Effects of chlorhexidine, essential oils and herbal medicines (Salvia, Chamomile, Calendula) on human fibroblast in vitro.

Antiseptic rinses have been successfully used in inflammatory states of the gums and oral cavity mucosa. Antibacterial effects of chlorhexidine, essential oils and some herbs are well documented. Reaction of host tissue to these substances has much poorer documentation. The aim of the study was to analyse the influence of chlorhexidine (CHX), essential oil (EO: thymol, 0.064%; eucalyptol, 0.092%; methyl salicylate, 0.060%; menthol, 0.042%) mouth rinses and salvia, chamomile and calendula brews on fibroblast biology in vitro. The human fibroblast CCD16 line cells were cultured in incubation media which contained the examined substances. After 24 and 48 hours, the cell morphology, relative growth and apoptosis were evaluated. Exposure of fibroblasts to CHX, EO or salvia caused various changes in cell morphology. Cells cultured for 48 hours with CHX revealed a noticeably elongated shape of while cells cultured in high EO concentration or with salvia were considerably smaller and contracted with fewer projections. Chlorhexidine, EO and salvia reduced the fibroblast proliferation rate and stimulated cell death. Both reactions to EO were dose dependent. Cells exposure to chamomile or calendula brews did not change morphology or proliferation of fibroblasts. The results of this in vitro study showed that in contrast to chamomile and calendula, the brews of EO, CHX or salvia had a negative influence on fibroblast biology.

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.

Characteristic of bacteriocines and their application.

Bacteriocines are small peptides with anti-bacterial properties. They are produced both by Gram-positive and Gram-negative bacteria. Until now, a few hundred bacteriocines were described. Classification of bacteriocines undergoes continuous alterations, as new developments regarding their structure, amino acid sequence and recognised mechanism of their action are available. Some of bacteriocins (lantibiotics) contain atypical amino acids, such as lantionine (Lan), methyllantionine (MeLan), dehydroalanine (Dha), dehydrobutyrine (Dhb), or D-alanine (D-Ala). The best recognized bacteriocines are produced by lactic acid bacteria, including nisine produced by strains of Lactococcus lactis. These bacteriocines have been recognized to be fully safe for humans. At present, nisine is used in food industry, as a preserving agent. Other lactic acid bacteria bacteriocines and probiotic preparations provide an alternative for antibiotics, and are used in food and in animal feed.

Selected Factors of Innate Immunity in Healthy Individuals with S. aureus Nasal Carriage.

Nasal carriage of Staphylococcus aureus represents a well-defined factor of risk involving community and hospital-acquired infections. Recently a significance of several host factors has been pointed out and, in particular, of immune determinants in nasal S. aureus colonization. Therefore, this study aimed at analysis of manifestation involving manifestation in the nasal secretions of important components of the host innate immunity - human beta-defensin-2 (HBD-2), lysozyme (Ly), and interferon-gamma (IFN-γ) in healthy individuals and in persons with persistent carriage of S. aureus. The studies were conducted in two groups of healthy volunteers, encompassing non-carriers (group 1) or persistent carriers of S. aureus (group 2). Elisa assays were employed to evaluate levels of HBD-2, Ly, and IFN-γ in nasal secretions of the examined donors. In S. aureus carriers a significant variability of HBD-2 levels was detected, corresponding to, respectively, the high (averaging at 1.46 ng/ml) and the low (averaging at 0.13 ng/ml) secretory response of the defensin. The level of Ly in S. aureus carriers averaged at 1.46 µg/ml and it manifested no significant difference as compared to that noted in non-carriers. In turn, concentrations of IFN-γ in nasal secretions in the group of carriers of S. aureus amounted on the average to 81.7 pg/ml and they were 1.3-fold higher that in the group of non-carriers. The obtained results allow to conclude that IFN-γ secretion by the nasal cavity-colonizing S. aureus remains quantitatively insufficient to eliminate the pathogen. Nevertheless, a significant increase in levels of this host factor may be important for restriction of the staphylococcal colonization and protection against development of an invasive infection. In turn, the role of HBD-2 and Ly in inactivation of the colonizing S. aureus remains doubtful.

Effect of novobiocin on the viability of human gingival fibroblasts (HGF-1).

BACKGROUND: Novobiocin is a coumarin antibiotic, which affects also eukaryotic cells inhibiting activity of Heat shock protein 90 (Hsp90). The Hsp90 represents a molecular chaperone critical for stabilization and activation of many proteins, particularly oncoproteins that drive cancer progression. Currently, Hsp90 inhibitors focus a significant attention since they form a potentially new class of drugs in therapy of cancer. However, in the process of tumorigenesis a significant role is played also by the microenvironment of the tumour, and, in particular, by cancer-associated fibroblasts (CAFs). This study aimed at examination of the effect played by novobiocin on viability of human gingival fibroblasts (HGF-1). METHODS: The studies were conducted using 24 h cultures of human gingival fibroblasts - HGF-1 (CRL-2014) in Chamber Slides, in presence of 0.1, 0.5, 1.0, 2.5 or 5.0 mM novobiocin. Cell viability was evaluated using fluorescence test, ATP assay and LDH release. RESULTS: Viability of HGF-1 was drastically reduced after 5 hour treatment with novobiocin in concentrations of 1 mM or higher. In turn, the percentage of LDH-releasing cells after 5 h did not differ from control value although it significantly increased after 10 h incubation with 1 mM and continued to increase till the 20th hour. CONCLUSIONS: The obtained data indicate that novobiocin may induce death of human gingival fibroblasts. Therefore, application of the Hsp90 inhibitor in neoplastic therapy seems controversial: on one hand novobiocin reduces tumour-associated CAFs but, on the other, it may induce a significant destruction of periodontium.

Effect of oral administration involving a probiotic strain of Lactobacillus reuteri on pro-inflammatory cytokine response in patients with chronic periodontitis.

This study aimed at evaluation of pro-inflammatory cytokine response (TNF-α, IL-1ß and IL-17) in patients with chronic periodontitis administered per os with a probiotic strain of Lactobacillus reuteri. In the 38 adult patients with moderate chronic periodontitis, professional cleaning of teeth was performed. Two weeks after performing the oral hygienization procedures, clinical examination permitted to distinguish a group of 24 patients (Group 1) in whom treatment with probiotic tablets containing L. reuteri strain, producing hydrogen peroxide (Prodentis), was conducted. In the remaining 14 patients, no probiotic tablet treatment was applied (the control group; Group 2). From all patients in two terms, gingival crevicular fluid (GCF) was sampled from all periodontal pockets. Estimation of TNF-α, IL-lß and IL-17 in GCF was performed using the ELISA method. After completion of the therapy with probiotic tablets, 18 (75%) of the patients of Group 1 have manifested a significant decrease in levels of studied pro-inflammatory cytokines (TNF-α, IL-1ß and IL-17). In parallel, we have detected an improvement of clinical indices [sulcus bleeding index (SBI), periodontal probing depth (PPD), clinical attachment level (CAL)]. At individuals of Group 2 levels of studies, pro-inflammatory cytokines and clinical indices (SBI, PPD, CAL) were significantly higher than in Group 1. Results obtained in this study indicate that application of oral treatment with tablets containing probiotic strain of L. reuteri induces in most patients with chronic periodontitis a significant reduction of pro-inflammatory cytokine response and improvement of clinical parameters (SBI, PPD, CAL). Therefore, such an effect may result in a reduced activity of the morbid process.

The evaluation of enamel matrix derivative on subgingival microbial environment in non-surgical periodontal therapy.

INTRODUCTION AND OBJECTIVE: Recent in vitro studies indicate that enamel matrix derivative (EMD) could modulate the growth of periodontopathogens. The aim of the presented study was an analysis of the influence of EMD on the presence of Porphyromonas and Prevotella bacteria in the periodontal pockets of patients with chronic periodontitis in non-surgical periodontal therapy. MATERIALS AND METHODS: The studies were conducted on 20 patients. The condition of the periodontium was evaluated by clinical indexes: API. SBI. PD. CAL before and 3 months after the therapy in two selected quadrants. The material was collected for investigation. The periodontopathogens were cultured and identified. Two days after EMD-scaling root planing (SRP) was applied into the pockets. RESULTS: In the group of patients under investigation before the EMD application the presence of P. gingivalis was found in 6 patients and P. intermedia in 8 patients. After root planing and EMD application no periodontopathogens were identified in those patients either in the periodontal pockets treated with EMD or in the periodontal pockets free from EMD (control). In the statistical analysis of changes in clinical indexes, the application of SRP and SRP combined with EMD was proved to significantly influence the improvement of the clinical state. However, no significant differences between the individual parameters were found in either group. CONCLUSIONS: The SRP is an effective method of limiting the development of periopathogens in periodontal pockets. The non-surgical therapy with EMD does not change the clinical parameters significantly, compared with the SRP. Simultaneously, the application of EMD inhibits the development of periopathogens, such as Porphyromonas gingivalis and Prevotella.