Huth KC, Quirling M, Maier S, Kamereck K, AlKhayer M, Paschos E, Welsch U, Miethke T, Brand K, Hickel R.Effectiveness of ozone against endodontopathogenic microorganisms in a root canal biofilm model. International Endodontic Journal, 42, 3-13, 2009. Aim To assess the antimicrobial efficacy of aqueous (1.25-20 lg mL )1 ) and gaseous ozone (1-53 g m )3 ) as an alternative antiseptic against endodontic pathogens in suspension and a biofilm model. Methodology Enterococcus faecalis, Candida albicans, Peptostreptococcus micros and Pseudomonas aeruginosa were grown in planctonic culture or in mono-species biofilms in root canals for 3 weeks. Cultures were exposed to ozone, sodium hypochlorite (NaOCl; 5.25%, 2.25%), chlorhexidine digluconate (CHX; 2%), hydrogen peroxide (H 2 O 2 ; 3%) and phosphate buffered saline (control) for 1 min and the remaining colony forming units counted. Ozone gas was applied to the biofilms in two experimental settings, resembling canal areas either difficult (setting 1) or easy (setting 2) to reach. Time-course experiments up to 10 min were included. To compare the tested samples, data were analysed by one-way anova.Results Concentrations of gaseous ozone down to 1 g m )3 almost and aqueous ozone down to 5 lg mL )1 completely eliminated the suspended microorganisms as did NaOCl and CHX. Hydrogen peroxide and lower aqueous ozone concentrations were less effective. Aqueous and gaseous ozone were dose-and strain-dependently effective against the biofilm microorganisms. Total elimination was achieved by high-concentrated ozone gas (setting 2) and by NaOCl after 1 min or a lower gas concentration (4 g m )3 ) after at least 2.5 min. High-concentrated aqueous ozone (20 lg mL )1 ) and CHX almost completely eliminated the biofilm cells, whilst H 2 O 2 was less effective. Conclusion High-concentrated gaseous and aqueous ozone was dose-, strain-and time-dependently effective against the tested microorganisms in suspension and the biofilm test model.

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Stimulation of the human monocytic cell line Mono Mac 6 with the synthetic lipopeptide (S)-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys4-OH, trihydrochloride (Pam3Cys) at 10 μg/ml induces a rapid expression of the TNF gene in a TLR2-dependent fashion. Preculture of the cells with Pam3Cys at 1 μg/ml leads to a reduced response after subsequent stimulation with Pam3Cys at 10 μg/ml, indicating that the cells have become tolerant to Pam3Cys. The CD14 and TLR2 expression is not decreased on the surface of the tolerant cells, but rather up-regulated. Analysis of the NF-κB binding in Pam3Cys-tolerant cells shows a failure to mobilize NF-κB-p50p65 heterodimers, while NF-κB-p50p50 homodimers remain unchanged. Pam3Cys-tolerant cells showed neither IκBα-Ser32 phosphorylation nor IκBα degradation but MyD88 protein was unaltered. However, IRAK-1 protein was absent in Pam3Cys-induced tolerance, while IRAK-1 mRNA was still detectable at 30% compared with untreated cells. In contrast, in LPS-tolerized cells, p50p50 homodimers were induced, IRAK-1 protein level was only partially decreased, and p50p65 mobilization remained intact. It is concluded that in Mono Mac 6 monocytic cells, inhibition of IRAK-1 expression at the mRNA and protein levels is the main TLR-2-dependent mechanism responsible for Pam3Cys-induced tolerance, but not for TLR-4-dependent LPS-induced tolerance.

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TNF is a major mediator of inflammation, immunity, and apoptosis. Pre-exposure to TNF reduces sensitivity to restimulation, a phenomenon known as tolerance, considered as protective in sepsis, but also as a paradigm for immunoparalysis. Earlier experiments in TNF-tolerant cells display inhibition of NF-κB-dependent IL-8 gene expression at the transcriptional level with potential involvement of C/EBPβ. In this study, we have shown that a κB motive was sufficient to mediate transcriptional inhibition under TNF tolerance conditions in monocytic cells. Furthermore, in tolerant cells, TNF-induced NF-κB p65 phosphorylation was markedly decreased, which was accompanied by the formation of C/EBPβ-p65 complexes. Remarkably, in C/EBPβ−/− cells incubated under the conditions of TNF tolerance, neither impairment of transcription nor inhibition of p65 phosphorylation was observed. Finally, we showed that C/EBPβ overexpression reduced p65-mediated transactivation and that association of C/EBPβ with p65 specifically prevented p65 phosphorylation. Our data demonstrate that C/EBPβ is an essential signaling component for inhibition of NF-κB-mediated transcription in TNF-tolerant cells and suggest that this is caused by blockade of p65 phosphorylation. These results define a new molecular mechanism responsible for TNF tolerance in monocytic cells that may contribute to the unresponsiveness seen in patients with sepsis.

Ozone has been proposed as an adjunct antiseptic in periodontitis therapy. The aim of this study was to investigate the antimicrobial effectiveness of gaseous/aqueous ozone, in comparison with that of the established antiseptic chlorhexidine digluconate (CHX), against periodontal microorganisms. Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, and Parvimonas micra in planktonic or biofilm cultures were exposed, for 1 min, to gaseous ozone, aqueous ozone, CHX, or phosphate-buffered saline (control). None of the agents was able to substantially reduce the A. actinomycetemcomitans count in biofilm cultures. In contrast, P. gingivalis, T. forsythia, and P. micra could be eliminated by 2% CHX or by ozone gas at 53 gm(-3) . Significantly greater antimicrobial effects were observed against planktonic cultures than against biofilm-associated bacteria. The rate of killing was influenced by the species of bacteria, and by the type and concentration of agent. There were no significant differences in the effectiveness of aqueous ozone (20 μg ml(-1) ) or gaseous ozone (≥ 4 gm(-3) ) compared with 2% CHX but they were more effective than 0.2% CHX. Therefore, high-concentrated gaseous and aqueous ozone merit further investigation as antiseptics in periodontitis therapy. A safe system for applying gaseous ozone into the periodontal pocket that avoids inhalation still needs to be developed.

Ozone has been proposed as an alternative oral antiseptic in dentistry, due to its antimicrobial power reported for gaseous and aqueous forms, the latter showing a high biocompatibility with mammalian cells. New therapeutic strategies for the treatment of periodontal disease and apical periodontitis should consider not only antibacterial effects, but also their influence on the host immune response. Therefore, our aim was to investigate the effect of aqueous ozone on the NF-κB system, a paradigm for inflammation-associated signaling/transcription. We showed that NF-κB activity in oral cells stimulated with TNF, and in periodontal ligament tissue from root surfaces of periodontally damaged teeth, was inhibited following incubation with ozonized medium. Under this treatment, IκBα proteolysis, cytokine expression, and κB-dependent transcription were prevented. Specific ozonized amino acids were shown to represent major inhibitory components of ozonized medium. In summary, our study establishes a condition under which aqueous ozone exerts inhibitory effects on the NF-κB system, suggesting that it has an anti-inflammatory capacity.

There is some evidence that the potent cytokine tumor necrosis factor (TNF) is able to induce tolerance after repeated stimulation of cells. To investigate the molecular mechanisms mediating this phenomenon, the expression of interleukin-8 (IL-8), which is regulated by transcription factors NF-B and C/EBP␤, was monitored under TNF tolerance conditions. Pretreatment of monocytic cells for 72 h with low TNF doses inhibited TNFinduced (restimulation with a high dose) IL-8 promoterdependent transcription as well as IL-8 production. Under these conditions neither activation of NF-B nor I B proteolysis was affected after TNF re-stimulation, albeit a slightly reduced I B-␣ level was found in the TNF pretreated but not re-stimulated sample. Remarkably, in tolerant cells an increased binding of C/EBP␤ to its IL-8 promoter-specific DNA motif as well as an elevated association of C/EBP␤ protein with p65-containing NF-B complexes was observed. Finally, overexpression of C/EBP␤, but not p65 or Oct-1, markedly prevented TNFinduced IL-8 promoter-dependent transcription. Taken together, these data indicate that the expression of IL-8 is inhibited at the transcriptional level in TNF-tolerant cells and C/EBP␤ is involved under these conditions in mediating the negative-regulatory effects, a mechanism that may play a role in inflammatory processes such as sepsis.