Effectiveness of ozone against periodontal pathogenic microorganisms.

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.

Involvement of NF-kappaB signalling in skin physiology and disease.

Transcription factors of the nuclear factor-kappaB (NF-kappaB)/Rel family play a crucial role in gene regulation during a variety of different cellular processes. This review focuses on the increasing knowledge of the role of NF-kappaB in skin physiology and pathology. Several studies demonstrate that NF-kappaB, or components of the system such as IkappaB kinase (IKK)-alpha, seem to be involved in epidermal development and differentiation. Furthermore, a dysregulation of NF-kappaB is suggested to play an important role in skin pathology, including proliferative disorders, e.g. psoriasis, inflammatory processes such as incontinentia pigmenti (IP), sunburn, Lyme disease, allergic contact dermatitis and autoimmune diseases, as well as also in skin carcinogenesis. However, although the knowledge concerning the role of NF-kappaB in the homeostasis of the skin is steadily increasing, many more questions need to be answered.

C/EBP beta blocks p65 phosphorylation and thereby NF-kappa B-mediated transcription in TNF-tolerant cells.

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-kappaB-dependent IL-8 gene expression at the transcriptional level with potential involvement of C/EBPbeta. In this study, we have shown that a kappaB motive was sufficient to mediate transcriptional inhibition under TNF tolerance conditions in monocytic cells. Furthermore, in tolerant cells, TNF-induced NF-kappaB p65 phosphorylation was markedly decreased, which was accompanied by the formation of C/EBPbeta-p65 complexes. Remarkably, in C/EBPbeta(-/-) cells incubated under the conditions of TNF tolerance, neither impairment of transcription nor inhibition of p65 phosphorylation was observed. Finally, we showed that C/EBPbeta overexpression reduced p65-mediated transactivation and that association of C/EBPbeta with p65 specifically prevented p65 phosphorylation. Our data demonstrate that C/EBPbeta is an essential signaling component for inhibition of NF-kappaB-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.

Tolerance induced by the lipopeptide Pam3Cys is due to ablation of IL-1R-associated kinase-1.

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)-Lys(4)-OH, trihydrochloride (Pam(3)Cys) at 10 microg/ml induces a rapid expression of the TNF gene in a TLR2-dependent fashion. Preculture of the cells with Pam(3)Cys at 1 microg/ml leads to a reduced response after subsequent stimulation with Pam(3)Cys at 10 microg/ml, indicating that the cells have become tolerant to Pam(3)Cys. The CD14 and TLR2 expression is not decreased on the surface of the tolerant cells, but rather up-regulated. Analysis of the NF-kappaB binding in Pam(3)Cys-tolerant cells shows a failure to mobilize NF-kappaB-p50p65 heterodimers, while NF-kappaB-p50p50 homodimers remain unchanged. Pam(3)Cys-tolerant cells showed neither IkappaBalpha-Ser(32) phosphorylation nor IkappaBalpha degradation but MyD88 protein was unaltered. However, IRAK-1 protein was absent in Pam(3)Cys-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 Pam(3)Cys-induced tolerance, but not for TLR-4-dependent LPS-induced tolerance.

Detection of IKKbeta-IKKgamma subcomplexes in monocytic cells and characterization of associated signaling.

The IkappaB kinase (IKK) complex is one major step in the regulation of the NF-kappaB/Rel system that is involved in inflammatory and immune responses as well as in proliferation and apoptosis. At present it is not clear whether besides the "classical" IKKalpha-IKKbeta-IKKgamma configuration additional complexes exist in vivo that solely contain IKKbeta and IKKgamma (without IKKalpha). In the current study we were able to demonstrate in monocytic cells that endogenous complexes, which only include IKKbeta as the kinase-active molecule do indeed exist in vivo and that these complexes contain IKKgamma as an additional component. Furthermore, we showed that these IKKbeta-IKKgamma complexes are involved in mainstream NF-kappaB activation cascades because they can be activated by tumor necrosis factor. In contrast, these subcomplexes appear not to participate in NIK-dependent pathways. As a next step we showed that exogenous IKKbeta-IKKgamma complexes can be formed in an intact cell by overexpression and that these artificial complexes fulfill the requirement for participation in regular signaling. Finally, in the absence of IKKalpha we found a retarded proteolysis of IkappaBalpha, but not of IkappaB in, which is associated with a reduced IKK activity. Differential pathways represented by various IKK subcomplexes may open attractive possibilities in treatment of inflammation or cancer allowing specific therapeutic intervention.

Transcriptional inhibition of interleukin-8 expression in tumor necrosis factor-tolerant cells: evidence for involvement of C/EBP beta.

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-kappaB and C/EBPbeta, was monitored under TNF tolerance conditions. Pretreatment of monocytic cells for 72 h with low TNF doses inhibited TNF-induced (restimulation with a high dose) IL-8 promoter-dependent transcription as well as IL-8 production. Under these conditions neither activation of NF-kappaB nor IkappaB proteolysis was affected after TNF re-stimulation, albeit a slightly reduced IkappaB-alpha level was found in the TNF pretreated but not re-stimulated sample. Remarkably, in tolerant cells an increased binding of C/EBPbeta to its IL-8 promoter-specific DNA motif as well as an elevated association of C/EBPbeta protein with p65-containing NF-kappaB complexes was observed. Finally, overexpression of C/EBPbeta, but not p65 or Oct-1, markedly prevented TNF-induced 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/EBPbeta is involved under these conditions in mediating the negative-regulatory effects, a mechanism that may play a role in inflammatory processes such as sepsis.