Oral Prevotella Species and Their Connection to Events of Clinical Relevance in Gastrointestinal and Respiratory Tracts

Prevotella is recognized as one of the core anaerobic genera in the oral microbiome. In addition, members of this genus belong to microbial communities of the gastrointestinal and respiratory tracts. Several novel Prevotella species, most of them of oral origin, have been described, but limited knowledge is still available of their clinical relevance. Prevotella melaninogenica is among the anaerobic commensals on oral mucosae from early months of life onward, and other early colonizing Prevotella species in the oral cavity include Prevotella nigrescens and Prevotella pallens. Oral Prevotella species get constant access to the gastrointestinal tract via saliva swallowing and to lower airways via microaspiration. At these extra-oral sites, they play a role as commensals but also as potentially harmful agents on mucosal surfaces. The aim of this narrative review is to give an updated overview on the involvement of oral Prevotella species in gastrointestinal and respiratory health and disease

Construction and characterization of a multilayered gingival keratinocyte culture model : the TURK-U model

In construction of epithelial cells as multilayers, the cells are grown submerged to confluence on fibroblast-embedded collagen gels and, then, lifted to air to promote their stratification. We recently demonstrated that gingival epithelial cells form uniform monolayers on semi-permeable nitrocellulose membranes, supported with a semi-solid growth medium, which allows the cells to grow at an air-liquid-solid interface from the beginning of the culturing protocol. In this study, the aim was to further develop our previous model to form a multilayered gingival epithelial culture model. Two different epithelial cell lines (HaCaT from skin and HMK from gingiva) were used in all experiments. Both cell lines were grown first as monolayers for 3 days. After that, keratinocytes were trypsinized, counted and seeded on a sterile semi-permeable nitrocellulose membrane placed on the top of a semi-solid growth medium, forming an air-liquid-solid interface for the cells to grow. At days 1, 4, and 7, epithelial cells were fixed, embedded in paraffin, and sectioned for routine Hematoxylin-Eosin staining and immunohistochemistry for cytokeratin (Ck). At day 1, HMK cells grew as monolayers, while HaCaT cells stratified forming an epithelium with two to three layers. At day 4, a stratified epithelium in the HMK model had four to five layers and its proliferation continued up to day 7. HaCaT cells formed a dense and weakly proliferating epithelium with three to four layers of stratification at day 4 but the proliferation disappeared at day 7. At all days, both models were strongly positive for Ck5, Ck7, and Ck 19, and weakly positive for Ck10. Gingival epithelial cells stratify successfully on semi-permeable nitrocellulose membranes, supported with a semi-solid growth medium. This technique allows researchers to construct uniform gingival epithelial cell multilayers at an air-liquid-solid interface, without using collagen gels, resulting in a more reproducible method.Peer reviewe

Salivary and serum levels of neutrophil proteases in periodontitis and rheumatoid arthritis

Objective: The aim was to profile serum and salivary levels of active-matrix metalloproteinase (aMMP)-8, tissue inhibitor MMP (TIMP)-1, aMMP-8/TIMP-1 ratio, total MMP (tMMP)-9, tMMP-9/TIMP-1 ratio, myeloperoxidase (MPO), and human neutrophil elastase (HNE) in periodontitis and rheumatoid arthritis (RA).Materials and Methods: Rheumatoid arthritis patients with periodontitis (RA + P, n = 26), periodontally healthy RA patients (RA, n = 23), systemically healthy periodontitis patients (P, n = 24), and controls (C, n = 24) were included. aMMP-8 levels were determined by a time-resolved immunofluorescence assay (IFMA), TIMP-1, tMMP-9, MPO, and HNE levels were measured by enzyme-linked immunosorbent (ELISA) assays.Results: Higher salivary aMMP-8 (p < 0.001), aMMP-8/TIMP-1 ratio (p = 0.043), tMMP-9 (p = 0.011), tMMP-9/TIMP-1 ratio (p = 0.022), MPO (p = 0.026) and HNE (p < 0.001) levels were detected in P relative to the controls. Salivary TIMP-1 was increased in RA patients regardless of periodontal status (RA + P vs. P: p = 0.038; RA vs. C: p = 0.020). Serum neutrophil proteases were increased in RA groups (RA + P, RA) compared to systemically healthy groups (P, C) (p < 0.05).Conclusions: Serum levels of neutrophil proteases were increased in RA study groups; however rheumatologic status seemingly does not affect salivary levels of these proteins

Understanding the roles of gingival beta-defensins

Gingival epithelium produces β-defensins, small cationic peptides, as part of its contribution to the innate host defense against the bacterial challenge that is constantly present in the oral cavity. Besides their functions in healthy gingival tissues, β-defensins are involved in the initiation and progression, as well as restriction of periodontal tissue destruction, by acting as antimicrobial, chemotactic, and anti-inflammatory agents. In this article, we review the common knowledge about β-defensins, coming from in vivo and in vitro monolayer studies, and present new aspects, based on the experience on three-dimensional organotypic culture models, to the important role of gingival β-defensins in homeostasis of the periodontium

Regulatory effects of PRF and titanium surfaces on cellular adhesion, spread, and cytokine expressions of gingival keratinocytes

Dental implant material has an impact on adhesion and spreading of oral mucosal cells on its surface. Platelet-rich fibrin (PRF), a second-generation platelet concentrate, can enhance cell proliferation and adhesion. The aim was to examine the regulatory effects of PRF and titanium surfaces on cellular adhesion, spread, and cytokine expressions of gingival keratinocytes. Human gingival keratinocytes were cultured on titanium grade 4, titanium grade 5 (Ti5), and HA discs at 37 degrees C in a CO2 incubator for 6h and 24h, using either elutes of titanium-PRF (T-PRF) or leukocyte and platelet-rich fibrin (L-PRF), or mammalian cell culture medium as growth media. Cell numbers were determined using a Cell Titer 96 assay. Interleukin (IL)-1 beta, IL-1Ra, IL-8, monocyte chemoattractant protein (MCP)-1, and vascular endothelial growth factor (VEGF) expression levels were measured using the Luminex((R)) xMAP (TM) technique, and cell adhesion and spread by scanning electron microscopy. Epithelial cell adhesion and spread was most prominent to Ti5 surfaces. L-PRF stimulated cell adhesion to HA surface. Both T-PRF and L-PRF activated the expressions of IL-1 beta, IL-8, IL-1Ra, MCP-1, and VEGF, T-PRF being the strongest activator. Titanium surface type has a regulatory role in epithelial cell adhesion and spread, while PRF type determines the cytokine response

Proteomic analysis of RAW macrophages treated with cGAMP or c-di-GMP reveals differentially activated cellular pathways

Global and quantitative analysis of the proteome help to reveal how host cells sense invading bacteria andrespond to bacterial signaling molecules. Here, we performed label free quantitative proteomic analysis ofRAW macrophages treated with host-derived cGAMP and bacterial-derived c-di-GMP, in an attempt toidentify cellular pathways impacted by these dinucleotides and determine if the host respondsdifferentially to these two cyclic dinucleotides. We identified a total of 3811 proteins of whichabundances of 404 proteins in cGAMP and 236 proteins in c-di-GMP treated cells were significantlydifferent compared to the control. Many of the proteins that were strongly and commonly upregulated,such as interferon-induced proteins 47, 202 and 204 (Ifi47, Ifi202, Ifi204), ubiquitin-activating enzyme E7(Uba7), interferon-induced protein with tetratricopeptide repeats 1, 2 or 3 (Ifit1, Ifit2, Ifit3), ubiquitin-likeprotein ISG15 (ISG15), might be due to the fact that both dinucleotides promote the production ofinterferons, which induce the expression of many proteins. However, there were also other proteins thatwere differentially affected by cGAMP or c-di-GMP treatment, including probable ATP-dependent RNAhelicase DHX58 (Dhx58), nuclear autoantigen Sp-100 (Sp100), MARCKS-related protein (Marcksl1) andantigen peptide transporter 2 (Tap2). This is probably due to the differential levels of IFNs produced bythe dinucleotides or may indicate that non-STING activation might also contribute to the host's responseto c-di-GMP and cGAMP. Interestingly Trex1, a nuclease that degrades DNA (an activator of cGAS toproduce cGAMP), was upregulated (3.22 fold) upon cGAMP treatment, hinting at a possible feedbackloop to regulate cGAMP synthesis. These results lay a foundation for future studies to better characterizeand understand the complex c-di-GMP and cGAMP signaling network.</p

NFE2L2/NRF2, OGG1, and cytokine responses of human gingival keratinocytes against oxidative insults of various origin

ObjectiveBacterial or tobacco-related insults induce oxidative stress in gingival keratinocytes. The aim of this study was to investigate anti-oxidative and cytokine responses of human gingival keratinocytes (HMK cells) against Porphyromonas gingivalis lipopolysaccharide (Pg LPS), nicotine, and 4-nitroquinoline N-oxide (4-NQO).Materials and methodsHMK cells were incubated with Pg LPS (1 mu l/ml), nicotine (1.54mM), and 4-NQO (1 mu M) for 24h. Intracellular and extracellular levels of interleukin (IL)-1, IL-1 receptor antagonist (IL-1Ra), IL-8, monocyte chemoattractant protein (MCP)-1, and vascular endothelial growth factor (VEGF) were measured with the Luminex (R) xMAP technique, and nuclear factor, erythroid 2 like 2 (NFE2L2/NRF2) and 8-oxoguanine DNA glycosylase (OGG1) with Western blots. Data were statistically analyzed by two-way ANOVA with Bonferroni correction.ResultsAll tested oxidative stress inducers increased intracellular OGG1 levels, whereas only nicotine and 4-NQO induced NFE2L2/NRF2 levels. Nicotine, 4-NQO, and their combinational applications with Pg LPS induced the secretions of IL-1 and IL-1Ra, while that of IL-8 was inhibited by the presence of Pg LPS. MCP-1 secretion was suppressed by nicotine, alone and together with Pg LPS, while 4-NQO activated its secretion. Treatment of HMK cells with Pg LPS, nicotine, 4-NQO, or their combinations did not affect VEGF levels.ConclusionPg LPS, nicotine, and 4-NQO induce oxidative stress and regulate anti-oxidative response and cytokine expressions in human gingival keratinocytes differently. These results may indicate that bacterial and tobacco-related insults regulate distinct pathways

NFE2L2/NRF2, OGG1, and cytokine responses of human gingival keratinocytes against oxidative insults of various origin

Objective: Bacterial or tobacco-related insults induce oxidative stress in gingival keratinocytes. The aim of this study was to investigate anti-oxidative and cytokine responses of human gingival keratinocytes (HMK cells) against Porphyromonas gingivalis lipopolysaccharide (Pg LPS), nicotine, and 4-nitroquinoline N-oxide (4-NQO).Materials and methodsHMK cells were incubated with Pg LPS (1 mu l/ml), nicotine (1.54mM), and 4-NQO (1 mu M) for 24h. Intracellular and extracellular levels of interleukin (IL)-1, IL-1 receptor antagonist (IL-1Ra), IL-8, monocyte chemoattractant protein (MCP)-1, and vascular endothelial growth factor (VEGF) were measured with the Luminex (R) xMAP technique, and nuclear factor, erythroid 2 like 2 (NFE2L2/NRF2) and 8-oxoguanine DNA glycosylase (OGG1) with Western blots. Data were statistically analyzed by two-way ANOVA with Bonferroni correction.ResultsAll tested oxidative stress inducers increased intracellular OGG1 levels, whereas only nicotine and 4-NQO induced NFE2L2/NRF2 levels. Nicotine, 4-NQO, and their combinational applications with Pg LPS induced the secretions of IL-1 and IL-1Ra, while that of IL-8 was inhibited by the presence of Pg LPS. MCP-1 secretion was suppressed by nicotine, alone and together with Pg LPS, while 4-NQO activated its secretion. Treatment of HMK cells with Pg LPS, nicotine, 4-NQO, or their combinations did not affect VEGF levels.ConclusionPg LPS, nicotine, and 4-NQO induce oxidative stress and regulate anti-oxidative response and cytokine expressions in human gingival keratinocytes differently. These results may indicate that bacterial and tobacco-related insults regulate distinct pathways.Sin financiación2.795 JCR (2019) Q3, 130/195 Cell Biology0.836 SJR (2019) Q2, 40/130 Clinical Biochemistry, 718/2754 Medicine (miscellaneous); Q3, 177/300 Cell Biology, 238/414 Molecular BiologyNo data IDR 2019UE