Witold Chmielewski

Whole cigarette smoke promotes human gingival epithelial cell apoptosis and inhibits cell repair processes.

BACKGROUND AND OBJECTIVE Smoking cigarettes increases the risk of developing various types of human diseases, including cancers and periodontitis. As gingival epithelial cells are known to play an active role in innate immunity via the secretion of a wide variety of mediators, and as these cells are the first ones exposed to environmental stimuli such as cigarette smoke, we sought to investigate the effects of whole cigarette smoke on normal human gingival epithelial cells and tissue. MATERIAL AND METHODS Human gingival epithelial cells were extracted from healthy nonsmokers and used either as a monolayer or as an engineered human oral mucosa to investigate the effect of whole cigarette smoke on cell growth, apoptosis and wound repair/migration. RESULTS Our findings show that when gingival epithelial cells were exposed once to whole cigarette smoke, this resulted in a significant inhibition of cell growth through an apoptotic pathway, as confirmed by an increase of Bax and a decrease of Bcl-xL and caspase-3 activity. Cigarette smoke also inhibited epithelial cell migration. These effects may explain the disorganization of the engineered human oral mucosa tissue when exposed to whole cigarette smoke. CONCLUSION Exposure to whole cigarette smoke markedly inhibits epithelial cell growth through an apoptosis/necrosis pathway that involves Bax and Bcl-xL proteins and caspase-3 activity. Cigarette smoke also disrupts epithelial cell migration, which may negatively affect periodontal wound healing.

Cigarette smoke condensate increases C. albicans adhesion, growth, biofilm formation, and EAP1 , HWP1 and SAP2 gene expression

BackgroundSmokers are more prone to oral infections than are non-smokers. Cigarette smoke reaches the host cells but also microorganisms present in the oral cavity. The contact between cigarette smoke and oral bacteria promotes such oral diseases as periodontitis. Cigarette smoke can also modulate C. albicans activities that promote oral candidiasis. The goal of this study was to investigate the effect of cigarette smoke condensate on C. albicans adhesion, growth, and biofilm formation as well as the activation of EAP1, HWP1 and secreted aspartic protease 2.ResultsCigarette smoke condensate (CSC) increased C. albicans adhesion and growth, as well as biofilm formation. These features may be supported by the activation of certain important genes. Using quantitative RT-PCR, we demonstrated that CSC-exposed C. albicans expressed high levels of EAP1, HWP1 and SAP2 mRNA and that this gene expression increased with increasing concentrations of CSC.ConclusionCSC induction of C. albicans adhesion, growth, and biofilm formation may explain the increased persistence of this pathogen in smokers. These findings may also be relevant to other biofilm-induced oral diseases.

Disruption of the ECM33 Gene in Candida albicans Prevents Biofilm Formation, Engineered Human Oral Mucosa Tissue Damage and Gingival Cell Necrosis/Apoptosis

In this study we demonstrated that ΔCaecm33 double mutant showed reduced biofilm formation and causes less damage to gingival mucosa tissues. This was confirmed by the reduced level of necrotic cells and Bax/Bcl2 gene expression as apoptotic markers. In contrast, parental and Caecm33 mutant strains decreased basement membrane protein production (laminin 5 and type IV collagen). We thus propose that ECM33 gene/protein represents a novel target for the prevention and treatment of infections caused by Candida.

The influence of the type of contraction on the masseter muscle EMG power spectrum

Different behaviours of the EMG power spectrum across increasing force levels have been reported for the masseter muscle. A factor that could explain these different behaviours may be the type of contraction used, as was recently shown for certain upper limb muscles(5). The purpose of this study was to compare, between two types of isometric contractions, the behaviour of EMG power spectrum statistics (median frequency (MF) and mean power frequency (MPF)) obtained across increasing force levels. Ten women exerted, while biting in the intercuspal position, three 5 s ramp contractions that increased linearly from 0 to 100% of the maximal voluntary contraction (MVC). They also completed three step contractions (constant EMG amplitude) at each of the following levels: 20, 40, 60 and 80% MVC. EMG signals from the masseter muscle were recorded with miniature surface electrodes. The RMS, as well as the MPF and MF of the power spectrum were calculated at 20, 40, 60 and 80% MVC for each type of contraction. As expected, the RMS values showed similar increases with increasing levels of effort for both types of contractions. Different behaviours for both MPF (contraction(∗)force interaction, ANOVA, P<0.05) and MF (contraction(∗)force interaction, ANOVA, P>0.05) across increasing levels of effort were found between the two types of contraction. The use of step contractions gave rise to a decrease of both MPF and MF with increasing force, while the use of ramp contractions gave rise to an increase in both statistics up to at least 40% MVC followed by a decrease at higher force levels. These findings suggest that the type of contraction used does influence the behaviour of the spectral statistics across increasing force levels and that this could explain the differences obtained in previous studies for the masseter muscle.

Diseases Associated with Oral Polymicrobial Biofilms

The human body can be defined as a symbiotic interaction between eukaryotic cells and prokaryotic cells. The body contains approximately ten times more microbial cells than mammalian cells. Fortunately, a large segment of the microbiome is both helpful and non-harmful, and constitutes the normal microbiome throughout the entire body. The digestive tract and the skin are the most microbial-rich sites containing approximately 1000 species of bacteria. The mouth is another site harbouring a diverse, abundant, and complex microbial community. Microorganisms in the mouth accumulate on both the hard and soft oral tissues and are frequently organised as microbial biofilm. This biofilm is usually harmless, yet under certain conditions, it may locally and systemically be the source of infection. This review focuses on oral biofilm formation and the diseases that may cause.

Cigarette Smoke-Exposed Candida albicans Increased Chitin Production and Modulated Human Fibroblast Cell Responses

The predisposition of cigarette smokers for development of respiratory and oral bacterial infections is well documented. Cigarette smoke can also contribute to yeast infection. The aim of this study was to investigate the effect of cigarette smoke condensate (CSC) on C. albicans transition, chitin content, and response to environmental stress and to examine the interaction between CSC-pretreated C. albicans and normal human gingival fibroblasts. Following exposure to CSC, C. albicans transition from blastospore to hyphal form increased. CSC-pretreated yeast cells became significantly (P < 0.01) sensitive to oxidation but significantly (P < 0.01) resistant to both osmotic and heat stress. CSC-pretreated C. albicans expressed high levels of chitin, with 2- to 8-fold recorded under hyphal conditions. CSC-pretreated C. albicans adhered better to the gingival fibroblasts, proliferated almost three times more and adapted into hyphae, while the gingival fibroblasts recorded a significantly (P < 0.01) slow growth rate but a significantly higher level of IL-1β when in contact with CSC-pretreated C. albicans. CSC was thus able to modulate both C. albicans transition through the cell wall chitin content and the interaction between C. albicans and normal human gingival fibroblasts. These findings may be relevant to fungal infections in the oral cavity in smokers.

E-Cigarette Vapor Induces an Apoptotic Response in Human Gingival Epithelial Cells Through the Caspase-3 Pathway†

Electronic cigarettes represent an increasingly significant proportion of todays consumable tobacco products. E‐cigarettes contain several chemicals which may promote oral diseases. The aim of this study was to investigate the effect of e‐cigarette vapor on human gingival epithelial cells. Results show that e‐cigarette vapor altered the morphology of cells from small cuboidal form to large undefined shapes. Both single and multiple exposures to e‐cigarette vapor led to a bulky morphology with large faint nuclei and an enlarged cytoplasm. E‐cigarette vapor also increased L‐lactate dehydrogenase (LDH) activity in the targeted cells. This activity was greater with repeated exposures. Furthermore, e‐cigarette vapor increased apoptotic/necrotic epithelial cell percentages compared to that observed in the control. Epithelial cell apoptosis was confirmed by TUNEL assay showing that exposure to e‐cigarette vapor increased apoptotic cell numbers, particularly after two and three exposures. This negative effect involved the caspase‐3 pathway, the activity of which was greater with repeated exposure and which decreased following the use of caspase‐3 inhibitor. The adverse effects of e‐cigarette vapor on gingival epithelial cells may lead to dysregulated gingival cell function and result in oral disease. J. Cell. Physiol. 232: 1539–1547, 2017.

Long-term exposure of human gingival fibroblasts to cigarette smoke condensate reduces cell growth by modulating Bax, caspase-3 and p53 expression.

BACKGROUND AND OBJECTIVE Smoking cigarettes increases the risk of oral tissue damage leading to periodontal disease. Gingival fibroblasts, the predominant cell type inhabiting gingival connective tissue, play a critical role in remodeling and maintaining gingival structure. The objective of this study was to investigate the effect of long-term exposure to cigarette smoke on human gingival fibroblast survival/apoptosis and the molecular pathways involved in these cell responses. MATERIAL AND METHODS Human gingival fibroblasts were extracted from healthy non-smokers and cultured in the presence of cigarette smoke condensate (CSC). At the end of each time point, cell growth was evaluated by means of MTT assay. Apoptotic and necrotic genes expression was investigated by polymerase chain reaction array and by annexin V/propidium iodide staining and cell cycle assays. Western blot was used to investigate Bax and p53 proteins. These tests were supported by caspase 3 activity analyses. RESULTS High levels of CSC decreased cell growth and deregulated cell cycle progression by increasing the G(0)/G(1) and reducing the S and G(2)/M phases of the gingival fibroblasts. Polymerase chain reaction arrays revealed the activation of several apoptotic genes by CSC, including TNF receptors, caspases, Bax and p53. This was supported by increases in the Bax and p53 protein levels as well as by an elevated activity of caspase-3 in the CSC-exposed cells. Immunofluorescence staining demonstrated that both Bax and caspase-3 displayed a cytosolic and mitochondrial distribution in the CSC-exposed gingival fibroblasts, compared to controls. The damaging effect of CSC on gingival fibroblast growth was also supported by the decrease in interleukin 6 and 8 secretion by the gingival fibroblasts. CONCLUSION These results suggest that CSC may contribute to deregulating fibroblast functions. This can compromise fibroblast-epithelial cell interactions, which ultimately increases the risk of gingival tissue damage and the onset of periodontitis.

Antagonistic effect of Candida albicans and IFNγ on E-cadherin expression and production by human primary gingival epithelial cells

Caused mainly by Candida albicans, oropharyngeal candidiasis is the most common oral complication associated with HIV disease worldwide. Host defenses against C. albicans essentially fall into two categories: specific immune mechanisms and local oral mucosal epithelial cell defenses. Since oral mucosa is the first line of defense in the form of a physical barrier against C. albicans invasion, and since epithelial cells are involved in anti-Candida innate immunity through different cytokines, we wanted to determine whether C. albicans alters E-cadherin expression and production, and whether interferon-γ (INFγ), a TH1 cytokine, is involved in the anti-Candida defense. Using primary human gingival epithelial cells, we demonstrated that C. albicans significantly decreased E-cadherin mRNA expression and protein production. This effect was basically obtained at later infective periods (24 and 48h). Interestingly, when IFNγ was added to C. albicans infected epithelial cell cultures, it prevented the side effect of C. albicans on E-cadherin mRNA expression and protein production and deposition. All together, these results suggested concomitant interactions between oral epithelial cells and IFNγ against C. albicans infection.

Evaluation of C. albicans Adhesion and Growth on Restorative Dental Materials Enriched or not with Fluoride

Candida albicans (C. albicans) is the most prevalent fungus in the human oral cavity and has been known as the primary cause of denture-related stomatitis. Candida cells have a high adhering potential to dental material in almost the same manner as to oral tissues, and they are known to form biofilm that leads to C. albicans resistance against antifungal drugs. The aim of this study was to investigate C. albicans adhesion and growth on different restorative dental materials and studied the effect of fluoride on C. albicans growth and morphological transition. To this end, C. albicans (Sc 5314) was cultured on acrylic resins, composite resin, and glass-ionomer materials. Growth was analyzed at various times using scanning electron microscopy analyses and cell proliferation assay. The effect of different concentrations (50 and 100 ppm) of exogenous fluoride on C. albicans growth and yeast-to-hyphae transition was investigated. Scanning electron microscopy showed that C. albicans adhered to all of the tested restorative materials. Adhesion was greater on diamond D and ivocap than on composite resin and glass ionomer. After 1 to 4 days, C. albicans growth on acrylic resins was two folds that of the composite resin and the glass-ionomer. The latter also displayed the lowest adhesion and growth which may be due to the release of antimicrobial molecules such as fluoride present in this material. This hypothesis is supported by our results showing that exogenous fluoride significantly inhibits C. albicans growth and its morphological changes from blastospore to hyphal form. This study clearly demonstrates that restorative materials are conducive to C. albicans adhesion and growth. Exogenous fluoride was also shown to down-regulate C. albicans growth and morphological changes. Overall data suggest the possible integration of fluoride into dental materials which may control oral microbial pathogenesis.