced by P. intermedia LPS, at the same time josamycin also failed to suppress the LPS-induced intracellular IL-1ß expression. Josamycin augmented HO-1 induction in cells exposed to P. intermedia LPS, and SnPP, an inhibitor of HO-1 activity, reversed the suppressive impact of josamycin upon NO generation induced by LPS. Josamycin diminished NF-κB transcriptional activity induced by P. intermedia LPS. Further, josamycin enhanced SOCS1 mRNA level in cells activated with LPS. SIGNIFICANCE: Josamycin suppressed P. intermedia LPS-induced generation of NO and IL-1ß in RAW264.7 macrophages via the inhibition of NF-κB activation as well as HO-1 and SOCS1 induction. Josamycin may have benefits as a host modulatory agent in treating periodontal disease.

Simultaneous Determination of Absolute Configuration and Quantity of Lipopeptides Using Chiral Liquid Chromatography/Mass Spectrometry and Diastereomeric Internal Standards.

Lipopeptides promote innate immune response and are related to disease pathology. To investigate the newly emerging roles of lipopeptides, accurate measurements of stereoisomers with multiple chiral centers are essential yet challenging. This work uses (3R)- and (3S)-(15-methyl-3-((13-methyltetradecanoyl)oxy)hexadecanoyl)glycyl-l-serine, abbreviated as l-serine-(R+S)-Lipid 654, to develop a method that combines chiral liquid chromatography, a diastereomeric mixture of isotopically labeled internal standards, and multiple reaction monitoring mass spectrometry. The new method allows for simultaneously determining the absolute configuration and quantity of stereoisomers of bacteria-derived lipopeptides. Total lipid extracts of nine evaluated bacteria strains had different amounts, but only the (R)-isoform of l-serine-Lipid 654. The developed method also allowed for the first quantitative analysis of hydrolysis of a nonphospholipid as a novel substrate of honey bee venom phospholipase A2.

The Black Bug Myth: Selective photodestruction of pigmented pathogens.

BACKGROUND AND OBJECTIVE: It is commonly believed that pigmented pathogens are selectively targeted by dental lasers. To test this notion optical diffuse reflection spectroscopy (DRS) was used to obtain absorption spectra for the periodontal pathogens, Porphyromonas gingivalis (Pg) and Prevotella intermedia (Pi). MATERIALS AND METHODS: Spectra from 400 to 1,100 nm wavelengths of Pg colonies cultured with different concentrations of hemin were obtained to test the hypothesis that "visual pigmentation" predicts absorption of near-infrared (IR) dental laser energy. Ablation threshold at 1,064 nm [1] was measured for the pathogenic fungus, Candida albicans (Ca). RESULTS: The hypothesis was demonstrated to be true at 810 nm, it was false at 1,064 nm. Diode laser (810 nm) efficacy and "depth of kill" is dependent on hemin availability from 400 to about 900 nm. Pg and Pi absorption at 1,064 nm (µa = 7.7 ± 2.6 cm(-1) ) is independent of hemin availability but is determined by another unknown chromophore. Ca is non-pigmented but very sensitive to 1,064 nm irradiation. CONCLUSIONS: The amount of visual pigmentation does not necessarily predict sensitivity to dental laser irradiation. Spectra in visible and near-IR wavelengths demonstrate a large difference in absorption between soft tissue and Pg or Pi. This difference represents a host/pathogen differential sensitivity to laser irradiation, the basis for selective photoantisepsis. Lasers Surg. Med. 48:706-714, 2016. © 2016 Wiley Periodicals, Inc.

Genistein suppresses Prevotella intermedia lipopolysaccharide-induced inflammatory response in macrophages and attenuates alveolar bone loss in ligature-induced periodontitis.

OBJECTIVE: Genistein is a major isoflavone subclass of flavonoids found in soybean and a potent tyrosine kinase inhibitor. The present study aimed to assess the effect of genistein on the production of proinflammatory mediators in murine macrophages stimulated with lipopolysaccharide (LPS) isolated from Prevotella intermedia, a pathogen associated with different forms of periodontal disease, and to evaluate its possible influence on alveolar bone loss in ligature-induced periodontitis using micro-computed tomography (micro-CT) analysis as well. DESIGN: LPS was isolated from P. intermedia ATCC 25611 by using the standard hot phenol-water method. Culture supernatants were analyzed for nitric oxide (NO) and interleukin-6 (IL-6). Inducible NO synthase (iNOS) protein expression was evaluated by immunoblot analysis. Real-time PCR was carried out to measure iNOS and IL-6 mRNA expression. In addition, effect of genistein on alveolar bone loss was evaluated in a rat model of experimental periodontitis using micro-CT analysis. RESULTS: Genistein significantly attenuated P. intermedia LPS-induced production of iNOS-derived NO and IL-6 with attendant decrease in their mRNA expression in RAW264.7 cells. In addition, when genistein was administered to rats, decreases in alveolar bone height and bone volume fraction induced by ligature placement were significantly inhibited. Genistein administration also prevented ligature-induced alterations in the microstructural parameters of trabecular bone, including trabecular thickness, trabecular separation, bone mineral density and structure model index. CONCLUSIONS: While additional studies are required, we suggest that genistein could be utilized for the therapy of human periodontitis in the future.

NCX 4040, a nitric oxide-donating aspirin derivative, inhibits Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages.

In this study, the effects and underlying mechanisms of NCX 4040, a nitric oxide (NO)-donating aspirin derivative, on the production of proinflammatory mediators were examined using murine macrophages exposed to lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in the etiology of periodontal disease. NCX 4040 significantly reduced P. intermedia LPS-induced production of inducible NO synthase (iNOS)-derived NO, IL-1ß and IL-6 as well as their mRNA expression in RAW264.7 cells. Notably, NCX 4040 was much more effective than the parental compound aspirin in reducing LPS-induced production of inflammatory mediators. NCX 4040 induced the expression of heme oxygenase-1 (HO-1) in cells treated with P. intermedia LPS, and the suppressive effect of NCX 4040 on LPS-induced NO production was significantly reversed by SnPP, a competitive HO-1 inhibitor. NCX 4040 did not influence LPS-induced phosphorylation of JNK and p38. IκB-α degradation as well as nuclear translocation and DNA-binding activities of NF-κB p65 and p50 subunits induced by P. intermedia LPS were significantly reduced by NCX 4040. Besides, LPS-induced phosphorylation of STAT1 and STAT3 was significantly down-regulated by NCX 4040. Further, NCX 4040 elevated the SOCS1 mRNA in cells stimulated with LPS. This study indicates that NCX 4040 inhibits P. intermedia LPS-induced production of NO, IL-1ß and IL-6 in murine macrophages through anti-inflammatory HO-1 induction and suppression of NF-κB, STAT1 and STAT3 activation, which is associated with the activation of SOCS1 signaling. NCX 4040 could potentially be a promising tool in the treatment of periodontal disease, although further studies are required to verify this.

Carbon monoxide-releasing molecule-3 suppresses Prevotella intermedia lipopolysaccharide-induced production of nitric oxide and interleukin-1ß in murine macrophages.

This study was performed to analyze the effect of carbon monoxide (CO)-releasing molecule-3 (CORM-3) in alleviating the production of proinflammatory mediators in macrophages treated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen associated with periodontal disease, and its possible mechanisms of action. LPS was isolated using the hot phenol-water method. Culture supernatants were assayed for nitric oxide (NO) and interleukin-1ß (IL-1ß). Gene expression was quantified by real-time PCR, and protein expression by immunoblotting. DNA-binding activities of NF-κB subunits were determined using an ELISA-based kit. CORM-3 suppressed the production of inducible NO synthase (iNOS)-derived NO and IL-1ß at both gene transcription and translation levels in P. intermedia LPS-activated RAW264.7 cells. CORM-3 enhanced heme oxygenase-1 (HO-1) expression in cells stimulated with P. intermedia LPS, and inhibition of HO-1 activity by SnPP notably reversed the suppressive effect of CORM-3 on LPS-induced production of NO. LPS-induced phosphorylation of p38 and JNK was not affected by CORM-3. CORM-3 did not influence P. intermedia LPS-induced degradation of IκB-α. Instead, nuclear translocation of NF-κB p65 and p50 subunits was blocked by CORM-3 in LPS-treated cells. In addition, CORM-3 reduced LPS-induced p65 and p50 binding to DNA. Besides, CORM-3 significantly suppressed P. intermedia LPS-induced phosphorylation of STAT1. Overall, this study indicates that CORM-3 suppresses the production of NO and IL-1ß in P. intermedia LPS-activated murine macrophages via HO-1 induction and inhibition of NF-κB and STAT1 pathways. The modulation of host inflammatory response by CORM-3 would be an attractive therapeutic approach to attenuate the progression of periodontal disease.

Real-time monitoring of the metabolic activity of periodontopathic bacteria.

Bacterial metabolic activity is associated with the onset and progression mechanisms of oral biofilm-mediated disease; however, at present no method to monitor bacterial metabolism exists, especially for periodontopathic bacteria. Therefore, we aimed to establish a novel method for monitoring the metabolic activity of periodontopathic bacteria, Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi) and Fusobacterium nucleatum (Fn), as well as Streptococcus mutans (Sm) for comparison. The method is based on the dye resazurin, which is converted to the fluorescent molecule resorufin by reducing molecules derived from bacterial metabolism. Additionally, the effects of antimicrobial substances on bacterial metabolic activity were evaluated using this method. When bacterial suspensions were incubated with tryptone, glutamate, aspartate or glucose in the presence of resazurin, the fluorescence intensity increased over time by these bacterial metabolic reactions, indicating that this method can be used to monitor the metabolic activity of periodontopathic bacteria. Chlorhexidine showed the 50% inhibitory concentration (IC50) of 15-49 µg/ml for tryptone metabolism by Pg, Pi, and Fn, and 7.1-18 µg/ml for glucose metabolism by Pi and Sm. The IC50s for cetylpyridinium chloride and sodium dodecyl sulfate were 0.8-2.1 and 28-44 µg/ml, respectively for all bacteria examined. Fluoride had no effect except the IC50 of 640 µg/ml for Sm, while minocycline hydrochloride had no effect on any of the bacteria. The present study established the method for real-time monitoring of the metabolic activity of periodontopathic bacteria, and the method might be useful for evaluating the effects of antimicrobial substances on the bacterial metabolic activity.

Effect of caffeic acid phenethyl ester on Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages.

BACKGROUND AND OBJECTIVE: Caffeic acid phenethyl ester (CAPE) has numerous potentially beneficial properties, including antioxidant, immunomodulatory and anti-inflammatory activities. However, the effect of CAPE on periodontal disease has not been studied before. This study was designed to investigate the efficacy of CAPE in ameliorating the production of proinflammatory mediators in macrophages activated by lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in periodontal disease. MATERIAL AND METHODS: LPS from P. intermedia ATCC 25611 was isolated by using the standard hot phenol-water method. Culture supernatants were assayed for nitric oxide (NO), interleukin (IL)-1ß and IL-6. We used real-time polymerase chain reaction to quantify inducible NO synthase, IL-1ß, IL-6, heme oxygenase (HO)-1 and suppressors of cytokine signaling (SOCS) 1 mRNA expression. HO-1 protein expression and levels of signaling proteins were assessed by immunoblot analysis. DNA-binding activities of NF-κB subunits were analyzed by using the enzyme-linked immunosorbent assay-based kits. RESULTS: CAPE exerted significant inhibitory effects on P. intermedia LPS-induced production of NO, IL-1ß and IL-6 as well as their mRNA expression in RAW264.7 cells. CAPE-induced HO-1 expression in cells activated with P. intermedia LPS, and selective inhibition of HO-1 activity by tin protoporphyrin IX attenuated the inhibitory effect of CAPE on LPS-induced NO production. CAPE did not interfere with IκB-α degradation induced by P. intermedia LPS. Instead, CAPE decreased nuclear translocation of NF-κB p65 and p50 subunits induced with LPS, and lessened LPS-induced p50 binding activity. Further, CAPE showed strong inhibitory effects on LPS-induced signal transducer and activator of transcription 1 and 3 phosphorylation. Besides, CAPE significantly elevated SOCS1 mRNA expression in P. intermedia LPS-stimulated cells. CONCLUSION: Modulation of host response by CAPE may represent an attractive strategy towards the treatment of periodontal disease. In vivo studies are required to appraise the potential of CAPE further as an immunomodulator in the treatment of periodontal disease.

Opposite effect of supernatants from selected periopathogens and oral lactobacilli cultures on ATP levels in human gingival fibroblasts.

Studies were performed on the effects of supernatants obtained from bacterial cultures, including cultures of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia and Lactobacillus acidophilus strains on ATP levels in human gingival fibroblasts (HGF-1) and on their viability. ATP levels were evaluated using luminescence test and cell viability was estimated using a fluorescence test. In control cultures mean levels of ATP in HGF-1 amounted to 4.90±0.32 mln RLU. Supernatants of P. gingivalis and A. actinomycetemcomitans cultures were found to significantly reduce ATP production in HGF-1 (mean levels of ATP amounted to 3.41±0.33 and 3.55±0.3 mln RLU respectively), which was not accompanied by an increased proportion of dead fibroblasts. Supernatants of P. intermedia induced no significant alterations in ATP level in HGF-1. In turn, supernatants of L. acidophilus H2O2 (+) and H2O2 (-) cultures significantly increased ATP levels in HGF-1 (the mean levels amounted to 5.94±0.31 mln RLU and 5.88±0.28 mln RLU respectively). The results indicate that extracellular products of P. gingivalis and A. actinomycetemcomitans most probably represent mitochondria-targeted peptides, which reduce synthesis of ATP in HGF-1. In turn, extracellular products of L. acidophilus seem to represent exopolysaccharides (EPS) with pro-oxidant activity, which stimulate synthesis of ATP in HGF-1.

Evidence of mutualism between two periodontal pathogens: co-operative haem acquisition by the HmuY haemophore of Porphyromonas gingivalis and the cysteine protease interpain A (InpA) of Prevotella intermedia.

Haem (iron protoporphyrin IX) is both an essential growth factor and a virulence regulator of the periodontal pathogens Porphyromonas gingivalis and Prevotella intermedia, which acquire it through the proteolytic degradation of haemoglobin and other haem-carrying plasma proteins. The haem-binding lipoprotein HmuY haemophore and the gingipain proteases of P. gingivalis form a unique synthrophic system responsible for capture of haem from haemoglobin and methaemalbumin. In this system, methaemoglobin is formed from oxyhaemoglobin by the activities of gingipain proteases and serves as a facile substrate from which HmuY can capture haem. This study examined the possibility of cooperation between HmuY and the cysteine protease interpain A (InpA) of Pr. intermedia in the haem acquisition process. Using UV-visible spectroscopy and polyacrylamide gel electrophoresis, HmuY was demonstrated to be resistant to proteolysis and so able to cooperate with InpA to extract haem from haemoglobin, which was proteolytically converted to methaemoglobin by the protease. Spectroscopic pH titrations showed that both the iron(II) and iron(III) protoporphyrin IX-HmuY complexes were stable over the pH range 4-10, demonstrating that the haemophore could function over a range of pH that may be encountered in the dental plaque biofilm. This is the first demonstration of a bacterial haemophore working in conjunction with a protease from another bacterial species to acquire haem from haemoglobin and may represent mutualism between P. gingivalis and Pr. intermedia co-inhabiting the periodontal pocket.

A common periodontal pathogen has an adverse association with both acute and stable coronary artery disease.

OBJECTIVE: The aim of this study was to investigate the association between angiographically verified coronary artery disease (CAD) and salivary levels of four major periodontal pathogens. METHODS: The study population (n = 492) was composed of 179 (36.4%) patients with stable CAD, 166 (33.7%) with acute coronary syndrome (ACS), and 119 (24.2%) showing no pathological findings by coronary angiography. All patients were subjected to a detailed oral health examination. The saliva samples were analyzed for lipopolysaccharide activity as well as for Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, and Tannerella forsythia by quantitative PCR. Serum antibodies levels against A. actinomycetemcomitans were analyzed. RESULTS: The level of bacterial burden was linearly associated with alveolar bone loss (p < 0.001) and bleeding on probing (p = 0.015). The median salivary levels of A. actinomycetemcomitans in pathogen-positive patients were significantly higher in the "Stable CAD" (p = 0.014) and the "ACS" (p = 0.044) groups when compared to "No significant CAD" patients. In logistic regression models, a 10-fold increase in the salivary A. actinomycetemcomitans levels was associated with a risk for stable CAD and ACS with odds ratios (ORs) of 7.47 (95% confidence interval [CI]: 1.57-35.5, p = 0.012) and 4.31 (95% CI: 1.06-17.5, p = 0.041), respectively. The OR for the association of IgA-class antibody levels against A. actinomycetemcomitans with ACS risk was 3.13 (95% CI: 1.38-7.12, p = 0.006)/log(10) unit increase. CONCLUSIONS: High salivary levels of A. actinomycetemcomitans and systemic exposure to the bacterium were associated with increased risk for CAD. These findings emphasize the importance of oral microbiota in cardiovascular risk assessment and therapeutics.

[Removal of oral Prevotella intermedia Endotoxin by octyl phenyl polyoxyethylene ether extraction method].

OBJECTIVE: To investigate an effective purification method for removing endotoxin from Prevotella intermedia. METHODS: The main protein ingredients of bacteria prepared from ammonium sulfate precipitation were further treated with octyl phenyl polyoxyethylene ether (Triton X-114), and then processed at 4°C, 37°C and 25°C. The obtained aqueous phase after at least two more cycle repeated operations was assayed for endotoxin by Western blotting, LAL-clotting method, in vitro cell stimulation and in vivo animal experiments. RESULTS: Western blotting and LAL-clotting method demonstrated that the reduction in endotoxin level was greater than 99.99% and recovery of the proteins after endotoxin removal was greater than 90% with Triton X-114 treatment for 3 cycles. The cytokines expression level was lower in both in vitro cell stimulation and in vivo animal experiments than in untreated group (P < 0.05). CONCLUSIONS: The extraction method provides a new choice for endotoxin removal from large volumes of the oral Prevotella intermedia.

The elicitation step of nickel allergy is promoted in mice by microbe-related substances, including some from oral bacteria.

Microbial components activate the host's innate immunity via interactions with molecules including TLRs and NODs. We previously reported that in mice (i) Escherichia coli lipopolysaccharide (LPS; TLR4 agonist) promotes Ni-allergy even in T-cell-deficient mice, (ii) E. coli LPS reduces the minimum allergy-inducing concentrations of Ni at both the sensitization and elicitation steps, and (iii) various microbe-related substances promote sensitization to Ni. Here, we examined the effects of microbe-related substances at the elicitation step. Mice (except for TLR4-mutated C3H/HeJ mice) were sensitized to Ni by intraperitoneal injection of NiCl(2) + E. coli LPS. Ten days later their ear-pinnas were challenged with 1 µM NiCl(2) with or without a test substance. Although NiCl(2) alone at this concentration does not induce Ni-allergy, its combination with the following substances induced Ni-allergy in BALB/c mice: LPS preparations from oral gram-negative bacteria (Prevotella intermedia and Porphyromonas gingivalis), a mannan preparation from a fungus (Saccharomyces cerevisiae), and synthetic NOD2 and TLR2 agonists. The effect of the mannan preparation was small in C3H/HeJ mice (sensitized with NiCl(2) + the P. intermedia preparation). The P. intermedia preparation promoted Ni-allergy in C3H/HeJ and nude mice, but not in mice deficient in either TLR2 or histidine decarboxylase. Intragingival injection of the P. intermedia preparation and later challenge with NiCl(2) alone to ear-pinnas also promoted Ni-allergy. These results indicate that (i) in Ni-allergy, a microbial milieu or innate immunity is important at the elicitation step, too, and (ii) some oral bacteria may promote Ni-allergy via TLR2-stimulant(s) production.

Effects of luteolin on the release of nitric oxide and interleukin-6 by macrophages stimulated with lipopolysaccharide from Prevotella intermedia.

BACKGROUND: Although a range of biologic and pharmacologic activities of luteolin has been reported, little is known about its potential as an agent to treat periodontal disease. In the present study, we investigated whether luteolin could downregulate the production of proinflammatory mediators in murine macrophage-like RAW264.7 cells stimulated with lipopolysaccharide (LPS) from Prevotella intermedia (Pi), a major cause of inflammatory periodontal disease, and we attempted to elucidate the possible mechanisms of action. METHODS: LPS was prepared from lyophilized Pi ATCC 25611 cells by the standard hot phenol-water method. Culture supernatants were collected and assayed for nitric oxide (NO) and interleukin (IL)-6. We used real-time polymerase chain reaction to detect inducible NO synthase (iNOS) and IL-6 mRNA expression. iNOS expression, phosphorylation of JNK and p38, IκB-α degradation, nuclear translocation of nuclear factor-kappaB (NF-κB) subunits, and signal transducer and activator of transcription-1 (STAT1) phosphorylation were characterized via immunoblotting. DNA-binding of NF-κB was also analyzed. RESULTS: Luteolin strongly suppressed the production of NO and IL-6 at both gene transcription and translation levels in Pi LPS-activated RAW264.7 cells. Mitogen-activated protein kinase pathways were not involved in the inhibition of Pi LPS-induced NO and IL-6 release by luteolin. Luteolin did not reduce NF-κB transcriptional activity at the level of IκB-α degradation. Luteolin blocked NF-κB signaling through inhibition of nuclear translocation and DNA binding activity of NF-κB p50 subunit and suppressed STAT1 signaling. CONCLUSIONS: Although further research is encouraged to clarify the detailed mechanism of action, flavonoid luteolin may contribute to blockade of the host-destructive processes mediated by these two proinflammatory mediators and could have potential use in the treatment of inflammatory periodontal disease.

Melatonin inhibits Prevotella intermedia lipopolysaccharide-induced production of nitric oxide and interleukin-6 in murine macrophages by suppressing NF-κB and STAT1 activity.

Although a range of biological and pharmacological activities of melatonin have been reported, little is known about its potential anti-inflammatory efficacy in periodontal disease. In this study, we investigated the effects of melatonin on the production of inflammatory mediators by murine macrophages stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a major cause of inflammatory reactions in the periodontium, and sought to determine the underlying mechanisms of action. Melatonin suppressed the production of nitric oxide (NO) and interleukin-6 (IL-6) at both gene transcription and translation levels in P. intermedia LPS-activated RAW264.7 cells. P. intermedia LPS-induced NF-κB-dependent luciferase activity was significantly inhibited by melatonin. Melatonin did not reduce NF-κB transcriptional activity at the level of IκB-α degradation. Melatonin blocked NF-κB signaling through the inhibition of nuclear translocation and DNA-binding activity of NF-κB p50 subunit and suppressed STAT1 signaling. Although further research is required to clarify the detailed mechanism of action, we conclude that melatonin may contribute to blockade of the host-destructive processes mediated by these two proinflammatory mediators and could be a highly efficient modulator of host response in the treatment of inflammatory periodontal disease.

Gene expression profile and pathogenicity of biofilm-forming Prevotella intermedia strain 17.

BACKGROUND: Prevotella intermedia (P. intermedia), a gram-negative, black-pigmented anaerobic rod, has been implicated in the development of chronic oral infection. P. intermedia strain 17 was isolated from a chronic periodontitis lesion in our laboratory and described as a viscous material producing strain. The stock cultures of this strain still maintain the ability to produce large amounts of viscous materials in the spent culture media and form biofilm-like structures. Chemical analyses of this viscous material showed that they were mainly composed of neutral sugars with mannose constituting 83% of the polysaccharides. To examine the biological effect of the extracellular viscous materials, we identified and obtained a naturally-occurring variant strain that lacked the ability to produce viscous materials in vitro from our stock culture collections of strain 17, designated as 17-2. We compared these two strains (strains 17 versus 17-2) in terms of their capacities to form biofilms and to induce abscess formation in mice as an indication of their pathogenicity. Further, gene expression profiles between these two strains in planktonic condition and gene expression patterns of strain 17 in solid and liquid cultures were also compared using microarray assays. RESULTS: Strain 17 induced greater abscess formation in mice as compared to that of the variant. Strain 17, but not 17-2 showed an ability to interfere with the phagocytic activity of human neutrophils. Expression of several genes which including those for heat shock proteins (DnaJ, DnaK, ClpB, GroEL and GroES) were up-regulated two to four-fold with statistical significance in biofilm-forming strain 17 as compared to the variant strain 17-2. Strain 17 in solid culture condition exhibited more than eight-fold up-regulated expression levels of several genes which including those for levanase, extracytoplasmic function-subfamily sigma factor (sigmaE; putative) and polysialic acid transport protein (KpsD), as compared to those of strain 17 in liquid culture media. CONCLUSION: These results demonstrate that the capacity to form biofilm in P. intermedia contribute to their resistance against host innate defence responses.