miRNAs from Inflamed Gingiva Link Gene Signaling to Increased MET Expression.

Several array-based microRNA (miRNA) expression studies independently showed increased expression of miRNAs hsa-miR-130a-3p, -142-3p, -144-3p, -144-5p, -223-3p, -17-5p, and -30e-5p in gingiva affected by periodontal inflammation. We aimed to determine direct target genes and signaling pathways regulated by these miRNAs to identify processes relevant to gingival inflammatory responses and tissue homeostasis. We transfected miRNA mimics (mirVana) for each of the 7 miRNAs separately into human primary gingival fibroblasts cultured from 3 different donors. Following RNA sequencing, differential gene expression and second-generation gene set enrichment analyses were performed. miRNA inhibition and upregulation was validated at the transcript and protein levels using quantitative reverse transcriptase polymerase chain reaction, Western blotting, and reporter gene assays. All 7 miRNAs significantly increased expression of the gene MET proto-oncogene, receptor tyrosine kinase (MET). Expression of known periodontitis risk genes CPEB1, ABCA1, and ATP6V1C1 was significantly repressed by hsa-miR-130a-3p, -144-3p, and -144-5p, respectively. The genes WASL, ENPP5, ARL6IP1, and IDH1 showed the most significant and strongest downregulation after hsa-miR-142-3p, -17-5p, -223-3p, and -30e-5p transfection, respectively. The most significantly regulated gene set of each miRNA related to cell cycle (hsa-miRNA-144-3p and -5p [Padj = 4 × 10-40 and Padj = 4 × 10-6], -miR-17-5p [Padj = 9.5 × 10-23], -miR-30e-5p [Padj = 8.2 × 10-18], -miR-130a-3p [Padj = 5 × 10-15]), integrin cell surface interaction (-miR-223-3p [Padj = 2.4 × 10-7]), and interferon signaling (-miR-142-3p [Padj = 5 × 10-11]). At the end of acute inflammation, gingival miRNAs bring together complex regulatory networks that lead to increased expression of the gene MET. This underscores the importance of mesenchymal cell migration and invasion during gingival tissue remodeling and proliferation in restoring periodontal tissue homeostasis after active inflammation. MET, a receptor of the mitogenic hepatocyte growth factor fibroblast secreted, is a core gene of this process.

Exome Sequencing of 5 Families with Severe Early-Onset Periodontitis.

Periodontitis is characterized by alveolar bone loss leading to tooth loss. A small proportion of patients develop severe periodontitis at the juvenile or adolescent age without exposure to the main risk factors of the disease. It is considered that these cases carry rare variants with large causal effects, but the specific variants are largely unknown. In this study, we performed exome sequencing of 5 families with children who developed stage IV, grade C, periodontitis between 3 and 18 y of age. In 1 family, we found compound heterozygous variants in the gene CTSC (p.R272H, p.G139R), 1 of which was previously identified in a family with prepubertal periodontitis. Subsequent targeted resequencing of the CTSC gene in 24 patients <25 y of age (stage IV, grade C) identified the known mutation p.I453V (odds ratio = 4.06, 95% CI = 1.6 to 10.3, P = 0.001), which was previously reported to increase the risk for adolescent periodontitis. An affected sibling of another family carried a homozygous deleterious mutation in the gene TUT7 (p.R560Q, CADD score >30 [Combined Annotation Dependent Depletion]), which is implicated in regulation of interleukin 6 expression. Two other affected siblings shared heterozygous deleterious mutations in the interacting genes PADI1 and FLG (both CADD = 36), which contribute to the integrity of the environment-tissue barrier interface. Additionally, we found predicted deleterious mutations in the periodontitis risk genes ABCA1, GLT6D1, and SIGLEC5. We conclude that the CTSC variants p.R272H and p.I453V have different expressivity and diagnostic relevance for prepubertal and adolescent periodontitis, respectively. We propose additional causal variants for early-onset periodontitis, which also locate within genes that carry known susceptibility variants for common forms. However, the genetic architecture of juvenile periodontitis is complex and differs among the affected siblings of the sequenced families.

BACH1 Binding Links the Genetic Risk for Severe Periodontitis with ST8SIA1.

Genome-wide association studies identified various loci associated with periodontal diseases, but assigning causal alleles remains difficult. Likewise, the generation of biological meaning underlying a statistical association has been challenging. Here, we characterized the genetic association at the gene ST8SIA1 that increases the risk for severe periodontitis in smokers. We used CRISPR/dCas9 activation and RNA-sequencing to identify genetic interaction partners of ST8SIA1 and to determine its function in the cell. We used reporter gene assays to identify regulatory elements at the associated single-nucleotide polymorphisms (SNPs) and to determine effect directions and allele-specific changes of enhancer activity. Antibody electrophoretic mobility shift assays proved allele-specific transcription factor binding at the putative causal SNPs. We found the reported periodontitis risk gene ABCA1 as the top upregulated gene following ST8SIA1 activation. Gene set enrichment analysis showed highest effects on integrin cell surface interactions (area under the curve [AUC] = 0.85; q = 4.9 × 10-6) and cell cycle regulation (AUC = 0.89; q = 1.6 × 10-5). We identified 2 associated repressor elements in the introns of ST8SIA1 that bind the transcriptional repressor BACH1. The putative causative variant rs2012722 decreased BACH1 binding by 40%. We also pinpointed ST8SIA1 as the target gene of the association. ST8SIA1 inhibits cell adhesion with extracellular matrix proteins, integrins, and cell cycle, as well as enhances apoptosis. Likewise, tobacco smoke reportedly results in an inhibition of cell adhesion and a decrease in integrin-positive cells and cell growth. We conclude that impaired ST8SIA1 repression, independently caused by reduced BACH1 binding at the effect T allele, as well as by tobacco smoke, contributes to higher ST8SIA1 levels, and in smokers who carry the effect T allele, both factors would be additive with damaging effects on the gingival barrier integrity. The activity of ST8SIA1 is also linked with the periodontitis risk gene ABCA1.

Periodontitis Risk Variants at SIGLEC5 Impair ERG and MAFB Binding.

Periodontitis is a common complex inflammatory disease of the oral cavity. It is characterized by inflammation of gingival tissues and alveolar bone loss. Recently, a genome-wide association study and 2 genome-wide association study meta-analyses found 2 associated regions (haplotype blocks) at the inhibitory immune receptor gene SIGLEC5 to increase the risk for periodontitis. The aims of the current study were the identification of the putative causal variants underlying these associations, characterization of their molecular biological effects, and validation of SIGLEC5 as the target gene. We mapped the associated single-nucleotide polymorphisms to DNA elements with predictive features of regulatory functions and screened the associated alleles for transcription factor (TF) binding sites. Antibody electrophoretic mobility shift assays (EMSAs) with allele-specific probes were used to identify TF binding and to quantify allele-specific effects on binding affinities. Luciferase reporter assays were used to quantify the effect directions and allele-specific strength of the associated regulatory elements. We used CRISPR-dCas9 gene activation to validate SIGLEC5 as a target of the association. EMSA in peripheral blood mononuclear cells showed that E-26 transformation-specific TF-related gene (ERG) binds at rs11084095, with almost complete loss of binding at the minor A-allele. Allele-specific reporter genes showed enhancer function of the DNA sequence at rs11084095, which was abrogated in the background of the A-allele. EMSA in B lymphocytes showed that TF MAF bZIP (MAFB) binds at the common G-allele of rs4284742, whereas the minor A-allele reduced TF binding by 69%, corresponding to 9-fold reduction of luciferase reporter gene activity by the A-allele. Using CRISPR-dCas9, we showed that the enhancer at rs4284742 strongly activated SIGLEC5 expression, validating this gene as the target gene of the association. We conclude that rs11084095 and rs4284742 are putatively causal for the genome-wide significant associations with periodontitis at SIGLEC5 that impair ERG and MAFB binding, respectively.

Entamoeba gingivalis Exerts Severe Pathogenic Effects on the Oral Mucosa.

The protozoan Entamoeba gingivalis colonizes the healthy oral mucosa with a prevalence of 15%. Colonization can be asymptomatic, and it is considered not pathogenic. However, it is able to invade lacerated oral mucosa, where it ingests fragments of live cells, suggesting pathogenous potential. Here, we characterized the transcriptomes of gingival cells after infection with E. gingivalis using RNA sequencing and observed pathogen interaction with the epithelial monolayer barrier by scanning electron microscopy. In epithelial and fibroblast cells, strongest differential expression showed gene set "chemokines and inflammatory molecules in myeloid cells" (area under the curve [AUC] = 0.9, effect size 5.15, adjusted P = 3.1 × 10-19) and "cell cycle and growth arrest" (AUC = 0.91, effect size = 4.56, adjusted P = 4.8 × 10-9), respectively. The most upregulated genes were TNF (fold change 430) and IL8 (fold change 359) in epithelial cells and ZN331 (fold change 18) in fibroblasts. We showed that E. gingivalis killed live epithelial cells by trogocytosis, demonstrating strong pathogenic potential.

Characterization of an ester-based core-multishell (CMS) nanocarrier for the topical application at the oral mucosa.

OBJECTIVES: Topical drug administration is commonly applied to control oral inflammation. However, it requires sufficient drug adherence and a high degree of bioavailability. Here, we tested the hypothesis whether an ester-based core-multishell (CMS) nanocarrier is a suitable nontoxic drug-delivery system that penetrates efficiently to oral mucosal tissues, and thereby, increase the bioavailability of topically applied drugs. MATERIAL AND METHODS: To evaluate adhesion and penetration, the fluorescence-labeled CMS 10-E-15-350 nanocarrier was applied to ex vivo porcine masticatory and lining mucosa in a Franz cell diffusion assay and to an in vitro 3D model. In gingival epithelial cells, potential cytotoxicity and proliferative effects of the nanocarrier were determined by MTT and sulphorhodamine B assays, respectively. Transepithelial electrical resistance (TEER) was measured in presence and absence of CMS 10-E-15-350 using an Endohm-12 chamber and a volt-ohm-meter. Cellular nanocarrier uptake was analyzed by laser scanning microscopy. Inflammatory responses were determined by monitoring pro-inflammatory cytokines using real-time PCR and ELISA. RESULTS: CMS nanocarrier adhered to mucosal tissues within 5 min in an in vitro model and in ex vivo porcine tissues. The CMS nanocarrier exhibited no cytotoxic effects and induced no inflammatory responses. Furthermore, the physical barrier expressed by the TEER remained unaffected by the nanocarrier. CONCLUSIONS: CMS 10-E-15-350 adhered to the oral mucosa and adhesion increased over time which is a prerequisite for an efficient drug release. Since TEER is unaffected, CMS nanocarrier may enter the oral mucosa transcellularly. CLINICAL RELEVANCE: Nanocarrier technology is a novel and innovative approach for efficient topical drug delivery at the oral mucosa.

Entamoeba gingivalis Causes Oral Inflammation and Tissue Destruction.

A metagenomics analysis showed a strongly increased frequency of the protozoan Entamoeba gingivalis in inflamed periodontal pockets, where it contributed the second-most abundant rRNA after human rRNA. This observation and the close biological relationship to Entamoeba histolytica, which causes inflammation and tissue destruction in the colon of predisposed individuals, raised our concern about its putative role in the pathogenesis of periodontitis. Histochemical staining of gingival epithelium inflamed from generalized severe chronic periodontitis visualized the presence of E. gingivalis in conjunction with abundant neutrophils. We showed that on disruption of the epithelial barrier, E. gingivalis invaded gingival tissue, where it moved and fed on host cells. We validated the frequency of E. gingivalis in 158 patients with periodontitis and healthy controls by polymerase chain reaction and microscopy. In the cases, we detected the parasite in 77% of inflamed periodontal sites and 22% of healthy sites; 15% of healthy oral cavities were colonized by E. gingivalis. In primary gingival epithelial cells, we demonstrated by quantitative real-time polymerase chain reaction that infection with E. gingivalis but not with the oral bacterial pathogen Porphyromonas gingivalis strongly upregulated the inflammatory cytokine IL8 (1,900 fold, P = 2 × 10-4) and the epithelial barrier gene MUC21 (8-fold, P = 7 × 10-4). In gingival fibroblasts, we showed upregulation of the collagenase MMP13 (11-fold, P = 3 × 10-4). Direct contact of E. gingivalis to gingival epithelial cells inhibited cell proliferation. We indicated the strong virulence potential of E. gingivalis and showed that the mechanisms of tissue invasion and destruction are similar to the colonic protozoan parasite E. histolytica. In conjunction with abundant colonization of inflamed periodontal sites and the known resistance of Entamoeba species to neutrophils, antimicrobial peptides, and various antibiotics, our results raise the awareness of this protozoan as a potential and, to date, underrated microbial driver of destructive forms of periodontitis.

Smoking Modifies the Genetic Risk for Early-Onset Periodontitis.

Periodontitis has low-prevalence, highly severe disease manifestations with an early onset and rapid progression. The diagnosis is based on severe destruction of the alveolar bone in adolescents and young adults. Genetic susceptibility variants and smoking are well-established risk factors, but their interactions in modifying disease susceptibility have not been studied. We aimed to identify genetic risk variants of early-onset periodontitis that unmask their effects on tobacco smoke exposure. To this end, we analyzed 79,780,573 common variants in 741 northwest Europeans diagnosed to have >30% bone loss at >2 teeth before 35 y of age, using imputed genotypes of the OmniExpress BeadChip. Never versus ever smokers were compared in a logistic regression analysis via a case-only approach. To explore the effect of tobacco smoke on the expression of the G×S-associated genes, cultures of primary gingival fibroblasts (n = 9) were exposed to cigarette smoke extract, and transcripts were quantified by reverse transcription polymerase chain reaction. We identified 16 loci for which our analysis suggested an association with G×S increased disease risk (P < 5 × 10-5). Nine loci had previously been reported to be associated with spirometric measures of pulmonary function by an earlier G×S genome-wide association study. Genome-wide significant cis expression quantitative trait loci were reported for G×S-associated single-nucleotide polymorphisms at ST8SIA1 and SOST, indicating a causal role of these genes in tobacco-related etiopathology. Notably, SOST is a negative regulator of bone growth, and ST8SIA1 has a role in tissue remodeling. Cigarette smoke extract significantly altered the expression of 2 associated genes: SSH1 (P = 5 × 10-07), which is required for NF-κB activation and innate immune responses to bacterial invasion, and ST8SIA1 (P = 0.0048). We conclude that the genetic predisposition to early-onset periodontitis is in part triggered by smoking and that tobacco smoke directly affects the expression of genes involved in bone homeostasis, tissue repair, and immune response.

Administration of systemic antibiotics during non-surgical periodontal therapy-a consensus report.

AIM: The aim of this meta-review was to evaluate whether there is a meaningful clinical benefit regarding the use of systemic adjunctive antibiotics in the treatment of patients with periodontitis. Additionally, a consensus regarding possible recommendations for future administration of antibiotics should be reached. METHODS: A structured literature search was performed by two independent investigators focusing on systematic reviews (SR) covering adjunctive systemic antibiosis during non-surgical periodontal therapy. Additionally, recent randomized clinical trials (RCT, July 2015 to July 2017) were searched systematically to update the latest SR. Results were summarized and discussed in a plenary to reach a consensus. RESULTS: Mostly, systematic reviews and RCTs showed a significant positive effect of adjunctive systematic antibiosis compared to controls. These positive effects gain clinical relevance in patients with severe periodontal disease aged 55 years and younger. CONCLUSION: Systemic antibiotics as an adjunct to non-surgical periodontal therapy should be sensibly administered and restrictively used. Only certain groups of periodontitis patients show a significant and clinically relevant benefit after intake of systemic antibiosis during periodontal therapy. CLINICAL RELEVANCE: Avoiding antibiotic resistance and possible side effects on the human microbiome should be a focus of dentists and physicians. Thus, a sensible administration of antibiotics is mandatory. This manuscript suggests guidelines for a reasonable use.

Integration of Murine and Human Studies for Mapping Periodontitis Susceptibility.

Periodontitis is one of the most common inflammatory human diseases with a strong genetic component. Due to the limited sample size of available periodontitis cohorts and the underlying trait heterogeneity, genome-wide association studies (GWASs) of chronic periodontitis (CP) have largely been unsuccessful in identifying common susceptibility factors. A combination of quantitative trait loci (QTL) mapping in mice with association studies in humans has the potential to discover novel risk loci. To this end, we assessed alveolar bone loss in response to experimental periodontal infection in 25 lines (286 mice) from the Collaborative Cross (CC) mouse population using micro-computed tomography (µCT) analysis. The orthologous human chromosomal regions of the significant QTL were analyzed for association using imputed genotype data (OmniExpress BeadChip arrays) derived from case-control samples of aggressive periodontitis (AgP; 896 cases, 7,104 controls) and chronic periodontitis (CP; 2,746 cases, 1,864 controls) of northwest European and European American descent, respectively. In the mouse genome, QTL mapping revealed 2 significant loci (-log P = 5.3; false discovery rate = 0.06) on chromosomes 1 ( Perio3) and 14 ( Perio4). The mapping resolution ranged from ~1.5 to 3 Mb. Perio3 overlaps with a previously reported QTL associated with residual bone volume in F2 cross and includes the murine gene Ccdc121. Its human orthologue showed previously a nominal significant association with CP in humans. Use of variation data from the genomes of the CC founder strains further refined the QTL and suggested 7 candidate genes ( CAPN8, DUSP23, PCDH17, SNORA17, PCDH9, LECT1, and LECT2). We found no evidence of association of these candidates with the human orthologues. In conclusion, the CC populations enabled mapping of confined QTL that confer susceptibility to alveolar bone loss in mice and larger human phenotype-genotype samples and additional expression data from gingival tissues are likely required to identify true positive signals.

Characterization of hyperbranched core-multishell nanocarriers as an innovative drug delivery system for the application at the oral mucosa.

BACKGROUND AND OBJECTIVES: In the oral cavity, the mucosal tissues may develop a number of different pathological conditions, such as inflammatory diseases (gingivitis, periodontitis) and autoimmune disorders (eg, oral lichen planus) that require therapy. The application of topical drugs is one common therapeutic approach. However, their efficacy is limited. Dilution effects due to saliva hinder the adherence and the penetration of drug formulations. Therefore, the bioavailability of oral topical drugs is insufficient, and patients may suffer from disease over years, if not life-long. MATERIAL AND METHODS: In the present study, we characterized core-multishell (CMS) nanocarriers for their potential use as drug delivery systems at oral mucosal tissues. For this purpose, we prepared porcine masticatory as well as buccal mucosa and performed Franz cell diffusion experiments. Penetration of fluorescently labeled CMS nanocarriers into the mucosal tissue was analyzed using confocal laser scanning microscopy. Upon exposure to CMS nanocarriers, the metabolic and proliferative activity of gingival epithelial cells was determined by MTT and sulforhodamine B assays, respectively. RESULTS: Here, we could show that the carriers penetrate into both mucosal tissues, while particles penetrate deeper into the masticatory mucosa. Electron paramagnetic resonance spectroscopy revealed that the 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy-labeled glucocorticoid dexamethasone loaded on to the CMS nanocarriers was released from the carriers in both mucosal tissues but with a higher efficiency in the buccal mucosa. The release from the nanocarriers is in both cases superior compared to the release from a conventional cream, which is normally used for the treatment of inflammatory conditions in the oral cavity. The CMS nanocarriers exhibited neither cytotoxic nor proliferative effects in vitro. CONCLUSION: These findings suggested that CMS nanocarriers might be an innovative approach for topical drug delivery in the treatment of oral inflammatory diseases.

Effect of photodynamic therapy in combination with various irrigation protocols on an endodontic multispecies biofilm ex vivo.

AIM: To analyse the antibacterial effect of photodynamic therapy (PDT) in combination with various irrigation protocols on a multispecies biofilm in root canals ex vivo. METHODOLOGY: A total of 160 extracted human single-rooted teeth were divided into four groups (n = 40). In group G1, root canals were instrumented up to size 60 (control group), whereas in G2 to G4 canals were enlarged up to size 40. All root canals were inoculated with a multispecies biofilm (Enterococcus faecalis, Streptococcus oralis, Prevotella intermedia) for 5 days. In G2 to G4, instrumentation up to size 60 was performed using 0.9% sodium chloride (NaCl) (G2), 1% sodium hypochlorite (NaOCl) (G3), 1% NaOCl and a final irrigation with 2% chlorhexidine (CHX) (G4), respectively. In all groups half of the specimens received adjunctive PDT using phenothiazine chloride as photosensitizer and a diode laser (wavelength 660 nm). Counts of colony-forming units (CFUs) in each group were analysed separately for planktonic and dentine-adherent bacteria immediately after therapy (T1) and after 5 days of further incubation (T2). Descriptive statistics and two-way analysis of variance were carried out to analyse reduction of planktonic bacteria and nonparametric tests were used to analyse dentine-adherent bacteria. RESULTS: CFU reduction in planktonic bacteria was significantly affected by the irrigation protocol at T1 and T2 (P < 0.0001), but PDT significantly reduced CFUs only at T2 (P = 0.01; anova). Irrigation using NaOCl, CHX and adjunctive PDT significantly reduced CFUs at T2 (P < 0.0001; Tukey HSD) compared to the control group. In 85.6% of all samples the same categories of CFU counts in both planktonic and dentine-adherent bacteria were detected at T1 and T2. CONCLUSIONS: Adjunctive photodynamic therapy in combination with an irrigation protocol including NaOCl and CHX was an effective method for reduction of bacterial biofilm inside the root canals of extracted teeth.

The PF4/PPBP/CXCL5 Gene Cluster Is Associated with Periodontitis.

Periodontitis is a common dysbiotic inflammatory disease with an estimated heritability of 50%. Due to the limited sample size of available periodontitis cohorts and the underlying trait heterogeneity, genome-wide association studies (GWAS) of chronic periodontitis (CP) have been unsuccessful in discovering susceptibility factors. A strategy that combines agnostic GWAS with a well-powered candidate-gene approach has the potential to discover novel loci. We combined RNA-seq data from gingival tissues with quantitative trait loci (QTLs) that were identified in a F2-cross of mice resistant and susceptible to infection with oral bacterial pathogens. Four genes, which were located within the mapped QTLs, showed differential expression. The chromosomal regions across the human orthologous were interrogated for putative periodontitis-associated variants using existing GWAS data from a German case-control sample of aggressive periodontitis (AgP; 651 cases, 4,001 controls), the most severe and early onset form of periodontitis. Two haplotype blocks, one upstream to the coding region of UGT2A1 (rs146712414, P = 9.1 × 10-5; odds ratio [OR], 1.34; 95% confidence interval [CI], 1.16-1.56) and one downstream of the genes PF4/PPBP/CXCL5 (rs1595009, P = 1.3 × 10-4; OR, 1.32; 95% CI, 1.15-1.52), were associated with AgP. The association of rs1595009 was validated in an independent cohort of CP of European Americans (1,961 cases and 1,864 controls; P = 0.03; OR, 1.45; 95% CI, 1.01-1.29). This association was further replicated in another sample of 399 German CP cases (disease onset <60 y of age) and 1,633 controls ( P = 0.03; OR, 1.75; 95% CI, 1.06-2.90). The combined estimates of association from all samples were P = 2.9 × 10-5 (OR, 1.2; 95% CI, 1.1-1.3). This study shows the strength of combining QTL mapping and RNA-Seq data from a mouse model with association studies in human case-control samples to identify genetic risk variants of periodontitis.

Expression of antimicrobial peptides and interleukin-8 during early stages of inflammation: An experimental gingivitis study.

BACKGROUND AND OBJECTIVES: In the oral cavity, the epithelial surface is constantly exposed to a number of different microorganisms that are organized in a well-structured biofilm. The aim of this study was to monitor gingival expression of antimicrobial peptides (AMPs) and interleukin-8 (IL-8) in an early gingivitis model. MATERIAL AND METHODS: Experimental gingivitis was allowed to develop in healthy volunteers (n = 17). Bleeding on probing (BOP%) and gingival crevicular fluid volume (GCF) were assessed at baseline and day 1, 3, 5, 7 and 14. Expression of AMPs (human beta-defensin-2, hBD-2; CC-chemokine ligand 20, CCL20; psoriasin, pso/S100A7) and IL-8 was analyzed by immunohistochemistry in gingival biopsies. In addition, hBD-2 and IL-8 protein expression was monitored in GCF using the ELISA technology. RESULTS: Experimental gingivitis gradually developed with an increase in BOP scores and GCF volume over time. In GCF, elevated concentrations of hBD-2 and IL-8 were monitored at day 1, 5 and 7 (p ≤ 0.0002). Immunohistochemical analysis of gingival sections demonstrated increased staining for hBD-2 at day 3, whereas the CCL20, pso/S100A7, and IL-8 expression was increased at later time points (p < 0.05). CONCLUSION: For the first time, this study showed the time-dependent regulation of AMPs, following clinical signs of experimentally induced gingival inflammation. Differential temporal expression for AMPs may ensure a constant antimicrobial activity against changes in the bacterial composition of the growing dental biofilm.

Influence of histamine on the expression of CCL20 in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Histamine plays an important role during allergic and inflammatory reactions, and it has been suggested to influence periodontal inflammation. The aim of this study was to investigate the effects of histamine on the expression of the antimicrobial peptide C-C chemokine ligand 20 (CCL20) in human gingival fibroblasts (HGFs) when exposed to toll-like receptor (TLR) agonists. MATERIAL AND METHODS: Monolayers of HGFs from three different donors were exposed to histamine, alone, and in combination with Pam3CSK4 (a TLR2 agonist) or lipopolysaccharide (LPS) from Escherichia coli (a TLR4 agonist), for 2, 4, 6 or 12 h. In another experimental group, cells were pretreated with a specific histamine-1 receptor antagonist (H1R) antagonist, cetirizine. Real-time PCR analysis was performed to detect expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), CCL20 and interleukin-8 (IL8) genes. The levels of CCL20 and IL-8 protein were determined by ELISA. RESULTS: In HGFs, histamine induced expression of CCL20 and IL8 genes in a time-dependent manner (p < 0.05). Combined stimulation with histamine and Pam3CSK4 or LPS led to a significant amplification in expression of CCL20 and IL-8 when compared with treatment with each stimulant alone (p < 0.05), and this effect was mediated via pathways involving the H1R (p < 0.05). CONCLUSION: The results of this study suggest a sensitizing effect of histamine on early innate immune responses of HGFs when simultaneously exposed to bacterial virulence factors.

Effect of growth factors on antimicrobial peptides and pro-inflammatory mediators during wound healing.

BACKGROUND: Antimicrobial peptides (AMPs), such as human beta-defensin-2 (hBD-2) and the CC-chemokine ligand 20 (CCL20), exhibit direct microbicidal effects and mediator-like activity. It was hypothesized that wounding induces the expression of AMPs and pro-inflammatory mediators and that endogenous mediators, such as insulin-like growth factor-1 (IGF-1) and transforming growth factor-alpha (TGF-alpha), modulate this induced expression. MATERIAL AND METHODS: Monolayers of gingival epithelial cells (GECs) and gingival fibroblast (HGFs) from three different donors were wounded using the scratch assay (in vitro wounding) in the presence (test group) or absence (control group) of IGF-1 and TGF-alpha. In vitro wound closure was monitored over time (0, 6, 24, 48, 72 h), and wound areas were microscopically analyzed (Axio-Vision® Software, Zeiss). Gene expression analysis of the GAPDH, hBD-2, CCL20, interleukin-1 beta (IL-1 beta), and interleukin-8 (IL-8) was performed by qPCR. RESULTS: In comparison to control cells, IGF-1 and TGF-alpha significantly enhanced in vitro wound closure (P < 0.05). In GECs, IGF-1 induced the gene expression of IL-1 beta and IL-8 when compared to control cells (P < 0.05). In HGFs, wounding per se induced the messenger RNA of hBD-2, CCL20, and IL-1 beta, whereas IGF-1 and TGF-alpha reversed this effect (P < 0.05). CONCLUSION: In gingival cells, the gene expression of AMPs was altered by injury, and endogenous growth factors further influenced the expression profiles, but with high interindividual differences.

A 3' UTR transition within DEFB1 is associated with chronic and aggressive periodontitis.

Periodontal diseases are complex inflammatory diseases and affect up to 20% of the worldwide population. An unbalanced reaction of the immune system toward microbial pathogens is considered as the key factor in the development of periodontitis. Defensins have a strong antimicrobial function and are important contributors of the immune system toward maintaining health. Here, we present the first systematic association study of DEFB1. Using a haplotype-tagging single nucleotide polymorphism (SNP) approach, including described promoter SNPs of DEFB1, we investigated the associations of the selected variants in a large population (N=1337 cases and 2887 ethnically matched controls). The 3' untranslated region SNP, rs1047031, showed the most significant association signal for homozygous carriers of the rare A allele (P=0.002) with an increased genetic risk of 1.3 (95% confidence interval: 1.11-1.57). The association was consistent with the specific periodontitis forms: chronic periodontitis (odds ratio=2.2 (95% confidence interval: 1.16-4.35), P=0.02), and aggressive periodontitis (odds ratio=1.3 (95% confidence interval 1.04-1.68), P=0.02). Sequencing of regulatory and exonic regions of DEFB1 identified no other associated variant, pointing toward rs1047031 as likely being the causative variant. Prediction of microRNA targets identified a potential microRNA-binding site at the position of rs1047031.

The immune response of oral epithelial cells induced by single-species and complex naturally formed biofilms.

INTRODUCTION: In the oral cavity, the surfaces are constantly exposed to a complex variety of microorganisms organized in biofilms. As part of a sophisticated local immune response, gingival epithelial cells (GECs) express antimicrobial peptides, such as human beta-defensin-2 (hBD-2), ribonuclease 7 (RNAase-7), and psoriasin (PSO), and pro-inflammatory mediators, such as interleukin-8 (IL-8) and 5-lipoxygenase (5-LO). The aim of the present study was to test whether GECs show a differential immune response to single-species biofilms compared with multi-species biofilms. METHODS: GECs were cultured from biopsies derived from three different healthy donors (n = 3). To obtain naturally formed biofilm (NFB), polymer disks were attached to prostheses and carried intraorally for 12, 24, 36, and 48 h. In addition, single-species biofilms (SSB; Streptococcus mutans and Streptococcus mitis) were cultured on polymer disks in vitro (12, 24, 36, and 48 h). The messenger RNA (mRNA) expression of hBD-2, RNAase-7, PSO, IL-8, 5-LO, and glycerylaldehyde-3-phosphate dehydrogenase was analysed using semi-quantitative reverse transcription-polymerase chain reaction. RESULTS: In GECs, the hBD-2 mRNA expression was significantly upregulated in response to S. mitis-biofilm stimulation compared with S. mutans-biofilm stimulation (P < 0.0001). In contrast, the RNAase-7 mRNA expression was significantly higher in GECs when responding to both S. mutans biofilms and naturally formed biofilms compared with S. mitis biofilms (P < 0.0001 and P < 0.001, respectively). The IL-8 and 5-LO mRNA was significantly upregulated in response to S. mutans biofilms (P < 0.0001 and P = 0.0002, respectively). CONCLUSION: This in vitro study found biofilm-dependent expression of antimicrobial peptides and inflammatory mediators in GECs.

SELDI-TOF-MS of gingival crevicular fluid--a methodological approach.

Human neutrophil peptides (HNPs) and the human cathelicidin LL-37 are antimicrobial peptides secreted by neutrophils, which play a crucial role in innate immune responses. The aim of this study was to establish a new method for ProteinChip arrays in combination with surface enhanced laser desorption/ionization (SELDI) technology and time-of-flight mass spectrometry to analyze gingival crevicular fluid (GCF) samples. To optimize experimental conditions, four different ProteinChip arrays (NP20; CM10, pH 4; CM10, pH 7; IMAC) along with corresponding binding buffers were tested. GCF samples were collected from patients showing healthy periodontal sites and sites with early signs of inflammation (gingivitis), but with no pocket depth greater than 4 mm. For GCF analysis, NP20 arrays and CM10 (pH 4) arrays showed specific and reproducible profiles in the range of 2.5-30.0 kDa. Donors that demonstrated significantly higher intensity peaks corresponding to the mass of LL-37 (p=0.01) also tended to show greater intensity peaks corresponding to the masses of HNP-1 and HNP-2 in samples from inflamed compared to healthy periodontal sites. The findings indicate that analysis of GCF samples by SELDI-TOF mass spectrometry is a useful approach to simultaneously analyze multiple markers, such as antimicrobial peptides, which may be beneficial for determination of new periodontal risk factors.

Nuclear hBD-1 accumulation in malignant salivary gland tumours.

BACKGROUND: Whereas the antimicrobial peptides hBD-2 and -3 are related to inflammation, the constitutively expressed hBD-1 might function as 8p tumour suppressor gene and thus play a key role in control of transcription and induction of apoptosis in malignant epithelial tumours. Therefore this study was conducted to characterise proteins involved in cell cycle control and host defence in different benign and malignant salivary gland tumours in comparison with healthy salivary gland tissue. METHODS: 21 paraffin-embedded tissue samples of benign (n = 7), and malignant (n = 7) salivary gland tumours as well as healthy (n = 7) salivary glands were examined immunohistochemically for the expression of p53, bcl-2, and hBD-1, -2, -3. RESULTS: HBD-1 was distributed in the cytoplasm of healthy salivary glands and benign salivary gland tumours but seems to migrate into the nucleus of malignant salivary gland tumours. Pleomorphic adenomas showed cytoplasmic as well as weak nuclear hBD-1 staining. CONCLUSION: HBD-1, 2 and 3 are traceable in healthy salivary gland tissue as well as in benign and malignant salivary gland tumours. As hBD-1 is shifted from the cytoplasm to the nucleus in malignant salivary gland tumours, we hypothesize that it might play a role in the oncogenesis of these tumours. In pleomorphic adenomas hBD-1 might be connected to their biologic behaviour of recurrence and malignant transformation.