Consensus report of the second European Consensus Workshop on Education in Periodontology.

BACKGROUND: The second European Consensus Workshop on Education in Periodontology was commissioned, as a result of the changes in the discipline and the advances in educational methods/technology, to update the 2009 Consensus report of the first European Federation of Periodontology (EFP) Workshop on the same topic that was jointly authored by the Association for Dental Education in Europe. AIM: To identify and propose changes necessary in periodontal education at three levels, namely undergraduate, specialist and continuing professional development (CPD), with respect to learning outcomes, competencies and methods of learning/training and evaluation. METHODS: Four working groups (WGs) considered education in periodontology at the undergraduate, specialist and CPD levels, and education methods. Four commissioned position papers, one per WG, summarized the relevant information. Workshop participants gathered at an in-person consensus meeting to discuss the individual reviews, and this consensus report summarizes the conclusions. RESULTS: The learning outcomes for undergraduate and specialist education in periodontology have been updated, and a proposal for learning outcomes for CPD programmes was made. Learning/teaching/training and evaluation methods were proposed for each level of education, which included face-to-face, virtual and blended learning methods. CONCLUSION: Developments in oral/dental medicine and in contemporary educational technologies have been translated into updated learning outcomes and learning/teaching/ training/evaluation methods relevant to education in periodontology.

Continuing Professional Development (CPD) and Vocational Education and Training (VET) in Periodontology and Implant Dentistry.

AIM: To (i) evaluate structured postgraduate part-time programs in periodontology, including those addressing peri-implant diseases, among members of the European Federation of Periodontology (EFP), (ii) the impact of the 2018 classification scheme and EFP clinical practice guidelines and (iii) propose a framework for periodontal vocational education and training. MATERIALS AND METHODS: A summary of relevant European guidelines for vocational education and training was compiled. In a survey and in a systematic review, current part-time programs in continuing professional education in periodontology as well as in prevention and management of peri-implant diseases were examined. The implementation and dissemination of the 2018 classification scheme and the EFP clinical practice guidelines were assessed by literature analysis. Based on these findings, a framework for periodontal vocational education and training was generated. RESULTS: Part-time programs for professional development in periodontology are established in nine EFP member countries. The systematic review identified lack of knowledge in prevention and management of peri-implant diseases among dental practitioners and hygienists. Continuing professional development was found to be important for education in prevention, classification and management of periodontal as well as peri-implant diseases. The proposed European framework consists of an escalator model with three levels (certificate, diploma and master). DISCUSSION: Considering the identified variation in the national programs, there is a need to improve education in periodontal and peri-implant diseases. The proposed frameworkmay will help harmonize the national structures. CONCLUSION: The proposed framework for part-time professional development is expected to enhance professional qualification.

Prevention and treatment of peri-implant diseases-The EFP S3 level clinical practice guideline.

BACKGROUND: The recently published Clinical Practice Guidelines (CPGs) for the treatment of stages I-IV periodontitis provided evidence-based recommendations for treating periodontitis patients, defined according to the 2018 classification. Peri-implant diseases were also re-defined in the 2018 classification. It is well established that both peri-implant mucositis and peri-implantitis are highly prevalent. In addition, peri-implantitis is particularly challenging to manage and is accompanied by significant morbidity. AIM: To develop an S3 level CPG for the prevention and treatment of peri-implant diseases, focusing on the implementation of interdisciplinary approaches required to prevent the development of peri-implant diseases or their recurrence, and to treat/rehabilitate patients with dental implants following the development of peri-implant diseases. MATERIALS AND METHODS: This S3 level CPG was developed by the European Federation of Periodontology, following methodological guidance from the Association of Scientific Medical Societies in Germany and the Grading of Recommendations Assessment, Development and Evaluation process. A rigorous and transparent process included synthesis of relevant research in 13 specifically commissioned systematic reviews, evaluation of the quality and strength of evidence, formulation of specific recommendations, and a structured consensus process involving leading experts and a broad base of stakeholders. RESULTS: The S3 level CPG for the prevention and treatment of peri-implant diseases culminated in the recommendation for implementation of various different interventions before, during and after implant placement/loading. Prevention of peri-implant diseases should commence when dental implants are planned, surgically placed and prosthetically loaded. Once the implants are loaded and in function, a supportive peri-implant care programme should be structured, including periodical assessment of peri-implant tissue health. If peri-implant mucositis or peri-implantitis are detected, appropriate treatments for their management must be rendered. CONCLUSION: The present S3 level CPG informs clinical practice, health systems, policymakers and, indirectly, the public on the available and most effective modalities to maintain healthy peri-implant tissues, and to manage peri-implant diseases, according to the available evidence at the time of publication.

Treatment of pulpal and apical disease: The European Society of Endodontology (ESE) S3-level clinical practice guideline.

BACKGROUND: The ESE previously published quality guidelines for endodontic treatment in 2006; however, there have been significant changes since not only in clinical endodontics but also in consensus and guideline development processes. In the development of the inaugural S3-level clinical practice guidelines (CPG), a comprehensive systematic and methodologically robust guideline consultation process was followed in order to produce evidence-based recommendations for the management of patients presenting with pulpal and apical disease. AIM: To develop an S3-level CPG for the treatment of pulpal and apical disease, focusing on diagnosis and the implementation of the treatment approaches required to manage patients presenting with pulpitis and apical periodontitis (AP) with the ultimate goal of preventing tooth loss. METHODS: This S3-level CPG was developed by the ESE, with the assistance of independent methodological guidance provided by the Association of Scientific Medical Societies in Germany and utilizing the GRADE process. A robust, rigorous and transparent process included the analysis of relevant comparative research in 14 specifically commissioned systematic reviews, prior to evaluation of the quality and strength of evidence, the formulation of specific evidence and expert-based recommendations in a structured consensus process with leading endodontic experts and a broad base of external stakeholders. RESULTS: The S3-level CPG for the treatment of pulpal and apical disease describes in a series of clinical recommendations the effectiveness of diagnosing pulpitis and AP, prior to investigating the effectiveness of endodontic treatments in managing those diseases. Therapeutic strategies include the effectiveness of deep caries management in cases with, and without, spontaneous pain and pulp exposure, vital versus nonvital teeth, the effectiveness of root canal instrumentation, irrigation, dressing, root canal filling materials and adjunct intracanal procedures in the management of AP. Prior to treatment planning, the critical importance of history and case evaluation, aseptic techniques, appropriate training and re-evaluations during and after treatment is stressed. CONCLUSION: The first S3-level CPG in endodontics informs clinical practice, health systems, policymakers, other stakeholders and patients on the available and most effective treatments to manage patients with pulpitis and AP in order to preserve teeth over a patient's lifetime, according to the best comparative evidence currently available.

Under pressure-mechanisms and risk factors for orthodontically induced inflammatory root resorption: a systematic review.

BACKGROUND: The application of orthodontic forces causes root resorption of variable severity with potentially severe clinical ramifications. OBJECTIVE: To systematically review reports on the pathophysiological mechanisms of orthodontically induced inflammatory root resorption (OIIRR) and the associated risk factors based on in vitro, experimental, and in vivo studies. SEARCH METHODS: We undertook an electronic search of four databases and a separate hand-search. SELECTION CRITERIA: Studies reporting on the effect of orthodontic forces with/without the addition of potential risk factors on OIIRR, including (1) gene expression in in-vitro studies, the incidence root resorption in (2) animal studies, and (3) human studies. DATA COLLECTION AND ANALYSIS: Potential hits underwent a two-step selection, data extraction, quality assessment, and systematic appraisal performed by duplicate examiners. RESULTS: One hundred and eighteen articles met the eligibility criteria. Studies varied considerably in methodology, reporting of results, and variable risk of bias judgements.In summary, the variable evidence identified supports the notion that the application of orthodontic forces leads to (1) characteristic alterations of molecular expression profiles in vitro, (2) an increased rate of OIIRR in animal models, as well as (3) in human studies. Importantly, the additional presence of risk factors such as malocclusion, previous trauma, and medications like corticosteroids increased the severity of OIIRR, whilst other factors decreased its severity, including oral contraceptives, baicalin, and high caffeine. CONCLUSIONS: Based on the systematically reviewed evidence, OIIRR seems to be an inevitable consequence of the application of orthodontic forces-with different risk factors modifying its severity. Our review has identified several molecular mechanisms that can help explain this link between orthodontic forces and OIIRR. Nevertheless, it must be noted that the available eligible literature was in part significantly confounded by bias and was characterized by substantial methodological heterogeneity, suggesting that the results of this systematic review should be interpreted with caution. REGISTRATION: PROSPERO (CRD42021243431).

Treatment of stage IV periodontitis: The EFP S3 level clinical practice guideline.

BACKGROUND: The recently published clinical practice guideline (CPG) for the treatment of periodontitis in stages I-III provided evidence-based recommendations for the treatment of periodontitis patients, defined according to the 2018 classification. Stage IV periodontitis shares the severity and complexity characteristics of stage III periodontitis, but includes the anatomical and functional sequelae of tooth and periodontal attachment loss (tooth flaring and drifting, bite collapse, etc.), which require additional interventions following completion of active periodontal therapy. AIM: To develop an S3 Level CPG for the treatment of stage IV periodontitis, focusing on the implementation of inter-disciplinary treatment approaches required to treat/rehabilitate patients following associated sequelae and tooth loss. MATERIALS AND METHODS: This S3 Level CPG was developed by the European Federation of Periodontology (EFP), following methodological guidance from the Association of Scientific Medical Societies in Germany and the Grading of Recommendations Assessment, Development and Evaluation (GRADE) process. A rigorous and transparent process included synthesis of relevant research in 13 specifically commissioned systematic reviews, evaluation of the quality and strength of evidence, the formulation of specific recommendations and a structured consensus process with leading experts and a broad base of stakeholders. RESULTS: The S3 Level CPG for the treatment of stage IV periodontitis culminated in recommendations for different interventions, including orthodontic tooth movement, tooth splinting, occlusal adjustment, tooth- or implant-supported fixed or removable dental prostheses and supportive periodontal care. Prior to treatment planning, it is critically important to undertake a definitive and comprehensive diagnosis and case evaluation, obtain relevant patient information, and engage in frequent re-evaluations during and after treatment. The periodontal component of therapy should follow the CPG for the treatment of periodontitis in stages I-III. CONCLUSIONS: The present S3 Level CPG informs clinical practice, health systems, policymakers and, indirectly, the public on the available and most effective modalities to treat patients with stage IV periodontitis and to maintain a healthy dentition over lifetime, according to the available evidence at the time of publication.

Differential DNA methylation and mRNA transcription in gingival tissues in periodontal health and disease.

AIM: We investigated differential DNA methylation in gingival tissues in periodontal health, gingivitis, and periodontitis, and its association with differential mRNA expression. MATERIALS AND METHODS: Gingival tissues were harvested from individuals and sites with clinically healthy and intact periodontium, gingivitis, and periodontitis. Samples were processed for differential DNA methylation and mRNA expression using the IlluminaEPIC (850 K) and the IlluminaHiSeq2000 platforms, respectively. Across the three phenotypes, we identified differentially methylated CpG sites and regions, differentially expressed genes (DEGs), and genes with concomitant differential methylation at their promoters and expression were identified. The findings were validated using our earlier databases using HG-U133Plus2.0Affymetrix microarrays and Illumina (450 K) methylation arrays. RESULTS: We observed 43,631 differentially methylated positions (DMPs) between periodontitis and health, and 536 DMPs between gingivitis and health (FDR < 0.05). On the mRNA level, statistically significant DEGs were observed only between periodontitis and health (n = 126). Twelve DEGs between periodontitis and health (DCC, KCNA3, KCNA2, RIMS2, HOXB7, PNOC, IRX1, JSRP1, TBX1, OPCML, CECR1, SCN4B) were also differentially methylated between the two phenotypes. Spearman correlations between methylation and expression in the EPIC/mRNAseq dataset were largely replicated in the 450 K/Affymetrix datasets. CONCLUSIONS: Concomitant study of DNA methylation and gene expression patterns may identify genes whose expression is epigenetically regulated in periodontitis.

Influence of soft tissue augmentation procedures around dental implants on marginal bone level changes-A systematic review.

OBJECTIVES: This systematic review assessed the influence of soft tissue augmentation procedures on marginal bone level changes in partial or fully edentulous patients. MATERIAL AND METHODS: We identified three relevant PICO questions related to soft tissue augmentation procedures and conducted a systematic search of four major electronic databases for clinical studies in systemically healthy patients receiving at least one dental implant and a minimum follow-up of one year after implant placement. The primary outcome was mean difference in marginal bone levels, and secondary outcomes were clinical and patient-related outcomes such as thickness of peri-implant mucosa, bleeding indices, and Pink Esthetic Score. RESULTS: We identified 20 publications reporting on 16 relevant comparisons. Studies varied considerably and thus only two meta-analyses could be performed. This systematic review showed that: Soft tissue augmentation either for augmentation of keratinized mucosa or soft tissue volume inconsistently had an effect on marginal bone level changes when compared to no soft tissue augmentation, but consistently improved secondary outcomes. The combination soft and hard tissue augmentation showed no statistically significant difference in terms of marginal bone level changes when compared to hard tissue augmentation alone, but resulted in less marginal soft tissue recession as shown by a meta-analysis. Soft or hard tissue augmentation performed as contour augmentations resulted in comparable marginal bone level changes. CONCLUSIONS: Peri-implant soft and hard tissues seem to have a bidirectional relationship: "Bone stands hard, but soft tissue is the guard".

Treatment of stage I-III periodontitis-The EFP S3 level clinical practice guideline.

BACKGROUND: The recently introduced 2017 World Workshop on the classification of periodontitis, incorporating stages and grades of disease, aims to link disease classification with approaches to prevention and treatment, as it describes not only disease severity and extent but also the degree of complexity and an individual's risk. There is, therefore, a need for evidence-based clinical guidelines providing recommendations to treat periodontitis. AIM: The objective of the current project was to develop a S3 Level Clinical Practice Guideline (CPG) for the treatment of Stage I-III periodontitis. MATERIAL AND METHODS: This S3 CPG was developed under the auspices of the European Federation of Periodontology (EFP), following the methodological guidance of the Association of Scientific Medical Societies in Germany and the Grading of Recommendations Assessment, Development and Evaluation (GRADE). The rigorous and transparent process included synthesis of relevant research in 15 specifically commissioned systematic reviews, evaluation of the quality and strength of evidence, the formulation of specific recommendations and consensus, on those recommendations, by leading experts and a broad base of stakeholders. RESULTS: The S3 CPG approaches the treatment of periodontitis (stages I, II and III) using a pre-established stepwise approach to therapy that, depending on the disease stage, should be incremental, each including different interventions. Consensus was achieved on recommendations covering different interventions, aimed at (a) behavioural changes, supragingival biofilm, gingival inflammation and risk factor control; (b) supra- and sub-gingival instrumentation, with and without adjunctive therapies; (c) different types of periodontal surgical interventions; and (d) the necessary supportive periodontal care to extend benefits over time. CONCLUSION: This S3 guideline informs clinical practice, health systems, policymakers and, indirectly, the public on the available and most effective modalities to treat periodontitis and to maintain a healthy dentition for a lifetime, according to the available evidence at the time of publication.

Biological factors involved in alveolar bone regeneration: Consensus report of Working Group 1 of the 15th European Workshop on Periodontology on Bone Regeneration.

BACKGROUND AND AIMS: To describe the biology of alveolar bone regeneration. MATERIAL AND METHODS: Four comprehensive reviews were performed on (a) mesenchymal cells and differentiation factors leading to bone formation; (b) the critical interplay between bone resorbing and formative cells; (c) the role of osteoimmunology in the formation and maintenance of alveolar bone; and (d) the self-regenerative capacity following bone injury or tooth extraction were prepared prior to the workshop. RESULTS AND CONCLUSIONS: This summary information adds to the fuller understanding of the alveolar bone regenerative response with implications to reconstructive procedures for patient oral rehabilitation. The group collectively formulated and addressed critical questions based on each of the reviews in this consensus report to advance the field. The report concludes with identified areas of future research.

The severity of human peri-implantitis lesions correlates with the level of submucosal microbial dysbiosis.

AIM: To cross-sectionally analyse the submucosal microbiome of peri-implantitis (PI) lesions at different severity levels. MATERIALS AND METHODS: Microbial signatures of 45 submucosal plaque samples from untreated PI lesions obtained from 30 non-smoking, systemically healthy subjects were assessed by 16s sequencing. Linear mixed models were used to identify taxa with differential abundance by probing depth, after correction for age, gender, and multiple samples per subject. Network analyses were performed to identify groups of taxa with mutual occurrence or exclusion. Subsequently, the effects of peri-implant probing depth on submucosal microbial dysbiosis were calculated using the microbial dysbiosis index. RESULTS: In total, we identified 337 different taxa in the submucosal microbiome of PI. Total abundance of 12 taxa correlated significantly with increasing probing depth; a significant relationship with lower probing depth was found for 16 taxa. Network analysis identified two mutually exclusive complexes associated with shallow pockets and deeper pockets, respectively. Deeper peri-implant pockets were associated with significantly increased dysbiosis. CONCLUSION: Increases in peri-implant pocket depth are associated with substantial changes in the submucosal microbiome and increasing levels of dysbiosis.

Periodontitis: Consensus report of workgroup 2 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions.

A new periodontitis classification scheme has been adopted, in which forms of the disease previously recognized as "chronic" or "aggressive" are now grouped under a single category ("periodontitis") and are further characterized based on a multi-dimensional staging and grading system. Staging is largely dependent upon the severity of disease at presentation as well as on the complexity of disease management, while grading provides supplemental information about biological features of the disease including a history-based analysis of the rate of periodontitis progression; assessment of the risk for further progression; analysis of possible poor outcomes of treatment; and assessment of the risk that the disease or its treatment may negatively affect the general health of the patient. Necrotizing periodontal diseases, whose characteristic clinical phenotype includes typical features (papilla necrosis, bleeding, and pain) and are associated with host immune response impairments, remain a distinct periodontitis category. Endodontic-periodontal lesions, defined by a pathological communication between the pulpal and periodontal tissues at a given tooth, occur in either an acute or a chronic form, and are classified according to signs and symptoms that have direct impact on their prognosis and treatment. Periodontal abscesses are defined as acute lesions characterized by localized accumulation of pus within the gingival wall of the periodontal pocket/sulcus, rapid tissue destruction and are associated with risk for systemic dissemination.

MicroRNAs Regulate Cytokine Responses in Gingival Epithelial Cells.

MicroRNAs (miRNAs) have been established as key regulators of various biological processes with possible involvement in the pathobiology of periodontal disease. Expanding our earlier observations of substantial differential expression of specific miRNAs between clinically healthy and periodontitis-affected gingival tissues, we used miRNA inhibitors (sponges) in loss-of-function experiments to investigate the involvement of specific miRNAs in the response of pocket epithelium-derived, telomerase-immortalized human gingival keratinocytes (TIGKs) to microbial infection. We constructed stable knockdown (KD) cell lines for five epithelium-expressed miRNAs (miR-126, miR-141, miR-155, miR-210, and miR-1246) and assessed their response to infection with periodontal pathogens using microarray analysis, quantitative PCR (qPCR), enzyme-linked immunosorbent assay (ELISA), and Western blot assay. miR-126 KD cells showed lower expression of interleukin 8 (IL-8) and CXCL1, both on the mRNA and protein levels, than did controls upon stimulation by heat-killed wild-type Porphyromonas gingivalis, live P. gingivalis protease-deficient mutant KDP128, and live Aggregatibacter actinomycetemcomitans In contrast, infection of miR-155 KD and miR-210 KD cells with the same organisms resulted in higher IL-8 and CXCL1 mRNA and protein expression. These effects appeared to be regulated by NF-κB, as suggested by altered transcription and/or phosphorylation status of components of the NF-κB system. Reduced neutrophil-like HL-60 cell chemotactic activity was observed in response to infection of miR-126 KD cells, indicating that miR-126 plays an important role in immune responses. Our findings indicate that specific miRNAs regulate the expression of inflammatory cytokines in human gingival epithelial cells in response to microbial infection.

Mini but mighty: microRNAs in the pathobiology of periodontal disease.

MicroRNAs (miRNAs) are a family of small, noncoding RNA molecules that negatively regulate protein expression either by inhibiting initiation of the translation of mRNA or by inducing the degradation of mRNA molecules. Accumulating evidence suggests that miRNA-mediated repression of protein expression is of paramount importance in a broad range of physiologic and pathologic conditions. In particular, miRNA-induced dysregulation of molecular processes involved in inflammatory pathways has been shown to contribute to the development of chronic inflammatory diseases. In this review, first of all we provide an overview of miRNA biogenesis, the main mechanisms of action and the miRNA profiling tools currently available. Then, we summarize the available evidence supporting a specific role for miRNAs in the pathobiology of periodontitis. Based on a review of available data on the differential expression of miRNAs in gingival tissues in states of periodontal health and disease, we address specific roles for miRNAs in molecular and cellular pathways causally linked to periodontitis. Our review points to several lines of evidence suggesting the involvement of miRNAs in periodontal tissue homeostasis and pathology. Although the intricate regulatory networks affected by miRNA function are still incompletely mapped, further utilization of systems biology tools is expected to enhance our understanding of the pathobiology of periodontitis.

Activation of invariant NK T cells in periodontitis lesions.

Periodontitis is one of the most prevalent human inflammatory diseases. The major clinical phenotypes of this polymicrobial, biofilm-mediated disease are chronic and aggressive periodontitis, the latter being characterized by a rapid course of destruction that is generally attributed to an altered immune-inflammatory response against periodontal pathogens. Still, the biological basis for the pathophysiological distinction of the two disease categories has not been well documented yet. Type I NKT cells are a lymphocyte subset with important roles in regulating immune responses to either tolerance or immunity, including immune responses against bacterial pathogens. In this study, we delineate the mechanisms of NKT cell activation in periodontal infections. We show an infiltration of type I NKT cells in aggressive, but not chronic, periodontitis lesions in vivo. Murine dendritic cells infected with aggressive periodontitis-associated Aggregatibacter actinomycetemcomitans triggered a type I IFN response followed by type I NKT cell activation. In contrast, infection with Porphyromonas gingivalis, a principal pathogen in chronic periodontitis, did not induce NKT cell activation. This difference could be explained by the absence of a type I IFN response to P. gingivalis infection. We found these IFNs to be critical for NKT cell activation. Our study provides a conceivable biological distinction between the two periodontitis subforms and identifies factors required for the activation of the immune system in response to periodontal bacteria.

Porcine dermal matrix in the treatment of dehiscence-type defects--an experimental split-mouth animal trial.

OBJECTIVES: To describe histometrical outcomes (tissue thickness, tissue height) of a porcine dermal matrix (PDX) and subepithelial connective tissue (CTG) in the treatment of dehiscence-type defects. MATERIAL AND METHODS: In five beagle dogs buccal dehiscence defects were created on both upper canines. The defects were covered in a split-mouth design either with a porcine dermal matrix or subepithelial connective tissue. After 4 months histometrical outcomes were evaluated using a nonparametric Brunner-Langer model. RESULTS: Neither in the test nor in the control specimen signs of inflammation or foreign body reaction was detected. Histometrically, no significant difference was found for tissue thickness and height between both treatment groups. CONCLUSIONS: Porcine dermal matrix can be used for grafting of dehiscence-type defects. Augmentation of tissue thickness seems to be comparable to subepithelial connective tissue.

On the relationship between gingival biotypes and gingival thickness in young Caucasians.

OBJECTIVES: To evaluate a possible relationship between gingival biotypes and gingival thickness, papilla height and gingival width. MATERIAL AND METHODS: Thirty-six adult subjects were stratified by their gingival biotype (GB), as defined by transparency of a periodontal probe through the buccal gingival margin, into "thin" (18 subjects) and "thick" (18 subjects) GB. Out of these, extreme cases (6 "very thin", 6 "very thick") were identified. Four different parameters were assessed: gingival thickness (GT), papilla height (PH), probing depth (PD) and gingival width (GW). RESULTS: When comparing "thin" and "thick" GB, midfacial GT (0.40 ± 0.07 vs. 0.72 ± 0.11 mm; P < 0.0001), PH (3.76 ± 0.50 vs. 3.95 ± 0.41 mm, P = 0.02) and GW (3.01 ± 1.26 vs. 4.63 ± 0.86 mm, P = 0.04) were lower in the "thin" GB group. Further stratification into moderately and extremely "thin"/"thick" GB eliminated the differences between the moderate groups. CONCLUSION: Our data support the traditional hypothesis that two different gingival biotypes with concomitant properties distinguishable by gingival transparency exist. In addition, we provide evidence that an alternative classification into "very thick", "moderate" and "very thin" biotypes might be advantageous, because the unique properties were seemingly primarily driven by subjects with extreme values.

Peripheral venous congestion causes inflammation, neurohormonal, and endothelial cell activation.

AIMS: Volume overload and venous congestion are typically viewed as a consequence of advanced and of acute heart failure (HF) and renal failure (RF) although it is possible that hypervolaemia itself might be a critical intermediate in the pathophysiology of these diseases. This study aimed at elucidating whether peripheral venous congestion is sufficient to promote changes in inflammatory, neurohormonal, and endothelial phenotype similar to those observed in HF and RF. METHODS: To experimentally model peripheral venous congestion, we developed a new method (so-called venous stress test) and applied the methodology on 24 healthy subjects (14 men, age 35 ± 2 years). Venous arm pressure was increased to ∼30 mmHg above the baseline level by inflating a tourniquet cuff around the dominant arm (test arm). Blood and endothelial cells (ECs) were sampled from test and control arm (lacking an inflated cuff) before and after 75 min of venous congestion, using angiocatheters and endovascular wires. Magnetic beads coated with EC-specific antibodies were used for EC separation; amplified mRNA was analysed by Affymetrix HG-U133 Plus 2.0 Microarray. RESULTS: Plasma interleukin-6 (IL-6), endothelin-1 (ET-1), angiotensin II (AII), vascular cell adhesion molecule-1 (VCAM-1), and chemokine (C-X-C motif) ligand 2 (CXCL2) were significantly increased in the congested arm. A total of 3437 mRNA probe sets were differentially expressed (P < 0.05) in venous ECs before vs. after testing, including ET-1, VCAM-1, and CXCL2. CONCLUSION: Peripheral venous congestion causes release of inflammatory mediators, neurohormones, and activation of ECs. Overall, venous congestion mimicked, notable aspects of the phenotype typical of advanced and of acute HF and RF.

Inhibition of endocannabinoid-degrading enzyme fatty acid amide hydrolase increases atherosclerotic plaque vulnerability in mice.

The role of endocannabinoids such as anandamide during atherogenesis remains largely unknown. Fatty acid amide hydrolase (FAAH) represents the key enzyme in anandamide degradation, and its inhibition is associated with subsequent higher levels of anandamide. Here, we tested whether selective inhibition of FAAH influences the progression of atherosclerosis in mice. Selective inhibition of FAAH using URB597 resulted in significantly increased plasma levels of anandamide compared to control, as assessed by mass spectrometry experiments in mice. Apolipoprotein E-deficient (ApoE(-/-)) mice were fed a high-fat, cholesterol-rich diet to induce atherosclerotic conditions. Simultaneously, mice received either the pharmacological FAAH inhibitor URB597 1mg/kg body weight (n=28) or vehicle (n=25) via intraperitoneal injection three times a week. After eight weeks, mice were sacrificed, and experiments were performed. Vascular superoxide generation did not differ between both groups, as measured by L012 assay. To determine whether selective inhibition of FAAH affects atherosclerotic plaque inflammation, immunohistochemical staining of the aortic root was performed. Atherosclerotic plaque formation, vascular macrophage accumulation, as well as vascular T cell infiltration did not differ between both groups. Interestingly, neutrophil cell accumulation was significantly increased in mice receiving URB597 compared to control. Vascular collagen structures in atherosclerotic plaques were significantly diminished in mice treated with URB597 compared to control, as assessed by picro-sirius-red staining. This was accompanied by an increased aortic expression of matrix metalloproteinase-9, as determined by quantitative RT-PCR and western blot analysis. Inhibition of fatty acid amide hydrolase does not influence plaque size but increases plaque vulnerability in mice.

Prognostic significance of phospho-histone H3 in prostate carcinoma.

PURPOSE: Prostate cancer is the second most common cancer in men and the sixth most common cause of death from cancer in men worldwide. Currently, a sufficient pathological distinction between patients requiring further treatment and those for which active surveillance remains an option is still lacking, which leads to the problem of overtreatment. Cell proliferation is routinely assessed by detecting Ki-67 antigen. While Ki-67 is expressed throughout the interphase of proliferating cells, phosphorylation of the chromatin constituent histone H3 occurs only during the late G2 phase and mitosis thus providing a more strict assessment of the mitotic activity. We undertook this study to test whether expression of the recently introduced proliferation marker phospho-histone H3 (pHH3) in prostate carcinoma tissue sections exhibits prognostic significance in comparison with Ki-67. METHODS: Protein expression of pHH3 and Ki-67 was assessed on TMA consisting of paraffin-embedded tissue from men that had undergone radical prostatectomy. The analysis included triplicate tissue cores of a total of 339 tumor foci. Immunohistochemical staining of pHH3 and Ki-67 was performed and analyzed using Definiens imaging software. RESULTS: Prostate cancer tissue exhibited a significantly higher frequency of pHH3-positive cells compared to benign prostate tissue. pHH3 expression was significantly correlated with Ki-67 expression. Furthermore, statistical analysis revealed positive correlation between pHH3 expression and PSA levels at diagnosis and in addition negatively correlated with overall survival. In contrast to Ki-67 staining, pHH3 expression did not correlate with Gleason grade. CONCLUSION: Our data point to a conceivable role of pHH3 as prognostic biomarker in prostate carcinoma.