Generation and functional characterization of recombinant Porphyromonas gingivalis W83 FimA.

Porphyromonas gingivalis (P. gingivalis) is regarded as a keystone pathogen in destructive periodontal diseases. It expresses a variety of virulence factors, amongst them fimbriae that are involved in colonization, invasion, establishment and persistence of the bacteria inside the host cells. The fimbriae also were demonstrated to affect the host immune-response mechanisms. The major fimbriae are able to bind specifically to different host cells, amongst them peripheral blood monocytes. The interaction of these cells with fimbriae induces release of cytokines such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α (TNF-α). The aim of this study was to generate recombinant major FimA protein from P. gingivalis W83 fimbriae and to prove its biological activity. FimA of P. gingivalis W83 was amplified from chromosomal DNA, cloned in a vector and transferred into Listeria innocua. (L. innocua).The expressed protein was harvested and purified using FPLC via a His trap HP column. The identity and purity was demonstrated by gel-electrophoresis and mass-spectrometry. The biological activity was assessed by stimulation of human oral epithelial cells and peripheral blood monocytes with the protein and afterwards cytokines in the supernatants were quantified by enzyme linked immunosorbent assay (ELISA) and cytometric bead array. Recombinant FimA could successfully be generated and purified. Gel-electrophoresis and mass-spectrometry confirmed that the detected sequences are identical with FimA. Stimulation of human monocytes induced the release of high concentrations of IL-1ß, IL-6, IL-10 and TNF-α by these cells. In conclusion, a recombinant FimA protein was established and its biological activity was proven. This protein may serve as a promising agent for further investigation of its role in periodontitis and possible new therapeutic approaches.

Porphyromonas gingivalis Cell Wall Components Induce Programmed Death Ligand 1 (PD-L1) Expression on Human Oral Carcinoma Cells by a Receptor-Interacting Protein Kinase 2 (RIP2)-Dependent Mechanism.

Programmed death-ligand 1 (PD-L1/B7-H1) serves as a cosignaling molecule in cell-mediated immune responses and contributes to chronicity of inflammation and the escape of tumor cells from immunosurveillance. Here, we investigated the molecular mechanisms leading to PD-L1 upregulation in human oral carcinoma cells and in primary human gingival keratinocytes in response to infection with Porphyromonas gingivalis (P. gingivalis), a keystone pathogen for the development of periodontitis. The bacterial cell wall component peptidoglycan uses bacterial outer membrane vesicles to be taken up by cells. Internalized peptidoglycan triggers cytosolic receptors to induce PD-L1 expression in a myeloid differentiation primary response 88 (Myd88)-independent and receptor-interacting serine/threonine-protein kinase 2 (RIP2)-dependent fashion. Interference with the kinase activity of RIP2 or mitogen-activated protein (MAP) kinases interferes with inducible PD-L1 expression.

Effect of Periodontal Treatment on HbA1c among Patients with Prediabetes.

Evidence is limited regarding whether periodontal treatment improves hemoglobin A1c (HbA1c) among people with prediabetes and periodontal disease, and it is unknown whether improvement of metabolic status persists >3 mo. In an exploratory post hoc analysis of the multicenter randomized controlled trial "Antibiotika und Parodontitis" (Antibiotics and Periodontitis)-a prospective, stratified, double-blind study-we assessed whether nonsurgical periodontal treatment with or without an adjunctive systemic antibiotic treatment affects HbA1c and high-sensitivity C-reactive protein (hsCRP) levels among periodontitis patients with normal HbA1c (≤5.7%, n = 218), prediabetes (5.7% < HbA1c < 6.5%, n = 101), or unknown diabetes (HbA1c ≥ 6.5%, n = 8) over a period of 27.5 mo. Nonsurgical periodontal treatment reduced mean pocket probing depth by >1 mm in both groups. In the normal HbA1c group, HbA1c values remained unchanged at 5.0% (95% CI, 4.9% to 6.1%) during the observation period. Among periodontitis patients with prediabetes, HbA1c decreased from 5.9% (95% CI, 5.9% to 6.0%) to 5.4% (95% CI, 5.3% to 5.5%) at 15.5 mo and increased to 5.6% (95% CI, 5.4% to 5.7%) after 27.5 mo. At 27.5 mo, 46% of periodontitis patients with prediabetes had normal HbA1c levels, whereas 47.9% remained unchanged and 6.3% progressed to diabetes. Median hsCRP values were reduced in the normal HbA1c and prediabetes groups from 1.2 and 1.4 mg/L to 0.7 and 0.7 mg/L, respectively. Nonsurgical periodontal treatment may improve blood glucose values among periodontitis patients with prediabetes (ClinicalTrials.gov NCT00707369).

Induction of B7-H1 receptor by bacterial cells fractions of Porphyromonas gingivalis on human oral epithelial cells: B7-H1 induction by Porphyromonas gingivalis fractions.

The immune-regulatory B7-H1 receptor, also known as programmed death-ligand 1 (PD-L1), plays an important role in cell-mediated immune response. It is a co-signaling molecule that mediates regulation of T cell activation and tolerance and is able to negatively regulate activated T cell functions and survival. High expression of B7-H1 in host cells may contribute to the chronicity of inflammatory disorders and represents a possible mechanism of immune evasion. Porphyromonas gingivalis is regarded as a keystone pathogen in periodontitis and is able to invade host cells and disposes a variety of virulence factors including lipopolysaccharide (LPS), fimbriae and proteases such as gingipains. Based on previous studies that demonstrated the capability of P. gingivalis to induce up-regulation of PD-L1 in malignant and non-malignant oral epithelial cells, the aim of the present work was to analyse the potential of various cellular components of P. gingivalis to induce the PD-L1 receptor. Human squamous carcinoma cells and primary gingival keratinocytes were stimulated with total, inner and outer membrane fractions, cytosolic proteins, as well as LPS and peptidoglycans. PD-L1 protein expression was investigated by Western blot analysis and RT-PCR. It was demonstrated that the total membrane fraction induced the highest up-regulation in B7-H1 expression, followed by the outer and inner membrane, whereas cytosolic proteins and LPS did not. In conclusion, we provide evidence that the membrane fraction of P. gingivalis is responsible for up-regulation of the immune-regulatory receptor PD-L1 in squamous carcinoma cells and gingival keratinocytes, and thus may support immune evasion of oral carcinomas.

Oral Squamous Carcinoma Cells Express B7-H1 and B7-DC Receptors in Vivo.

B7-H1 and B7-DC ligands are members of the B7 family with important regulatory functions in cell-mediated immune response. Both receptors are ligands of the programmed death receptor PD-1. B7-H1 expression has been detected in the majority of human carcinomas in vivo. B7-H1 mediated signals are able to negatively regulate activated T cell functions and survival, and enable tumor cells to overcome host response. The aim of this study was to investigate the expression of B7-H1 and B7-DC proteins in oral squamous cell carcinomas (OSCC) in vivo. Tissues from 15 samples were cryo-sected and following histological routine staining (HE), incubated with antibodies against human B7-H1 and B7-DC. Immuno-staining of pan-cytokeratin was performed to ascertain the epithelial origin of the tissue and CK 19 to demonstrate the proliferating stage. Confocal laser scanning microscopy confirmed the presence of both B7-H1 and B7-DC in all 15 OSCC. The B7-H1 and B7-DC staining was located in areas of the tissue that were identified as cancerous lesions in the previously stained HE sections before. Staining with Pan-CK and CK19 provided evidence for the epithelial origin and the proliferating stage of the tissue. The in vivo expression of the B7-H1 and B7-DC receptors in oral squamous cell carcinomas suggest that general mechanisms for immune evasion of tumors are also found in OSCC.

Influence of retinoic acid on human gingival epithelial barriers.

BACKGROUND AND OBJECTIVES: The gingival epithelium plays an important role in the protection of oral tissues from microbial challenge. Oral keratinocytes form a barrier and show various cellular contacts, including tight junctions (TJ). To analyse the barrier function in vitro the transepithelial electrical resistance (TER) is commonly used. Retinoic acid (RA) is an important signalling molecule in most tissues, including epithelial differentiation. RA signalling is mediated through three RA receptors. The aim of the study was to investigate the influence of RA on human gingival barriers in vitro. MATERIAL AND METHODS: Immortalized human gingival keratinocytes were seeded on culture plate inserts. The effect of RA with and without infection with Porphyromonas gingivalis W83 on the barrier was analysed by TER measurements. The expression of TJ proteins was investigated by western blot. RESULTS: During differentiation, mean TER increased from 16 (1 h), 43 (4 h) to 62 (6 h) Ohm × cm2 . Addition of 15 µm RA increased TER by +19 after 1 h, +25 after 4 h and +16 Ohm × cm2 after 6 h. The pan-RA receptor inhibitor BMS 493 resulted in TER values comparable to the control. The mean established TER of the control was approximately 110 Ohm × cm2 . Addition of 15 µm RA elevated TER to 127 Ohm × cm2 after 1 h, 150 Ohm × cm2 after 4 h and 189 Ohm × cm2 after 6 h (p ≤ 0.01). RA plus infection with P. gingivalis W83 further increased the TER increasing effect but could not prevent the destruction of TER induced by bacterial infection. The protein expression of the TJ proteins claudin 4 and occludin was enhanced while ZO-1 was downregulated after 1 h of RA incubation. CONCLUSION: RA provides barrier-positive elements to the gingival epithelial cell model that is accompanied by altered expression of TJ proteins.

Peri-implant tissue destruction. The Third EAO Consensus Conference 2012.

OBJECTIVE: The task of this working group was to update the existing knowledge base regarding the prevalence of peri-implant tissue destruction, the role of occlusal overload, and the outcome of non-surgical and surgical treatment. MATERIALS AND METHODS: The literature was systematically searched and critically reviewed. Four manuscripts were presented in key areas deemed to be essential for the current understanding of the magnitude of the clinical entity peri-implantitis. The role of overload as an etiological component was reviewed. Also available data on the results from non-surgical and surgical interventions for the control of tissue destruction were presented. RESULTS: The consensus statements following plenary session approval, clinical implications, and directions for future research based on the group discussions are presented in this article. The results and conclusions of the systematic review process are presented by the respective authors in the subsequent papers.

Oral mucosa model based on a collagen-elastin matrix.

BACKGROUND AND OBJECTIVE: The collagen-elastin matrix (Matriderm(®)) is used to treat deep and full-thickness burns and was recently described as a suitable scaffold for tissue engineering. The aim of the present study was to investigate the biocompatibility of Matriderm(®) for gingival use through creation of an oral mucosa model ex vivo. MATERIAL AND METHODS: Gingival fibroblasts and keratinocytes were cultured. A dermal area on the base of the collagen-elastin matrix was repopulated with fibroblasts. After 14 days, keratinocytes were seeded on this dermal area to engineer a multilayered mucosa. Analysis of the architecture was performed using light and electron microscopy. Immunohistochemical detection of collagen IV and cytokeratin was carried out. RESULTS: Based on this scaffold we generated a multilayered oral mucosa-like structure. Histological, immunohistochemical and electron microscopic analysis of the dermal/epidermal junction showed a typical basement membrane and hemidesmosomal structures. Neighboring keratinocytes formed desmosomes in the epidermal sections. Cytokeratin was detectable in all epidermal layers. These experiments revealed that the collagen-elastin matrix was highly biocompatible with gingival cells under ex vivo conditions. CONCLUSION: Employing tissue-engineering techniques with dermal and epidermal cells from the gingiva, a multilayered oral mucosa was generated and characterized with respect to biocompatibility for Matriderm(®). The results indicate that Matriderm(®) is suitable for the ex vivo growth of gingival tissue cells and is a useful scaffold with possible applications in periodontal therapy.

[Diabetes mellitus and periodontitis. Bidirectional relationship and clinical implications. A consensus document]. / Diabetes mellitus und Parodontitis. Wechselbeziehung und klinische Implikationen. Ein Konsensuspapier.

Diabetes and periodontitis are chronic diseases with an increasing prevalence in the German population. There is a bi-directional relationship between both diseases. Diabetes promotes the occurrence, the progression and the severity of periodontitis. Periodontitis complicates the glycemic control of diabetes, increases the risk of diabetes-associated complications and possibly even of its onset. In view of the existing evidence, that is still not sufficiently communicated within the medical community, an expert panel consisting of four diabetologists and four periodontists has addressed the following questions: What is the effect of diabetes mellitus on periodontitis and on periodontal therapy? What is the effect of periodontitis on diabetes mellitus? What are the practical consequences, that result for interdisciplinary treatment strategies? The treatment of periodontal infections should become an integral part of the management of diabetes, whereas glycemic control is a prerequisite for successful periodontal therapy.

Production of interleukin-13 is influenced by the interleukin-4 -34TT and -590TT genotype in patients with aggressive periodontitis.

Aggressive periodontitis (AgP) is a specific form of periodontal disease, with rapid destruction of the tissues supporting the teeth in otherwise young healthy individuals. We recently showed a higher frequency of the interleukin-4 (IL-4) -34TT and -590TT genotype in AgP patients compared to controls (P<0.05). Herein, we demonstrated that this specific IL-4 genotype exerts its function by increasing expression of IL-4 and STAT6, and producing higher concentrations of IL-4 in activated CD4+ cells of patients with AgP. In the present study, we investigated the effects of the IL-4-specific genotype on IL-13, IL-2 and IFN-γ expression and production in activated CD4+ cells of patients with AgP and healthy controls. Results revealed higher IFN-γ and IL-2 expression and significantly increased IL-13 production in the cells of the patients who were homozygous for the -34T and -590T alleles in comparison with the patients who were homozygous for the -34C and -590C alleles (P<0.05). Results of controls with the -34C and -590C alleles were similar to those of AgP with the same genotype. To our knowledge, the present study is the first to show an effect of the -34TT and -590TT genotype on IL-13 production. There is an increased production of IL-13 by the T cells of aggressive periodontitis patients with the IL-4 genotype.

Scope, competences, learning outcomes and methods of periodontal education within the undergraduate dental curriculum: a consensus report of the 1st European Workshop on Periodontal Education--position paper 2 and consensus view 2.

The explosion in new knowledge in the last three decades has imposed important challenges in the training of all health workers, including dentists. These breakthroughs do not rapidly permeate educational cirricula. This is particularly relevant in Periodontology, because of recent breakthroughs in diagnostics and therapeutic approaches and by the advent of new knowledge on the implications of periodontal and peri-implant infections on systemic health and on ageing populations. Periodontology as one of the major oral health sciences requires a broad understanding of a spectrum of healthcare and basic sciences, together with specific education. In preparation for graduation, students must demonstrate a variety of acquired learning out-comes, which, in turn demand variety in learning and teaching methods. Based on the ADEE principles for dental education and in full respect of the Bolonga process, this paper describes the competencies and learning outcomes which are requested in periodontology for the dentist at graduation.

Use of platelet-rich plasma in periodontal surgery--a prospective randomised double blind clinical trial.

The aim of this prospective controlled randomized clinical trial was to evaluate the additional effect of platelet-rich plasma (PRP) in attachment gain. Twenty-two patients showing contralateral intrabony defects were included. Defects were randomized to beta-TCP (Cerasorb) in combination with PRP (test) or alone (control). Probing pocket depth (PPD), clinical attachment level (CAL), and relative AL (RAL) were assessed at the first, initial, re-evaluation (or basis examinations) and 6 months after surgery. Defect dimensions were recorded at baseline surgery (day 0) and during re-entry surgery (after 6 months), with vertical depth of the defect as primary outcome variable. An early healing index (EHI) was assessed 3 days, 1, 2 and 4 weeks after surgery. Both treatments led to clinical improvements. The median reduction of open vertical depth was 1.9 mm (interquartile intervals, 0.75 and 2.5 mm) at test sites, compared with 2.6 mm (1.8 and 3.5 mm) at control sites (p = 0.19, Wilcoxon). The median reductions of PPD and CAL at the four sites in close proximity to the defect in the interproximal area at test sites were 0.8 and 0.28 mm, and at control sites 0.4 and 0.13 mm, respectively. The EHI showed a reduction from grade 3 after 3 days to grade 1 after 4 weeks. PRP did not improve the results achieved with beta-TCP in the treatment of intrabony defects.

Establishment and characterization of immortalized human gingival keratinocyte cell lines.

BACKGROUND AND OBJECTIVE: Primary human keratinocytes are used to analyze the properties of the oral epithelium and the early stages of oral bacterial infections. In vitro, these cells are characterized by their short life span and restricted availability. Approaches for culturing these cells will end after approximately 6-10 passages as a result of entry into apoptosis. For this reason, it is important to generate cell lines suitable for obtaining an unlimited source of cells. Therefore, the aim of the present study was to generate gingival keratinocyte cell lines and to compare their in vitro behaviour with those of primary human gingival keratinocytes. MATERIAL AND METHODS: Primary human gingival keratinocytes were immortalized with a combination of the human papilloma virus onkoproteins E6 and E7. The pattern of the cytokeratins, involucrin and filaggrin was investigated by intracellular staining using flow cytometry. This method allows quantitative analysis of the expression of a variety of intracellular or extracellular markers. RESULTS: The immortalized cell lines showed many morphological similarities, expressing a cytokeratin pattern that is comparable with that of primary gingival keratinocytes. Furthermore, they developed transepithelial electrical resistance, which is a marker for the generation of tight junctions. These results indicate that the cells might be able to act as an epithelial barrier, reflecting the reaction of primary human cells. CONCLUSION: The establishment of a continuous line of human gingival epithelial cells with functional characteristics of the epithelial barrier provides a valuable in vitro model for using to study the early steps of gingival/periodontal infections.

Clinical outcomes after treatment of intra-bony defects with an EMD/synthetic bone graft or EMD alone: a multicentre randomized-controlled clinical trial.

OBJECTIVES: Comparison of the outcomes of a combination of an enamel matrix derivative and a synthetic bone graft (EMD/SBC) with EMD alone in wide intra-bony defects. MATERIAL AND METHODS: Seventy-three patients with chronic periodontitis were recruited in five centres in Germany. All patients had one wide intra-bony defect of >/=4 mm. Surgical procedures involved microsurgical technique and the modified papilla preservation flap. After debridement, defects were randomly assigned to EMD/SBC (test) or EMD (control). Assessments at baseline and after 6 months included bone sounding, attachment levels, probing pocket depths, bleeding on probing and recessions. Early wound-healing, adverse effects and patients' perceptions were also recorded. RESULTS: Both treatment modalities led to significant clinical improvements. Change in bone fill 6 months after surgery was 2.0 mm (+/- 2.1) in the test group and 2.1 mm (+/- 1.2) in the control group. A gain in clinical attachment of 1.3 mm (+/- 1.8) in the test group and 1.8 mm (+/- 1.6) in the control group was observed. One week after surgery, primary closure was maintained in 95% of the test sites and 100% of the control sites. No differences in patients' perceptions were found. CONCLUSION: The results of the present study showed similar clinical outcomes following both treatment modalities.

Single-nucleotide polymorphisms in the IL-4 and IL-13 promoter region in aggressive periodontitis.

INTRODUCTION: IL-4 and IL-13 polymorphisms have been shown to influence the susceptibility to systemic diseases. In this study, possible associations between the IL-4 -590 C-->T, IL-4 -34 C-->T, IL-13 -1112 C-->T and IL-13 -1512 A-->C promoter polymorphisms were investigated in subjects with generalized aggressive periodontitis (AgP) compared with healthy individuals. MATERIAL AND METHODS: Fifty-eight patients with diagnosis of generalized AgP and 51 matched healthy controls participated in the study. Blood samples were collected and DNA isolated. Molecular analyses were performed by PCR-RFLP in a blind fashion. Genotype and allele frequencies among study groups were compared using Fisher's exact test (alpha value: 0.05). Pearson's chi(2) test was used for analysis of Hardy-Weinberg equilibrium. RESULTS: The frequency of the IL-4 -590 T/T and IL-4 -34 T/T genotypes differed significantly between groups (p=0.05, 0.02, respectively), although the allele frequencies were similar. There was a higher frequency of the IL-4 -590 T/T and IL-4 -34 T/T genotypes in patients with AgP compared with controls. The genotype and allele frequencies of the IL-13 polymorphisms did not differ between groups. CONCLUSIONS: This study demonstrated an association between the IL-4 -590 T/T and IL-4 -34 T/T genotypes and AgP. Further research is necessary to prove if there is an association of these polymorphisms with AgP, and if the polymorphisms have a functional effect.

Interleukin-4 gene polymorphisms in Japanese and Caucasian patients with aggressive periodontitis.

OBJECTIVES: Recently, interleukin (IL) 4 gene polymorphisms have been analyzed in association with periodontitis. Genetic differences between Caucasian and Japanese patients with periodontitis have previously been detected. The aim of the present study was to analyze IL-4 genotypes in Caucasian and Japanese patients with aggressive periodontitis (AgP). MATERIAL AND METHODS: One hundred and twenty-four subjects were included in the study, 31 Japanese and 30 Caucasian patients with generalized AgP, plus 30 Japanese and 33 Caucasian healthy controls. IL-4 polymorphisms were determined by polymerase chain reaction. A logistic regression was used to investigate the possible association of the genotypes with the disease in both populations. Odds ratio (OR) estimates were analyzed for allele frequencies. RESULTS: No significant association of IL-4 polymorphisms with the risk of AgP was determined in either population. However, the allele frequencies showed different results between populations. The carriage of the polymorphism in intron 2 was higher in Caucasian patients compared with controls (OR: 2.0, 95% confidence interval: [1.0;4.2]. Furthermore, the frequency of the IL-4 promoter/intron 2 composite genotype (PP+/IP+) in patients and controls, respectively, was found to be approximately 25% and 60% higher in the Japanese population than in the Caucasian population. CONCLUSION: There was no evidence of an association of IL-4 genotypes and AgP in either population, although the frequencies of the IL-4 genotypes in the Japanese and the Caucasians were different.

Analysis of genetic polymorphisms at the interleukin-10 loci in aggressive and chronic periodontitis.

BACKGROUND/AIMS: Different cytokine genotypes have been described in periodontal disease. The aim of the present study was to investigate the genetic association of two previously described interleukin-10 (IL-10) polymorphisms in patients with aggressive (AP) and chronic periodontitis (CP) and to investigate possible associations with clinical manifestations. METHODS: Based on clinical parameters and radiographs, 23 patients with CP and 18 patients with AP were included in the study. Additionally, 21 age-matched healthy subjects served as a control group. Genomic DNA was isolated from whole blood samples and the IL-10 promoter sequences from positions - 597 to - 824 were amplified by polymerase chain reaction (PCR). Polymorphisms were detected by restriction-enzyme cleavage. The A and C alleles at the - 597 position were associated with the T and C alleles at the - 824 position, respectively. Fisher's exact test was used for the statistical analysis. RESULTS: No significant differences were observed in the allele frequencies between controls and AP patients (p = 0.70) or CP patients (p = 0.43), although the previously reported association between allele A at position - 597 and allele T at position - 824 was observed in our population. CONCLUSION: We conclude that the investigated polymorphisms are not associated with periodontal disease.

Influences of systemic diseases on periodontitis in children and adolescents.

Systemic diseases affecting the host response as primary immunodeficiencies or secondary defects caused by lack of nutrients or changes in the local tissues are very often accompanied by early-onset prepubertal periodontitis. Local treatment in combination with systemic antibiotics may in milder forms improve the situation, but in many cases the success is questionable and premature loss of teeth occurs. Since the genetic basis of many of the diseases has been identified, future developments permit the correction of at least some of these defects by gene therapy.

Interleukin-4 polymorphisms in early onset periodontitis.

BACKGROUND, AIMS: Periodontitis is the result of a complex interplay between oral bacteria and the host response, often modulated by behavioral factors. Early-onset periodontitis (EOP) is defined by the age of onset, the distribution of lesions and specific microbial pathogens. METHOD: Studies in twins suggested a genetic contribution to the pathogenesis of periodontitis. EOP heritable factors may be related to immune mechanisms which could enhance the pathogenic potential of plaque bacteria in susceptible individuals. Among others, Interleukin 4 (IL-4) is a potent cytokine in the immune response and is a potent down regulator of macrophage function. In the present study, we report a specific genotype of the IL-4 gene, which was detected by specific primers and PCR analysis. RESULTS: In the EOP-group 27.8% were IL-4 promotor- and intron polymorphism positive (PP+ and IP+). None of the age-matched healthy controls or patients with adult periodontitis (n=25) carried the markers. Moreover, serum IL-4 levels of PP+ and IP+ patients were below the detection limit and significantly different (p<0.01) from the IL-4 concentrations of healthy controls and PP- and IP- patients.