Human periodontal ligament cells exhibit no endotoxin tolerance upon stimulation with Porphyromonas gingivalis lipopolysaccharide.

BACKGROUND/OBJECTIVES: Endotoxin tolerance is characterized by a state of hyporesponsiveness after confrontation with endotoxins such as lipopolysaccharides (LPS) at low concentrations. The aim of this study was to investigate, whether pretreatment with Porphyromonas gingivalis leads to endotoxin tolerance induction and possible alterations in toll-like receptor (TLR) 2- and 4-induced response in human periodontal ligament cells (hPDLCs). MATERIAL AND METHODS: Primary hPDLCs were pretreated with P. gingivalis (0.1 or 0.3 µg/mL) LPS for 24 hours and afterwards treated with one of the following stimuli: P. gingivalis LPS (1 µg/mL); TLR4 agonist Escherichia coli LPS (0.1 µg/mL; 1 µg/mL); TLR2 agonist Pam3CSK4 (0.1 µg/mL; 1 µg/mL). The protein expression of interleukin (IL)-6, IL-8 and monocyte chemotactic protein-1 was analyzed with quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Gene expression levels of TLR2 and TLR4 were determined by quantitative polymerase chain reaction. RESULTS: Pretreatment of cells with low concentrations of P. gingivalis LPS did not result in lower production of IL-6, IL-8 and monocyte chemotactic protein-1 compared to control group. In some cases, pretreated cells exhibited lower gene expression levels of TLR2 and TLR4 compared to non-pretreated cells. CONCLUSION: The results of this study implicate that hPDLCs do not develop endotoxin tolerance. Furthermore, the amplitude of the inflammatory response shows no significant dependency on TLR2 and TLR4 expression levels.

Outer membrane vesicles of Tannerella forsythia: biogenesis, composition, and virulence.

Tannerella forsythia is the only 'red-complex' bacterium covered by an S-layer, which has been shown to affect virulence. Here, outer membrane vesicles (OMVs) enriched with putative glycoproteins are described as a new addition to the virulence repertoire of T. forsythia. Investigations of this bacterium are hampered by its fastidious growth requirements and the recently discovered mismatch of the available genome sequence (92A2 = ATCC BAA-2717) and the widely used T. forsythia strain (ATCC 43037). T. forsythia was grown anaerobically in serum-free medium and biogenesis of OMVs was analyzed by electron and atomic force microscopy. This revealed OMVs with a mean diameter of ~100 nm budding off from the outer membrane while retaining the S-layer. An LC-ESI-TOF/TOF proteomic analysis of OMVs from three independent biological replicates identified 175 proteins. Of these, 14 exhibited a C-terminal outer membrane translocation signal that directs them to the cell/vesicle surface, 61 and 53 were localized to the outer membrane and periplasm, respectively, 22 were predicted to be extracellular, and 39 to originate from the cytoplasm. Eighty proteins contained the Bacteroidales O-glycosylation motif, 18 of which were confirmed as glycoproteins. Release of pro-inflammatory mediators from the human monocytic cell line U937 and periodontal ligament fibroblasts upon stimulation with OMVs followed a concentration-dependent increase that was more pronounced in the presence of soluble CD14 in conditioned media. The inflammatory response was significantly higher than that caused by whole T. forsythia cells. Our study represents the first characterization of T. forsythia OMVs, their proteomic composition and immunogenic potential.

[Mechanisms of nephrotoxicity of novel anticancer compound maleimide derivative MI-1].

The features of the impact of the maleimide derivative 1-(4-Cl-benzyl)-3-chloro-4-(CF3-fenilamino)-1H-pyrrole-2,5-dione (MI-1) on the viability and apoptosis-induced cell death of renal proximal and distal tubular epithelial cells and the amount of total and phosphorylated ERK1/2 were studied in order to establish possible mechanisms of nephrotoxicity induced by of MI-1. The viability and apoptosis of renal epithelial tubular cells after incubation with MI-1 were perfomed by 3,4,5-dymetyltiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT)-test and by flow cytometry after staining with specific antibodies to annexin V, respectively. The amount of ERK 1/2 was determined by Western blotting. The data indicate that MI-1 was more toxic with respect to the epithelial cells of distal than proximal tubule cells. The apoptosis-induced cell death pathway is involved in the mechanisms of MI-1 cytotoxicity. One of the possible mechanisms of MI-1 nephrotoxicity is increase in phosphorylation of ERK1/2 in the distal tubules. At the same time the increase amount of total ERK1/2 in proximal tubules under the influence of MI-1 may contribute to the survival of proximal tubular epithelial cells under the impact of a toxic factor or oxidative stress.

Smoking influences salivary histamine levels in periodontal disease.

OBJECTIVES: Histamine, a potent vasoactive amine, is increased in saliva of periodontitis patients. The present study aimed to further investigate the diagnostic potential of histamine for periodontal disease and assessed smoking, a major risk factor of periodontitis, as a possible influencing factor. METHODS: Salivary and serum samples of 106 participants (60 periodontitis patients, 46 controls) were collected. Salivary histamine was determined by a commercially available ELISA kit, and serum C-reactive protein was measured by a routine laboratory test. Cigarettes per day and packyears were assessed as smoking exposure parameters. RESULTS: Statistically significantly increased levels of salivary histamine and serum C-reactive protein were detected between the patient and control group (P = 0.022 and P = 0.001). Salivary histamine levels were significantly higher in smoking compared with non-smoking patients (P < 0.001), and salivary histamine as well as serum C-reactive protein correlated significantly positively with smoking exposure parameters (P < 0.05). CONCLUSIONS: Smoking, an established and common risk factor of periodontitis, was assessed as a possible influencing factor for salivary histamine. Most interestingly, salivary histamine differed highly significantly between smoking and non-smoking periodontitis patients. Our results suggest a possible involvement of histamine in tobacco-exacerbated periodontal disease, but do not suggest salivary histamine as a reliable diagnostic marker for periodontitis.

Potential of the Tannerella forsythia S-layer to delay the immune response.

UNLABELLED: The periodontal pathogen Tannerella forsythia possesses a glycosylated S-layer as an outermost cell decoration. While the S-layer provides a selection advantage to the bacterium in the natural habitat, its virulence potential remains to be investigated. In the present study, the immune responses of human macrophages and gingival fibroblasts upon stimulation with wild-type T. forsythia and an S-layer-deficient mutant were investigated. The mRNA expression levels of the pro-inflammatory mediators IL-1ß, TNF-α, and IL-8 were analyzed by qPCR, and the production of the corresponding cytokines was investigated by ELISA. The S-layer-deficient T. forsythia mutant induced significantly higher levels of pro-inflammatory mediators compared with wild-type T. forsythia, especially at the early phase of response. Analysis of these data suggests that the S-layer of T. forsythia is an important virulence factor that attenuates the host immune response to this pathogen by evading the bacterium's recognition by the innate immune system. ABBREVIATIONS: DMSO, dimethylsulfoxide; FBS, fetal bovine serum; GAPDH, glycerinaldehyde-3-phosphate-dehydrogenase; HGFs, human gingival fibroblasts; LPS, lipopolysaccharide; MEM, minimal essential medium; MTT, 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide; OD, optical density; PBS, phosphate-buffered saline; qPCR, quantitative polymerase chain-reaction; SD, standard deviation; Tannerella forsythia ATCC 43037, Tf wt; Tannerella forsythia ATCC 43037 S-layer mutant, Tf ΔtfsAB.

[Effect of nitric oxide on 5'-nucleotidase activity of the membrane rafts in the smooth muscle cells].

We investigated the effect of nitric oxide on the catalytic activity of 5'-nucleotidase associated with insoluble membrane domains (rafts) of pig stomach smooth muscle. The low concentration (0.1-10.0 microM) of nitric oxide donor sodium nitroprusside led to essential increase of catalytic activity of 5'-nucleotidase. Maximal increase was observed at concentration of sodium nitroprusside of 1 microM. The enzyme's catalytic activity decreased to about control value at higher concentration of this substance. The catalytic activity of 5'-nucleotidase was also increased at presence of NaNO2, but only at high concentration (10 mM). The specific thiol-alkylating agent N-ethylmaleinimide (1-100 microM) led to essential decrease of enzyme catalytic activity. Our data shows that nitric oxide changes the AMP-ase activity of 5'-nucleotidase, that is thought to be due to direct effect of this substance on protein. We suppose, that such effect of nitric oxide could be physiologicaly important in functioning of smooth muscle.

[Catalytic characteristics of 5'-nucleotidase in the structure of cell membrane rafts in smooth muscles]. / Katalitychni vlastyvosti 5'-nukleotydazy u skladi membrannykh raftiv klityn hladen'kykh m'iaziv.

5'-nucleotidase (EN 3.1.3.5) is widely distributed enzyme occurring in vertebrate, bacterial and plant cells. The main physiological function of 5'-nucleotidase is hydrolysis of 5'-AMP to adenosine and Pi. It was found that the detergent-insoluble membrane domains (rafts) are enriched by proteins possessing high 5'-AMPase activity. This study is aimed to investigate some physical and chemical properties of 5'-nucleotidase, which is present in detergent insoluble membrane domains isolated from pig stomach and lung. It was shown for the first time that catalytic properties of the raft-associated 5'-nucleotidase and of the pure enzyme described in literature differ. Our results demonstrate that the greatest activity of the raft-associated enzyme takes place in the physiological conditions contrary to the pure enzyme. Our data suggest that such changes of 5'-nucleotidase catalytic activity might be due to the disruption of its interaction with membrane rafts.

[Effect of nitric oxide on ATPase activity of smooth muscle actomyosin]. / Vplyv oksydu azotu na ATP-aznu aktyvnist' aktomiozynu hladen'kikh m'iaziv.

The effects of nitric oxide donor sodium nitroprusside (SNP) on ATPase activities of smooth muscle actomyosin and myosin were investigated. The effect of SNP on actomyosin ATPase activity was biphasic: the low concentration of this reagent increased the actomyosin ATPase activity while the high concentration exerted opposite effect. These effects were similar to those induced by the specific thiol-alkylating agent N-ethylmaleimide. These data demonstrate that nitric oxide exert the direct effect on smooth muscle contractile proteins. Such effect may be involved in physiological action of NO on smooth muscle.

[Mechanism of nitric oxide's effect on the sensitivity of smooth muscle contractions to calcium]. / Mekhanizm vplyvu oksydu azotu na chutlyvist' skorochuval'noho aparatu hladen'kykh m'iaziv do Ca2+.

It was investigated whether the SH-groups of contractile proteins are involved in NO-induced relaxation of saponin-skinned smooth muscle strips. Both, the thiol-specific alkylating agent N-ethylmaleamide (NEM), and thiol-reducing agent dithiotreitol (DTT) induced relaxation of maximally activated skinned rat portal vein preparations. The relaxing effects of DTT and nitric oxide donor sodium nitroprusside were not additive. After the relaxation, induced by one of this agent, the effect of the other one was negligible. We suggest that NO-induced smooth muscle relaxation be due to decreasing of force sensitivity to Ca2+. This effect of nitric oxide is realized by its interaction with critical thiol groups of smooth muscle contractile proteins.

[The effect of nitric oxide on the contractile activity of skinned preparations of the smooth muscles of the rat portal vein]. / Vplyv oksydu azotu na skorochuval'nu aktyvnist' skinovanykh prepartiv hladen'kykh m'iaziv voritnoï veny shchura,

The effects of nitric oxide donors sodium nitroprusside and nitroglycerin on the contractile activity of the skinned rat portal vein were investigated. The skinned strips of rat portal vein were prepared by treatment 0.1 mg/ml saponin for 20 min. Maximally pCa 4.0 activated strips were exposed to nitric oxide donors. Both sodium nitroprusside and nitroglycerin induced dose-dependent decrease of the isometric force of the contraction. The inhibitor of soluble guanylyl cyclase methylene blue (50 microM) did not blocked this effect. This result shows the existence of the mechanism of NO actions on vascular smooth muscle which is not associated with the changes of intracellular Ca2+ and the activation of the soluble guanil cyclase.

[The effect of annexins on the contractile activity of the smooth muscles in the rat portal vein]. / Vplyv aneksyniv na skorochuval'nu aktiyvnist' hladen'kykh m'iaziv voritnoi veny shchura.

This study examines the effects of the family of calcium-dependent phospholipids binding proteins (annexins) on the contractile properties of rat portal vein. We developed the new method of annexin's purification from pig stomach muscle. It found that in nanomolar concentrations annexins caused dose-dependent effects on phasic and tonic component of rat portal vein contractile activity. The frequency of spontaneous contractions was increased whereas the amplitude was decreased. At the same time we observed the rise of basal tone level of contractions. We suggested that annexins may change the contractile properties of vascular smooth muscle.