Stannous fluoride forms aggregates between outer and inner membranes leading to membrane rupture of Porphyromonas gingivalis and Prevotella pallens.

Objective: Stannous has been shown to bind to free lipopolysaccharides, thus preventing them from binding to TLR receptors. This study was undertaken to determine the histomorphological mechanism of stannous binding to anaerobic bacteria. Methods: Two bacteria associated with gingivitis and advanced periodontal disease, Porphyromonas gingivalis (P. gingivalis) and Prevotella pallens (P. pallens), were cultured in 25-1,000 µM of stannous fluoride and stannous chloride for 48 h. The growth rate was estimated using absorbance OD600. Bacterial cells were then fixed and processed for transmission electron microscopy (TEM) analysis. Results: Stannous fluoride inhibited proliferation of both P. gingivalis and P. pallens in a dose-dependent manner. There was a statistically significant suppression of the growth curve starting at 100 µM for P. pallens (P = 0.050) and 200 µM for P. gingivalis (P = 0.039). TEM analysis revealed a thick layer of polysaccharides (19.8 nm) in P. gingivalis. The outer and inner membranes were clearly visible with low electron densities in both bacteria. Stannous diffused into bacterial membranes and formed precipitates in the areas spanning outer and inner membranes and below inner membranes. Precipitates varied in size ranging from 46.4 to 84.5 nm in length, and 18.4 to 35.9 nm in width. The membranes were disintegrated in the region where stannous formed precipitates. Cytosolic contents were leaked out, and in several cases, small vesicles were formed. Stannous precipitates were more abundant in numbers and larger in size in bacteria treated with high concentrations (100-300 µM) than in low concentrations (25-50 µM) of stannous fluoride. Furthermore, most of the bacteria were disintegrated in the groups treated with 100-300 µM stannous fluoride. At low concentrations (25 µM), stannous fluoride formed complexes primarily around outer membranes, to which lipopolysaccharides are anchored. Stannous chloride results showed similar trends, but it was less potent than stannous fluoride. Conclusion: Stannous fluoride can penetrate bacteria, bind to the constituents of the membrane and form precipitates between outer and inner membranes and beneath inner membranes. These large precipitates damaged the integrity of membranes and allowed cytosolic contents to be leaked out. Stannous complexes formed at the outer membranes, even at low concentrations (25 µM).

Aging-Associated Changes in the Adult Human Skin Microbiome and the Host Factors that Affect Skin Microbiome Composition.

Understanding the changes in the skin microbiome and their relationship to host skin factors during aging remains largely unknown. To better understand this phenomenon, we collected samples for metagenomic and host skin factor analyses from the forearm, buttock, and facial skin from 158 Caucasian females aged 20‒24, 30‒34, 40‒44, 50‒54, 60‒64, and 70‒74 years. Metagenomics analysis was performed using 16S ribosomal RNA gene sequencing, whereas host sebocyte gland area, skin lipids, natural moisturizing factors, and antimicrobial peptides measurements were also performed. These analyses showed that skin bacterial diversity increased at all the skin sites with increasing age. Of the bacterial genera with an average relative abundance >1%, only Lactobacillus and Cutibacterium demonstrated a significant change (decrease) in abundance at all sampled skin sites with increasing age. Additional bacterial genera demonstrated significant age- and site-specific changes in abundance. Analysis of sebocyte area, natural moisturizing factors, lipids, and antimicrobial peptides showed an age-related decrease in sebocyte area and increases in natural moisturizing factors/antimicrobial peptides/skin lipids, all of which correlated with changes in specific bacterial genera. In conclusion, the human skin microbiome undergoes age-associated alterations that may reflect underlying age-related changes in cutaneous biology.

Clinical effects of stannous fluoride dentifrice in reducing plaque microbial virulence III: Lipopolysaccharide and TLR2 reporter cell gene activation.

PURPOSE: This study expanded the analysis of subgingival dental plaques from previous research to include the evaluation of cohort, site and treatment effects on chemically measured endotoxin and activation of Toll-like receptor (TLR) based gene expression in two additional reporter cell lines: a TLR2 specific cell line and a THP-1 (multi TLR reporter) cell line. METHODS: Participants from high and low bleeding cohorts were sampled at baseline for both supra and subgingival dental plaque at both healthy as well as clinically diseased sites and then provided with intervention hygiene products including a stabilized SnF2 dentifrice and a new soft bristle manual toothbrush. Following 2 and 4 weeks of assigned dentifrice use, participants returned for a re-evaluation of gingival inflammation and bleeding and repeat samplings of dental plaque. Subgingival sampled plaques were chemically analyzed for endotoxin concentration using a Thermo Scientific Pierce LAL chromogenic endotoxin quantitation kit. Samples were also used for inoculation of two reporter cell assays (an HEK293 TLR2 reporter cell line and a THP-1 monocyte cell line). Reporter cell activation was analyzed via luminescence changes of secreted embryonic alkaline phosphatase. RESULTS: The endotoxin content of subgingival plaque could be measured directly with dye assays and plaque isolates activated gene expression in both TLR reporter cell lines. Higher disease cohorts and sites with gingival inflammation generally showed more endotoxins and higher levels of plaque virulence as compared to low disease cohorts or plaque sampled from clinically healthy sites. SnF2 dentifrice treatment was associated with broad scale reductions in endotoxin content and virulence potentiation properties of dental plaque samples collected subgingivally from patients. CLINICAL SIGNIFICANCE: These results collectively support the use of dye or various reporter cell lines in the characterization of plaque virulence in diseased populations and as a potential route for analysis in clinical evaluations of treatment interventions. Subgingival plaque 'detoxification' including effects on microbial pathogenicity as well as metabolic activity may be considered important mechanisms contributing to clinical benefits of SnF2 dentifrice.

Clinical Effects of Stabilized Stannous Fluoride Dentifrice in Reducing Plaque Microbial Virulence II: Metabonomic Changes.

OBJECTIVES: The clinical effects of stannous fluoride (SnF2) dentifrice in reducing symptoms of gingivitis and reducing the virulence of subgingival plaque through suppression of activation of gene expression in toll receptor based reporter cells were previously reported. This study expanded analysis of the clinical study to include evaluation of dentifrice effects on salivary metabolites using 1H Nuclear Magnetic Resonance (1H NMR) systems biology-based metabonomics. METHODS: The clinical design was reported previously (J Clin Dent2017;28:16-26). Participants included a cohort exhibiting high and low levels of gingival disease as presented at initiation of the study. Participants provided morning lavage saliva samples at baseline. Following this, participants were provided with a hygiene intervention, including a stabilized SnF2 dentifrice and a new soft bristle manual toothbrush. Following two and four weeks of assigned dentifrice use, participants again collected morning lavage saliva samples. Samples were analyzed by 1HNMR spectroscopy on a Bruker 600MHz NMR spectrometer. System-wide analyses were carried out by partial least squared (PLS) comparisons of aggregate spectra, and discrete metabolites with established spectral signatures were likewise directly compared. RESULTS: PLS analysis showed significant differences in saliva composition for saliva collected from high bleeding and low bleeding cohorts. Clear shifts in saliva composition were observed in system-wide PLS analysis following intervention of SnF2 dentifrice for both cohorts. A number of discrete spectral changes were consistently observed with SnF2 dentifrice intervention, most notably including reductions in propionic acid and butyric acid, key short chain fatty acids associated with anaerobic metabolism in dental plaques. CONCLUSIONS: These results collectively demonstrate that SnF2 dentifrice treatment was associated with broad scale modifications in saliva composition following intervention in both high and low diseased cohorts. Changes in overall salivary composition and specific reductions in saliva concentrations of propionic and butyric acid reductions occurred coincident with clinical improvements in gingivitis and gingival bleeding. These results provide support for the hypothesis that the effectiveness of SnF2 dentifrice in improving gingival health is associated with a modification of microbiome metabolism, including suppression of short chain fatty acid metabolites.

Clinical Effects of Stabilized Stannous Fluoride Dentifrice in Reducing Plaque Microbial Virulence I: Microbiological and Receptor Cell Findings.

OBJECTIVES: Lipopolysaccharides (LPSs) and lipoteichoic acids (LTAs), or bacterial endotoxins, bind with Toll-like receptors (TLRs) that are expressed on host cells of the periodontium, thereby contributing to the periodontal pathogenicity of oral bacteria. Stannous fluoride (SnF2), an antibacterial fluoride that treats and controls gingivitis, has been shown to react with lipophilic domains/anionic charges in LPS and LTA. The effects of bacterial species and dental plaque on toll receptors can be studied using genetically engineered cell lines containing linked toll receptors on their surfaces. This randomized, examiner-blinded study examined the clinical effects of stabilized SnF2 dentifrice intervention on gingivitis and dental plaque virulence in populations exhibiting high and low levels of clinical gingivitis. METHODS: Recruited populations were evaluated for gingival inflammation (MGI) and gingival bleeding (GBI) at baseline and assigned into two cohorts of 20 each, those with high (GBI > 20 sites) and low (GBI < 3 sites) levels of observed bleeding/gingivitis. Participants were sampled at baseline for both supra- and subgingival dental plaque at both healthy (no bleeding, PD = 2 mm), as well as clinically diseased sites (bleeding, PD = 3-4 mm), and then provided with an intervention hygiene product including a stabilized SnF2 dentifrice and a new soft bristle manual toothbrush. Following two and four weeks of assigned dentifrice use, participants returned for a re-evaluation of gingival inflammation and bleeding and repeat samplings of dental plaque. Plaque samples were analyzed by anaerobic culturing of gram negative anaerobes (GNA), as well as by incubation of subgingival sampled plaques with TLR4 transfected HEK293 cells, where gene expression was assessed by measurement of a SEAP alkaline phosphatase reporter as a marker of toll receptor activation. RESULTS: Clinical assessments showed statistically significant reductions in MGI (24-26%) and GBI (42-53%) gingivitis in both diseased and healthy cohorts following four weeks of dentifrice intervention. For all clinical examinations, MGI and bleeding sites were statistically significantly different (lower) in the low bleeding versus the higher bleeding cohort. Supragingival and subgingival GNAs were significantly reduced (p < 0.05) in both high and low disease cohorts at bleeding and healthy sites following four weeks of stabilized SnF2 dentifrice use. TLR activation from subgingival sampled plaque was reduced following four weeks of stabilized SnF2 dentifrice use in both high and low disease cohorts and in both healthy, as well as diseased sites. CONCLUSIONS: Collectively, these results support the potential for stabilized SnF2 dentifrice to provide clinical gingivitis benefits via mechanisms beyond control of plaque mass, potentially directly decreasing the pathogenicity of plaque biofilms by blocking reactivity of LPS and LTA ligands with tissue receptors associated with inflammation. Importantly, benefits could be seen in both diseased sites, as well as sites not yet exhibiting symptoms of inflammation, supporting the activity of SnF2 not just in treating diseased sites, but in preventing disease development. These learnings may influence treatment planning for patients susceptible to gingivitis.

Quantitation of endotoxin and lipoteichoic acid virulence using toll receptor reporter gene.

PURPOSE: To apply quantitative Toll-like receptors (TLR) cell assays to compare lipopolysaccharides (LPSs) and lipoteichoic acids (LTAs) from different oral bacterial strains for potential pathogenicity in vitro. METHODS: The potency of LPS and LTA from different bacteria on activation of TLR reporter genes in HEK-tlr cell lines was examined. P. gingivalis LPS mix, P. gingivalis 1690 LPS, P. gingivalis 1435/50 LPS, E. coli LPS (E. coli K12), B. subtilis LTA, S. aureus LTA, E. hirae LTA and S. pyogenes LTA were examined in both TLR2 and TLR4 HEK cell line reporter assays. Solutions of LPS and LTA from selected bacteria were applied in a dose response fashion to the TLR reporter cells under standard culture conditions for mammalian cells. Reporter gene secreted-embryonic-alkaline-phosphatase (SEAP) was measured, and half maximal effective concentration (EC50) was determined for each sample. Concentration dependent TLR activation was compared to similar responses to LPS and LTA for commercial BODIPY-TR-Cadaverine and LAL biochemical (non cell based) assays. RESULTS: All LPS from P. gingivalis activated both TLR2 and TLR4 responses. E. coli LPS is a strong activator for TLR4 but not for TLR2 responses. In contrast, both B. subtilis and S. aureus LTA provoked responses only in TLR2, but not in the TLR4 assay. Interestingly, E. hirae LTA and S. pyogenes LTA did not stimulate strong TLR2 responses. Instead, both E. hirae LTA and S. pyogenes LTA mounted a reasonable response in TLR4 reporter gene assay. Both LPS and LTA showed deactivation of fluorescence in BODIPY-TR-Cadaverine while only LPS was active in LAL. As with biochemical assays, an EC50 could be determined for LPS and LTA from various bacterial strains. The EC50 is defined as a concentration of LPS or LTA that provokes a response halfway between the baseline and maximum responses. Lower EC50 means higher potency in promoting TLR responses, and in principle indicates greater toxicity to the host. CLINICAL SIGNIFICANCE: InvivoGen TLR2 and TLR4 assays distinguish specific types of microbial products, such as LPS and LTA from different bacteria. Application of EC50 determinations creates a means for quantitative and comparisons of LPS and LTA virulence in a cellular-based assay and combinations of TLR reporter cell assays along with biochemical evaluation of LPS#47;LTA in BODIPY-TR-Cadaverine and LPS in LAL assays provides a means to quantitate virulence of plaque samples with respect to both LPS and LTA. These learnings have long-term implications for patient care in that understanding the virulence of patients' plaque provides important information to assess risk of oral diseases.

Lipopolysaccharide and lipoteichoic acid binding by antimicrobials used in oral care formulations.

PURPOSE: To study the reactivity of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) with the cationically charged agents cetylpyridinium chloride, stannous fluoride, and the non-cationic agent triclosan. We also assessed the effect of these agents to inhibit LPS and LTA binding to cellular Toll-like Receptors (TLRs) in vitro. METHODS: The ability of these antimicrobials to bind with LPS and/or LTA was assessed in both the Limulus amebocyte lysate and BODIPY-TR-cadaverine dye assays. Mass spectroscopy was then used to confirm that stannous fluoride directly binds with LPS and to determine stoichiometry. Lastly, we looked for possible inhibitory effects of these antimicrobial agents on the ability of fluorescently conjugated LPS to bind to TLR4 expressed on HEK 293 cells. RESULTS: Cetylpyridinium chloride (CPC) and stannous salts including stannous fluoride interfered with LPS and LTA reactivity in both dye assays, while triclosan had no effect. Mass spectroscopy revealed direct binding of stannous fluoride with E. Coli LPS at 1:1 stoichiometric ratios. In the cellular assay, cetylpyridinium chloride and stannous fluoride, but not triclosan, inhibited LPS binding to TLR4. CLINICAL SIGNIFICANCE: These results support a potential mechanism of action for stannous fluoride and CPC formulated in oral products in which these ingredients bind bacterial toxins and potentially render them less toxic to the host. These results may influence home care recommendations for patients at risk for plaque-related diseases.

Lipopolysaccharide and Lipoteichoic Acid Virulence Deactivation by Stannous Fluoride.

OBJECTIVES: Oral bacterial pathogens promote gingivitis and periodontal disease. Bacterial endotoxins, also known as lipopolysaccharides (LPSs) and lipoteichoic acids (LTAs), are known to enhance bacterial pathogenicity through binding with Toll-like receptors (TLRs), a group of pattern recognition receptors critical to the activation of innate immunity, that are expressed on host cells. Both LPS and LTA contain lipophilic domains and anionic charges that may be susceptible to reactivity with stannous fluoride, a commonly used ingredient clinically proven for the treatment and prevention of gingivitis. This study examined the effects of stannous fluoride on Toll-like receptor activation in response to bacterially derived LPS and LTA in select cell lines and secretion of inflammatory cytokines from human primary peripheral monocytes likewise exposed to LPS. METHODS: TLR4 and TLR2 transfected HEK293 cells and THP1-Dual™ cells were exposed to bacterial LPS and LTA in the presence of increasing concentrations of stannous fluoride. Gene expression was assessed by measurement of secreted embryonic alkaline phosphatase (SEAP) reporter gene for HEK293 cells and SEAP and luciferase for THP-1 cells. Cell viability was confirmed using PrestoBlue. Human primary monocytes were treated with LPS with various concentrations of supplemented stannous fluoride, and cytokine expression was directly measured. RESULTS: Stannous fluoride inhibited gene expression response of TLR4 and TLR2 in HEK293 cells and THP1-Dual™ cells in a dose response fashion, producing complete inhibition at micromolar concentrations. The addition of stannous fluoride suppressed production of TNF-a, IFN-g, IL12p70, IL10, IL-1b, IL2, and IL-6, and also increased secretion of Il-8 in dose response fashion. Viability assays confirmed no effects of LPS or stannous fluoride on viability of HEK293, THP-1, and primary human monocytes. CONCLUSIONS: These results support the potential for stannous fluoride to provide clinical gingivitis benefits by directly decreasing the pathogenicity of plaque biofilms by blocking reactivity of LPS and LTA ligands with tissue receptors associated with inflammation. These learnings may influence recommendations for patients at risk for plaque-related diseases.

Response of chronic gingivitis to hygiene therapy and experimental gingivitis. Clinical, microbiological and metabonomic changes.

PURPOSE: To compare the clinical, microbiological and metabonomic profiles of subjects with high and low levels of chronic gingival bleeding during a controlled oral hygiene regimen intervention including sequential phases of rigorous therapeutic oral hygiene followed by experimental gingivitis (EG). METHODS: Two cohorts of qualified study subjects with differences in gingival bleeding on probing levels at their baseline clinical examination were entered into the study. These two cohorts were followed through three separate study phases including a 1-week baseline phase, a 2-week phase of rigorous oral hygiene including dental prophylaxis, and a 3-week EG phase of no oral hygiene to encourage relapse of gingivitis. The 58 subjects were assessed during each phase of the study for clinical presentation of gingivitis and concurrently had plaque sampled for real-time polymerase chain reaction (RTPCR) microbiological characterization and salivary lavage samples for 'systems biology' metabonomics assessment by 1H-NMR. RESULTS: Subjects presenting with different levels of gingival bleeding on probing when they entered the study responded differently to rigorous oral hygiene and EG. Specifically, the high bleeding cohort responded sluggishly to rigorous oral hygiene and exhibited markedly greater relapse to gingivitis during EG. RTPCR analysis showed changes in bacterial populations that were associated with study phases, particularly the increases in putative periodontal pathogens during EG. However, the microbiological profiles of high- and low-susceptibility gingival bleeding patients were largely similar. Metabonomic analysis likewise revealed significant changes in metabolite composition during study phases associated with differences in plaque toxicity, especially the short chain carboxylic acids propionate and n-butyrate, which tracked clinical changes in gingivitis severity. Systems analysis of metabonomic changes suggested differences between cohorts, although analysis to date has not elucidated whether these differences are causative (population predictive) or simply diagnostic of clinical status within populations.

Quantitative proteogenomic profiling of epidermal barrier formation in vitro.

BACKGROUND: The barrier function of the epidermis is integral to personal well-being, and defects in the skin barrier are associated with several widespread diseases. Currently there is a limited understanding of system-level proteomic changes during epidermal stratification and barrier establishment. OBJECTIVE: Here we report the quantitative proteogenomic profile of an in vitro reconstituted epidermis at three time points of development in order to characterize protein changes during stratification. METHODS: The proteome was measured using data-dependent "shotgun" mass spectrometry and quantified with statistically validated label-free proteomic methods for 20 replicates at each of three time points during the course of epidermal development. RESULTS: Over 3600 proteins were identified in the reconstituted epidermis, with more than 1200 of these changing in abundance over the time course. We also collected and discuss matched transcriptomic data for the three time points, allowing alignment of this new dataset with previously published characterization of the reconstituted epidermis system. CONCLUSION: These results represent the most comprehensive epidermal-specific proteome to date, and therefore reveal several aspects of barrier formation and skin composition. The limited correlation between transcript and protein abundance underscores the importance of proteomic analysis in developing a full understanding of epidermal maturation.